The new front-line in hepatitis B/D research: Identification and blocking of a functional receptor

Authors

  • Nadia Warner B.Sc. (Hons), Ph.D.,

    Corresponding author
    1. WHO Regional Reference Laboratory for Hepatitis B, Victorian Infectious Diseases Reference Laboratory, North Melbourne, Australia
    • Victorian Infectious Diseases Reference Laboratory, 10 Wreckyn Street, North Melbourne, Victoria 3051, Australia===

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    • fax: +61-39342-2666

  • Stephen Locarnini M.B.B.S., B.Sc. (Hons), Ph.D., F.R.C. [PATH]

    1. WHO Regional Reference Laboratory for Hepatitis B, Victorian Infectious Diseases Reference Laboratory, North Melbourne, Australia
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  • See Articles on Pages 31 and 43

  • Potential conflict of interest: Dr. Locarnini consults for Gilead and Bristol-Myers Squibb.

Treatment for chronic hepatitis B (CHB) over the last two decades has drawn on immune-based interferon-α (IFN-α) or direct-acting antiviral agents in the category of nucleos(t)ide analogues (NAs). Over this time, various combinations of these two treatment approaches have been submitted to trials, but with disappointing gains over the respective monotherapies. This has been offset in part by the positive impact that these therapies have had on the lives of patients with CHB in significantly reducing the risk of development of progressive liver disease and hepatocellular carcinoma.1 Equally dramatic has been the observed reversal of hepatitis B virus (HBV)-associated fibrosis and cirrhosis, with a commensurate decrease in the need for liver transplantation. Unfortunately, current therapies remain less than ideal. Pegylated IFN-α, despite a finite duration of therapy, has a substantial adverse event profile, and patients struggle to stay on treatment for the full 48 weeks. In contrast, NAs require long-term therapy, perhaps lifelong, in order to achieve the benefits outlined above. This is because the NAs have little effect on the virological goal of eradicating HBV covalently closed circular DNA (cccDNA) from infected hepatocytes and markers of active viral replication, including HBV DNA, hepatitis B e antigen (HBeAg) and hepatitis B surface antigen (HBsAg), leading to a key endpoint for achieving cure through HBsAg antibody (anti-HBs) seroconversion. Recent mathematical modeling has estimated the time to HBsAg loss/anti-HBs seroconversion with the existing NAs at over 30 years.2 Thus, problems of compliance and resistance, even with the most potent NAs, will almost certainly emerge.

In the human immunodeficiency virus (HIV)-1/acquired immune deficiency syndrome (AIDS) treatment armamentarium, there are over 20 drugs from six major classes3 directed against multiple targets in the HIV life cycle,4 including entry, enzyme action, assembly, and release. These drugs are used very effectively in synergistic combinations that form the basis of successful highly active antiretroviral therapy regimens.5 From this level of control of active HIV replication, patients can be expected to have a normal lifespan, and HIV-AIDS researchers are preparing new strategies to eradicate HIV from the infected host. This goal has been given the highest priority by national funding agencies. In contrast, in the hepatitis B treatment arena, more drugs targeted to other parts of the viral life cycle are desperately needed if HBV control and eradication are to be achieved. Fortunately, the news from the front line in the battle against HBV and its satellite virusoid, hepatitis delta virus (HDV), is encouraging.

Abbreviations

aa, amino acid; AIDS, acquired immune deficiency syndrome; anti-HBs, HBsAg antibody; cccDNA, covalently closed circular DNA; CHB, chronic hepatitis B; HBeAg, hepatitis B e antigen; HBsAg, hepatitis B surface antigen; HBV, hepatitis B virus; HDV, hepatitis delta virus; HIV, human immunodeficiency virus; IFN-α, interferon-α; NA, nucleos(t)ide analogue; NTCP, sodium taurocholate cotransporting polypeptide.

In this issue of HEPATOLOGY, two papers from the University Hospital Heidelberg group led by Stephan Urban report some critical next steps.6, 7 The investigators focused on early events, both in vitro and in vivo, in the HBV life cycle, namely attachment followed by specific binding to a receptor usually expressed on the cell surface. These steps account for the striking host species specificity (humans, higher primates, and Tupaia belangeri) and tissue tropism (liver) of HBV. Earlier studies from this group and their collaborators demonstrated that myristoylated pre-S1 peptides efficiently blocked and thereby inhibited HBV infection in in vitro models of primary Tupaia hepatocytes and cultures of differentiated HepaRG cells.8, 9 By using these model systems, the specific receptor binding site of HBV has been narrowed down to a critical region of the pre-S1 protein spanning amino acid (aa) residues 9-18, with aa residues 29-48 enhancing infection inhibition, whereas aa residues 19-28 and 1-8 were dispensable.8 Interestingly, HDV, which replicates in HBV-infected hepatocytes and packages its ribonucleoprotein in the HBV envelope, can also be inhibited by these acylated HBV pre-S derived peptides (e.g., preS/2-48myr) with the same specificity.10 This shows that HDV has at least one step in common with HBV for entering hepatocytes. Despite not knowing the identity of the receptor, Urban and colleagues managed to develop a way to block it, based on a detailed understanding of the receptor's binding partner, the HBV pre-S1 protein. From these observations, the investigators have developed a novel therapeutic against HBV and HDV: Myrcludex B based on pre S/2-48myr (Fig. 1). This drug has entered clinical trials after showing efficient blocking of both HBV and HDV infection as well as inhibition of replication in a uPA-SCID mouse model.11 The therapeutic implications of using a drug like Myrcludex B for treating CHB and chronic HDV infection are profound. Blocking entry protects new hepatocytes from de novo infection, whereas preinfected hepatocytes are not subjected to multiple rounds of reinfection (Fig. 1). This latter pathway may turn out to be an even more important mechanism of persistence in chronic HBV infection than previously recognized, possibly accounting for the failure of NAs to affect HBV cccDNA levels or promoting its accelerated decay or silencing.12-14 Thus, one would anticipate that blocking re-entry would result in a significant decrease in the number of infected cells, reducing the intrahepatic burden of HBV cccDNA, with associated significant declines in viremia and HBs antigenemia. The flow-on effects in CHB would be substantial, especially in combination with IFN-α or highly potent, high genetic barrier NAs. Myrcludex B could also have application for preventing perinatal transmission or reinfection after liver transplantations in HBV-infected individuals. Because there is no effective therapy for HDV infection, except long-term IFN-α, a drug like Myrcludex B would represent the first selective therapy for this debilitating disease.

