Hepatitis B Virus Transgenic Mice Research Articles

Single-cell RNA sequencing reveals reduced intercellular adhesion molecule crosstalk between activated hepatic stellate cells and neutrophils alleviating liver fibrosis in hepatitis B virus transgenic mice post menstrual blood-derived mesenchymal stem cell transplantation.

Liver fibrosis can cause hepatitis B virus (HBV)-associated hepatocellular carcinoma. Menstrual blood-derived mesenchymal stem cells (MenSCs) can ameliorate liver fibrosis through paracrine. Single-cell RNA sequencing (scRNA-seq) may be used to explore the roadmap of activated hepatic stellate cell (aHSC) inactivation to target liver fibrosis. This study established HBV transgenic (HBV-Tg) mouse model of carbon tetrachloride (CCl4)-induced liver fibrosis and demonstrated that MenSCs migrated to the injured liver to improve serological indices and reduce fibrotic accumulation. RNA-bulk analysis revealed that MenSCs mediated extracellular matrix accumulation and cell adhesion. Liver parenchymal cells and nonparenchymal cells were identified by scRNA-seq in the control, CCl4, and MenSC groups, revealing the heterogeneity of fibroblasts/HSCs. A CellChat analysis revealed that diminished intercellular adhesion molecule (ICAM) signaling is vital for MenSC therapy. Specifically, Icam1 in aHSCs acted on Itgal/Itgb2 and Itgam/Itgb2 in neutrophils, causing decreased adhesion. The expression of Itgal, Itgam, and Itgb2 was higher in CCl4 group than in the control group and decreased after MenSC therapy in neutrophil clusters. The Lcn2, Pglyrp1, Wfdc21, and Mmp8 had high expression and may be potential targets in neutrophils. This study highlights interacting cells, corresponding molecules, and underlying targets for MenSCs in treating HBV-associated liver fibrosis.

HBV-related HCC development in mice is STAT3 dependent and indicates an oncogenic effect of HBx

Background&Aims: Chronic Hepatitis B with high risk for hepatocellular carcinoma (HCC) remains a major challenge, worldwide. Although many HCC cases are driven by hepatitis and liver cirrhosis, a subset of patients develops HCC in the absence of advanced liver disease, indicating the oncogenic potential of Hepatitis B Virus (HBV). We investigated the role of HBV-transcripts and proteins on HCC development in the absence of inflammation in HBV-transgenic mice. MethodsHBV-transgenic mice replicating HBV and expressing all HBV proteins from a single integrated 1.3-fold HBV genome in presence or absence of wildtype HBx (HBV1.3/HBVxfs) were analyzed. Flow cytometry, molecular, histological and in vitro analyses using human cell lines were performed. Hepatocyte-specific Stat3- and Socs3-knockout was analyzed in HBV1.3 mice. Results∼38% of HBV1.3 mice developed liver tumors. Protein expression patterns, histology and mutational landscape analyses indicated that tumors resembled human HCC. HBV1.3 mice showed no signs of active hepatitis up to the time-point of HCC development but STAT3-activation. HBV-RNAs covering HBx sequence, 3.5kb HBV-RNA and HBx-protein were detected in HCC tissue. Interestingly, HBVxfs mice expressing all HBV proteins but a C-terminally truncated HBx (without the ability to bind DDB1) showed reduced signs of DNA damage response and had a significantly reduced HCC incidence. Importantly, intercrossing HBV1.3 mice with a hepatocyte-specific STAT3-knockout abrogated HCC development. ConclusionExpression of HBV-proteins is sufficient to cause HCC in the absence of detectable inflammation. This indicates the oncogenic potential of HBV and in particular HBx. In our model, HBV-driven HCC was STAT3-dependent. Our study highlights the immediate oncogenic potential of HBV, challenging the notion of a benign highly replicative phase of HBV infection and indicating the necessity for an HBV “cure”. Impact and implicationsAlthough most HCC cases in chronically HBV infected patients occur after a sequence of liver damage and fibrosis, a subset of patients develop HCC without any signs of advanced liver damage. We demonstrate that the expression of all viral transcripts in HBV-transgenic mice suffices to induce HCC development independent of inflammation and fibrosis. These data indicate the direct oncogenic effects of HBV and emphasize the notion of early antiviral treatment in the “immune-tolerant” phase (HBeAg positive chronic HBV infection).

