Hepatitis D virus (HDV) super-infection of hepatitis B virus (HBV)-infected patients is associated with rapid progression to liver cirrhosis and hepatocellular carcinoma. Treatment options are limited, and no vaccine is available. Although HDV-specific CD8+ T cells are thought to mediate viral control, little is known about the repertoire of targeted epitopes, and it remains unclear why HDV-specific CD8+ T cells ultimately fail during persistent infection. We aimed to define how viral escape impacts the efficacy of HDV-specific CD8+ T-cells.

Myrcludex B (MyrB) is a first-in-class entry inhibitor, which blocks the HBV/HDV receptor sodium taurocholate co-transporter NTCP. While the results of the phase 2b clinical trial (MYR202) on chronically HBV/HDV co-infected individuals receiving MyrB daily in combination with TDF show a dose-dependent serum HDV RNA decline associated with improvements of biochemical activity and liver stiffness, here we investigated the impact of MyrB treatment in paired liver biopsies obtained at baseline (BL) and week 24. Methods 120 HBeAg-negative patients with chronic Hepatitis D were randomized in 4 treatment arms. TDF treatment (245 mg/day) started at least 12w prior to MyrB. MyrB was administered s.c. once daily at 2 (A), 5 (B) or 10 (C) mg for 24w. Patients in arm D received TDF alone. BL biopsies were available for 31 patients, paired liver biopsies for 22 patients. Virological parameters and expression of inflammatory genes were assessed by qPCR, RNA in situ hybridization (RNA-ISH) and immunohistochemistry. Results At w24, intrahepatic HDV RNA declined in all MyrB arms with median reductions from BL by 0.9log in A (n = 7), 1.1log in B (n = 5) and by 1.4log in C (n = 7). TDF treatment (D) showed modest HDV RNA reductions (0.3log IU/ml, n = 3). HBV infection levels declined only in some patients with the strongest median reduction of HBV pgRNA (0.9log) and total HBV DNA (0.6log) in group C. Of note, immunohistochemistry revealed a substantial and dose-dependent decrease of HDAg+ cells. The strong reduction of HDV replication was also confirmed by RNA-ISH on exemplary biopsy sections. Furthermore, transcriptional levels of proinflammatory cytokines (CXCL10, CXCL8, IL18, TGFB1) decreased in all MyrB treatment arms. The reduction was more pronounced in patients with intrahepatic HDVRNA decline of ≥1log. Conclusions Intrahepatic levels of HDV RNA and of HDAg+ cells strongly declined after 24 weeks of MyrB treatment in a dose-dependent manner. The concomitant reduction of ALT and inflammatory cytokines suggests that the drop of HDV infection can diminish liver inflammation. Because of the strong decline of intrahepatic HDV infection levels induced by blocking HDV entry and turnover, HDV clearance might be achievable upon prolonged treatment durations.

Patienten mit chronischer Hepatitis B (HBV) Infektion bedürfen in den meisten Fällen einer regelmäßigen lebenslangen hepatologischen Begleitung, oftmals einer medikamentösen Dauertherapie und werden in unterschiedlichen Lebensbereichen von ihrer Krankheit affektiert. Zusätzlich sind die Therapieprinzipien einer HBV Therapie für Laien nicht immer einfach verständlich. Bisher ist wenig über das generelle Krankheitsverständnis sowie die Lebensqualität von Patienten mit chronischer HBV Infektion bekannt.

Hintergrund: Interferon-alpha (IFN) ist zurzeit die einzige zugelassene und empfohlene HDV-Therapie. In den bisherigen kontrollierten HDV-Therapiestudien, insbesondere der HIDIT II-Studie, zeigte sich eine negative HDV-PCR bis > 30% nach 96 Wochen IFN-Therapie. Im Gegensatz hierzu gibt es nur wenig “real-life” -Daten zum klinischen outcome von IFN-Therapien HBV-HDV-koinfizierter Patienten.

Type III interferons (IFN-lambda 1,2 and 3 or IL-29, IL28a and IL-28b, respectively) and Type I interferons (IFN-alpha/beta) activate similar signaling cascades although via different receptors. While the IFN-alpha/beta receptor is ubiquitously expressed, the IFN-lambda receptor, which is a heterodimer consisting of IL-10 and IL-28 receptor subunits, is mainly expressed on epithelial cells. Despite the convergence of IFN-lambda signaling cascades with that of type I and II IFNs, the cytokine families are known to differ in their antiviral activities. In this study we therefore aimed at dissecting at which step IFN-lambda blocks HBV replication after infection. For this, HepaRG cells differentiated in 1.8% DMSO were treated for different time periods with IFN-lambda1, IFN-lambda2 or IFN-alpha and mRNA expression of interferon-stimulated genes (ISG) was analyzed by qRT-PCR. Differentiated HepaRG cells were infected with HBV at an MOI of 200 DNA containing particles/cell for 10 d and treated for another 7 d with 1 – 1000 U IFN-lambda1 or IFN-lambda2. CccDNA amounts and secreted HBeAg were measured by qPCR and ELISA, respectively. Hereby, we found that Type III interferons IFN-lambda1 and IFN-lambda2 induce interferon-stimulated genes (f.e. Mx1 and OAS) as well as cytidine deaminases (f.e. Apobec3A, Apobec3B and Apobec 3G) in HepaRG cells with a similar efficacy as IFN-alpha but with distinct kinetics. IFN-lambda1 and IFN-lambda2 did not only reduce HBV replication, progeny and HBeAg secretion, but also reduced levels of intracellular cccDNA. Analysis of cccDNA amplicons indicated sequence alterations from GC to AT-rich regions as a consequence of IFN-lambda induced deamination of HBV cccDNA. Conclusions: IFN-lambda1 and IFN-lambda2 efficiently suppress HBV replication and reduce intracellular cccDNA similar to IFN-alpha but with distinct kinectics. Thus, both cytokine families seem to operate through a common molecular mechanism of cccDNA deamination and subsequent degradation.

