Multiscale modeling of HBV infection integrating intra- and intercellular viral propagation to analyze extracellular viral markers.

Chronic infection with hepatitis B virus (HBV) is caused by the persistence of closed circular DNA (cccDNA) in the nucleus of infected hepatocytes. Despite available therapeutic anti-HBV agents, eliminating the cccDNA remains challenging. Thus, quantifying and understanding the dynamics of cccDNA are essential for developing effective treatment strategies and new drugs. However, such study requires repeated liver biopsy to measure the intrahepatic cccDNA, which is basically not accepted because liver biopsy is potentially morbid and not common during hepatitis B treatment. We here aimed to develop a noninvasive method for quantifying cccDNA in the liver using surrogate markers in peripheral blood. We constructed a multiscale mathematical model that explicitly incorporates both intracellular and intercellular HBV infection processes. The model, based on age-structured partial differential equations, integrates experimental data from in vitro and in vivo investigations. By applying this model, we roughly predicted the amount and dynamics of intrahepatic cccDNA within a certain range using specific viral markers in serum samples, including HBV DNA, HBsAg, HBeAg, and HBcrAg. Our study represents a significant step towards advancing the understanding of chronic HBV infection. The noninvasive quantification of cccDNA using our proposed method holds promise for improving clinical analyses and treatment strategies. By comprehensively describing the interactions of all components involved in HBV infection, our multiscale mathematical model provides a valuable framework for further research and the development of targeted interventions.

Blocking viral entry with bulevirtide reduces the number of HDV-infected hepatocytes in human liver biopsies.

BACKGROUND & AIMS: Bulevirtide (BLV) is a first-in-class entry inhibitor and the only approved treatment for patients chronically infected with HDV in Europe. We aimed to investigate the efficacy of BLV treatment in paired liver biopsies obtained at baseline and after 24 or 48 weeks of treatment. METHODS: We performed a combined analysis of 126 paired liver biopsies derived from three clinical trials. In the phase II clinical trial MYR202, patients with chronic hepatitis D were randomised to receive 24 weeks of BLV at 2 mg, 5 mg or 10 mg/day. Patients in MYR203 (phase II) and MYR301 (phase III) received 48 weeks of BLV at 2 mg or 10 mg/day. Tenofovir disoproxil fumarate monotherapy or delayed treatment served as comparators. Virological parameters and infection-related host genes were assessed by qPCR and immunohistochemistry. RESULTS: At week 24, median intrahepatic HDV RNA decline from baseline was 0.9Log10 with 2 mg (n = 7), 1.1Log10 with 5 mg (n = 5) and 1.4 Log10 with 10 mg (n = 7) of BLV. At week 48, median reductions were 2.2Log10 with 2 mg (n = 27) and 2.7Log10 with 10 mg (n = 37) of BLV, while HDV RNA levels did not change in the comparator arms. Notably, a drastic decline in the number of hepatitis delta antigen-positive hepatocytes and a concomitant decrease in transcriptional levels of inflammatory chemokines and interferon-stimulated genes was determined in all BLV-treatment arms. Despite the abundance of HBsAg-positive hepatocytes, replication and covalently closed circular DNA levels of the helper virus HBV were low and remained unaffected by BLV treatment. CONCLUSION: Blocking viral entry diminishes signs of liver inflammation and promotes a strong reduction of HDV infection within the liver, thus suggesting that some patients may achieve HDV cure with long-term treatment. IMPACT AND IMPLICATIONS: Chronic infection with HDV causes the most severe form of viral hepatitis, affecting approximately 12 million people worldwide. The entry inhibitor bulevirtide (BLV) is the only recently approved anti-HDV drug, which has proven efficacious and safe in clinical trials and real-word data. Here, we investigated paired liver biopsies at baseline and after 24 or 48 weeks of treatment from three clinical trials to understand the effect of the drug on viral and host parameters in the liver, the site of viral replication. We found that BLV treatment strongly reduces the number of HDV-infected cells and signs of liver inflammation. This data implies that blocking viral entry ameliorates liver inflammation and that prolonged treatment regimens might lead to HDV cure in some patients. This concept will guide the further development of therapeutic strategies and combination treatments for patients with CHD. CLINICAL TRIAL NUMBERS: NCT03546621, NCT02888106, NCT03852719.

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.

Multiscale modeling of HBV infection integrating intra- and intercellular viral propagation for analyzing extracellular viral markers.

