Prediction of hepatocellular carcinoma and liver-related events in anti-HDV positive individuals.

BACKGROUND AND AIMS: Chronic hepatitis D (CHD) is the most severe form of chronic viral hepatitis, with a high risk of developing hepatocellular carcinoma (HCC) and liver-related mortality. Risk stratification is needed to guide HCC surveillance strategies and to prioritize treatment with antiviral agents. METHODS: We conducted a multicenter retrospective cohort of anti-HDV positive individuals managed at sites in the Netherlands and the United Kingdom. We studied the 5-year cumulative incidences of HCC and liver-related events (first of HCC, liver transplantation and liver-related mortality), in the overall cohort and among relevant subgroups. RESULTS: We analyzed 269 anti-HDV positive individuals with a median follow-up of 4.3 years in which 47 first events occurred. The 5-year cumulative incidences of HCC and liver-related events were 3.8% and 15.6% in the overall cohort. The 5-year cumulative incidence of HCC and liver-related events for individuals without cirrhosis was 0% and 0.9% compared to 12% and 41.3% for individuals with cirrhosis (p<0.001). The 5-year cumulative incidence of HCC and liver-related events was 0% and 2.1% among individuals with low PAGE-B scores, compared to 3.2% and 21.1% with intermediate and 25.4% and 45.5% with high risk scores (p<0.001). We found comparable results for the FIB-4 score. Findings were consistent regardless of cirrhosis or detectable HDV RNA (p<0.001). CONCLUSION: Anti-HDV positive individuals are at high risk of adverse liver-related outcomes. The incidences of HCC was negligible among individuals without cirrhosis and among individuals with low baseline PAGE-B and/or FIB-4 scores. Therefore, these score can be used to guide HCC surveillance strategies and potentially also for treatment prioritization.

Hepatitis delta genotype 5 is associated with favourable disease outcome and better response to treatment compared to genotype 1.

BACKGROUND & AIMS: Coinfection with HDV causes rapid progression to liver cirrhosis and hepatic decompensation in patients with chronic hepatitis B. Factors that are associated with disease progression are poorly understood. In this study we aim to identify risk factors associated with disease progression and better characterise clinical differences and treatment response between HDV genotype 1 and 5. METHODS: In this retrospective study, all patients under our care between 2005 and 2016 with HBV/HDV coinfection (HBsAg+, anti-HDV antibodies positive) were analysed. Patients were excluded if follow-up was less than 6 months, if they had HCV and/or HIV coinfection or an acute HDV infection. Demographic data, stage of liver disease, development of liver complications and treatment response were recorded. RESULTS: One-hundred seven patients (mean age 36.0 years, 57% male) were followed for a median period of 4.4 years (range 0.6-28.1 years); 64% were of African origin and 17% were of European origin, with 28% of patients being cirrhotic at first visit; 43% patients had actively replicating HDV virus (anti-HDV-IgG+, anti-HDV-IgM+ or HDV RNA+) and 57% of patients were HDV exposed (anti-HDV-IgG+, HDV RNA-). Patients with actively replicating HDV more often developed liver complications than HDV-exposed patients (p = 0.002), but no differences in baseline characteristics were observed. Patients with HDV genotype 5 less often developed cirrhosis or hepatic decompensation compared to patients with HDV genotype 1. Twenty-four patients were treated with peg-IFN and post-treatment response was significantly better in patients infected with genotype 5 (10% GT1 vs. 64% GT5, p = 0.013). CONCLUSION: Patients infected with HDV genotype 5 appear to have a better prognosis with fewer episodes of hepatic decompensation and better response to peg-IFN treatment than patients infected with HDV genotype 1. LAY SUMMARY: Hepatitis delta is a virus that affects the liver. The virus is known to have different subtypes, called genotypes. With this research we discovered that hepatitis delta virus genotype 1 behaves differently than genotype 5 and causes faster development of liver disease. This is important for education of our patients and to determine how often we need to check our patients.

Immunological Analysis During Interferon-Free Therapy for Chronic Hepatitis C Virus Infection Reveals Modulation of the Natural Killer Cell Compartment.

