Virus entry and its inhibition to prevent and treat hepatitis B and hepatitis D virus infections

doi:10.1016/j.coviro.2018.04.004

Current Opinion in Virology

Volume 30, June 2018, Pages 68-79

https://doi.org/10.1016/j.coviro.2018.04.004 Get rights and content

Highlights

•
Hepatitis B virus (HBV) and hepatitis D virus (HDV) share common entry steps.
•
Current entry inhibitors (e.g. immunoglobulins) are only used for prophylaxis.
•
New HBV/HDV entry inhibitors have been developed, including Myrcludex B.
•
These novel drugs reduce virus persistence, inflammation, and HBV DNA integration.
•
Thus, use of entry inhibitors should extend to the treatment of chronic infections.

While chronic infection with the human hepatitis B virus (HBV) and hepatitis delta virus (HDV) inflict major health burdens worldwide, current therapies cannot cure patients. One possible novel approach is blocking virus entry to prevent the establishment of infection in naïve hepatocytes. As HBV and HDV use identical viral envelope proteins and the same entry mechanisms, such a strategy would target both viruses. Entry inhibitors (e.g. neutralizing antibodies) have been relegated to the limited role of prophylaxis. However, recent clinical data and infection studies show that new viral entry inhibitors could play a major role in eliminating intrahepatic HBV/HDV genomes. We highlight the consequences on viral persistence after preventing virus entry and the therapeutic benefits entry inhibitors could achieve.

Graphical abstract

Introduction

Despite the availability of vaccines hepatitis B virus (HBV) infection represents a major global health threat and cure remains an unachievable goal with current therapies for most chronically infected patients. Chronic HBV infection is associated with a 25% lifetime risk of developing cirrhosis and hepatocellular carcinoma (HCC), which claims ∼600 000 lives annually. Patient disease progression even increases 2–3-fold if the patient is co-infected with hepatitis Delta virus (HDV), a satellite viroid of HBV affecting an estimated 15–25 million HBV carriers. Current treatment regimens for chronic HBV/HDV infection include interferons and reverse transcriptase inhibitors to suppress intrahepatic virus replication without eliminating the viral templates cccDNA (HBV) or circular ssRNA (HDV). Thus, novel strategies to treat chronic HBV/HDV patients are urgently required.

The causative agent for HBV infection is an enveloped, partially double-stranded DNA virus (Figure 1a). HBV represents the prototype of the growing family of hepadnaviruses [1, 2]. The satellite virus HDV is genetically distinct from HBV with a single-stranded circular RNA genome complexed with the HDV antigen (HDAg) forming a ribonucleoprotein complex [3]. HDV has functionally adapted to HBV by using the HBV-encoded envelope proteins for its spread (Figure 1a). The three HBV envelope proteins — small (S), middle (M), large (L) — are encoded in one commonly used open reading frame (ORF) in the HBV genome with three in-frame start codons [1] (Figure 1b). Using the same envelope proteins implies that HDV and HBV share common attachment and entry steps, which can be inhibited by compounds interfering with crucial viral or cellular factors involved in attachment, specific receptor binding or membrane fusion.

Section snippets

Virus entry and replication of HBV and HDV

Entry and replication of HBV and HDV occurs exclusively in hepatocytes (Figure 2). This tissue specificity but also the host prevalence is predominantly governed by the hepatocyte-specific expression of the species specific cellular receptor sodium taurocholate co-transporting polypeptide (NTCP) [4••, 5••, 6].

Entry

HBV/HDV initially attaches to the cell surface via low-specificity interactions with heparin sulphate proteoglycans (HSPG) [7, 8, 9]. HSPG binding likely induces conformational changes in the surface proteins inducing a highly-specific interaction with NTCP, a bile salt transporter that acts as a HBV/HDV receptor [4••, 5••]. Key to virus infectivity is the large hepatitis B virus surface protein (L-protein), particularly the myristoylated N-terminal 75 residues of the PreS1 domain of the