Analysis of the pharmacokinetics and efficacy of RBD1016 – A GalNAc-siRNA targeting Hepatitis B Virus X gene using semi-mechanistic PK/PD model

Small interference RNA (siRNA) is a class of short double-stranded RNA molecules that cause mRNA degradation through an RNA interference mechanism and is a promising therapeutic modality. RBD1016 is a siRNA drug in clinical development for the treatment of chronic Hepatitis B Virus (HBV) infection, which contains a conjugated with N-acetylglucosamine moiety that can facilitate its hepatic delivery. We aimed to construct a semi-mechanistic model of RBD1016 in pre-clinical animals, to elucidate the pharmacokinetic/pharmacodynamic (PK/PD) profiles in mice and PK profiles in monkeys, which can lay the foundation for potential future translation of RBD1016 PK and PD from the pre-clinical stage to the clinic stage. The proposed semi-mechanistic PK/PD model fitted PK and PD data in HBV transgenic mice well and described plasma and liver concentrations in the monkeys well. The simulation results showed that our model has a reasonable predictive ability for Hepatitis B surface antigen (HBsAg) levels after multiple dosing in mice. Further PK and PD data for RBD1016, including clinical data, will assist in refining the model presented here. Our current effort focused on model building for RBD1016, we anticipate that the model could apply to other GalNAc-siRNA drugs.

Galectin-3-ITGB1 Signaling Mediates Interleukin 10 Production of Hepatic Conventional Natural Killer Cells in Hepatitis B Virus Transgenic Mice and Correlates with Hepatocellular Carcinoma Progression in Patients.

The outcomes of HBV infections are related to complex immune imbalances; however, the precise mechanisms by which HBV induces immune dysfunction are not well understood. HBV transgenic (HBs-Tg) mice were used to investigate intrahepatic NK cells in two distinct subsets: conventional NK (cNK) and liver-resident NK (LrNK) cells during a chronic HBV infection. The cNK cells, but not the LrNK cells, were primarily responsible for the increase in the number of bulk NK cells in the livers of ageing HBs-Tg mice. The hepatic cNK cells showed a stronger ability to produce IL-10, coupled with a higher expression of CD69, TIGIT and PD-L1, and lower NKG2D expression in ageing HBs-Tg mice. A lower mitochondrial mass and membrane potential, and less polarized localization were observed in the hepatic cNK cells compared with the splenic cNK cells in the HBs-Tg mice. The enhanced galectin-3 (Gal-3) secreted from HBsAg+ hepatocytes accounted for the IL-10 production of hepatic cNK cells via ITGB1 signaling. For humans, LGALS3 and ITGB1 expression is positively correlated with IL-10 expression, and negatively correlated with the poor clinical progression of HCC. Gal-3-ITGB1 signaling shapes hepatic cNK cells but not LrNK cells during a chronic HBV infection, which may correlate with HCC progression.

Paclitaxel-induced Immune Dysfunction and Activation of Transcription Factor AP-1 Facilitate Hepatitis B Virus Replication.

Hepatitis B virus (HBV) reactivation is commonly observed in individuals with chronic HBV infection undergoing antineoplastic drug therapy. Paclitaxel (PTX) treatment has been identified as a potential trigger for HBV reactivation. This study aimed to uncover the mechanisms of PTX-induced HBV reactivation in vitro and in vivo, which may inform new strategies for HBV antiviral treatment. The impact of PTX on HBV replication was assessed through various methods including enzyme-linked immunosorbent assay, dual-luciferase reporter assay, quantitative real-time PCR, chromatin immunoprecipitation, and immunohistochemical staining. Transcriptome sequencing and 16S rRNA sequencing were employed to assess alterations in the transcriptome and microbial diversity in PTX-treated HBV transgenic mice. PTX enhanced the levels of HBV 3.5-kb mRNA, HBV DNA, HBeAg, and HBsAg both in vitro and in vivo. PTX also promoted the activity of the HBV core promoter and transcription factor AP-1. Inhibition of AP-1 gene expression markedly suppressed PTX-induced HBV reactivation. Transcriptome sequencing revealed that PTX activated the immune-related signaling networks such as IL-17, NF-κB, and MAPK signaling pathways, with the pivotal common key molecule being AP-1. The 16S rRNA sequencing revealed that PTX induced dysbiosis of gut microbiota. PTX-induced HBV reactivation was likely a synergistic outcome of immune suppression and direct stimulation of HBV replication through the enhancement of HBV core promoter activity mediated by the transcription factor AP-1. These findings propose a novel molecular mechanism, underscoring the critical role of AP-1 in PTX-induced HBV reactivation.