Chronic infection of hepatitis B virus (HBV) is caused by the persistence of closed circular DNA (cccDNA) in the nucleus of infected hepatocytes. Despite available therapeutic anti-HBV agents, eliminating the cccDNA remains challenging. The quantifying and understanding dynamics of cccDNA are essential for developing effective treatment strategies and new drugs. However, it requires a liver biopsy to measure the intrahepatic cccDNA, which is basically not accepted because of the ethical aspect. We here aimed to develop a non-invasive method for quantifying cccDNA in the liver using surrogate markers present in peripheral blood. We constructed a multiscale mathematical model that explicitly incorporates both intracellular and intercellular HBV infection processes. The model, based on age-structured partial differential equations (PDEs), integrates experimental data from in vitro and in vivo investigations. By applying this model, we successfully predicted the amount and dynamics of intrahepatic cccDNA using specific viral markers in serum samples, including HBV DNA, HBsAg, HBeAg, and HBcrAg. Our study represents a significant step towards advancing the understanding of chronic HBV infection. The non-invasive quantification of cccDNA using our proposed methodology holds promise for improving clinical analyses and treatment strategies. By comprehensively describing the interactions of all components involved in HBV infection, our multiscale mathematical model provides a valuable framework for further research and the development of targeted interventions.

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.

A 3-Year Course Of Bulevirtide Monotherapy May Cure Hdv Infection In Cirrhotics.

Bulevirtide has been recently conditionally approved by the European Medicines Agency for the treatment of Chronic Hepatitis Delta, but the ideal duration of therapy is unknown. Here we describe the first case of cure of Hepatitis Delta following 3 years of Bulevirtide monotherapy in a patient with compensated cirrhosis and esophageal varices. During the 72-week off-Bulevirtide follow-up, virological and biochemical responses were maintained. In the off-therapy liver biopsy, intrahepatic HDV RNA and Hepatitis D antigen were undetectable, <1% hepatocytes were Hepatitis B surface antigen positive while hepatitis B core antigen was negative. Grading and staging improved compared to pre-treatment biopsy.

Quantification of the hepatitis B virus cccDNA: evidence-based guidelines for monitoring the key obstacle of HBV cure.

OBJECTIVES: A major goal of curative hepatitis B virus (HBV) treatments is the reduction or inactivation of intrahepatic viral covalently closed circular DNA (cccDNA). Hence, precise cccDNA quantification is essential in preclinical and clinical studies. Southern blot (SB) permits cccDNA visualisation but lacks sensitivity and is very laborious. Quantitative PCR (qPCR) has no such limitations but inaccurate quantification due to codetection of viral replicative intermediates (RI) can occur. The use of different samples, preservation conditions, DNA extraction, nuclease digestion methods and qPCR strategies has hindered standardisation. Within the ICE-HBV consortium, available and novel protocols for cccDNA isolation and qPCR quantification in liver tissues and cell cultures were compared in six laboratories to develop evidence-based guidance for best practices. DESIGN: Reference material (HBV-infected humanised mouse livers and HepG2-NTCP cells) was exchanged for cross-validation. Each group compared different DNA extraction methods (Hirt extraction, total DNA extraction with or without proteinase K treatment (+PK/-PK)) and nuclease digestion protocols (plasmid-safe ATP-dependent DNase (PSD), T5 exonuclease, exonucleases I/III). Samples were analysed by qPCR and SB. RESULTS: Hirt and -PK extraction reduced coexisting RI forms. However, both cccDNA and the protein-free relaxed circular HBV DNA (pf-rcDNA) form were detected by qPCR. T5 and Exo I/III nucleases efficiently removed all RI forms. In contrast, PSD did not digest pf-rcDNA, but was less prone to induce cccDNA overdigestion. In stabilised tissues (eg, Allprotect), nucleases had detrimental effects on cccDNA. CONCLUSIONS: We present here a comprehensive evidence-based guidance for optimising, controlling and validating cccDNA measurements using available qPCR assays.

A roadmap for serum biomarkers for hepatitis B virus: current status and future outlook.

Globally, 296 million people are infected with hepatitis B virus (HBV), and approximately one million people die annually from HBV-related causes, including liver cancer. Although there is a preventative vaccine and antiviral therapies suppressing HBV replication, there is no cure. Intensive efforts are under way to develop curative HBV therapies. Currently, only a few biomarkers are available for monitoring or predicting HBV disease progression and treatment response. As new therapies become available, new biomarkers to monitor viral and host responses are urgently needed. In October 2020, the International Coalition to Eliminate Hepatitis B Virus (ICE-HBV) held a virtual and interactive workshop on HBV biomarkers endorsed by the International HBV Meeting. Various stakeholders from academia, clinical practice and the pharmaceutical industry, with complementary expertise, presented and participated in panel discussions. The clinical utility of both classic and emerging viral and immunological serum biomarkers with respect to the course of infection, disease progression, and response to current and emerging treatments was appraised. The latest advances were discussed, and knowledge gaps in understanding and interpretation of HBV biomarkers were identified. This Roadmap summarizes the strengths, weaknesses, opportunities and challenges of HBV biomarkers.