Formation and maintenance of episomal viral genomes

Following receptor-mediated entry, HBV nucleocapsids (NC) detach (by as-yet unknown mechanisms) from their envelope and are transported to the nuclear pore complex, where relaxed circular (rc)DNA genomes are released and ‘repaired’ to form HBV cccDNA [1]. This episomal DNA acts as a template for HBV gene products and subsequent formation of new virions. By contrast, HDV contains a circular RNA embedded in a HDAg-containing RNP, which also traffics to the nucleus but initiates a double

Envelopment and particle assembly

L-protein, M-protein, and S-protein (encoded in the HBV cccDNA) are required to envelop the mature rcDNA-containing HBV NC and so to assemble viruses. The NC-binding sequence encoded by the PreS1/2-domain (aa 103–124 of the HBV surface L-protein) interacts with motifs in the assembled capsid [19], forming a tight an well-structured complex. Moreover, in contrast to other enveloped viruses, the HBV surface S-protein self-assembles and forms sub-viral particles (SVP), which are secreted in excess

Virus entry: viral and cellular determinants of infectivity

Determinants of infectivity had been initially investigated in primary human hepatocytes (PHH) infected with cell culture-derived recombinant HBV carrying mutated envelope proteins. Amino acid exchanges and deletions were introduced in the PreS1, PreS2 and S domains [23, 24, 25]. Further, HDV has been used as a ‘pseudotyped’ virus to assign additional sequence elements, which interfere with virion assembly of HBV (e.g. mutations in the NC-binding site) [26, 27]. A detailed description of the

Strategies to interfere with viral entry

Building upon these fundamental discoveries on the entry mechanism of HBV and HDV, the inhibition of this process by different agents (Table 1) came into focus of research (reviewed in detail [53]).

Attachment and membrane fusion inhibitors

Therapeutic approaches targeting the initial virus-cell binding interactions are scarce. An early clinical approach in HBV-infected patients using the highly-charged molecule suramin (which inhibits entry of DHBV and HDV [54]) did not show promising results in a clinical trial due to toxic side-effects [55]. Most clinically-applied antibodies recognize the HBV antigenic loop in the S-domain that participates in HSPG binding [9], so they presumably interfere with viral attachment and thereby

NTCP inhibitors

As the specific receptor for HBV/HDV, NTCP has become the primary target for inhibitory drugs. All NTCP-binding molecules can be sub-divided into two principally differently acting groups: (i) substrates that are transported by NTCP and (ii) compounds that bind NTCP but block function in an allosteric manner without being transported into the cell. Compounds of the former group likely inhibit HBV infection due to mutual exclusion between virus-binding and substrate-binding. Thus, blockade of

Conventional uses for HBV/HDV entry inhibitors

The only clinically approved ‘entry inhibitor’ for HBV are immunoglobulins (HBIG) that prevent the virus from engagement to its receptors. So far, the clinical uses of HBIGs are centred on the prevention or limiting the early intrahepatic spread. Current guidelines recommend the use of HBIGs to protect people at risk of exposure [76]. For example, HBIG is administered to newborn children of HBV-positive pregnant women to neutralize virions and thereby prevent vertical transmission. Further,

Novel uses for entry inhibitors in chronic HBV/HDV patients

Recent molecular insights into the HBV and HDV replication cycle and the results of clinical trials with Myrcludex B have uncovered previously unforeseen aspects of HBV and HDV persistence. One important finding showed that dividing hepatocytes in vivo very poorly propagate HBV cccDNA directly to their daughter cells [80^••] but require de novo entry of virions via NTCP [81^••]. Accordingly, viral entry inhibitors (either alone or in combination with drugs that accelerate the turnover of infected

Entry inhibitors in the disruption of the intrahepatic viral genome persistence

The goal of the therapeutic treatments against chronic HBV/HDV infection is the elimination (or permanently silencing) of replication-competent viral episomes (HBV cccDNA and HDV circular ssRNA) in the liver. By eliminating of hepatocytes that express viral antigens, the hope is to limit chronic inflammation, the main driver of liver disease progression [82, 83].

