Lunzhi Yuan

Pharmacological targeting of kinases MST1 and MST2 augments tissue repair and regeneration

Manipulation of the Hippo signaling pathway with a reversible and selective small-molecule inhibitor of Hippo kinase MST1/2 provides a therapeutic option for tissue injury and repair. Drug-induced regeneration Popping a pill to repair an organ may eventually become reality. Turning away from conventional scaffolds, materials, and cell-based regenerative medicine strategies, Fan and colleagues sought a small molecule that could specifically target a critical signaling molecule in the Hippo pathway. Loss of kinases in this pathway, MST1/2, increases cell proliferation during development; thus, the authors hypothesized that inhibiting their activity in mature organs could help repair any damage. They discovered a drug, XMU-MP-1, that blocked MST1/2 activity and found that it promoted liver repair and regeneration in four different mouse models of acute and chronic injuries, including acetaminophen-induced injury, which is a common cause of liver failure worldwide. Such a pharmacological strategy could make tissue regeneration easier for many, compared to complex biomaterial and cell therapies. Tissue repair and regenerative medicine address the important medical needs to replace damaged tissue with functional tissue. Most regenerative medicine strategies have focused on delivering biomaterials and cells, yet there is the untapped potential for drug-induced regeneration with good specificity and safety profiles. The Hippo pathway is a key regulator of organ size and regeneration by inhibiting cell proliferation and promoting apoptosis. Kinases MST1 and MST2 (MST1/2), the mammalian Hippo orthologs, are central components of this pathway and are, therefore, strong target candidates for pharmacologically induced tissue regeneration. We report the discovery of a reversible and selective MST1/2 inhibitor, 4-((5,10-dimethyl-6-oxo-6,10-dihydro-5H-pyrimido[5,4-b]thieno[3,2-e][1,4]diazepin-2-yl)amino)benzenesulfonamide (XMU-MP-1), using an enzyme-linked immunosorbent assay–based high-throughput biochemical assay. The cocrystal structure and the structure-activity relationship confirmed that XMU-MP-1 is on-target to MST1/2. XMU-MP-1 blocked MST1/2 kinase activities, thereby activating the downstream effector Yes-associated protein and promoting cell growth. XMU-MP-1 displayed excellent in vivo pharmacokinetics and was able to augment mouse intestinal repair, as well as liver repair and regeneration, in both acute and chronic liver injury mouse models at a dose of 1 to 3 mg/kg via intraperitoneal injection. XMU-MP-1 treatment exhibited substantially greater repopulation rate of human hepatocytes in the Fah-deficient mouse model than in the vehicle-treated control, indicating that XMU-MP-1 treatment might facilitate human liver regeneration. Thus, the pharmacological modulation of MST1/2 kinase activities provides a novel approach to potentiate tissue repair and regeneration, with XMU-MP-1 as the first lead for the development of targeted regenerative therapeutics.

Prolonged suppression of HBV in mice by a novel antibody that targets a unique epitope on hepatitis B surface antigen

Objective This study aimed to investigate the therapeutic potential of monoclonal antibody (mAb) against HBV as a novel treatment approach to chronic hepatitis B (CHB) in mouse models. Methods Therapeutic effects of mAbs against various epitopes on viral surface protein were evaluated in mice mimicking persistent HBV infection. The immunological mechanisms of mAb-mediated viral clearance were systematically investigated. Results Among 11 tested mAbs, a novel mAb E6F6 exhibited the most striking therapeutic effects in several HBV-persistent mice. Single-dose administration of E6F6 could profoundly suppress the levels of hepatitis B surface antigen (HBsAg) and HBV DNA for several weeks in HBV-transgenic mice. E6F6 regimen efficiently prevented initial HBV infection, and reduced viral dissemination from infected hepatocytes in human-liver-chimeric mice. E6F6-based immunotherapy facilitated the restoration of anti-HBV T-cell response in hydrodynamic injection (HDI)-based HBV carrier mice. Immunological analyses suggested that the Fcγ receptor-dependent phagocytosis plays a predominant role in E6F6-mediated viral suppression. Molecular analyses suggested that E6F6 recognises an evolutionarily conserved epitope (GPCK(R)TCT) and only forms a smaller antibody–viral particle immune complex with limited interparticle crosslinking when it binds to viral particles. This unique binding characteristic of E6F6 to HBV was possibly associated with its effective in vivo opsonophagocytosis for viral clearance. Conclusions These results provided new insight into understanding the therapeutic role and mechanism of antibody against persistent viral infection. The E6F6-like mAbs may provide a novel immunotherapeutic agent against human chronic HBV infection.