BACKGROUND: Chronic hepatitis C virus (HCV) infection is a global health problem, resulting in liver failure, hepatocellular carcinoma, and liver-related death. Natural killer (NK) cells are innate immune cells, and their activity is known to correlate to viral treatment response of HCV. In this study, we investigate the immune effects of viral load decline with direct-acting antivirals (DAAs) in blood. METHODS: Twelve patients with chronic HCV were treated with asunaprevir and daclatasvir, and peripheral blood was analyzed at various time points during therapy. RESULTS: In line with previous studies, we confirmed restoration of HCV-specific T-cell frequency upon viral load decline. In addition, we show that serum interferon (IFN)-γ inducible-protein 10, interleukin (IL)-12p40, and IL-18 levels decreased early after start of therapy. Surface expression of activation receptors NKp30, NKp46, and inhibitory receptor NKG2A on blood NK cells reduced during therapy. In addition, the expression of TRAIL on NK cells was reduced during IFN-free therapy, suggesting a decrease in TRAIL-mediated killing by NK cells. CONCLUSIONS: We show that viral load decline as a consequence of treatment with novel DAAs in chronic HCV patients reduces serum levels of NK cell-stimulating cytokines and causes correction of the altered NK cell phenotype observed in chronic HCV patients. CLINICAL TRIALS REGISTRATION: NCT02282709.

The Intrahepatic T Cell Compartment Does Not Normalize Years After Therapy-Induced Hepatitis C Virus Eradication.

Little is known about the immune status in liver and blood of patients with chronic hepatitis C virus (HCV) infection long after therapy-induced viral clearance. In this study, we demonstrate that, 4 years after clearance, regulation of HCV-specific immunity in blood by regulatory T cells (Tregs) and the immunosuppressive cytokines interleukin 10 and transforming growth factor ß is still ongoing. Importantly, analysis of liver specimens collected 4 years after HCV clearance shows that intrahepatic Tregs are still present in all patients, suggesting that liver T cells remain regulated. Identifying mechanisms that regulate HCV-specific memory T-cell responses after clearance is highly relevant for the development of protective vaccines, especially in patients at high risk of reinfection.

CD4+ CXCR5+ T cells in chronic HCV infection produce less IL-21, yet are efficient at supporting B cell responses.

BACKGROUND & AIMS: During chronic HCV infection, T cell dependent virus-specific antibodies are produced. However, the role of B-T cell interaction in chronic HCV is largely unknown. CD4(+)CXCR5(+) T follicular helper (TFH)-cells activate B cells and are important for clearance of various chronic viral infections. We investigated the function of TFH cells and B cells in liver and in peripheral blood of chronic HCV patients. METHODS: T cells from chronic HCV patients and healthy individuals were analysed for expression of CXCR5, PD-1, ICOS, and IL-21 and IFN-γ production by flow cytometry. CD19(+) B cell subpopulations were identified on the basis of CD27 and IgD expression. In order to assess the frequency and function of T cells and B cells in liver follicles, immunohistochemistry was performed for CD3, CXCR5, Bcl6, IL-21, CD20, IgD, IgM, and IgG. RESULTS: The frequency of IL-21-producing CXCR5(+)CD4(+) T cells in blood was lower in HCV patients compared to healthy individuals (p=0.002), which was reflected by lower serum IL-21 levels (p<0.001). Nonetheless, CXCR5(+)CD4(+) T cells from HCV patients and healthy individuals were equally capable to stimulate CD19(+)CD27(+) memory B cells into IgG and IgM-producing plasmablasts. Importantly, human intrahepatic TFH cells and their related function were identified by immunohistochemistry on liver biopsies for CD3, Bcl6, and CD20 within portal areas and follicles. CONCLUSIONS: The specific localization of TFH cells and IgG and IgD/IgM-producing B cells suggests a functional B-T cell environment in liver follicles during HCV infection. The decreased frequency of IL-21-producing CXCR5(+)CD4(+) T cells and lower serum IL-21 levels in chronic HCV patients did not lead to an altered TFH-B cell interaction.

