Gregor Ebert

Eliminating hepatitis B by antagonizing cellular inhibitors of apoptosis

Significance Current antiviral treatments for chronic hepatitis B virus (HBV) infection are effective in suppressing production of virus, but they have poor efficacy in promoting the elimination of infection. Hence, most patients with chronic HBV infection are maintained on antiviral therapies indefinitely. There is much interest in identifying treatments that promote the clearance of infected hepatocytes, thus purging the HBV DNA reservoir in the liver. Here, we show that the clinical-stage drug birinapant, which antagonizes host cell inhibitor of apoptosis proteins (cIAPs), preferentially promotes the killing of HBV-infected hepatocytes in a mouse model of HBV. Therefore, birinapant and other antagonists of cIAPs may be efficacious in the treatment of chronic HBV infection and may promote elimination of virus. We have shown that cellular inhibitor of apoptosis proteins (cIAPs) impair clearance of hepatitis B virus (HBV) infection by preventing TNF-mediated killing/death of infected cells. A key question, with profound therapeutic implications, is whether this finding can be translated to the development of drugs that promote elimination of infected cells. Drug inhibitors of cIAPs were developed as cancer therapeutics to promote TNF-mediated tumor killing. These drugs are also known as Smac mimetics, because they mimic the action of the endogenous protein Smac/Diablo that antagonizes cIAP function. Here, we show using an immunocompetent mouse model of chronic HBV infection that birinapant and other Smac mimetics are able to rapidly reduce serum HBV DNA and serum HBV surface antigen, and they promote the elimination of hepatocytes containing HBV core antigen. The efficacy of Smac mimetics in treating HBV infection is dependent on their chemistry, host CD4+ T cells, and TNF. Birinapant enhances the ability of entecavir, an antiviral nucleoside analog, to reduce viral DNA production in HBV-infected animals. These results indicate that birinapant and other Smac mimetics may have efficacy in treating HBV infection and perhaps, other intracellular infections.

Cellular inhibitor of apoptosis proteins prevent clearance of hepatitis B virus

Significance Hepatitis B virus (HBV) causes substantial morbidity and mortality. A large proportion of infected individuals controls infection but does not completely eradicate HBV DNA from the liver, and flares in hepatitis can be precipitated by immunosuppression. A proportion of individuals never controls infection, and these people are at substantial risk of developing liver failure and liver cancer. Current therapies are not effective at eliminating virus, and there is a major interest in developing functional cures for HBV infection. We identified host cell signaling molecules that can restrict the ability to eradicate infected cells. These molecules can be therapeutically targeted, and drugs that interfere with the function of these host cell proteins may be useful therapies to promote clearance of HBV infection. Hepatitis B virus (HBV) infection can result in a spectrum of outcomes from immune-mediated control to disease progression, cirrhosis, and liver cancer. The host molecular pathways that influence and contribute to these outcomes need to be defined. Using an immunocompetent mouse model of chronic HBV infection, we identified some of the host cellular and molecular factors that impact on infection outcomes. Here, we show that cellular inhibitor of apoptosis proteins (cIAPs) attenuate TNF signaling during hepatitis B infection, and they restrict the death of infected hepatocytes, thus allowing viral persistence. Animals with a liver-specific cIAP1 and total cIAP2 deficiency efficiently control HBV infection compared with WT mice. This phenotype was partly recapitulated in mice that were deficient in cIAP2 alone. These results indicate that antagonizing the function of cIAPs may promote the clearance of HBV infection.

ARIH2 is essential for embryogenesis, and its hematopoietic deficiency causes lethal activation of the immune system

The E3 ligase ARIH2 has an unusual structure and mechanism of elongating ubiquitin chains. To understand its physiological role, we generated gene-targeted mice deficient in ARIH2. ARIH2 deficiency resulted in the embryonic death of C57BL/6 mice. On a mixed genetic background, the lethality was attenuated, with some mice surviving beyond weaning and then succumbing to an aggressive multiorgan inflammatory response. We found that in dendritic cells (DCs), ARIH2 caused degradation of the inhibitor IκBβ in the nucleus, which abrogated its ability to sequester, protect and transcriptionally coactivate the transcription factor subunit p65 in the nucleus. Loss of ARIH2 caused dysregulated activation of the transcription factor NF-κB in DCs, which led to lethal activation of the immune system in ARIH2-sufficent mice reconstituted with ARIH2-deficient hematopoietic stem cells. Our data have therapeutic implications for targeting ARIH2 function.