Effects of Citrus depressa Hayata juice on high-fat diet-induced obesity in HBV transgenic mice

The present study investigated the potential anti-obesity properties of Citrus depressa Hayata (CDH) juice in HBV transgenic mice, as well as the impact of fermentation on the effectiveness of the juice. The results revealed that fermentation increased the levels of polyphenols and hesperidin in CDH juice. The animal study demonstrated that both juices were effective in mitigating the weight gain induced by a high-fat diet by correcting metabolic parameter imbalances, reducing hepatic lipid accumulation, and reversing hepatic immune suppression. Furthermore, fermented juice exhibited superior efficacy in managing body weight and inhibiting the expansion of white adipose tissue (WAT). Fermented juice significantly enhanced adiponectin production and PPARγ expression in WAT, while also reducing hypertrophy. This study offers valuable insights into the potential role of CDH juices in combating obesity associated with high fat consumption and underscores the promise of CDH juice as a functional beverage.

Tg1.4HBV-s-rec mice, a crossbred hepatitis B virus-transgenic model, develop mild hepatitis

Hepatitis B virus (HBV)-transgenic mice exhibit competent innate immunity and are therefore an ideal model for considering intrinsic or cell-based mechanisms in HBV pathophysiology. A highly replicative model that has been little used, let alone characterized, is the Tg1.4HBV-s-rec strain derived from cross breeding of HBV-transgenic mouse models that either accumulate (Alb/HBs, Tg[Alb1-HBV]Bri44) or lack (Tg1.4HBV-s-mut) the hepatitis B surface antigen (HBsAg). Tg1.4HBV-s-rec hepatocytes secreted HBsAg, Hepatitis B extracellular antigen (HBeAg) and produced HBV virions. Transmission electron microscopy visualised viral particles (Tg1.4HBV-s-rec), nuclear capsid formations (Tg1.4HBV-s-mut and Tg1.4HBV-s-rec) and endoplasmic reticulum malformations (Alb/HBs). Viral replication in Tg1.4HBV-s-rec and Tg1.4HBV-s-mut differed in HBsAg expression and interestingly in the distribution of HBV core antigen (HBcAg) and HBV × protein. While in Tg1.4HBV-s-mut hepatocytes, the HBcAg was located in the cytoplasm, in Tg1.4HBV-s-rec hepatocytes, the HBcAg appeared in the nuclei, suggesting a more productive replication. Finally, Tg1.4HBV-s-rec mice showed symptoms of mild hepatitis, with reduced liver function and elevated serum transaminases, which appeared to be related to natural killer T cell activation. In conclusion, the study of Alb/HBs, Tg1.4HBV-s-mut and their F1 progeny provides a powerful tool to elucidate HBV pathophysiology, especially in the early HBeAg-positive phases of chronic infection and chronic hepatitis.

Antiviral therapy reduces hepatocellular carcinoma through suppressing hepatitis B virus replication may improve ER stress, mitochondrial and metabolic dysfunctions and decrease p62 in hybridized mice with single HBV transgene and miR-122.

