Potent broadly neutralizing antibody VIR-3434 controls hepatitis B and D virus infection and reduces HBsAg in humanized mice.

BACKGROUND & AIMS: Chronic hepatitis B is a global public health problem, and coinfection with hepatitis delta virus (HDV) worsens disease outcome. Here, we describe a hepatitis B virus (HBV) surface antigen (HBsAg)-targeting monoclonal antibody (mAb) with the potential to treat chronic hepatitis B and chronic hepatitis D. METHODS: HBsAg-specific mAbs were isolated from memory B cells of HBV vaccinated individuals. In vitro neutralization was determined against HBV and HDV enveloped with HBsAg representing eight HBV genotypes. Human liver-chimeric mice were treated twice weekly with a candidate mAb starting 3 weeks post HBV inoculation (spreading phase) or during stable HBV or HBV/HDV coinfection (chronic phase). RESULTS: From a panel of human anti-HBs mAbs, VIR-3434 was selected and engineered for pre-clinical development. VIR-3434 targets a conserved, conformational epitope within the antigenic loop of HBsAg and neutralized HBV and HDV infection with higher potency than hepatitis B immunoglobulins in vitro. Neutralization was pan-genotypic against strains representative of HBV genotypes A-H. In the spreading phase of HBV infection in human liver-chimeric mice, a parental mAb of VIR-3434 (HBC34) prevented HBV dissemination and the increase in intrahepatic HBV RNA and covalently closed circular DNA. In the chronic phase of HBV infection or co-infection with HDV, HBC34 treatment decreased circulating HBsAg by >1 log and HDV RNA by >2 logs. CONCLUSIONS: The potently neutralizing anti-HBs mAb VIR-3434 reduces circulating HBsAg and HBV/HDV viremia in human liver-chimeric mice. VIR-3434 is currently in clinical development for treatment of patients with chronic hepatitis B or D. IMPACT AND IMPLICATIONS: Chronic infection with hepatitis B virus and co-infection with hepatitis D virus place approximately 290 million individuals worldwide at risk of severe liver disease and cancer. Available treatments result in low rates of functional cure or require lifelong therapy that does not eliminate the risk of liver disease. We isolated and characterized a potent human antibody that neutralizes hepatitis B and D viruses and reduces infection in a mouse model. This antibody could provide a new treatment for patients with chronic hepatitis B and D.

Strain-specific responsiveness of hepatitis D virus to interferon-alpha treatment.

Background & Aims: Pegylated interferon alpha (pegIFNα) is commonly used for the treatment of people infected with HDV. However, its mode of action in HDV-infected cells remains elusive and only a minority of people respond to pegIFNα therapy. Herein, we aimed to assess the responsiveness of three different cloned HDV strains to pegIFNα. We used a previously cloned HDV genotype 1 strain (dubbed HDV-1a) that appeared insensitive to interferon-α in vitro, a new HDV strain (HDV-1p) we isolated from an individual achieving later sustained response to IFNα therapy, and one phylogenetically distant genotype 3 strain (HDV-3). Methods: PegIFNα was administered to human liver chimeric mice infected with HBV and the different HDV strains or to HBV/HDV infected human hepatocytes isolated from chimeric mice. Virological parameters and host responses were analysed by qPCR, sequencing, immunoblotting, RNA in situ hybridisation and immunofluorescence staining. Results: PegIFNα treatment efficiently reduced HDV RNA viraemia (∼2-log) and intrahepatic HDV markers both in mice infected with HBV/HDV-1p and HBV/HDV-3. In contrast, HDV parameters remained unaffected by pegIFNα treatment both in mice (up to 9 weeks) and in isolated cells infected with HBV/HDV-1a. Notably, HBV viraemia was efficiently lowered (∼2-log) and human interferon-stimulated genes similarly induced in all three HBV/HDV-infected mouse groups receiving pegIFNα. Genome sequencing revealed highly conserved ribozyme and L-hepatitis D antigen post-translational modification sites among all three isolates. Conclusions: Our comparative study indicates the ability of pegIFNα to lower HDV loads in stably infected human hepatocytes in vivo and the existence of complex virus-specific determinants of IFNα responsiveness. Impact and implications: Understanding factors counteracting HDV infections is paramount to develop curative therapies. We compared the responsiveness of three different cloned HDV strains to pegylated interferon alpha in chronically infected mice. The different responsiveness of these HDV isolates to treatment highlights a previously underestimated heterogeneity among HDV strains.