CRISPR/Cas9—The ultimate weapon to battle infectious diseases?

Infectious diseases are a leading cause of death worldwide. Novel therapeutics are urgently required to treat multidrug‐resistant organisms such as Mycobacterium tuberculosis and to mitigate morbidity and mortality caused by acute infections such as malaria and dengue fever virus as well as chronic infections such as human immunodeficiency virus‐1 and hepatitis B virus.

Vaccines licensed and in clinical trials for the prevention of dengue.

ABSTRACT Dengue has become a major global public health threat with almost half of the worlds population living in at-risk areas. Vaccination would likely represent an effective strategy for the management of dengue disease in endemic regions, however to date there is only one licensed preventative vaccine for dengue infection. The development of a vaccine against dengue virus (DENV) has been hampered by an incomplete understanding of protective immune responses against DENV. The most clinically advanced dengue vaccine is the chimeric yellow fever-dengue vaccine (CYD) that employs the yellow fever virus 17D strain as the replication backbone (Chimerivax-DEN; CYD-TDV). This vaccine had an overall pooled protective efficacy of 65.6% but was substantially more effective against severe dengue and dengue hemorrhagic fever. Several other vaccine approaches have been developed including live attenuated chimeric dengue vaccines (DENVax and LAV Delta 30), DEN protein subunit V180 vaccine (DEN1–80E) and DENV DNA vaccines. These vaccines have been shown to be immunogenic in animals and also safe and immunogenic in humans. However, these vaccines are yet to progress to phase III trials to determine their protective efficacy against dengue. This review will summarize the details of vaccines that have progressed to clinical trials in humans.

The role of tumour necrosis factor in hepatitis B infection: Jekyll and Hyde.

Chronic hepatitis B (CHB) is a major health problem worldwide and is associated with significant long‐term morbidity and mortality. The hepatitis B virus (HBV) is a hepatotropic virus that is capable of integrating in the host nucleus permanently resulting in lifelong infection. To date, there is no definitive cure for HBV, as our current treatments cannot eradicate the viral reservoir that has integrated in the liver. Elucidating the immunopathogenesis is key to finding a therapeutic target for HBV as the virus is not in itself cytopathic but the immune response to the virus causes the majority of the cellular injury. In most cases, the virus reaches a state of equilibrium with low viral replication constrained by host immunity. Multiple cytokines have been implicated in the pathogenesis of CHB. Tumor necrosis factor (TNF) has emerged as a key player; on one hand it can facilitate immune‐mediated virological control but on the other hand it can cause collateral hepatocyte damage, cirrhosis and possibly promote hepatocellular carcinoma. In this review, we discuss the current understanding of the immunopathogenesis of HBV, focusing on TNF and whether it can be harnessed in therapeutic strategies to cure HBV infection.

A fully humanized IgG-like bispecific antibody for effective dual targeting of CXCR3 and CCR6

Chemokines and their receptors are pivotal for the trafficking of leukocytes during immune responses, and host defense. However, immune cell migration also contributes to a wide variety of autoimmune and chronic inflammatory diseases. Compelling evidence suggests that both CXCR3 and CCR6 chemokine receptors play crucial roles in the migration of pathological Th1 and Th17 cells during the course of certain inflammatory diseases. The use of two or more receptors by pathogenic cells may explain why targeting of individual receptors has proven disappointing in the clinic. We therefore hypothesized that simultaneous targeting of both CXCR3 and CCR6 with a bispecific antibody (BsAb) might result in decreased chemotaxis and/or specific depletion of pro-inflammatory T cell subsets. In this study, we designed and characterized a fully humanized BsAb. We show that the BsAb binds to both chemokine receptors, as demonstrated by Flow Cytometry and Surface Plasmon Resonance analysis. Furthermore, we demonstrate that the BsAb effectively blocks cell chemotaxis and induces specific antibody-dependent cell-mediated cytotoxicity (ADCC) in vitro. Therefore, we propose that dual targeting of CXCR3 and CCR6 with a fully humanized BsAb may display a potent interventional approach for the treatment of inflammatory and autoimmune diseases.