Hepatitis B virus (HBV) hijacks autophagy for its replication. Nucleos(t)ide analogs (NUCs) treatment suppressed HBV replication and reduced hepatocellular carcinoma (HCC) incidence. However, the use of NUCs in chronic hepatitis B (CHB) patients with normal or minimally elevated serum alanine aminotransferase (ALT) levels is still debated. Animal models are crucial for studying the unanswered issue and evaluating new therapies. MicroRNA-122 (miR-122), which regulates fatty acid and cholesterol metabolism, is downregulated during hepatitis and HCC progression. The reciprocal inhibition of miR-122 with HBV highlights its role in HCC development as a tumor suppressor. By crossbreeding HBV-transgenic mice with miR-122 knockout mice, we generated a hybrid mouse model with a high incidence of HCC up to 89% and normal ALT levels before HCC. The model exhibited early-onset hepatic steatosis, progressive liver fibrosis, and impaired late-phase autophagy. Metabolomics and microarray analysis identified metabolic signatures, including dysregulation of lipid metabolism, inflammation, genomic instability, the Warburg effect, reduced TCA cycle flux, energy deficiency, and impaired free radical scavenging. Antiviral treatment reduced HCC incidence in hybrid mice by approximately 30-35% compared to untreated mice. This effect was linked to the activation of ER stress-responsive transcription factor ATF4, clearance of autophagosome cargo p62, and suppression of the CHOP-mediated apoptosis pathway. In summary, this study suggests that despite minimal ALT elevation, HBV replication can lead to liver injury. Endoplasmic reticulum stress, reduced miR-122 levels, mitochondrial and metabolic dysfunctions, blocking protective autophagy resulting in p62 accumulation, apoptosis, fibrosis, and HCC. Antiviral may improve the above-mentioned pathogenesis through HBV suppression.

Engineered extracellular vesicles for delivering functional Cas9/gRNA to eliminate hepatitis B virus cccDNA and integration

ABSTRACT The persistence of HBV covalently closed circular DNA (cccDNA) and HBV integration into the host genome in infected hepatocytes pose significant challenges to the cure of chronic HBV infection. Although CRISPR/Cas9-mediated genome editing shows promise for targeted clearance of viral genomes, a safe and efficient delivery method is currently lacking. Here, we developed a novel approach by combining light-induced heterodimerization and protein acylation to enhance the loading efficiency of Cas9 protein into extracellular vesicles (EVs). Moreover, vesicular stomatitis virus-glycoprotein (VSV-G) was incorporated onto the EVs membrane, significantly facilitating the endosomal escape of Cas9 protein and increasing its gene editing activity in recipient cells. Our results demonstrated that engineered EVs containing Cas9/gRNA and VSV-G can effectively reduce viral antigens and cccDNA levels in the HBV-replicating and infected cell models. Notably, we also confirmed the antiviral activity and high safety of the engineered EVs in the HBV-replicating mouse model generated by hydrodynamic injection and the HBV transgenic mouse model. In conclusion, engineered EVs could successfully mediate functional CRISPR/Cas9 delivery both in vitro and in vivo, leading to the clearance of episomal cccDNA and integrated viral DNA fragments, and providing a novel therapeutic approach for curing chronic HBV infection.

Increased liver stiffness promotes hepatitis B progression by impairing innate immunity in CCl4-induced fibrotic HBV+ transgenic mice.

Hepatitis B virus (HBV) infection develops as an acute or chronic liver disease, which progresses from steatosis, hepatitis, and fibrosis to end-stage liver diseases such as cirrhosis and hepatocellular carcinoma (HCC). An increased stromal stiffness accompanies fibrosis in chronic liver diseases and is considered a strong predictor for disease progression. The goal of this study was to establish the mechanisms by which enhanced liver stiffness regulates HBV infectivity in the fibrotic liver tissue. For in vitro studies, HBV-transfected HepG2.2.15 cells were cultured on polydimethylsiloxane gels coated by polyelectrolyte multilayer films of 2 kPa (soft) or 24 kPa (stiff) rigidity mimicking the stiffness of the healthy or fibrotic liver. For in vivo studies, hepatic fibrosis was induced in C57Bl/6 parental and HBV+ transgenic (HBVTg) mice by injecting CCl4 twice a week for 6 weeks. We found higher levels of HBV markers in stiff gel-attached hepatocytes accompanied by up-regulated OPN content in cell supernatants as well as suppression of anti-viral interferon-stimulated genes (ISGs). This indicates that pre-requisite "fibrotic" stiffness increases osteopontin (OPN) content and releases and suppresses anti-viral innate immunity, causing a subsequent rise in HBV markers expression in hepatocytes. In vitro results were corroborated by data from HBVTg mice administered CCl4 (HBVTg CCl4). These mice showed higher HBV RNA, DNA, HBV core antigen (HBcAg), and HBV surface antigen (HBsAg) levels after liver fibrosis induction as judged by a rise in Col1a1, SMA, MMPs, and TIMPs mRNAs and by increased liver stiffness. Importantly, CCl4-induced the pro-fibrotic activation of liver cells, and liver stiffness was higher in HBVTg mice compared with control mice. Elevation of HBV markers and OPN levels corresponded to decreased ISG activation in HBVTg CCl4 mice vs HBVTg control mice. Based on our data, we conclude that liver stiffness enhances OPN levels to limit anti-viral ISG activation in hepatocytes and promote an increase in HBV infectivity, thereby contributing to end-stage liver disease progression.