A Broadly Cross-protective Vaccine Presenting the Neighboring Epitopes within the VP1 GH Loop and VP2 EF Loop of Enterovirus 71

Human enterovirus 71 (EV71) and coxsackievirus A16 (CA16) are the major etiological agents of hand, foot and mouth disease (HFMD) and are often associated with neurological complications. Currently, several vaccine types are being developed for EV71 and CA16. In this study, we constructed a bivalent chimeric virus-like particle (VLP) presenting the VP1 (aa208-222) and VP2 (aa141-155) epitopes of EV71 using hepatitis B virus core protein (HBc) as a carrier, designated HBc-E1/2. Immunization with the chimeric VLPs HBc-E1/2 induced higher IgG titers and neutralization titers against EV71 and CA16 in vitro than immunization with only one epitope incorporated into HBc. Importantly, passive immunization with the recombinant HBc-E2 particles protected neonatal mice against lethal EV71 and CA16 infections. We demonstrate that anti-VP2 (aa141-155) sera bound authentic CA16 viral particles, whereas anti-VP1 (aa208-222) sera could not. Moreover, the anti-VP2 (aa141-155) antibodies inhibited the binding of human serum to virions, which demonstrated that the VP2 epitope is immunodominant between EV71 and CA16. These results illustrated that the chimeric VLP HBc-E1/2 is a promising candidate for a broad-spectrum HFMD vaccine, and also reveals mechanisms of protection by the neighboring linear epitopes of the VP1 GH and VP2 EF loops.

A Chimeric Humanized Mouse Model by Engrafting the Human Induced Pluripotent Stem Cell-Derived Hepatocyte-Like Cell for the Chronic Hepatitis B Virus Infection

Humanized mouse model generated by grafting primary human hepatocytes (PHHs) to immunodeficient mouse has contributed invaluably to understanding the pathogenesis of hepatitis B virus (HBV). However, the source of PHHs is limited, which necessitates the search for alternatives. Recently, hepatocyte-like cells (HLCs) generated from human induced pluripotent stem cells (hiPSCs) have been used for in vitro HBV infection. Herein, we developed a robust human liver chimeric animal model to study in vivo HBV infection by engrafting the hiPSC-HLCs to Fah-/-Rag2-/-IL-2Rγc-/- SCID (FRGS) mice. After being optimized by a small molecule, XMU-MP-1, the hiPSC-HLCs engrafted FRGS (hHLC-FRGS) mice displayed approximately 40% liver chimerism at week 6 after engraftment and maintained at this level for at least 14 weeks. Viremia and HBV infection markers include antigens, RNA, DNA, and covalently closed circular DNA were detectable in HBV infected hHLC-FRGS mice. Furthermore, hiPSC-HLCs and hHLC-FRGS mice were successfully used to evaluate different antivirals. Therefore, we established a humanized mouse model for not only investigating HBV pathogenesis but also testing the effects of the anti-HBV drugs. Highlights: (1) The implanted hiPSC-HLCs established a long-term chimerism in FRGS mice liver. (2) hHLC-FRGS mice are adequate to support chronic HBV infection with a full viral life cycle. (3) hiPSC-HLCs and hHLC-FRGS mice are useful tools for evaluation of antivirals against HBV infection in vitro and in vivo. Research in Context To overcome the disadvantages of using primary human hepatocytes, we induced human pluripotent stem cells to hepatocyte-like cells (hiPSC-HLCs) that developed the capability to express important liver functional markers and critical host factors for HBV infection. The hiPSC-HLCs were permissive for the HBV infection and supported a full HBV replication. The hiPSC-HLCs were then engrafted to immunodeficient mouse to establish a chimeric liver mouse model, which was capable of supporting HBV infection in vivo and evaluating the effects of antiviral drugs. Our results shed light into improving the cellular and animal models for studying HBV and other hepatotropic viruses.

A novel toolbox for the in vitro assay of hepatitis D virus infection.

Hepatitis D virus (HDV) is a defective RNA virus that requires the presence of hepatitis B virus (HBV) for its life cycle. The in vitro HDV infection system is widely used as a surrogate model to study cellular infection with both viruses owing to its practical feasibility. However, previous methods for running this system were less efficient for high-throughput screening and large-scale studies. Here, we developed a novel method for the production of infectious HDV by adenoviral vector (AdV)-mediated transduction. We demonstrated that the AdV-based method yields 10-fold higher viral titers than the transient-transfection approach. The HDV-containing supernatant derived from AdV-infected Huh7 cells can be used as the inoculum in infectivity assays without requiring further concentration prior to use. Furthermore, we devloped a chemiluminescent immunoassay (HDV-CLEIA) to quantitatively determine intracellular HDAg with a dynamic range of 5–11,000 pg/mL. HDV-CLEIA can be used as an alternative approach to assess HDV infection. The advantages of our updated methodology were demonstrated through in vitro HDV infection of HepaRG cells and by evaluating the neutralization activity using antibodies that target various regions of the HBV/HDV envelope proteins. Together, the methods presented here comprise a novel toolbox of in vitro assays for studying HDV infection.