Therapeutic shutdown of HBV transcripts promotes reappearance of the SMC5/6 complex and silencing of the viral genome in vivo.

OBJECTIVE: Therapeutic strategies silencing and reducing the hepatitis B virus (HBV) reservoir, the covalently closed circular DNA (cccDNA), have the potential to cure chronic HBV infection. We aimed to investigate the impact of small interferring RNA (siRNA) targeting all HBV transcripts or pegylated interferon-α (peg-IFNα) on the viral regulatory HBx protein and the structural maintenance of chromosome 5/6 complex (SMC5/6), a host factor suppressing cccDNA transcription. In particular, we assessed whether interventions lowering HBV transcripts can achieve and maintain silencing of cccDNA transcription in vivo. DESIGN: HBV-infected human liver chimeric mice were treated with siRNA or peg-IFNα. Virological and host changes were analysed at the end of treatment and during the rebound phase by qualitative PCR, ELISA, immunoblotting and chromatin immunoprecipitation. RNA in situ hybridisation was combined with immunofluorescence to detect SMC6 and HBV RNAs at single cell level. The entry inhibitor myrcludex-B was used during the rebound phase to avoid new infection events. RESULTS: Both siRNA and peg-IFNα strongly reduced all HBV markers, including HBx levels, thus enabling the reappearance of SMC5/6 in hepatocytes that achieved HBV-RNA negativisation and SMC5/6 association with the cccDNA. Only IFN reduced cccDNA loads and enhanced IFN-stimulated genes. However, the antiviral effects did not persist off treatment and SMC5/6 was again degraded. Remarkably, the blockade of viral entry that started at the end of treatment hindered renewed degradation of SMC5/6. CONCLUSION: These results reveal that therapeutics abrogating all HBV transcripts including HBx promote epigenetic suppression of the HBV minichromosome, whereas strategies protecting the human hepatocytes from reinfection are needed to maintain cccDNA silencing.

Clinical establishment of a laboratory developed quantitative HDV PCR assay on the cobas6800 high-throughput system.

BACKGROUND & AIMS: Currently available HDV PCR assays are characterized by considerable run-to-run and inter-laboratory variability. Hence, we established a quantitative reverse transcription real-time PCR (RT-qPCR) assay on the open channel of a fully automated PCR platform (cobas6800, Roche) offering improved consistency and reliability. METHODS: A primer/probe-set targeting a highly conserved region upstream of the HDV antigen was adapted for use on the cobas6800. The lower limit of detection (LLOD) was determined using a dilution panel of the HDV WHO standard (n = 21/dilution). Linearity and inclusivity were tested by preparing 10-fold dilution series of cell culture-derived virus (genotype [GT]1-8; n = 5/dilution). Patient samples containing a variety of bloodborne viral pathogens were tested to confirm exclusivity (n = 60). RESULTS: The LLOD of the HDV utility-channel (HDV_UTC) assay was determined as 3.86 IU/ml (95% CI 2.95-5.05 IU/ml) with a linear range from 10-10ˆ8 IU/ml (GT1). Linear relationships were observed for all HDV GTs with slopes ranging from -3.481 to -4.134 cycles/log and R2 from 0.918 to 0.994. Inter-run and intra-run variability were 0.3 and 0.6 Ct (3xLLOD), respectively. No false-positive results were observed. To evaluate clinical performance, 110 serum samples of anti-HDV-Ab+ patients were analyzed using the HDV_UTC and CE-IVD RoboGene assays. 58/110 and 49/110 samples were concordant positive or negative, respectively (overall agreement 97.3%). Quantitative comparison demonstrated a strong correlation (R2 0.8733; 95% CI 0.8914-0.9609; p value <0.0001). CONCLUSION: The use of highly automated, sample-to-result solutions for molecular diagnostics holds many inherent benefits over manual workflows, including improved reliability, reproducibility and dynamic scaling of testing capacity. The assay we established showed excellent analytical and clinical performance, with inclusivity for all HDV GTs and a limit of quantification of 10 IU/ml, making it a sensitive new tool for HDV screening and viral load monitoring. LAY SUMMARY: The hepatitis delta virus (HDV) causes a severe form of inflammation in the liver. We developed a tool for molecular diagnostics, a polymerase chain reaction HDV assay that showed great performance. It can be used to improve diagnosis of HDV, as well as for monitoring treatment responses. The assay allows for quantification of the virus in the tested samples and is performed on a fully automated platform (cobas6800), which provides various benefits including less hands-on time and excellent comparability of test results.