High Rates of Liver Cirrhosis and Hepatocellular Carcinoma in Chronic Hepatitis B Patients with Metabolic and Cardiovascular Comorbidities.

BACKGROUND: The prevalence of metabolic and cardiovascular diseases is rising worldwide. However, little is known about the impact of such disorders on hepatic disease progression in chronic hepatitis B (CHB) during the era of potent nucleo(s)tide analogues (NAs). METHODS: We retrospectively analyzed a single-center cohort of 602 CHB patients, comparing the frequency of liver cirrhosis at baseline and incidences of liver-related events during follow-up (hepatocellular carcinoma, liver transplantation and liver-related death) between CHB patients with a history of diabetes, obesity, hypertension or coronary heart disease (CHD). RESULTS: Rates of cirrhosis at baseline and liver-related events during follow-up (median follow-up time: 2.51 years; NA-treated: 37%) were substantially higher in CHB patients with diabetes (11/23; 3/23), obesity (6/13; 2/13), CHD (7/11; 2/11) or hypertension (15/43; 4/43) compared to CHB patients without the indicated comorbidities (26/509; 6/509). Multivariate analysis identified diabetes as the most significant predictor for cirrhosis (p = 0.0105), while comorbidities did not correlate with liver-related events in pre-existing cirrhosis. CONCLUSION: The combination of metabolic diseases and CHB is associated with substantially increased rates of liver cirrhosis and secondary liver-related events compared to CHB alone, indicating that hepatitis B patients with metabolic comorbidities warrant particular attention in disease surveillance and evaluation of treatment indication.

In Vivo Models of HDV Infection: Is Humanizing NTCP Enough?

The discovery of sodium taurocholate co-transporting polypeptide (NTCP) as a hepatitis B (HBV) and delta virus (HDV) entry receptor has encouraged the development of new animal models of infection. This review provides an overview of the different in vivo models that are currently available to study HDV either in the absence or presence of HBV. By presenting new advances and remaining drawbacks, we will discuss human host factors which, in addition to NTCP, need to be investigated or identified to enable a persistent HDV infection in murine hepatocytes. Detailed knowledge on species-specific factors involved in HDV persistence also shall contribute to the development of therapeutic strategies.

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.

cccDNA Maintenance in Chronic Hepatitis B - Targeting the Matrix of Viral Replication.

Chronic hepatitis B is a numerically important cause of cirrhosis and hepatocellular carcinoma, despite an effective prophylactic vaccine and well-tolerated and effective oral antivirals. Both the incapacity of the immune system to clear hepatitis B virus (HBV) infection and the unique replication strategies adopted by HBV are considered key determinants of HBV chronicity. In this regard, the formation of the HBV DNA minichromosome, the covalently closed circular DNA (cccDNA), in the nucleus of infected hepatocytes, is essential not only for the production of all viral proteins but also for HBV persistence even after long-term antiviral therapy. Licensed polymerase inhibitors target the HBV reverse transcriptase activity, control the disease with long-term therapy but fail to eliminate the cccDNA. Consequently, the production of viral RNAs and proteins, including the hepatitis B surface antigen (HBsAg), is not abolished. Novel therapeutic efforts that are in the pipeline for early clinical trials explore novel targets and molecules. Such therapeutic efforts focus on achieving a functional cure, which is defined by the loss of HBsAg and undetectable HBV DNA levels in serum. Since a true cure of HBV infection requires the elimination of the cccDNA from infected cells, comprehension of the mechanisms implicated in cccDNA biogenesis, regulation and stability appears necessary to achieve HBV eradication. In this review, we will summarize the state of knowledge on cccDNA metabolism, focusing on insights suggesting potential weak points of the cccDNA that may be key for the development of therapeutic approaches and design of clinical trials aiming at lowering cccDNA loads and activity.

A novel method to precisely quantify hepatitis B virus covalently closed circular (ccc)DNA formation and maintenance.