Optimized HepaRG is a suitable cell source to generate the human liver chimeric mouse model for the chronic hepatitis B virus infection

The human liver chimeric mouse with primary human hepatocytes (PHHs) engraftment has been demonstrated to be a useful animal model to study hepatitis B virus (HBV) pathogenesis and evaluate anti-HBV drugs. However, the disadvantages of using PHHs include the inability for cellular expansion in vitro, limited donor availability, individual differences, and ethical issues, necessitating the development of alternatives. To obtain in vitro expandable hepatocytes, we optimized the hepatic differentiation procedure of the human liver progenitor cell line, HepaRG, using four functional small molecules (4SM) and enriched the precursor hepatocyte-like cells (HLCs). HepaRG cells of different hepatic differentiation states were engrafted to immunodeficient mice (FRGS) with weekly 4SM treatment. The HepaRG-engrafted mice were challenged with HBV and/or treated with several antivirals to evaluate their effects. We demonstrated that the 4SM treatment enhanced hepatic differentiation and promoted cell proliferation capacity both in vitro and in vivo. Mice engrafted with enriched HepaRG of prehepatic differentiation and treated with 4SM displayed approximately 10% liver chimerism at week 8 after engraftment and were maintained at this level for another 16 weeks. Therefore, we developed a HepaRG-based human liver chimeric mouse model: HepaRG-FRGS. Our experimental results showed that the liver chimerism of the mice was adequate to support chronic HBV infection for 24 weeks and to evaluate antivirals. We also demonstrated that HBV infection in HepaRG cells was dependent on their hepatic differentiation state and liver chimerism in vivo. Overall, HepaRG-FRGS mice provide a novel human liver chimeric mouse model to study chronic HBV infection and evaluate anti-HBV drugs.

ER stress regulating protein phosphatase 2A-B56γ, targeted by hepatitis B virus X protein, induces cell cycle arrest and apoptosis of hepatocytes

Hepatitis B virus X (HBx) protein contributes to the progression of hepatitis B virus (HBV)-related hepatic injury and diseases, but the exact mechanism remains unclear. Protein phosphatase 2 A (PP2A) is a major serine/threonine phosphatase involved in regulating many cellular phosphorylation signals that are important for regulation of cell cycle and apoptosis. Does HBx target to PP2A-B56γ and therefore affect HBx-induced hepatotoxicity? In the present study, the expression of B56γ positively correlated with the level of HBx in HBV-infected primary human hepatocytes in human-liver-chimeric mice, HBx-transgenic mice, HBV-infected cells, and HBx-expressing hepatic cells. B56γ promoted p53/p21-dependent cell cycle arrest and apoptosis. Mechanistically, B56γ was transactivated by AP-1, which was under the regulation of endoplasmic reticulum (ER) stress induced CREBH signaling in HBx-expressing hepatic cells. B56γ dephosphorylated p-Thr55-p53 to trigger p53/p21 pathway-dependent cell cycle G1 phase arrest, resulting in apoptosis of hepatic cells. In conclusion, this study provides a novel insight into a mechanism of B56γ mediating cell cycle arrest and apoptosis of HBx-expressing hepatic cells and a basis for B56γ being a potential therapeutic target for HBV-infected hepatic cells.

Detection and analysis of tupaia hepatocytes via mAbs against tupaia serum albumin

On the basis of its close phylogenetic relationship with primates, the development of Tupaia belangeri as an infection animal model and drug metabolism model could provide a new option for preclinical studies, especially in hepatitis virus research. As a replacement for primary human hepatocytes (PHHs), primary tupaia hepatocytes (PTHs) have been widely used. Similar to human serum albumin, tupaia serum albumin (TSA) is the most common liver synthesis protein and is an important biomarker for PTHs and liver function. However, no detection or quantitative method for TSA has been reported. In this study, mouse monoclonal antibodies (mAbs) 4G5 and 9H3 against TSA were developed to recognize PTHs, and they did not show cross-reactivity with serum albumin from common experimental animals, such as the mouse, rat, cow, rabbit, goat, monkey, and chicken. The two mAbs also exhibited good performance in fluorescence activated cell sorting (FACS) analysis and immunofluorescence (IF) detection of PTHs. A chemiluminescent enzyme immune assay method using the two mAbs, with a linear range from 96.89 pg/ml to 49,609.38 pg/ml, was developed for the quantitative detection of TSA. The mAbs and the CLEIA method provide useful tools for research on TSA and PTHs.

An HBV-tolerant immunocompetent model that effectively simulates chronic hepatitis B virus infection in mice

Hepatitis B virus (HBV) is the leading cause of liver disease and hepatic carcinoma (HCC). Approximately 350 million people worldwide are infected with HBV and at risk of chronicity. An efficient HBV-tolerant murine model that mimics HBV infection in humans is desirable for HBV-related research. In this study, we investigated and established a murine model by hydrodynamic injection (HDI) of pAAV/HBV into the tail vein of AAVS1 site element-transgenic mice. In 80% of the injected mice, the serum level of HBsAg reached 103-4 IU/ml and persisted for more than half a year. Next, the model was used to evaluate RNA interference (RNAi)-based antiviral therapy. Data obtained using the model demonstrated that this model will facilitate the elucidation of the mechanisms underlying chronic HBV infection and will also be useful for evaluating new antiviral drugs.