Strong Replication Interference Between Hepatitis Delta Viruses in Human Liver Chimeric Mice.

BACKGROUND: Hepatitis D Virus (HDV) is classified into eight genotypes with distinct clinical outcomes. Despite the maintenance of highly conserved functional motifs, it is unknown whether sequence divergence between genotypes, such as HDV-1 and HDV-3, or viral interference mechanisms may affect co-infection in the same host and cell, thus hindering the development of HDV inter-genotypic recombinants. We aimed to investigate virological differences of HDV-1 and HDV-3 and assessed their capacity to infect and replicate within the same liver and human hepatocyte in vivo. METHODS: Human liver chimeric mice were infected with hepatitis B virus (HBV) and with one of the two HDV genotypes or with HDV-1 and HDV-3 simultaneously. In a second set of experiments, HBV-infected mice were first infected with HDV-1 and after 9 weeks with HDV-3, or vice versa. Also two distinct HDV-1 strains were used to infect mice simultaneously and sequentially. Virological parameters were determined by strain-specific qRT-PCR, RNA in situ hybridization and immunofluorescence staining. RESULTS: HBV/HDV co-infection studies indicated faster spreading kinetics and higher intrahepatic levels of HDV-3 compared to HDV-1. In mice that simultaneously received both HDV strains, HDV-3 became the dominant genotype. Interestingly, antigenomic HDV-1 and HDV-3 RNA were detected within the same liver but hardly within the same cell. Surprisingly, sequential super-infection experiments revealed a clear dominance of the HDV strain that was inoculated first, indicating that HDV-infected cells may acquire resistance to super-infection. CONCLUSION: Infection with two largely divergent HDV genotypes could be established in the same liver, but rarely within the same hepatocyte. Sequential super-infection with distinct HDV genotypes and even with two HDV-1 isolates was strongly impaired, suggesting that virus interference mechanisms hamper productive replication in the same cell and hence recombination events even in a system lacking adaptive immune responses.

Murine hepatocytes do not support persistence of Hepatitis D virus mono-infection in vivo.

BACKGROUNDS & AIMS: As a result of the limited availability of in vivo models for hepatitis D virus (HDV), treatment options for HDV chronically infected patients are still scant. The discovery of sodium taurocholate cotransporting polypeptide (NTCP) as HDV entry receptor has enabled the development of new infection models. AIM: To comparatively assess the efficacy and persistence of HDV mono-infection in murine and human hepatocytes in vivo. METHODS: Mice with humanized NTCP (hNTCPed84-87 mice) were generated by editing amino acid residues 84-87 of murine NTCP in C57BL/6J mice. HDV infection was assessed in hNTCPed84-87 mice and in immune deficient uPA/SCID/beige (USB) mice, whose livers were reconstituted with human or murine (hNTCPed84-87 ) hepatocytes. Livers were analysed between 5 and 42 days post-HDV inoculation by qRT-PCR, immunofluorescence and RNA in situ hybridization (ISH). RESULTS: hNTCPed84-87 mice could be infected with HDV genotype 1 or 3. ISH analysis demonstrated the presence of antigenomic HDV RNA positive murine hepatocytes with both genotypes, proving initiation of HDV replication. Strikingly, murine hepatocytes cleared HDV within 21 days both in immunocompetent hNTCPed84-87 mice and in immunodeficient USB mice xenografted with murine hepatocytes. In contrast, HDV infection remained stable for at least 42 days in human hepatocytes. Intrinsic innate responses were not enhanced in any of the HDV mono-infected cells and livers. CONCLUSION: These findings suggest that in addition to NTCP, further species-specific factors limit HDV infection efficacy and persistence in murine hepatocytes. Identifying such species barriers may be crucial to develop novel potential therapeutic targets of HDV.

Hepatitis Delta Virus Acts as an Immunogenic Adjuvant in Hepatitis B Virus-Infected Hepatocytes.