Hepatitis B virus (HBV) is the major cause of virus-associated liver disease. Persistent HBV infection is maintained by its episomal genome (covalently closed circular DNA, cccDNA), which acts as a template for viral transcripts. The formation of cccDNA is poorly characterised due to limited ability to quantify it accurately in the presence of replicative intermediates. Here, we describe a novel cccDNA quantification assay (cccDNA inversion quantitative PCR, cinqPCR), which uses restriction enzymes to invert a DNA sequence close to the gap region of Genotype D HBV strains, including the isolate widely used in experimental studies. Importantly, cinqPCR allows simultaneous normalisation to cellular DNA in a single reaction, provides absolute copy numbers without requiring a standard curve, and has high precision, sensitivity, and specificity for cccDNA compared to previous assays. We first established that cinqPCR gives values consistent with classical approaches in both in vitro and in vivo (humanised mice) HBV infections. We then used cinqPCR to find that cccDNA is formed within 12 h post-inoculation (hpi). cccDNA formation slowed by 28 hpi despite de novo synthesis of HBV DNA, indicating inefficient conversion of new viral genomes to cccDNA within infected cells. Finally, we show that cinqPCR can be used as a 96-well screening assay. Thus, we have developed an ideal method for testing current and future anti-cccDNA therapeutics with high precision and sensitivity.

Epigenetic modulation in chronic hepatitis B virus infection.

The human hepatitis B virus (HBV) is a small-enveloped DNA virus causing acute and chronic hepatitis. Despite the existence of an effective prophylactic vaccine and the strong capacity of approved antiviral drugs to suppress viral replication, chronic HBV infection (CHB) continues to be a major health burden worldwide. Both the inability of the immune system to resolve CHB and the unique replication strategy employed by HBV, which forms a stable viral covalently closed circular DNA (cccDNA) minichromosome in the hepatocyte nucleus, enable infection persistence. Knowledge of the complex network of interactions that HBV engages with its host is still limited but accumulating evidence indicates that epigenetic modifications occurring both on the cccDNA and on the host genome in the course of infection are essential to modulate viral activity and likely contribute to pathogenesis and cancer development. Thus, a deeper understanding of epigenetic regulatory processes may open new venues to control and eventually cure CHB. This review summarizes major findings in HBV epigenetic research, focusing on the epigenetic mechanisms regulating cccDNA activity and the modifications determined in infected host cells and tumor liver tissues.

High rates of cirrhosis and severe clinical events in patients with HBV/HDV co-infection: longitudinal analysis of a German cohort.

BACKGROUND: Chronic hepatitis delta virus (HDV) infection causes severe liver disease which often leads to cirrhosis and hepatocellular carcinoma (HCC). Aim of this study was to establish the disease severity and prognostic factors for disease outcome by analysing frequencies of clinical events and their correlation with baseline virological and biochemical parameters as well as interferon and nucleos(t)ide analogue treatment choice. METHODS: We studied a single-centre cohort of 49 anti-HDAg-positive patients with HBsAg persistence for at least 6 months. Virological and biochemical parameters, interferon and nucleos(t)ide analogue treatment choice as well as clinical events during follow-up were analysed by retrospective chart review (mean follow-up time 3 years, range 0.25-7.67 years). RESULTS: Severe clinical events occurred in 11/49 hepatitis D patients, including HCC (8/49), death (8/49) or liver transplantation (2/49). HCCs only occurred secondary to liver cirrhosis and their event rates in this cohort of hepatitis D patients did not differ from a matched HBV mono-infected cohort with comparable frequency of liver cirrhosis. A stepwise multivariate logistic regression revealed low platelet count (p = 0. 0290) and older age (p = 0.0337) correlating most strongly with overall clinical events, while serum HDV RNA positivity at baseline did not correlate with any clinical outcome. Interferon-free but not nucleos(t)ide analogue-free patient care correlated with the occurrence of HCC at logistic regression, although only 3/18 interferon-treated patients demonstrated repeatedly negative HDV PCR results post therapy. CONCLUSIONS: Our data indicate that progressive liver disease at baseline plays a major role as predictive factor for overall clinical outcome of hepatitis D patients. In particular, HCC risk may not be underestimated in hepatitis D virus RNA negative hepatitis D patients with advanced liver fibrosis.

Hepatitis B Virus Particles Activate Toll-Like Receptor 2 Signaling Initially Upon Infection of Primary Human Hepatocytes.

