Search

Objective: HCV is characterised by its ability to establish chronic infection in hepatocytes and to replicate in the presence of an inflammation. We mimicked this situation in vivo in immune-competent mice by syngeneic transplantation of HCV replicon-containing mouse hepatoma cells., Design: A total of 5 million H-2 b positive Hep56.1D cells, carrying a subgenomic genotype (gt) 2a replicon (HCV replicon cells) or stably expressing comparable levels of the HCV NS3/4A protease/helicase complex (NS3/4A hepatoma cells), were injected subcutaneously into syngeneic H-2 b -restricted mice. Kinetics of tumour growth, HCV RNA replication levels and HCV-specific immune responses were monitored. For immune monitoring, new H-2 b -restricted cytotoxic T cell epitopes within the gt2a NS3/4A region were mapped. Immune mice were generated by DNA-based vaccination., Results: HCV replicon and NS3/4A hepatoma cells generated solid tumours in vivo. Similar to what is seen in human HCV infection did HCV RNA replicate in the presence of inflammation. NS3/4A-specific CD8+ T cells seemed to transiently reduce HCV RNA levels. Both CD4+ and CD8+ T cells were required for protection against tumour growth. Vaccine-induced NS3/4A(gt2a)-specific T cells protected against HCV replicon tumours in wild-type, but not in HCV NS3/4A(gt1a)-transgenic mice with dysfunctional HCV-specific T cells. Importantly, as in human HCV infection, HCV replicon cells neither primed nor boosted a strong NS3/4A-specific T cell response., Conclusion: Syngeneic transplantation of mouse HCV replicon cells into immune-competent animals mirrors many in vivo events in humans. This system is versatile and can be applied to any genetically modified H-2 b -restricted mouse strain., Competing Interests: Competing interests: MS and LF are cofounders of Svenska Vaccinfabriken, which holds commercial rights to vaccine patents. RB is a cofounder of ReBLikoN GmbH, which holds commercial rights to HCV replicon technology., (© Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2018. All rights reserved. No commercial use is permitted unless otherwise expressly granted.)

HCV infection typically induces liver injury and inflammation, which appears to be responsible for the associated fibrogenesis. To date, the mechanism underlying the different rates of disease progression remains unclear. The aim of the study is to understand the possible role of the HCV non-structural (NS) 3/4A protein in the fibrosis progression. We used NS3/4A-expressing transgenic mice (NS3/4A-Tg) to accomplish the goals of the study. Different stages of liver fibrosis were induced in wild-type and NS3/4A-Tg mice by single carbon tetrachloride (acute) or multiple injections for 4 (intermediate) or 8 (chronic) weeks. Fibrotic parameters, inflammatory responses and hepatocyte turnover were extensively examined. Hepatic expression of HCV NS3/4A did not induce spontaneous liver damage. However, NS3/4A expression exerted contrasting effects during acute and chronic liver damage. During early fibrogenesis and intermediate fibrosis (4 weeks), NS3/4A-Tg mice exhibited enhanced liver damage whereas reduced fibrosis was observed in NS3/4A-Tg during chronic liver fibrosis (8 weeks). Furthermore, attenuated inflammation was observed in NS3/4A-Tg during chronic fibrosis with increase in M2 macrophages, hepatocyte proliferation, decreased hepatocyte apoptosis and decreased ductular reaction. In conclusion, during early fibrogenesis, HCV NS3/4A contributes to liver damage. While, during chronic liver fibrosis, NS3/4A dampens inflammation and induces hepatocyte regeneration thereby contributing to slow fibrosis progression to promote its survival or persistence.