Hepatitis C virus (HCV) infection is a major cause of liver cirrhosis and hepatocellular carcinoma. Within days post infection, innate immunity is activated by HCV reflected by an induction of IFN-stimulated genes (ISGs). The precise cell type and cellular pathogen pattern recognition receptors (PRRs) involved in innate immune sensing of HCV, however, still remain unknown.

ing viral interference in this model. HDV infection was associated with an increased expression of ISGs that was correlated with the increasing MOI (fold changes compared to mock: USP18 5X, CXCL10 12X, ISG56 14X, OAS 15X, ISG15 15X, MxA 52X, RSAD2 177X), but did not induce an increase of IL-6 or IL-8 expression. No induction of ISGs’ or cytokines’ expression was associated with HBV infection, even at increasing MOIs. We established a relevant in vitro model to further characterize HBV/HDV interplay and to evaluate the hepatocyte innate immune response to HBV and HDV infections. Here, a suppressive role of HDV on HBV replication could clearly be confirmed. Moreover, we could identify a strong induction of ISGs associated with HDV but not with HBV infection. A possible link between the interferon response induced by HDV and the suppression of HBV replication is currently being investigated.

Chronic hepatitis B virus (HBV) infection is characterized by dysfunctional innate and adaptive immune responses and recent in vitro studies indicated that the interferon (IFN) signaling is impaired in HBV-transfected hepatoma cell lines. Aim of this study was to investigate whether HBV can directly interfere with the hepatocellular IFN alpha response in chronically infected human hepatocytes repopulating the livers of uPA/SCID mice. Methods: Using primers specific for human transcripts and not cross-reacting with murine genes, we comparatively analysed responsiveness of transplanted hepatocytes to treatment with human IFN alpha (1350 IU/g daily) both in uninfected and in HBV-chronically infected (median viremia 3×107 copies/ml) chimeric mice transplanted with human hepatocytes isolated from the same liver donor. To determine kinetics of induction of human IFN responsive genes (IRGs), animals treated either with IFN or PBS were sacrificed at 4, 8 or 24h after the last injection, respectively. Results: Five days of IFN alpha administration induced moderate reduction of HBV-DNA serum titres (median 0.75 log), rcDNA and pgRNA amounts compared to untreated control animals, demonstrating a direct antiviral effect of IFN alpha in human hepatocytes. As expected, IFN treatment induced significant up-regulation of human OAS or MxA RNA levels in uninfected chimeric animals (median 3-fold OAS and 10-fold MXA, 8 hr after injection). However, we found that induction of the same RNA transcripts was clearly impaired in the livers of IFN-treated HBV-infected mice. Similarly, IFN treatment was able to increase Myd88 and TLRs RNA levels in uninfected human hepatocytes, while in HBV-infected hepatocytes expression levels remained unchanged. Conclusions: These results provide direct evidence that HBV can specifically hinder the cellular response to IFN in chronically infected human hepatocytes by interfering with the activation of several genes of the innate immune system.

Hintergrund: Interaktionen zwischen dem Hepatitis B Virus (HBV) und dem Interferonsystem des Wirtes sind noch immer unzureichend untersucht.

had no effect of infectivity of HBV. Macropinocytosis could be ruled out as entry pathway of HBV since uptake of the pinocytic marker substrate horseradish-peroxidase (HRP) was efficiently blocked with Ethylisopropylamiloride (EIPA), but had no impact on HBV infection. Agents that prevent endosomal acidification, e.g. Bafilomycin and NH4Cl, were not able to inhibit HBV infection, but blocked infection with SFV very efficiently. Conclusions: These results suggest that in contrast to other viruses, HBV does not use classical clathrinor caveolin-dependent endocytic mechanism for infection, but an unusual endosomal entry pathway that needs to be determined. A low pH within endosomal compartments, important for infection of many other enveloped viruses is not necessary to establish an HBV infection in primary hepatocytes.

Conclusions: MF provide Hh paracrine signals that promote the migration of NKT cells towards reactive cholangiocytes.These findings identify a novel mechanism for recruitment of NKT cells into PBC portal tracts.

Einleitung: Neueste Forschungsergebnisse weisen darauf hin, dass mehrere Viren Mechanismen entwickelt haben, um die Aktivierung der Toll-Like Rezeptor Signalkaskade (TLR) zu verhindern. Es ist aber derzeit unklar, ob das Hepatitis B Virus (HBV) ebenfalls das angeborene Immunsystem in Rahmen einer chronischen HBV Infektion beeinflussen kann. Ziel dieser Studie war es daher zu untersuchen, ob die Expression von TLR Signalkaskaden mit der intrahepatischen viralen Aktivität in Patienten mit chronischer HBV (CH-B) korreliert.