Inhibitory receptor molecules in chronic hepatitis B and C infections: novel targets for immunotherapy?

Chronic HBV and HCV infections are the leading cause of liver-related morbidity and mortality. For effective antiviral immunity, virus-specific T cells are required, but these cells have been shown to be weak or absent in chronic HBV and HCV patients. One of the mechanisms that underlies the impaired T-cell response is the result of the continuously high viral load that causes HBV-specific and HCV-specific T cells to become exhausted, which is characterized by impaired proliferation, cytokine production and cytotoxic activity of T cells as well as high susceptibility to apoptosis. In vitro studies from chronic HBV and HCV patients as well as in vivo studies in animal models demonstrated a reversible state of T-cell exhaustion, which can be manipulated to reinvigorate the specific antiviral immune responses. In chronic HCV infection, this concept has been explored in clinical trials by administration of specific antibody to block the inhibitory pathways. The manipulation of inhibitory receptors is a promising and potential strategy for immunotherapeutic interventions in chronic HBV and HCV patients to facilitate complete elimination of the viruses or sustained viral control.

The effects of nucleoside/nucleotide analogues on host immune cells: the baseline for future immune therapy for HBV?

HBV is a non-cytopathic virus and the progression of liver fibrosis is attributed to the host immune response. Complete suppression of viral replication using nucleotide or nucleoside analogues (NUCs) can prevent most complications related to chronic HBV infection. Unfortunately, antiviral treatment has to be administered lifelong to the majority of patients as HBV persists in the hepatocytes. However, although NUCs are very frequently administered in clinical practice, their effects on vital parts of the host immune response to HBV are not well established. In this review we summarize the currently available data gathered from longitudinal studies that investigated treatment-associated alterations of HBV-specific CD4+ and CD8+ T-cells, regulatory T-cells and natural killer (NK) cells. These observations are important, as they can guide the design of studies that investigate the efficacy of new immune therapeutic agents. Novel experimental compounds will likely be added to ongoing NUC treatment, which leads to a functional cure in only a small minority of patients.

Interleukin 15 upregulates the expression of PD-1 and TIM-3 on CD4+ and CD8+ T cells.

Virus-specific T cell-mediated immunity is severely impaired in chronic hepatitis B virus (HBV) patients. HBV-specific T cells in chronic HBV patients show a low ability to produce cytokines and to exert their cytotoxic activity. A prominent characteristic of these exhausted T cells is overexpression of inhibitory receptor molecules which negatively regulate T cell function. In this study, we examined in vitro regulation of two inhibitory receptor expressions, programmed death 1 (PD-1) and T cell immunoglobulin mucin domain-containing molecule 3 (TIM-3). Peripheral blood mononuclear cells (PBMCs) obtained from healthy individuals were in vitro stimulated with a panel of cytokines. PD-1 and TIM-3 expression levels on CD4+ and CD8+ T cells were examined at days 2 and 7 post stimulation. We demonstrated that PD-1 and TIM-3 were induced via polyclonal (anti-CD3) and cytokine (interleukin 15 [IL-15]) stimulations. Noteworthy, there was a significantly increased induction of TIM-3 on CD8+ T cells as compared to CD4+ T cells. Our study thus contributes to further understanding the regulation of T cell exhaustion markers PD-1 and TIM-3.

Healthy human salivary glands contain a DHEA-sulphate processing intracrine machinery, which is deranged in primary Sjögren's syndrome.