Hepatitis delta virus (HDV) requires hepatitis B virus (HBV) to complete its infection cycle and causes severe hepatitis, with limited therapeutic options. To determine the prospect of T cell therapy in HBV/HDV co-infection, we study the impact of HDV on viral antigen processing and presentation. Using in vitro models of HBV/HDV co-infection, we demonstrate that HDV boosts HBV epitope presentation, both in HBV/HDV co-infected and neighboring mono-HBV-infected cells through the upregulation of the antigen processing pathway mediated by IFN-ß/λ. Liver biopsies of HBV/HDV patients confirm this upregulation. We then validate in vitro and in a HBV/HDV preclinical mouse model that HDV infection increases the anti-HBV efficacy of T cells with engineered T cell receptors. Thus, by unveiling the effect of HDV on HBV antigen presentation, we provide a framework to better understand HBV/HDV immune pathology, and advocate the utilization of engineered HBV-specific T cells as a potential treatment for HBV/HDV co-infection.

T cell receptor grafting allows virological control of Hepatitis B virus infection.

T cell therapy is a promising means to treat chronic HBV infection and HBV-associated hepatocellular carcinoma. T cells engineered to express an HBV-specific T cell receptor (TCR) may achieve cure of HBV infection upon adoptive transfer. We investigated the therapeutic potential and safety of T cells stably expressing high affinity HBV envelope- or core-specific TCRs recognizing European and Asian HLA-A2 subtypes. Both CD8+ and CD4+ T cells from healthy donors and from chronic hepatitis B patients became polyfunctional effector cells when grafted with HBV-specific TCRs and eliminated HBV from infected HepG2-NTCP cell cultures. A single transfer of TCR-grafted T cells into HBV-infected, humanized mice controlled HBV infection and virological markers declined 4-5 log or below detection limit. When - as in a typical clinical setting - only a minority of hepatocytes were infected, engineered T cells specifically cleared infected hepatocytes without damaging non-infected cells. Cell death was compensated by hepatocyte proliferation and alanine amino transferase levels peaking at day 5 to 7 normalized again thereafter. Co-treatment with the entry inhibitor Myrcludex B ensured long-term control of HBV infection. Thus, T cells stably transduced with highly functional TCRs have the potential to mediate clearance of HBV-infected cells causing limited liver injury.

Hepatitis delta virus persists during liver regeneration and is amplified through cell division both in vitro and in vivo.

OBJECTIVE: Hepatitis delta virus (HDV) was shown to persist for weeks in the absence of HBV and for months after liver transplantation, demonstrating the ability of HDV to persevere in quiescent hepatocytes. The aim of the study was to evaluate the impact of cell proliferation on HDV persistence in vitro and in vivo. DESIGN: Genetically labelled human sodium taurocholate cotransporting polypeptide (hNTCP)-transduced human hepatoma(HepG2) cells were infected with HBV/HDV and passaged every 7 days for 100 days in the presence of the entry inhibitor Myrcludex-B. In vivo, cell proliferation was triggered by transplanting primary human hepatocytes (PHHs) isolated from HBV/HDV-infected humanised mice into naïve recipients. Virological parameters were measured by quantitative real time polymerase chain reaction (qRT-PCR). Hepatitis delta antigen (HDAg), hepatitis B core antigen (HBcAg) and cell proliferation were determined by immunofluorescence. RESULTS: Despite 15 in vitro cell passages and block of viral spreading by Myrcludex-B, clonal cell expansion permitted amplification of HDV infection. In vivo, expansion of PHHs isolated from HBV/HDV-infected humanised mice was confirmed 3 days, 2, 4 and 8 weeks after transplantation. While HBV markers rapidly dropped in proliferating PHHs, HDAg-positive hepatocytes were observed among dividing cells at all time points. Notably, HDAg-positive cells appeared in clusters, indicating that HDV was transmitted to daughter cells during liver regeneration even in the absence of de novo infection. CONCLUSION: This study demonstrates that HDV persists during liver regeneration by transmitting HDV RNA to dividing cells even in the absence of HBV coinfection. The strong persistence capacities of HDV may also explain why HDV clearance is difficult to achieve in HBV/HDV chronically infected patients.

A humanized mouse model of liver fibrosis following expansion of transplanted hepatic stellate cells.