The maintenance of viral templates in a chronically-infected liver remains the subject of intense study and is still not fully

Normalization of ALTs

An unexpected outcome of the Myrcludex B clinical trials was that inhibition of virus entry normalizes serum ALT levels in HBV and HBV/HDV co-infected patients. The reason behind this remains unclear. One hypothesis is that that newly infected hepatocytes are recognized or otherwise targeted by the antiviral immune system more readily compared to cells that are chronically infected. Indeed, this could explain the similar phenomenon of ALT normalization in response to nucleo(t/s)ide analogue

Inhibition of de novo HBV DNA integration

Finally, HBV entry inhibitors are likely to suppress the rate of new HBV DNA integration in the liver. The integrated form of HBV DNA cannot produce new virions due to its inability to produce pregenomic RNA (reviewed by us [99]). However, integrated HBV DNA can produce HBsAg (and has been reported as a major source of HBsAg in HBeAg-negative patients [100^••]). The role of integrated HBV DNA in chronic infection is still unknown, but has now been considered of great importance with some

Summary

In conclusion, novel understanding of the molecular mechanisms involved in HBV/HDV entry and intrahepatic maintenance open up novel treatment strategies. We show here that new entry inhibitors have a potential role in not only adjuncts or substitutes for the current conventional strategies of preventing virus infection, but could also play a crucial role in disrupting the maintenance of an established chronic infection, limiting liver damage and inhibiting ongoing HBV DNA integration.

Conflict of interest statement

SU is co-applicant and co-inventor on patents protecting Myrcludex B as an HBV/HDV entry inhibitor. TT has no conflicts of interest to declare.

References and recommended reading

Papers of particular interest, published within the period of review, have been highlighted as:

• of special interest
•• of outstanding interest

Acknowledgments

This work received funding from the German Centre for Infection Research (DZIF), TTU Hepatitis Projects 5.807 and 5.704, and the Deutsche Forschungsgemeinschaft (DFG) TRR179 (TP 15), and the Australian Centre for HIV and Hepatitis Virology Research. We are grateful to Miriam Kleinig for proofreading and Prof. Ralf Bartenschlager for continuous support.

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Hepatitis B is an infectious disease caused by the HBV virus. It presents a significant challenge for treatment due to its chronic nature and the potential for developing severe complications, including hepatocirrhosis and hepatocellular carcinoma. These complications not only cause physical and psychological distress to patients but also impose substantial economic and social burdens on both individuals and society as a whole. The internalization of HBV relies on endocytosis and necessitates the involvement of various proteins, including heparin sulfate proteoglycans, epidermal growth factor receptors, and NTCP. Among these proteins, NTCP is pivotal in HBV internalization and is primarily located in the liver's basement membrane. As a transporter of bile acids, NTCP also serves as a receptor facilitating HBV entry into cells. Numerous molecules have been identified to thwart HBV infection by stifling NTCP activity, although only a handful exhibit low IC₅₀ values. In this systematic review, our primary focus dwells on the structure and regulation of NTCP, as well as the mechanism involved in HBV internalization. We underscore recent drug breakthroughs that specifically target NTCP to combat HBV infection. By shedding light on these advances, this review contributes novel insights into developing effective anti-HBV medications.
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Citation Excerpt :
Among them, the peptide entry-inhibitor bulevirtide (BLV) previously known as myrcludex B blocks the binding of HBsAg-enveloped particles to the NTCP (sodium taurocholate co-transporting polypeptide), which is the cell entry receptor for both HBV and HDV. It thus prevents the entry of HDV into hepatocytes and subsequent spreading of the virus.6 The favourable therapeutic potential and the good tolerability of BLV were reported in a phase II trial,7 while a phase III trial is ongoing.
Chronic hepatitis D (CHD) is the most severe form of viral hepatitis, characterised by the greatest increase in risk of cirrhosis, hepatic decompensation and hepatocellular carcinoma. Pegylated-interferon-α (pegIFNα), the only off-label therapeutic option, has been available for the last 30 years but is associated with suboptimal response rates and poor tolerability. Among the new treatment strategies under clinical evaluation, the entry inhibitor bulevirtide (BLV) is the only one that has received conditional approval from the European Medicines Agency (EMA); approval was granted in July 2020 for the treatment of adult patients with compensated CHD at a dose of 2 mg daily. Phase II studies and the week 24 interim analysis of a phase III study demonstrated the efficacy and safety of this treatment as a monotherapy or combined with pegIFNα. This favourable profile has been confirmed by recent real-world studies performed in Europe. As a long-term monotherapy, BLV has been successfully used to treat patients with advanced compensated cirrhosis. These encouraging yet preliminary findings must be viewed with caution as many critical issues related to this new antiviral strategy are still poorly understood, as summarised in this review. While waiting for new anti-HBV and anti-HDV drugs to become available for combination studies, BLV treatment is currently the only available anti-HDV therapeutic option that might improve the long-term prognosis of difficult-to-manage patients with CHD.
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Virus entry and its inhibition to prevent and treat hepatitis B and hepatitis D virus infections