Sjögren's syndrome (SS) patients have low salivary dehydroepiandrosterone (DHEA) and androgen biomarker levels, but high salivary oestrogen levels. The hypothesis was that the healthy glands contain DHEA-sulphate processing intracrine machinery; the local androgen/oestrogen imbalance suggests that this is disarranged in SS. Indirect immunofluorescence and quantitative real-time PCR (qRT-PCR) of steroid sulphatase, sulfotransferase, 3beta- and 17beta-hydroxysteroid dehydrogenases (3beta- and 17beta-HSD), 5alpha-reductase and aromatase were performed for labial salivary glands of healthy controls and persons with SS. In control acini steroid sulphatase and sulfotransferase immunoreactivities were located in the basolateral cell parts. 3Beta- and 17beta-HSD formed strong, interrupted bands along the basal cell parts. 5alpha-reductase was mainly located in acinar cell nuclei and aromatase in the apical cell membrane. All enzymes were more widespread in ducts. In SS, steroid sulphatase was weak and deranged, 3beta- and 17beta-HSD had lost their strict basal acinar cell localization and 5alpha-reductase was mainly found in the cytoplasm of the acinar cells, whereas aromatase showed similar staining in SS and controls. qRT-PCR of labial salivary glands disclosed all corresponding enzyme mRNAs with the levels of 3beta-HSD in SS being the lowest. Healthy tubuloacinar epithelial cells contain complete intracrine machineries for DHEA(-sulphate) pro-hormone processing. These enzymes have in healthy acini an organized architecture, which corresponds with DHEA uptake from the circulation, nuclear site of production of the active dihydrotestosterone (DHT) end product and production of oestrogens into saliva for export to ductal and oral epithelial cells. SS is characterized by low 3beta-HSD levels, which together with impaired subcellular compartmentalization of HSDs and 5alpha-reductase may explain the low local DHT and androgen biomarker levels in SS.

The role of anti-HBs in hepatitis B reactivation during direct-acting antiviral therapy for chronic hepatitis C.

BACKGROUND: Hepatitis B reactivation in patients with resolved HBV can occur during hepatitis C treatment with direct-acting antivirals, but only a few cases have been described. It is not clear which patients are at risk for HBV reactivation and how to manage them. METHODS: Three patients (all hepatitis B surface antigen [HBsAg]-negative, antibody to hepatitis B core [anti-HBc] positive and HBV DNA negative) experienced a late HBV reactivation 12 weeks post-treatment but were able to control their viraemia. HCV RNA, HBV DNA, anti-HBc and anti-HBs were measured in these patients and in 37 HBsAg-negative, anti-HBc positive, HBV DNA negative control patients during direct-acting antiviral therapy for chronic hepatitis C. RESULTS: Baseline anti-HBs do not differ between patients with and without HBV reactivation. Patients with HBV reactivation, however, significantly increased their anti-HBs at time of reactivation, while patients without HBV reactivation show stable anti-HBs during therapy (P=0.007). CONCLUSIONS: Anti-HBs might be an important marker to delineate patients at risk for clinically significant HBV reactivation.

Frequencies of Circulating MAIT Cells Are Diminished in Chronic HCV, HIV and HCV/HIV Co-Infection and Do Not Recover during Therapy.

OBJECTIVE: Mucosal-associated invariant T (MAIT) cells comprise a subpopulation of T cells that can be activated by bacterial products and cytokines to produce IFN-γ. Since little is known on MAIT cells during HCV infection, we compared their phenotype and function in comparison to HIV and HCV/HIV co-infected patients, and determined the effect of IFN-α-based and direct-acting antiviral therapy on MAIT cells of HCV patients. METHODS: Blood samples from patients with chronic HCV (CHCV), virologically suppressed HIV, acute HCV/HIV co-infection (AHCV/HIV) and healthy individuals were examined by flowcytometry for phenotype and function of MAIT and NK cells. RESULTS AND CONCLUSIONS: Compared to healthy individuals, the frequency of CD161+Vα7.2+ MAIT cells was significantly decreased in patients with CHCV, HIV and AHCV/HIV co-infection. CD38 expression on MAIT cells was increased in AHCV/HIV patients. MAIT cells were responsive to IFN-α in vitro as evidenced by enhanced frequencies of IFN-γ producing cells. IFN-α-based therapy for CHCV decreased the frequency of IFN-γ+ MAIT cells, which was still observed 24 weeks after successful therapy. Importantly, even after successful IFN-α-based as well as IFN-α-free therapy for CHCV, decreased frequencies of MAIT cells persisted. We show that the frequencies of MAIT cells are reduced in blood of patients with CHCV, HIV and in AHCV/HIV co-infection compared to healthy individuals. Successful therapy for CHCV did not normalize MAIT cell frequencies at 24 weeks follow up. The impact of HIV and HCV infection on the numbers and function of MAIT cells warrant further studies on the impact of viral infections and the antimicrobial function of MAIT cells.

