Moving Fast Toward Hepatitis B Virus Elimination

Abstract

Currently, there are two safe and effective therapeutic strategies for chronic hepatitis B treatment, namely, nucleoside analogs and interferon alpha (pegylated or non-pegylated). These treatments can control viral replication and improve survival; however, they do not eliminate the virus and therefore require long-term continued therapy. In addition, there are significant concerns about virus rebound on discontinuation of therapy and the development of fibrosis and hepatocellular carcinoma despite therapy. Therefore, the search for new, more effective, and safer antiviral agents that can cure hepatitis B virus (HBV) continues. Anti-HBV drug discovery and development is fundamentally impacted by our current understanding of HBV replication, disease physiopathology, and persistence of HBV covalently closed circular DNA (cccDNA). Several HBV replication targets are the basis for novel anti-HBV drug development strategies. Many of them are already in clinical trial phase 1 or 2, while others with promising results are still in preclinical stages. As research intensifies, potential HBV curative therapies and modalities in the pipeline are now on the horizon.

5.1. Introduction

Chronic hepatitis B virus (HBV) infection affects approximately 300 million people worldwide [1], and while prophylactic vaccines and antiviral therapies are currently in use, they do not provide a cure. Therefore, safe antiviral agents that target the HBV replication cycle and sites of virus persistence are urgently needed to prevent the nearly one million human deaths annually due to liver diseases associated with hepatitis B. HBV is a hepadnavirus that replicates its DNA in the liver through two main steps: formation of covalently closed circular DNA (cccDNA) and the reverse transcription of a pregenomic RNA (pgRNA).

With current available antiviral therapies for chronic hepatitis B, it is possible to control HBV replication. However, treatment is non-curative and therefore requires long-term continued use which has resulted in concerns for the development of antiviral resistance and adverse events, such as renal impairment or gastrointestinal disorders (important issue when considering adherence to treatment) [2–4]. The clinical endpoints now are focused on suppressing viral replication and alanine aminotransferase (ALT) normalization. This desirable endpoint of a functional cure (loss of HBsAg) is unlikely with current nucleoside analogs or pegylated interferons [5]. This may be due to cccDNA that persists in the nuclei of infected hepatocytes where it forms the template for all viral transcripts and HBV integration. New HBV targets and immune therapies are being sought, and we aim to review them according to their stage in clinical development, focusing on medicinal chemistry and/or biochemistry/molecular biology [6]. In addition, this review focuses on the outcomes of antiviral drugs newly developed or in clinical evaluation, as well as novel experimental drugs.

5.2. HBV Pathogenicity (Immunological Background)

HBV is a hepatotropic virus and most of the time does not cause a cytopathic effect [7]. The host immune response determines whether the virus persists (chronic infection) or not (cleared infection). In the natural history of chronic hepatitis B infection, initially there is an immunotolerant phase characterized by the presence of HBeAg, high rates of HBV DNA replication, and absence of inflammatory liver disease progression [3]. In this phase, the innate immune system is poorly activated due to an intrinsic ability of the virus to escape recognition [8].

In contrast, a persistent immune response to HBV-infected hepatocytes is the determinant of chronic liver disease, with inflammation (with or without HBeAg) leading to progression of fibrosis and cirrhosis, and ultimately hepatocellular carcinoma [3, 9, 10]. Individuals who have resolved HBV infection, with HBsAg clearance with or without HBs antibody, undetectable HBV DNA, and normal levels of ALT, are in the so-called functional cure phase [11]. In this phase, HBV is not fully eliminated, with a few hepatocytes remaining with the cccDNA form under a repressed translational control by innate and adaptive immune mechanisms [9].

In this regard, several immune pathways with the potential to suppress HBV replication in infected hepatocytes are currently under consideration as targets for the development of new therapeutic strategies for chronic hepatitis B infection. For example, retinoic acid-inducible gene-I (RIG-I) and apolipoprotein B mRNA editing catalytic polypeptide-like (APOBEC) [9] are among other pathways that will be discussed below.