HMMR alleviates endoplasmic reticulum stress by promoting autophagolysosomal activity during endoplasmic reticulum stress-driven hepatocellular carcinoma progression.

The mechanism of hepatitis B virus (HBV)-induced carcinogenesis remains an area of interest. The accumulation of hepatitis B surface antigen in the endoplasmic reticulum (ER) of hepatocytes stimulates persistent ER stress. Activity of the unfolded protein response (UPR) pathway of ER stress may play an important role in inflammatory cancer transformation. How the protective UPR pathway is hijacked by cells as a tool for malignant transformation in HBV-related hepatocellular carcinoma (HCC) is still unclear. Here, we aimed to define the key molecule hyaluronan-mediated motility receptor (HMMR) in this process and explore its role under ER stress in HCC development. An HBV-transgenic mouse model was used to characterize the pathological changes during the tumor progression. Proteomics and transcriptomics analyses were performed to identify the potential key molecule, screen the E3 ligase, and define the activation pathway. Quantitative real-time PCR and Western blotting were conducted to detect the expression of genes in tissues and cell lines. Luciferase reporter assay, chromatin immunoprecipitation, coimmunoprecipitation, immunoprecipitation, and immunofluorescence were employed to investigate the molecular mechanisms of HMMR under ER stress. Immunohistochemistry was used to clarify the expression patterns of HMMR and related molecules in human tissues. We found sustained activation of ER stress in the HBV-transgenic mouse model of hepatitis-fibrosis-HCC. HMMR was transcribed by c/EBP homologous protein (CHOP) and degraded by tripartite motif containing 29 (TRIM29) after ubiquitination under ER stress, which caused the inconsistent expression of mRNA and protein. Dynamic expression of TRIM29 in the HCC progression regulated the dynamic expression of HMMR. HMMR could alleviate ER stress by increasing autophagic lysosome activity. The negative correlation between HMMR and ER stress, positive correlation between HMMR and autophagy, and negative correlation between ER stress and autophagy were verified in human tissues. This study identified the complicated role of HMMR in autophagy and ER stress, that HMMR controls the intensity of ER stress by regulating autophagy in HCC progression, which could be a novel explanation for HBV-related carcinogenesis.

Avasimibe can cooperate with a DC-targeting and integration-deficient lentivector to induce stronger HBV specific T cytotoxic response by regulating cholesterol metabolism