Longitudinal analysis of peripheral and intrahepatic NK cells in chronic HCV patients during antiviral therapy.

INTRODUCTION: A strong immune response is integral to the clearance of HCV infection. NK cells are specialized cells that are able to inhibit replication of HCV in infected hepatocytes. Previous studies have correlated therapy outcome in HCV to the expression of various markers on NK cells. However, the effect of viral load reduction on NK cell function during therapy is still largely unknown, particularly in the liver. Therefore we investigated NK cell phenotype and effector function in both the peripheral and intrahepatic compartments during the course of antiviral therapy in chronic HCV patients. METHODS: Chronic HCV patients were treated for 24 or 48weeks with triple therapy consisting of telaprevir, pegIFN-α and ribavirin. Blood and fine needle aspiration (FNA) biopsies of the liver were collected at start and 6h, 1week and 12weeks during therapy. Flowcytometry was performed for expression of different markers (NKG2A, NKG2D, NKp46, and CD69). RESULTS: Our results demonstrate a highly activated phenotype of NK cells in liver compared to blood in chronic HCV patients. Six hours after start of triple therapy, no activation of intrahepatic NK cells was observed in the liver as compared to baseline. At 1week after start of triple therapy, the frequency of NK cells with the activating receptor NKp46 was increased in blood, whereas at week 12, the frequencies of the inhibitory receptor NKG2A was increased. No alterations were observed during therapy in liver NK cell phenotype. CONCLUSION: IFN-based therapy for chronic HCV affects NK cell phenotype in peripheral blood more than in the liver.

Erythropoietin administration suppresses human monocyte function in vitro and during therapy-induced anemia in HCV patients.

Erythropoietin (EPO) is a hormone that controls red blood cell production. Binding of EPO to the EPO-receptor results in increased numbers of red blood cells in the circulation, which makes EPO a potent molecule to treat anemia in various groups of patients. Although numerous studies have examined the clinical effects of EPO, its immunological effects have received less attention. In this study, we examined the immunological effects of EPO on human monocytes. We show that human monocytes express EPO receptor mRNA, and are responsive to EPO in cell culture. In vitro exposure of PBMC from individuals to EPO and the TLR4 ligand LPS showed a significant reduction of monocytes producing IL-6 and TNF, while the frequencies of IL-12p40, IL-10, MIP-1ß and IL-8-producing cells did not change upon incubation with EPO. In addition, EPO did increase the phagocytic activity but did not affect the ability to produce ROS by monocytes. Moreover, we studied eight chronic HCV patients undergoing treatment with peg-IFN and ribavirin, who were administered EPO for treatment-induced anemia. Blood was collected before and 7 days after EPO injection. In 7 patients, we observed a significant decline at day 7 after EPO administration of the frequency of monocytes producing various pro-inflammatory cytokines following stimulation with the TLR4 ligand LPS and the TLR7/8 ligand R848, which is in line with our in vitro findings. Our findings demonstrate an inhibitory effect of EPO on the secretion of effector molecules by monocytes and a stimulatory effect on the phagocytic activity by monocytes.

Immunology of hepatitis C virus infections.

The immune response in patients chronically infected with HCV plays a unique role during the infection because of its potential to contribute not only to viral clearance and, in some cases, protective immunity, but also to liver injury. A detailed understanding of the immunological mechanisms involved in persistence to HCV is essential to fully appreciate the complexity of the disease. In recent years, enormous progress has been made to characterize the dysfunctional natural killer cells and T cells during the chronic phase of infection. This information is important to further optimize treatment strategies based on the strengthening antiviral and immunomodulatory activities in patients chronically infected with HCV.