5.3. HBV Replication

HBV is a 3200 bp partially double-stranded DNA (rcDNA, relaxed circular DNA) from Hepadnaviridae family. Difference greater than 8% in nucleotide sequence across the complete HBV genotype determines ten major genotypes (A to J) with differences in replication, natural history, pathogenesis, and treatment response [12, 13]. HBV genome encodes four overlapping genes. The HBV RNA transcripts are translated into seven proteins: HBsAg (surface large [preS1+preS2+S domains], middle [preS2+S domain], small [S domain]), HBeAg, HBcAg (core), RT-polymerase, and X protein. The HBV virion particles have an outside envelope composed with three forms (large, middle, and small) of surface proteins that encloses the capsid with the double-stranded DNA genome (Fig. 5.1). An important intermediate form (occurring in the nucleus of infected cells) is the covalently closed circular DNA (cccDNA) that is the template for pregenomic RNA (pgRNA) transcription and produces the template for reverse transcription and viral genome replication [14].

Fig. 5.1.

(a, b) HBV replication mechanism, genome structure, and schematic representation of inhibition sites. (a) The HBV has an envelope composed with three forms (large, middle, and small) of surface proteins that encloses the capsid with the double-stranded DNA genome. (b) Replication starts with HBV binding to the hepatocyte at the NTCP receptor. After entry, the viral particles are uncoated, and the nucleocapsid particle goes to the cellular nucleus. HBV protein free rcDNA (Pf-rcDNA) is converted to an episomal cccDNA, which is the transcription template for all four viral RNAs. The pgRNA is encapsidated together with viral polymerase and subsequently reverse-transcribed into viral minus strand DNA, followed by degradation of the RNA by RNAseH. Then, the plus-stranded DNA is synthesized to form the partially double-stranded relaxed circular DNA. Mature nucleocapsid can either be recycled back to the nucleus to maintain the pool of cccDNA or packed with envelope proteins and exported as infectious virions to infect other cells. pgRNA containing nucleocapsid and empty nucleocapsids are also packed with envelope proteins and released. S small, M medium, L large, DS double stranded, NTCP sodium taurocholate cotransporting polypeptide, CRISPR clustered regularly interspaced short palindrome repeats; CAS9 CRISPR associated 9

5.3.1. Replication Cycle

HBV binds to the hepatocyte at the sodium taurocholate cotransporting polypeptide (NTCP) receptor and enters into the cells. HBV attachment is believed to be mediated through the preS1 domain [15]. After entry, the viral particles containing the relaxed circular DNA (rcDNA) are uncoated, and the nucleocapsid particle must be directed into the cellular nucleus. HBV rcDNA is converted to an episomal cccDNA (see detailed information below). HBV cccDNA is the transcription template for all four viral RNAs (Fig. 5.1):

1. A 2.4-kb mRNA for the large (L) envelope protein, a 2.1-kb mRNA for the middle (M), and major surface (S) proteins

2. A 0.7-kb mRNA for the X protein

3. A 3.5-kb pre-core mRNA that encodes the pre-core protein

4. A 3.5-kb pregenomic RNA (pgRNA) that encodes the core and the polymerase

The pgRNA, upon being exported to the cytoplasm, is encapsidated together with viral polymerase and subsequently reverse-transcribed into viral minus strand DNA. Then, the plus-stranded DNA is synthesized to form the partially double-stranded relaxed circular DNA. The mature nucleocapsid can either be recycled back to the nucleus to maintain the pool of cccDNA or packed with envelope proteins and exported as infectious virions to infect other cells [14, 16] (Fig. 5.1).