We have reported a lentivector which could effectively induce HBV-specific cytotoxic T lymphocytes (CTLs). Avasimibe is an inhibitor of acetyl-CoA acetyltransferase-1 (ACAT1), and has been shown to enhance T lymphocyte cytotoxicity on tumor cells. However, the role of avasimibe in lentivector-induced HBV-specific T cytotoxic response remains unknown. Based on previous study, we constructed an integration-deficient lentivector LVDC-ID-HBV (harboring HBcAg expression), and the in vitro experiments showed that the combination of avasimibe exhibited better efficacy in inducing HBV-specific CTL responses including cell proliferation, production of cytokines, as well as CTL killing activities. Mechanism experiments showed that increasing cell membrane cholesterol levels by MβCD-coated cholesterol or ACAT1 inhibition efficiently promoted TCR clustering, signaling transduction and immunological synapse formation, thereby mediating augmented CTL responses. Nevertheless, the depletion of plasma membrane cholesterol with MβCD led to obviously decreased CTL responses. The avasimibe-mediated strengthened immune effects were also determined in animal experiments and the results were in agreement with those from the in vitro research. In particular, the in vivo CTL killing activities were identified by the CFSE or BV-labeled splenocyte lysis assay. Moreover, the experiments in HBV transgenic mice showed that the LVDC-ID-HBV plus avasimibe group demonstrated the lowest serum HBsAg and HBV DNA levels, as well as the lowest expression of HBsAg and HBcAg in liver tissues. We concluded that the HBV-specific CTL immune responses could be potentiated by avasimibe through regulating plasma membrane cholesterol levels. Avasimibe may be a potential adjuvant for lentivector vaccine against HBV infection.

A hepatitis B virus-derived peptide combined with HBsAg exerts an anti-HBV effect in an HBV transgenic mouse model as a therapeutic vaccine.

For complete or functional cure of hepatitis B virus (HBV) infection, application of immunotherapy is now being attempted. Recently, we reported that a 6-mer hepatitis B virus (HBV)-derived peptide, Poly6, exerts a strong anticancer effect in tumor-implanted mice through inducible nitric oxide synthase (iNOS)-producing DCs (Tip-DCs) in a type 1 interferon (IFN-I)-dependent manner, suggesting its potential as a vaccine adjuvant. In this study, we explored the potential of Poly6 in combination with HBsAg as a therapeutic vaccine against hepatitis B virus infection. We investigated the immunotherapeutic potential of Poly6 combined with HBsAg vaccination against hepatitis B virus infection in C57BL/6 mice or an HBV transgenic mouse model. In C57BL/6 mice, Poly6 enhanced DC maturation and DC migration capacity in an IFN-I-dependent manner. Moreover, the addition of Poly6 to alum in combination with HBsAg also led to enhanced HBsAg-specific cell-mediated immune (CMI) responses, suggesting its potential as an adjuvant of HBsAg-based vaccines. In HBV transgenic mice, vaccination with Poly6 combined with HBsAg exerted a strong anti-HBV effect via induction of HBV-specific humoral and cell-mediated immune responses. In addition, it also induced HBV-specific effector memory T cells (TEM). Our data indicated that vaccination with Poly6 in combination with HBsAg exerts an anti-HBV effect in HBV transgenic mice, which is mainly mediated by HBV-specific CMI and humoral immune responses via IFN-I-dependent DC activation, suggesting the feasibility of Poly6 as an adjuvant for an HBV therapeutic vaccine.

Hepatitis B virus hijacks TSG101 to facilitate egress via multiple vesicle bodies.

Hepatitis B virus (HBV) chronically infects 296 million individuals and there is no cure. As an important step of viral life cycle, the mechanisms of HBV egress remain poorly elucidated. With proteomic approach to identify capsid protein (HBc) associated host factors and siRNA screen, we uncovered tumor susceptibility gene 101 (TSG101). Knockdown of TSG101 in HBV-producing cells, HBV-infected cells and HBV transgenic mice suppressed HBV release. Co-immunoprecipitation and site mutagenesis revealed that VFND motif in TSG101 and Lys-96 ubiquitination in HBc were essential for TSG101-HBc interaction. In vitro ubiquitination experiment demonstrated that UbcH6 and NEDD4 were potential E2 ubiquitin-conjugating enzyme and E3 ligase that catalyzed HBc ubiquitination, respectively. PPAY motif in HBc and Cys-867 in NEDD4 were required for HBc ubiquitination, TSG101-HBc interaction and HBV egress. Transmission electron microscopy confirmed that TSG101 or NEDD4 knockdown reduces HBV particles count in multivesicular bodies (MVBs). Our work indicates that TSG101 recognition for NEDD4 ubiquitylated HBc is critical for MVBs mediated HBV egress.

Poly(I:C) Induces Distinct Liver Cell Type-Specific Responses in Hepatitis B Virus-Transgenic Mice In Vitro, but Fails to Induce These Signals In Vivo.