Figure 1.

Myrcludex B based on pre S/2-48myr as a therapeutic against HBV and HDV.

The two papers by Urban and colleagues build on existing data establishing that the myristoylated N terminus of the HBV L-protein mediates the highly specific interaction of HBV with susceptible hepatocytes in vitro and in vivo. Furthermore, potential applications of liver targeting in vivo are also explored. In the first study, Meier and colleagues6 used fluorescently labeled myristoylated pre-S1 lipopeptides (representing the hepatocyte binding domains of HBV, including the pre S/2-48myr) and elegantly visualized in vitro the presence of a specific HBV receptor/ligand complex on the surface of susceptible hepatocytes and quantified its turnover kinetics. The investigators then went on to demonstrate the presence of this highly specific HBV receptor on the plasma membrane of susceptible primary human and primary Tupaia hepatocytes, HepaRG cells, and, intriguingly, on the hepatocytes from nonsusceptible species such as mouse, rat, rabbit, and dog but not pig, cynomolgus monkey, or rhesus monkey. As expected, this HBV-specific receptor was not detectable on HepG-2 or Huh-7 cells. The presence of this receptor required the maintenance of hepatocytes in a differentiated state in order for specific pre-S1 binding to occur, and receptor turnover on the hepatocyte membrane was slow. This in vitro study further confirmed the potent antiviral activity of pre-S/2-48myr by inhibiting viral entry as well as HBeAg secretion.

In the paper by Schieck et al.,7 the targeting of these N-terminally myristoylated pre-S1 peptidic receptor ligands to the liver was demonstrated clearly. As with the in vitro study, hepatocytes from the same nonsusceptible species also bound the labeled lipopeptides and were enriched in the liver, suggesting that the block in HBV infection of these cells is not due to the lack of receptor binding, but rather a lack of a critical coreceptor, a block in entry, or a post-binding step such as nuclear transport or cccDNA generation and processing. These in vivo studies also have important implications regarding the excellent pharmacokinetic properties of drugs like Myrcludex B, potentially the first entry inhibitor for HBV/HDV, and furthermore, provide a basis for the application of these peptides as vehicles for hepatocyte-specific drug targeting.15

Both studies from the Urban group provide tantalizing clues to the identity of the elusive HBV/HDV receptor(s), but the discovery seemed to remain just out of reach until scientists from the National Institute of Biological Sciences in Beijing, China, led by Professor Wenhui Li and colleagues, identified the sodium taurocholate cotransporting polypeptide (NTCP) as a functional receptor for HBV and HDV.16 In their extensive experimental study, the investigators drew directly upon the existing knowledge that the HBV pre-S–derived lipopeptides, including HBV pre-S/2-48myr, blocked infection by binding to a putative viral receptor.17 By using zero distance photo-affinity cross-linking and mass spectrometry, the investigators identified NTCP as a receptor for the HBV pre-S1 peptide.16, 18 The NTCP, also known as SLC10A1, is an integral membrane protein normally involved in bile acid transport in the liver.19 NTCP is localized to the basolateral plasma membrane of hepatocytes (Fig. 1), consistent with its role in “capturing” blood-borne HBV and HDV.20 The Beijing group used a number of different experimental approaches to confirm the role of NTCP as a receptor, including knockdown of NTCP messenger RNA by small interfering RNA, which rendered primary human hepatocytes and primary Tupaia hepatocytes resistant to HBV infection. Transfection of nonsusceptible human hepatoma cell lines with an expression plasmid of human NTCP rendered Huh-7 and HepG-2 cells permissive to infection with HBV and HDV. Furthermore, sequence swapping of nine amino acids in the NTCP taken from nonsusceptible monkeys with the corresponding sequence from the human form of this protein converted the monkey NTCP into a functional receptor for both viruses. These results have implications for the mouse hepatocytes and other animal data presented by the Urban group cited above, and further studies are required to clarify these observations.

The discovery of NTCP as a receptor for HBV and HDV is an important step forward in our attempts to control and eliminate HBV/HDV, but there are some caveats. Transfection of Huh-7/Hep G-2 with NTCP did render them susceptible to HBV/HDV infection in vitro, but only 10% of the cell cultures were positive, and the extracellular yield of virus and subviral particles was disappointingly low. This is in stark contrast to clinical HBV and HDV infection, where nearly 100% of hepatocytes can be infected and the cells express extremely high titers of viral nucleic acids and proteins. As discussed by Schieck et al.,7 host components or conditions that permit efficient viral infection and replication or block any restriction factors in vivo have yet to be identified fully. Together, these landmark studies herald an exciting and vibrant new era in HBV virology, cell biology, and pathogenesis and should accelerate the discovery and development of a new class of HBV and HDV inhibitors. Hopefully, the eradication of both viruses and the curing of patients will now become a very real possibility.