Hepatic stellate cells (HSCs) are major contributors to liver fibrosis, as hepatic injuries may cause their transdifferentiation into myofibroblast-like cells capable of producing excessive extracellular matrix proteins. Also, HSCs can modulate engraftment of transplanted hepatocytes and contribute to liver regeneration. Therefore, understanding the biology of human HSCs (hHSCs) is important, but effective methods have not been available to address their fate in vivo. To investigate whether HSCs could engraft and repopulate the liver, we transplanted GFP-transduced immortalized hHSCs into immunodeficient NOD/SCID mice. Biodistribution analysis with radiolabeled hHSCs showed that after intrasplenic injection, the majority of transplanted cells rapidly translocated to the liver. GFP-immunohistochemistry demonstrated that transplanted hHSCs engrafted alongside hepatic sinusoids. Prior permeabilization of the sinusoidal endothelial layer with monocrotaline enhanced engraftment of hHSCs. Transplanted hHSCs remained engrafted without relevant proliferation in the healthy liver. However, after CCl4 or bile duct ligation-induced liver damage, transplanted hHSCs expanded and contributed to extracellular matrix production, formation of bridging cell-septae and cirrhosis-like hepatic pseudolobules. CCl4-induced injury recruited hHSCs mainly to zone 3, whereas after bile duct ligation, hHSCs were mainly in zone 1 of the liver lobule. Transplanted hHSCs neither transdifferentiated into other cell types nor formed tumors in these settings. In conclusion, a humanized mouse model was generated by transplanting hHSCs, which proliferated during hepatic injury and inflammation, and contributed to liver fibrosis. The ability to repopulate the liver with transplanted hHSCs will be particularly significant for mechanistic studies of cell-cell interactions and fibrogenesis within the liver.

A novel orally available small molecule that inhibits hepatitis B virus expression.

BACKGROUND & AIMS: The hallmarks of chronic HBV infection are a high viral load (HBV DNA) and even higher levels (>100-fold in excess of virions) of non-infectious membranous particles containing the tolerogenic viral S antigen (HBsAg). Currently, standard treatment effectively reduces viremia but only rarely results in a functional cure (defined as sustained HBsAg loss). There is an urgent need to identify novel therapies that reduce HBsAg levels and restore virus-specific immune responsiveness in patients. We report the discovery of a novel, potent and orally bioavailable small molecule inhibitor of HBV gene expression (RG7834). METHODS: RG7834 antiviral characteristics and selectivity against HBV were evaluated in HBV natural infection assays and in a urokinase-type plasminogen activator/severe combined immunodeficiency humanized mouse model of HBV infection, either alone or in combination with entecavir. RESULTS: Unlike nucleos(t)ide therapies, which reduce viremia but do not lead to an effective reduction in HBV antigen expression, RG7834 significantly reduced the levels of viral proteins (including HBsAg), as well as lowering viremia. Consistent with its proposed mechanism of action, time course RNA-seq analysis revealed a fast and selective reduction in HBV mRNAs in response to RG7834 treatment. Furthermore, oral treatment of HBV-infected humanized mice with RG7834 led to a mean HBsAg reduction of 1.09 log10 compared to entecavir, which had no significant effect on HBsAg levels. Combination of RG7834, entecavir and pegylated interferon α-2a led to significant reductions of both HBV DNA and HBsAg levels in humanized mice. CONCLUSION: We have identified a novel oral HBV viral gene expression inhibitor that blocks viral antigen and virion production, that is highly selective for HBV, and has a unique antiviral profile that is clearly differentiated from nucleos(t)ide analogues. LAY SUMMARY: We discovered a novel small molecule viral expression inhibitor that is highly selective for HBV and unlike current therapy inhibits the expression of viral proteins by specifically reducing HBV mRNAs. RG7834 can therefore potentially provide anti-HBV benefits and increase HBV cure rates, by direct reduction of viral agents needed to complete the viral life cycle, as well as a reduction of viral agents involved in evasion of the host immune responses.

Efficacy of NVR 3-778, Alone and In Combination With Pegylated Interferon, vs Entecavir In uPA/SCID Mice With Humanized Livers and HBV Infection.