BACKGROUND AND AIMS: To date, conflicting data exist as to whether hepatitis B virus (HBV) has the ability to induce innate immune responses. Here, we investigated cellular changes after the first contact between HBV and primary human hepatocytes (PHH) in vitro and in vivo. APPROACH AND RESULTS: The exposure of PHH to HBV particles resulted in nuclear translocation of NFκB, followed by the expression and secretion of inflammatory cytokines (IL [interleukin] 1B, IL6, and TNF [tumor necrosis factor]). Ultraviolet irradiation of viral particles suppressed HBV infectivity but not the induction of cytokines in PHH, suggesting that the inoculum contains the immune-inducing agent. Purified HBV particles on the whole, which were prepared from HBV DNA-positive and protein-rich fractions after heparin column separation, still had immune-inducing capacity in PHH. The HBV-induced gene expression profile was similar to that induced by toll-like receptor 2 (TLR2) ligand Pam3Cys, but different from those induced by the viral sensors TLR3 or TLR7-9. Treatment of PHH with both HBV particles and Pam3Cys led to phosphorylation of ERK (extracellular signal-regulated kinase), JNK, and p38 mitogen-activated protein kinases as well as NFκB (nuclear factor kappa B). Finally, HBV-induced gene expression could be neutralized by TLR2-specific antibodies. Of note, pretreatment with an HBV entry inhibitor attenuated the TLR2-mediated response to HBV, suggesting a receptor binding-related mechanism. In liver-humanized uPA/severe combined immunodeficient (SCID)/beige mice challenged with HBV in vivo, immune induction could only marginally be seen. CONCLUSIONS: PHHs are able to sense HBV particles through TLR2, leading to an activation of anti-HBV immune responses in vitro. These findings challenge the previously described stealth properties of HBV.

Down-regulation of cell membrane localized NTCP expression in proliferating hepatocytes prevents hepatitis B virus infection.

Hepatocyte proliferation could result in the loss of covalently closed circular DNA (cccDNA) and the emergence of cccDNA-cleared nascent hepatocytes, which appear refractory to hepatitis B virus (HBV) reinfection with unknown mechanism(s). Sodium taurocholate cotransporting polypeptide (NTCP) is the functional receptor for HBV entry. In this study, down-regulation of cell membrane localized NTCP expression in proliferating hepatocytes was found to prevent HBV infection in HepG2-NTCP-tet cells and in liver-humanized mice. In patients, lower NTCP protein expression was correlated well with higher levels of hepatocyte proliferation and less HBsAg expression in HBV-related focal nodular hyperplasia (FNH) tissues. Clinically, significantly lower NTCP protein expression was correlated with more active hepatocyte proliferation in CHB patients with severe active necroinflammation and better antiviral treatment outcome. Mechanistically, the activation of cell cycle regulatory genes p53, S-phase kinase-associated protein 2 (SKP2) and cyclin D1 during cell proliferation, as well as proliferative and inflammatory cytokine Interleukin-6 (IL-6) could transcriptionally down-regulate NTCP expression. From these aspects, we conclude that within the milieu of hepatocyte proliferation, down-regulation of cell membrane localized NTCP expression level renders nascent hepatocytes resistant to HBV reinfection. This may accelerate virus clearance during immune-mediated cell death and compensatory proliferation of survival hepatocytes.

CD49a Expression Identifies a Subset of Intrahepatic Macrophages in Humans.

Macrophages play central roles in inflammatory reactions and initiation of immune responses during infections. More than 80% of total tissue macrophages are described to be located in the liver as liver-resident macrophages, also named Kupffer cells (KCs). While studies in mice have established a central role of liver-resident KCs in regulating liver inflammation, their phenotype and function are not well-characterized in humans. Comparing paired human liver and peripheral blood samples, we observed significant differences in the distribution of macrophage (Mφ) subsets, with lower frequencies of CD14hiCD16lo and higher frequencies of CD14int-hiCD16int Mφ in human livers. Intrahepatic Mφ consisted of diverse subsets with differential expression of CD49a, a liver-residency marker previously described for human and mice NK cells, and VSIG4 and/or MARCO, two recently described human tissue Mφ markers. Furthermore, intrahepatic CD49a+ Mφ expressed significantly higher levels of maturation and activation markers, exhibited higher baseline levels of TNF-α, IL-12, and IL-10 production, but responded less to additional in vitro TLR stimulation. In contrast, intrahepatic CD49a- Mφ were highly responsive to stimulation with TLR ligands, similar to what was observed for CD49a- monocytes (MOs) in peripheral blood. Taken together, these studies identified populations of CD49a+, VSIG4+, and/or MARCO+ Mφ in human livers, and demonstrated that intrahepatic CD49a+ Mφ differed in phenotype and function from intrahepatic CD49a- Mφ as well as from peripheral blood-derived monocytes.

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.