Highlights

Graphical abstract

Introduction

Section snippets

Virus entry and replication of HBV and HDV

Entry

Formation and maintenance of episomal viral genomes

Envelopment and particle assembly

Virus entry: viral and cellular determinants of infectivity

Strategies to interfere with viral entry

Attachment and membrane fusion inhibitors

NTCP inhibitors

Conventional uses for HBV/HDV entry inhibitors

Novel uses for entry inhibitors in chronic HBV/HDV patients

Entry inhibitors in the disruption of the intrahepatic viral genome persistence

Normalization of ALTs

Inhibition of de novo HBV DNA integration

Summary

Conflict of interest statement

References and recommended reading

Acknowledgments

Virology

Gut

Gut

Science

J Virol

J Virol

J Virol

Int Immunol

PLoS Pathog

Virology

J Virol

PLoS One

J Hepatol

J Virol

Sci Rep

J Hepatol

Expert Opin Invest Drugs

J Hepatol

Transpl Proc

PLoS Pathog

Viruses

Gastroenterology

Gastroenterology

Immunity

Cell Rep

Deciphering the origin and evolution of hepatitis B viruses by means of a family of non-enveloped fish viruses

Cell Host Microbe

Hepatitis delta virus: insights into a peculiar pathogen and novel treatment options

Nat Rev Gastroenterol Hepatol

Sodium taurocholate cotransporting polypeptide is a functional receptor for human hepatitis B and D virus

Elife

Hepatitis B and D viruses exploit sodium taurocholate co-transporting polypeptide for species-specific entry into hepatocytes

Gastroenterology

The role of the sodium-taurocholate cotransporting polypeptide (NTCP) and of the bile salt export pump (BSEP) in physiology and pathophysiology of bile formation

Handb Exp Pharmacol

Hepatitis B virus infection initiates with a large surface protein-dependent binding to heparan sulfate proteoglycans

Hepatology

Role of glycosaminoglycans for binding and infection of hepatitis B virus

Cell Microbiol

A conformational heparan sulfate binding site essential to infectivity overlaps with the conserved hepatitis B virus a-determinant

Hepatology

Hepatitis delta virus

Virology

The hepatitis delta virus: replication and pathogenesis

J Hepatol

Treatment of chronic hepatitis D with the entry inhibitor myrcludex B: first results of a phase Ib/IIa study

J Hepatol

A proof-of-concept Phase 2a clinical trial with HBV/HDV entry inhibitor Myrcludex B: LB-20

Interim results of a multicenter, open-label phase 2b clinical trial to assess the safety and efficacy of Mycrludex B in combination with tenofovir in patients with HBV/HDV coinfection

Interim results of a multicenter, open-label phase 2b clinical trial to assess safety and efficacy of Myrcludex B in combination with Tenofovir in patients with chronic HBV/HDV co-infection

A short linear sequence in the pre-S domain of the large hepatitis B virus envelope protein required for virion formation

J Virol

Hepatitis B virus morphogenesis

World J Gastroenterol

Role of the pre-S2 domain of the large envelope protein in hepatitis B virus assembly and infectivity

J Virol

Infection process of the hepatitis B virus depends on the presence of a defined sequence in the pre-S1 domain

J Virol