BACKGROUND & AIMS: NVR3-778 is a capsid assembly modulator in clinical development. We determined the in vivo antiviral efficacy and effects on innate and endoplasmic reticulum (ER) stress responses of NVR3-778 alone or in combination with pegylated interferon alpha (peg-IFN) and compared with entecavir. METHODS: We performed 2 studies, with a total of 61 uPA/SCID mice with humanized livers. Mice were infected with a hepatitis B virus (HBV) genotype C preparation; we waited 8 weeks for persistent infection of the human hepatocytes in livers of mice. Mice were then randomly assigned to groups (5 or 6 per group) given vehicle (control), NVR3-778, entecavir, peg-IFN, NVR3-778 + entecavir, or NVR3-778 + peg-IFN for 6 weeks. We measured levels of HB surface antigen, HB e antigen, HBV RNA, alanine aminotransferase, and human serum albumin at different time points. Livers were collected and analyzed by immunohistochemistry; levels of HBV DNA, covalently closed circular DNA, and HBV RNA, along with markers of ER stress and IFN response, were quantified. RESULTS: Mice given NVR3-778 or entecavir alone for 6 weeks had reduced serum levels of HBV DNA compared with controls or mice given peg-IFN. The largest reduction was observed in mice given NVR3-778 + peg-IFN; in all mice in this group, the serum level of HBV DNA was below the limit of quantification. NVR3-778 and peg-IFN, but not entecavir, also reduced serum level of HBV RNA. The largest effect was obtained in the NVR3-778 + peg-IFN group, in which serum level of HBV RNA was below the limit of quantification. Levels of HB surface antigen and HB e antigen were reduced significantly in only the groups that received peg-IFN. Levels of covalently closed circular DNA did not differ significantly among groups. NVR3-778 was not associated with any significant changes in level of alanine aminotransferase, the ER stress response, or IFN-stimulated genes. CONCLUSIONS: NVR3-778 has high antiviral activity in mice with humanized livers and stable HBV infection, reducing levels of serum HBV DNA and HBV RNA. Entecavir reduced levels of serum HBV DNA, but had no effect on HBV RNA. The combination of NVR3-778 and peg-IFN prevented viral replication and HBV RNA particle production to a greater extent than each compound alone or entecavir.

Proliferation of primary human hepatocytes and prevention of hepatitis B virus reinfection efficiently deplete nuclear cccDNA in vivo.

OBJECTIVE: The stability of the covalently closed circular DNA (cccDNA) in nuclei of non-dividing hepatocytes represents a key determinant of HBV persistence. Contrarily, studies with animal hepadnaviruses indicated that hepatocyte turnover can reduce cccDNA loads but knowledge on the proliferative capacity of HBV-infected primary human hepatocytes (PHHs) in vivo and the fate of cccDNA in dividing PHHs is still lacking. This study aimed to determine the impact of human hepatocyte division on cccDNA stability in vivo. METHODS: PHH proliferation was triggered by serially transplanting hepatocytes from HBV-infected humanised mice into naïve recipients. Cell proliferation and virological changes were assessed by quantitative PCR, immunofluorescence and RNA in situ hybridisation. Viral integrations were analysed by gel separation and deep sequencing. RESULTS: PHH proliferation strongly reduced all infection markers, including cccDNA (median 2.4 log/PHH). Remarkably, cell division appeared to cause cccDNA dilution among daughter cells and intrahepatic cccDNA loss. Nevertheless, HBV survived in sporadic non-proliferating human hepatocytes, so that virological markers rebounded as hepatocyte expansion relented. This was due to reinfection of quiescent PHHs since treatment with the entry inhibitor myrcludex-B or nucleoside analogues blocked viral spread and intrahepatic cccDNA accumulation. Viral integrations were detected both in donors and recipient mice but did not appear to contribute to antigen production. CONCLUSIONS: We demonstrate that human hepatocyte division even without involvement of cytolytic mechanisms triggers substantial cccDNA loss. This process may be fundamental to resolve self-limiting acute infection and should be considered in future therapeutic interventions along with entry inhibition strategies.

Lymphocytes transiently expressing virus-specific T cell receptors reduce hepatitis B virus infection.