Immunopathology in hepatitis B virus (HBV) infection is driven by innate and adaptive immunity. Whether the hepatitis B surface antigen (HBsAg) affects hepatic antiviral signalling was investigated in HBV-transgenic mouse models that either accumulate (Alb/HBs, Tg[Alb1HBV]Bri44), lack (Tg1.4HBV-s-mut3) or secrete (Tg1.4HBV-s-rec (F1, Tg1.4HBV-s-mut × Alb/HBs) the HBsAg. Herein, the responsiveness of TLR3 and RIG-I in primary parenchymal and non-parenchymal liver cells was determined in vitro and in vivo. Cell type-specific and mouse strain-dependent interferon, cytokine and chemokine expression were observed by LEGENDplex™ and validated by quantitative PCR. In vitro, the hepatocytes, liver sinusoidal endothelial cells and Kupffer cells of Tg1.4HBV-s-rec mice showed poly(I:C) susceptibilities similar to the wild-type controls, while in the remaining leucocyte fraction the interferon, cytokine and chemokine induction was reduced. On the contrary, poly(I:C)-injected 1.4TgHBV-s-rec mice showed suppressed interferon, cytokine and chemokine levels in hepatocytes but increased levels in the leucocyte fraction. Thus, we concluded that liver cells of Tg1.4HBV-s-rec mice, which produce HBV particles and release the HBsAg, responded to exogenous TLR3/RIG-I stimuli in vitro but exhibited a tolerogenic environment in vivo.

Co-delivery of Cas9 mRNA and guide RNAs edits hepatitis B virus episomal and integration DNA in mouse and tree shrew models

With 296 million chronically infected individuals worldwide, hepatitis B virus (HBV) causes a major health burden. The major challenge to cure HBV infection lies in the fact that the source of persistence infection, viral episomal covalently closed circular DNA (cccDNA), could not be targeted. In addition, HBV DNA integration, although normally results in replication-incompetent transcripts, considered as oncogenic. Though several studies evaluated the potential of gene-editing approaches to target HBV, previous in vivo studies have been of limited relevance to authentic HBV infection, as the models do not contain HBV cccDNA or feature a complete HBV replication cycle under competent host immune system. In this study, we evaluated the effect of in vivo codelivery of Cas9 mRNA and guide RNAs (gRNAs) by SM-102-based lipid nanoparticles (LNPs) on HBV cccDNA and integrated DNA in mouse and a higher species. CRISPR nanoparticle treatment decreased the levels of HBcAg, HBsAg and cccDNA in AAV-HBV1.04 transduced mouse liver by 53%, 73% and 64% respectively. In HBV infected tree shrews, the treatment achieved 70% reduction of viral RNA and 35% reduction of cccDNA. In HBV transgenic mouse, 90% inhibition of HBV RNA and 95% inhibition of DNA were observed. CRISPR nanoparticle treatment was well tolerated in both mouse and tree shrew, as no elevation of liver enzymes and minimal off-target was observed. Our study demonstrated that SM-102-based CRISPR is safe and effective in targeting HBV episomal and integration DNA in vivo. The system delivered by SM-102-based LNPs may be used as a potential therapeutic strategy against HBV infection.

Discovery, optimization and biological evaluation of novel HBsAg production inhibitors

Hepatitis B virus (HBV) infection is a major global health problem. HBsAg inhibitors are expected to reduce the production of HBsAg via inhibiting host proteins PAPD5 & PAPD7 and finally achieve the ideal goal of “functional cure”. In this work, a series of tetrahydropyridine (THP) derivatives with a bridged ring were synthesized and evaluated for their inhibiting HBsAg production and HBV DNA activity. Among them, compound 17i was identified as potent HBsAg production inhibitor with excellent in vitro anti-HBV potency (HBV DNA EC50 = 0.018 μM, HBsAg EC50 = 0.044 μM) and low toxicity (CC50 > 100 μM). Moreover, 17i exhibited favorable in vitro/in vivo DMPK properties in mice. 17i could also significantly reduce serum HBsAg and HBV DNA levels (1.08 and 1.04 log units, respectively) in HBV transgenic mice.

