Search

Objective: Chronic HBV/HDV infections are a major cause of liver cancer. Current treatments can only rarely eliminate HBV and HDV. Our previously developed preS1-HDAg immunotherapy could induce neutralising antibodies to HBV in vivo and raise HBV/HDV-specific T-cells. Here, we further investigate if a heterologous prime-boost strategy can circumvent T-cell tolerance and preclude HDV superinfection in vivo., Design: A DNA prime-protein boost strategy was evaluated for immunogenicity in mice and rabbits. Its ability to circumvent T-cell tolerance was assessed in immunocompetent hepatitis B surface antigen (HBsAg)-transgenic mice. Neutralisation of HBV and HDV was evaluated both in vitro and in immunodeficient human-liver chimeric mice upon adoptive transfer., Results: The prime-boost strategy elicits robust HBV/HDV-specific T-cells and preS1-antibodies that can effectively prevent HBV and HDV (co-)infection in vitro and in vivo. In a mouse model representing the chronic HBsAg carrier state, active immunisation primes high levels of preS1-antibodies and HDAg-specific T-cells. Moreover, transfer of vaccine-induced antibodies completely protects HBV-infected human-liver chimeric mice from HDV superinfection., Conclusion: The herein described preS1-HDAg immunotherapy is shown to be immunogenic and vaccine-induced antibodies are highly effective at preventing HBV and HDV (super)infection both in vitro and in vivo. Our vaccine can complement current and future therapies for the control of chronic HBV and HDV infection., Competing Interests: Competing interests: MS and LF are founders and shareholders of Svenska Vaccinfabriken, which holds the IP to the immunogens described in this manuscript. All other authors report no potential conflicts. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed., (© Author(s) (or their employer(s)) 2023. Re-use permitted under CC BY. Published by BMJ.)

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.)

Background: The non-structural (NS) 3/4A protease/helicase of the hepatitis C virus is known to modulate signalling pathways in the infected hepatocyte by cleaving CARD adaptor inducing IFNβ (Cardif), T-cell protein tyrosine phosphatase (TC-PTP) and TIR domain-containing adaptor inducing IFNβ (TRIF), but the effects of NS3/4A in vivo still remain unclear., Aim: To investigate the influence of NS3/4A on intracellular and intercellular signalling in vivo by analysing the intrahepatic inflammatory response of naïve, lipopolysaccharide (LPS)/D-galactosamine (D-galN) or tumour necrosis factor-α (TNFα)/D-galN-treated NS3/4A-transgenic (Tg) mice., Methods: The intrahepatic immunity of naïve and LPS/D-galN- or TNFα/D-galN-treated NS3/4A-Tg mice was determined using western blot, ELISA, real-time PCR, flow cytometry and survival monitoring. The injection of cytokines or antibodies against signalling components was performed to analyse the relevance of the respective pathways for the investigated issues. A Tg mouse lineage expressing an inactivated NS3/4A protease (NS3/4A(Ile1073Ala)-Tgs) was generated to examine if protective effects were NS3/4A protease dependent., Results: The activation of hepatic signal transducer and activator of transcription 1 and 2 was impaired in NS3/4A-Tg mice after treatment with LPS/D-galN or TNFα/D-galN. This was paralleled by a reduction in hepatic interferon-γ (IFNγ). Reconstitution of IFNγ reverted the resistance to LPS/TNFα in NS3/4A-Tg mice. Subsequently, blocking IFNγ in vivo rendered wild-type mice resistant against treatment with LPS/TNFα. A new Tg mouse expressing an inactivated NS3/4A protease had the same phenotype as wild-type mice with respect to hepatic IFNγ levels and sensitivity to LPS/d-galN. Finally, the chemokine profile was altered in the NS3/4A-Tg mice towards an anti-inflammatory state, which helps to explain the altered immune cell subsets and reduction in hepatic IFNγ production., Conclusions: Our data demonstrate that the NS3/4A protease reduces the intrahepatic production of IFNγ and alters TNFα-mediated effects, thereby impairing the hepatic inflammatory response. This may contribute to viral persistence.

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.

Background: The hepatitis C virus (HCV) establishes chronic infection by incompletely understood mechanisms. The non-structural (NS) 3/4A protease/helicase has been proposed as a key complex in modulating the infected hepatocyte, although nothing is known about the effects this complex exerts in vivo., Aim: To generate mice with stable and transient hepatocyte expression of the HCV NS3/4A proteins to study its effects in vivo., Methods: NS3/4A expression was determined by western blot and immunohistochemistry. Two independent pathologists determined the liver histology. Hepatic immunity was characterised by quantifying intrahepatic immune cell subsets. Liver damage was induced using carbon tetrachloride (CCl(4)), lipopolysaccaride (LPS), tumour necrosis factor alpha (TNFalpha), and anti-Fas antibody., Results: Expression of NS3/4A was restricted to the cytoplasm of hepatocytes, and did not cause liver cancer or any spontaneous liver pathology. However, the presence of NS3/4A modulated the intrahepatic immunity, as follows: first, the CD4+ T cell and type I/II dendritic cell subsets were reduced in transgenic livers; second, NS3/4A protected hepatocytes from liver damage mediated in vivo by CCl(4), LPS, TNFalpha, but not FAS; and third, both stable and transiently NS3/4A transgenic mice were resistant to lethal doses of liver targeted TNFalpha, and the resistance could be reverted by treatment with a p38 mitogen activated protein kinase inhibitor (MAPK)., Conclusions: Hepatic expression of NS3/4A does not induce spontaneous liver disease. NS3/4A does, however, alter the intrahepatic immune cell subsets and protects hepatocytes against TNFalpha induced liver damage in vivo. The TNFalpha resistance can be reverted by treatment with a p38 MAPK inhibitor. This represents a new immune evasion strategy conferred by NS3/4A.

Background: The hepatitis C virus (HCV) mutates within human leucocyte antigen (HLA) class I restricted immunodominant epitopes of the non-structural (NS) 3/4A protease to escape cytotoxic T lymphocyte (CTL) recognition and promote viral persistence. However, variability is not unlimited, and sometimes almost absent, and factors that restrict viral variability have not been defined experimentally., Aims: We wished to explore whether the variability of the immunodominant CTL epitope at residues 1073-1081 of the NS3 protease was limited by viral fitness., Patients: Venous blood was obtained from six patients (four HLA-A2+) with chronic HCV infection and from one HLA-A2+ patient with acute HCV infection., Methods: NS3/4A genes were amplified from serum, cloned in a eukaryotic expression plasmid, sequenced, and expressed. CTL recognition of naturally occurring and artificially introduced escape mutations in HLA-A2-restricted NS3 epitopes were determined using CTLs from human blood and genetically immunised HLA-A2-transgenic mice. HCV replicons were used to test the effect of escape mutations on HCV protease activity and RNA replication., Results: Sequence analysis of NS3/4A confirmed low genetic variability. The major viral species had functional proteases with 1073-1081 epitopes that were generally recognised by cross reactive human and murine HLA-A2 restricted CTLs. Introduction of mutations at five positions of the 1073-1081 epitope prevented CTL recognition but three of these reduced protease activity and RNA replication., Conclusions: Viral fitness can indeed limit the variability of HCV within immunological epitopes. This helps to explain why certain immunological escape variants never appear as a major viral species in infected humans.