Adoptive transfer of T cells engineered to express a hepatitis B virus-specific (HBV-specific) T cell receptor (TCR) may supplement HBV-specific immune responses in chronic HBV patients and facilitate HBV control. However, the risk of triggering unrestrained proliferation of permanently engineered T cells raises safety concerns that have hampered testing of this approach in patients. The aim of the present study was to generate T cells that transiently express HBV-specific TCRs using mRNA electroporation and to assess their antiviral and pathogenetic activity in vitro and in HBV-infected human liver chimeric mice. We assessed virological and gene-expression changes using quantitative reverse-transcriptase PCR (qRT-PCR), immunofluorescence, and Luminex technology. HBV-specific T cells lysed HBV-producing hepatoma cells in vitro. In vivo, 3 injections of HBV-specific T cells caused progressive viremia reduction within 12 days of treatment in animals reconstituted with haplotype-matched hepatocytes, whereas viremia remained stable in mice receiving irrelevant T cells redirected toward hepatitis C virus-specific TCRs. Notably, increases in alanine aminotransferase levels, apoptotic markers, and human inflammatory cytokines returned to pretreatment levels within 9 days after the last injection. T cell transfer did not trigger inflammation in uninfected mice. These data support the feasibility of using mRNA electroporation to engineer HBV TCR-redirected T cells in patients with chronic HBV infection.

Both interferon alpha and lambda can reduce all intrahepatic HDV infection markers in HBV/HDV infected humanized mice.

Co-infection with hepatitis B (HBV) and D virus (HDV) is associated with the most severe course of liver disease. Interferon represents the only treatment currently approved. However, knowledge about the impact of interferons on HDV in human hepatocytes is scant. Aim was to assess the effect of pegylated interferon alpha (peg-IFNα) and lambda (peg-IFNλ), compared to the HBV-polymerase inhibitor entecavir (ETV) on all HDV infection markers using human liver chimeric mice and novel HDV strand-specific qRT-PCR and RNA in situ hybridization assays, which enable intrahepatic detection of HDV RNA species. Peg-IFNα and peg-IFNλ reduced HDV viremia (1.4 log and 1.2 log, respectively) and serum HBsAg levels (0.9-log and 0.4-log, respectively). Intrahepatic quantification of genomic and antigenomic HDV RNAs revealed a median ratio of 22:1 in untreated mice, resembling levels determined in HBV/HDV infected patients. Both IFNs greatly reduced intrahepatic levels of genomic and antigenomic HDV RNA, increasing the amounts of HDAg- and antigenomic RNA-negative hepatocytes. ETV-mediated suppression of HBV replication (2.1-log) did not significantly affect HBsAg levels, HDV productivity and/or release. In humanized mice lacking adaptive immunity, IFNs but not ETV suppressed HDV. Viremia decrease reflected the intrahepatic reduction of all HDV markers, including the antigenomic template, suggesting that intracellular HDV clearance is achievable.

Haem oxygenase-1 polymorphisms can affect HCV replication and treatment responses with different efficacy in humanized mice.

BACKGROUND & AIMS: Enhancement of host anti-oxidant enzymes, such as haemoxygenase-1, may attenuate virus-mediated hepatocyte injury, while the induction of HO-1 by cobalt-protoporphyrin-IX (CoPP) administration, as the application of its haem degradation product biliverdin (BV), was shown to hinder HCV replication in vitro. In addition, (GT)n -repeats length in the polymorphic region of the HO-1 promoter may affect HO-1 expression and responsiveness to infection and disease severity. Aim of this study was to investigate the antiviral and hepatoprotective effects of CoPP-mediated HO-1 induction, alone or in combination with interferon alpha (peg-IFNα), in HCV-infected mice harbouring hepatocytes from donors with different HO-1-promoter polymorphisms. METHODS: Upon establishment of HCV infection, CoPP, BV and peg-IFNα were given alone or in combination. Viraemia changes and intrahepatic human gene expression were determined by qRT-PCR and immunohistochemistry. RESULTS: CoPP administration increased human HO-1 expression and significantly reduced viraemia, although changes correlated with promoter length (Δ0.5log and Δ2log reduction with medium- and short-polymorphism respectively). Polymorphisms did not influence BV-mediated antiviral effects (Δ1log). Notably, HO-1 induction attenuated basal HCV-driven enhancement of interferon genes and pro-inflammatory cytokines, both in cells with short- or medium-polymorphisms. Moreover, simultaneous administration of CoPP and peg-IFNα reduced viraemia even stronger (median 3log), whereas 1log viraemia reduction was determined in mice receiving peg-IFNα monotherapy. CONCLUSIONS: Although the protective function of HO-1 could be elicited in vivo with both host polymorphisms, the strength of HO-1 induction and suppression of HCV occurred in a polymorphism-dependent manner, indicating that host-genetic determinants may affect disease progression and infection outcome.