Exosomes loading Tapasin enhance T cell immune response by autophagy to inhibit HBV replication.

Hepatitis B virus (HBV) specific T cell immune response plays a vital role in viral clearance. Dendritic cell derived exosomes (Dexs) can activate T cell immunity effectively. Tapasin (TPN) is involved in antigen processing and specific immune recognition. In the present study, we elucidated that Dexs loading TPN (TPN-Dexs) could enhance CD8+ T cell immune response and inhibit virus replication in HBV transgenic mice. T cell immune response and the ability of inhibiting HBV replication were measured in HBV transgenic mice immunized with TPN-Dexs. Meanwhile, CD8+ T cell autophagy and specific T cell immune responses were measured in vitro and vivo, and the mechanisms probably involved in were explored. Purified TPN-Dexs could be taken up into the cytoplasm of DCs and upregulate CD8+ T cell autophagy to enhance specific T cell immune response. In addition, TPN-Dexs could increase the expression of AKT and decrease the expression of mTOR in CD8+ T cells. Further research confirmed that TPN-Dexs could inhibit virus replication and decrease the expression of HBsAg in the liver of HBV transgenic mice. Nevertheless, those also could elicit mice hepatocytes damage. In conclusion, TPN-Dexs could enhance specific CD8+ T cell immune responses via the AKT/mTOR pathway to regulate the autophagy and exert the antiviral effect in HBV transgenic mice.

Synthesis and evaluation of N-sulfonylpiperidine-3-carboxamide derivatives as capsid assembly modulators inhibiting HBV in vitro and in HBV-transgenic mice

The hepatitis B virus (HBV) capsid assembly modulators (CAMs) have been developed as effective anti-HBV agents in the treatment of chronic HBV infection by targeting the HBV core protein and inducing the formation of aberrant or morphologically normal capsid. However, some CAMs have been observed adverse events such as ALT flares and rash. Therefore, finding new CAMs is of great importance. In this report, we synthesized N-sulfonylpiperidine-3-carboxamides (SPCs) derivatives and evaluated their anti-HBV activities. Among the SPC derivatives, compound C-49 notably suppressed HBV replication in HepAD38, HepG2-HBV1.3 and HepG2-NTCP cells. Moreover, treatment with C-49 for 12 days exhibited potent anti-HBV activity (100 mg/kg; 2.42 log reduction of serum HBV DNA) in HBV-transgenic mice without apparent hepatotoxicity. Our findings provided a new SPC derivative as HBV capsid assembly modulator for developing safe and efficient anti-HBV therapy.

Evaluation of the Effect of CD70 Co-Expression on CD8 T Cell Response in Protein-Prime MVA-Boost Vaccination in Mice.

Here, we investigate the potential of CD70 co-expression during viral vector boost vaccination to improve an antigen-specific T cell response. To determine the chance of activating antigen-specific T cells by CD70, we used the HBV core antigen as a model antigen in a heterologous protein-prime, Modified Vaccinia virus Ankara (MVA) boost vaccination scheme. Both the HBV core and a CD70 expression cassette were co-expressed upon delivery by an MVA vector under the same promoter linked by a P2A site. To compare immunogenicity with and without CD70 co-expression, HBV-naïve, C57BL/6 (wt) mice and HBV-transgenic mice were prime-vaccinated using recombinant HBV core antigen followed by the MVA vector boost. Co-expression of CD70 increased the number of vaccine-induced HBV core-specific CD8 T cells by >2-fold and improved their effector functions in HBV-naïve mice. In vaccinated HBV1.3tg mice, the number and functionality of HBV core-specific CD8 T cells was slightly increased upon CD70 co-expression in low-viremic, but not in high-viremic animals. CD70 co-expression did not impact liver damage as indicated by ALT levels in the serum, but increased the number of vaccine-induced, proliferative T cell clusters in the liver. Overall, this study indicates that orchestrated co-expression of CD70 and a vaccine antigen may be an interesting and safe means of enhancing antigen-specific CD8 T cell responses using vector-based vaccines, although in our study it was not sufficient to break immune tolerance.