Is Receptor-Interacting Protein Kinase 3 a Viable Therapeutic Target for Mycobacterium tuberculosis Infection?

The dwindling list of antimicrobial agents exhibiting broad efficacy against clinical strains of Mycobacterium tuberculosis (Mtb) has forced the medical community to redefine current approaches to the treatment of tuberculosis (TB). Host receptor-interacting protein kinase 3 (RIPK3) has been flagged recently as a potential target, given that it is believed to regulate necroptosis-independent signaling pathways, which have been implicated in exacerbating several inflammatory conditions and which reportedly play a role in the necrosis of Mtb-infected macrophages. To examine the therapeutic potential of inhibiting RIPK3, we infected RIPK3-deficient mice with aerosolized Mtb. We found that the loss of RIPK3 did not alter overall disease outcomes, with deficient animals harboring similar bacterial numbers in the lungs and spleens compared to their wild-type counterparts. Mtb-infected macrophages were not rescued from dying by Ripk3 deletion, nor did this affect production of the pro-inflammatory cytokine IL-1β, both in vitro and in vivo. Infiltration of immune cells into the lungs, as well as the activation of adaptive immunity, similarly was not overtly affected by the loss of RIPK3 signaling. Collectively, our data argue against a role of RIPK3 in mediating pathological inflammation or macrophage necrosis during Mtb disease pathogenesis and thus suggest that this host protein is unlikely to be an attractive therapeutic target for TB.

Hepatitis B virus and inhibitor of apoptosis proteins – a vulnerable liaison

HEPATITIS B INFECTION AND CURRENT THERAPIES The treatment for hepatitis C virus (HCV) infection has been revolutionized by the introduction of highly potent antiviral drugs that can successfully cure most patients. A major distinguishing feature between HCV and HBV infection is that the latter forms a persistent mini-chromosome and it integrates into the host genome, whereas HCV is localised solely to the cytoplasmic compartment. Our current HBV antiviral drugs are highly effective in suppressing viral replication, but they cannot cure the infection that is present in 42 billion people worldwide. The immune system of most of these people is capable of effectively controlling the acute phase of HBV infection. However, a proportion of people with HBV infection have persistent viral replication and chronic inflammation that predisposes them to cirrhosis and hepatocellular carcinoma (HCC). A vaccine has been available since the early 1980s that is highly effective in preventing HBV infection, but it has no therapeutic efficacy for the 360 million people who are chronically infected with HBV and have persistent disease. The replicative episomal form of HBV DNA, called covalently closed circular (ccc) DNA, prevents current therapies, including antiviral drugs, from being curative. These drugs need to be taken indefinitely to prevent viral relapse. Strengthened by the advent of HCV curative treatments, there is a huge interest in developing curative therapies for HBV infection. Potential new therapies for chronic hepatitis B include direct-acting antivirals, such as viral assembly inhibitors, gene silencing approaches and viral entry inhibitors. All of these therapies primarily target the virus itself and indeed many, if not most of our anti-infective agents rely on interfering with microbial proteins or the microbial genome to interrupt the ability of the pathogens to replicate. A less explored avenue for the treatment of chronic infections, which may offer enormous potential, is targeting host cell factors that modulate cell signalling, innate or adaptive immune responses. Interferon therapy is an established method of modulating host responses to HBV infection and toll-like receptor agonists (e.g., TLR7 agonist) are currently being tested. However, to date none of these treatments have shown great efficacy in curing HBV infection.

Hydrodynamic Injection as a Method of Gene Delivery in Mice: A Model of Chronic Hepatitis B Virus Infection

Gene delivery methods are important for both therapeutic intervention and as tools in research to address specific questions. Hydrodynamic injection (HDI) is a method that facilitates the delivery and expression of genetic material in target cells, namely hepatocytes, through an intravenous injection. HDI has great utility for research involving cell death and signaling pathways essential in the processes of cancer, inflammation, and transplant therapy, as well as representing a valuable technique to establish hepatitis B virus (HBV) expression in hepatocytes. This chapter describes in detail how to generate a model of chronic HBV infection in immunocompetent mice using HDI as a delivery method.