Great progress has been made in understanding the development of non-alcoholic fatty liver disease (NAFLD) but less is known about the mechanisms underlying the progress from steatosis to steatohepatitis (NASH). Our aim was to evaluate if the amount and type of storage of fat in hepatocytes is of importance for hepatocyte injury. We also wanted to show if not only the innate immunity but also the adaptive immunity is involved in NASH. Thirty-one patients with NASH or borderline NASH and 18 non-NASH patients were investigated. Liver biopsies were scored for NASH according to Kleiner et al. Paraffin-embedded liver biopsies were stained with antibodies against CD3, TLR4, CD68, Cleaved Caspase-3, ICAM1, Foxp3 and ApopTag by immunohistochemistry. Serum soluble ICAM-1 (sICAM-1) were analysed by ELISA. The volume density of fat was 59% in the NASH patients and microvesicular fat, increased in high NAS score patients. ICAM-1 positive hepatocytes were seen in NASH patients and were localized in areas with microvesicular fat. Non-NASH biopsies were negative for ICAM-1 positive hepatocytes. The sICAM-1 were significantly higher in NASH-patients (339.8 ± 34.07) than in non-NASH patients (229.5 ± 12.14), p = 0.0015. Patients with NAS score over four had higher area of CD68 positive cells p = 0.0011 and Foxp3 positive cells (p = 0.024) than non-NASH patients. In liver tissue with NASH, hepatocytes with microvesicular steatosis seem to be expressing more inflammatory markers, and in this liver tissue an increased number of CD68 cells and regulatory T-cells (Tregs, e.g. Foxp3+ cells) were seen, indicating an involvement of, both the innate and the adaptive immunity., (© 2011 The Authors. APMIS © 2011 APMIS.)

Background: Hepatitis C virus (HCV) effectively establishes persistent infection in human livers. The non-structural (NS) 3/4A complex participates in this process by cleavage of interferon beta (IFN beta) promoter stimulator-1 (IPS-1; also termed Cardif/MAVS/VISA), which inhibits responses to double stranded (ds) RNA. However, it is not known whether this effect extends beyond innate responses., Aims: To test if HCV NS3/4A affects innate and adaptive immune responses in vivo., Methods: NS3 levels were semi-quantified in human liver biopsies, transfected cells, and in transgenic (Tg) mouse livers by western blot. The effect of NS3/4A on dsRNA-mediated signalling and on the integrity of IPS-1 was analysed using in vitro translation, transfected cells and Tg mice. Cytotoxic T cell (CTL)-mediated clearance of transient firefly luciferase (FLuc)- and/or NS3/4A-Tg hepatocytes was determined using in vivo imaging and western blot., Results: NS3 protein levels were in a comparable range (0.1-49 microg/g tissue) in infected human livers and Tg mouse livers. Importantly, these levels of NS3/4A reduced murine innate responses to synthetic dsRNA in vivo, supporting the possibility that this occurs also in infected humans. The likely explanation for this was the NS3/4A-mediated cleavage of mouse IPS-1, albeit less efficiently than human IPS-1. Despite this, FLuc- and/or NS3/4A-expressing murine hepatocytes were effectively eliminated by hepatic CTLs, utilising the classical molecules for virus-infected cell lysis, including CD8, IFN gamma, perforin and FasL., Conclusions: Although HCV NS3/4A inhibits the innate immunity, this does not prevent CTL-mediated clearance of NS3/4A-expressing hepatocytes in vivo. Thus, other HCV proteins are most likely responsible for interfering with the adaptive immunity.

A key question in the development of a therapeutic vaccine against hepatitis C virus (HCV) is whether vaccine-primed T cells enter the liver and eliminate HCV-expressing hepatocytes. In the absence of an infectious small-animal model, we evaluated liver homing of vaccine-primed T cells in mice with transient hepatic transgene expression of the HCV nonstructural 3/4A (NS3/4A) protein. We found that T cells primed by transdermal DNA-based vaccination entered the liver and cleared NS3/4A-expressing hepatocytes in transiently transgenic CD8(+/+) mice but not in CD8(-/-) mice. Hence, peripherally primed NS3/4A-specific CD8(+) T cells home to the liver and clear HCV protein-expressing hepatocytes.