Multi-color RGB marking enables clonality assessment of liver tumors in a murine xenograft model.

We recently introduced red-green-blue (RGB) marking for clonal cell tracking based on individual color-coding. Here, we applied RGB marking to study clonal development of liver tumors. Immortalized, non-tumorigenic human fetal hepatocytes expressing the human telomerase reverse transcriptase (FH-hTERT) were RGB-marked by simultaneous transduction with lentiviral vectors encoding mCherry, Venus, and Cerulean. Multi-color fluorescence microscopy was used to analyze growth characteristics of RGB-marked FH-hTERT in vitro and in vivo after transplantation into livers of immunodeficient mice with endogenous liver damage (uPA/SCID). After initially polyclonal engraftment we observed oligoclonal regenerative nodules derived from transplanted RGB-marked FH-hTERT. Some mice developed monochromatic invasive liver tumors; their clonal origin was confirmed both on the molecular level, based on specific lentiviral-vector insertion sites, and by serial transplantation of one tumor. Vector insertions in proximity to the proto-oncogene MCF2 and the transcription factor MITF resulted in strong upregulation of mRNA expression in the respective tumors. Notably, upregulated MCF2 and MITF expression was also observed in 21% and 33% of 24 human hepatocellular carcinomas analyzed. In conclusion, liver repopulation with RGB-marked FH-hTERT is a useful tool to study clonal progression of liver tumors caused by insertional mutagenesis in vivo and will help identifying genes involved in liver cancer.

A Slow Maturation Process Renders Hepatitis B Virus Infectious.

Hepatitis B virus (HBV) replication is strictly limited to the liver. Virions attach to hepatocytes through interactions of the viral PreS envelope protein domain with heparan sulfate proteoglycans (HSPGs). However, HSPG is ubiquitously present on many cell types, suggesting that HBV employs mechanisms to avoid attachment at extrahepatic sites. We demonstrate that HBV particles are released from cells in an inactive form with PreS hidden in the interior. These HSPG-non-binding (N-type) particles develop receptor binding competence by translocating PreS across the envelope onto their surface. Conversion into HSPG-binding (B-type) particles occurs spontaneously and renders HBV infectious. Low-dose inoculation of mice with human liver xenografts demonstrates superiority of N-type particles in establishing infections, while mature B-type virions, generated via N-type conversion, are profoundly impaired, correlating with non-selective accumulation in extrahepatic tissues. This dynamic topology switch represents a maturation process utilized by HBV to most likely avoid non-productive docking outside the liver.

Primary Human Hepatocytes Repopulate Livers of Mice After In Vitro Culturing and Lentiviral-Mediated Gene Transfer.

Cell-based therapies represent a promising alternative to orthotopic liver transplantation. However, therapeutic effects are limited by low cell engraftment rates. We recently introduced a technique creating human hepatocyte spheroids for potential therapeutic application. The aim of this study was to evaluate whether these spheroids are suitable for engraftment in diseased liver tissues. Intrasplenic spheroid transplantation into immunodeficient uPA/SCID/beige mice was performed. Hepatocyte transduction ability prior to transplantation was tested by lentiviral labeling using red-green-blue (RGB) marking. Eight weeks after transplantation, animals were sacrificed and livers were analyzed by immunohistochemistry and immunofluorescence. To investigate human hepatocyte-specific gene expression profiles in mice, quantitative real-time-PCR was applied. Human albumin and alpha-1-antitrypsin concentrations in mouse serum were quantified to assess the levels of human chimerism. Precultured human hepatocytes reestablished their physiological liver tissue architecture and function upon transplantation in mice. Positive immunohistochemical labeling of the proliferating cell nuclear antigen revealed that human hepatocytes retained their in vivo proliferation capacity. Expression profiles of human genes analyzed in chimeric mouse livers resembled levels determined in native human tissue. Extensive vascularization of human cell clusters was detected by demonstration of von Willebrand factor activity. To model gene therapy approaches, lentiviral transduction was performed ex vivo and fluorescent microscopic imaging revealed maintenance of RGB marking in vivo. Altogether, this is the first report demonstrating that cultured and retroviral transduced human hepatocyte spheroids are able to engraft and maintain their regenerative potential in vivo.