5.3.2. Role of cccDNA

Intrahepatic cccDNA is the episomal virus template in the nucleus of HBV-infected hepatocytes. It is considered an important cause of viral persistence and a key obstacle for a cure of chronic hepatitis B [17]. This is especially true because current antiviral therapies including nucleoside analogs do not eliminate HBV mini-chromosome (cccDNA) or integrated HBV; therefore, continued virus gene expression from these templates will drive pathogenesis toward hepatocellular carcinoma, one of the main complications of chronic hepatitis B. Another currently used treatment for chronic hepatitis B, interferon alpha, upregulates the expression of APOBEC3 nuclear deaminase resulting in a modest reduction in cccDNA copy number via deamination [18].

Because cccDNA elimination is a major goal for the future HBV antiviral agents for the treatment of chronic hepatitis B, it is important to monitor and study this particular HBV form. However, the amount of cccDNA compared to pgRNA is very low (median 1.5 copies and 6.5 per cell, respectively) [19]. Therefore, to detect HBV cccDNA unambiguously is a great challenge [17]. Southern blotting is the gold standard test for detection and quantification of HBV intermediates and cccDNA; however, few samples can be tested at a time, and it requires high amounts of infected cells to detect cccDNA. Because it is not a high-throughput system, other tests including cccDNA-specific PCR have been assessed using specific primers located at each side of the gap region of rcDNA together with the appropriate HBV DNA purification or nucleus enrichment and the use of appropriate enzymes to selectively remove HBV rcDNA without degrading cccDNA [20]. Because liver biopsy is required to quantify cccDNA in vivo, measurements of HBV RNA and HBcAg in the serum may serve as surrogate biomarkers for cccDNA [11].

5.4. Overview of Current Therapies

Interferon alpha 2b (FDA approved in 1991) and peginterferon alpha-2a (approved in 2005) are immunomodulators administered subcutaneously, but due to adverse effects treatment duration varies up to 48 weeks (Table 5.1) [26, 27]. There are reports that HBV genotype A may present a higher response rate considering HBeAg seroconversion [28, 29].

Table 5.1.

Antivirals approved for chronic hepatitis B

Drug	Route	Class	HBV DNA EC50, μM	Company	FDA approval year
Lamivudine	Oral	Nucleoside analog	0.56 [21]	GlaxoSmithKline	1998
Adefovir dipivoxil	Oral	Nucleotide analog	0.58 [21]	Gilead Sciences	2002
Entecavir	Oral	Nucleoside analog	0.00036 [21]	BMS	2005
Tenofovir disoproxil fumarate	Oral	Nucleotide analog	0.1 [22]	Gilead Sciences	2008
Telbivudine	Oral	Nucleoside analog	1.3 [23]	Novartis	2006
Tenofovir alafenamide	Oral	Nucleotide analog	0.0347 [24]	Gilead Sciences	2016
Interferon alpha 2b	Parenteral	Immunomodulator		Merck	1991
Peginterferon alpha 2a	Parenteral	Immunomodulator		Genentech	2005
Clevudine	Oral	Nucleoside analog	0.053 [21]	Bukwang/Esai Pharmaceuticals	2009
Besifovir (LB80380)	Oral	Nucleotide analog	0.5 [25]	LG Chem Ltd.	n/a
Thymosin alpha-1	Parenteral	Immunomodulator		SciClone Pharmaceuticals

EC₅₀ median effective concentration to inhibit HBV DNA replication, n/a not applicable

Lamivudine (approved in 1998), adefovir (approved in 2002), entecavir (approved in 2005), telbivudine (approved in 2006), tenofovir (approved in 2008), and tenofovir alafenamide (approved in 2016) are nucleoside analogs used orally, with fewer adverse events compared to immunomodulators and very efficient to reduce viral load (Table 5.1 and Fig. 5.2) [27]. However, a functional cure (loss of HBsAg) is rarely seen with these therapies. Duration of treatment varies, most of the time lasting several years. Because of the long-term need for these medications, adhesion to treatment is a concern, together with the development of drug resistance [21, 30].

Fig. 5.2.

Chemical structures of nucleoside/nucleotide analogs and year of FDA approval. *Approved in South Korea

Other nucleoside analogs approved are clevudine (approved for HBV in South Korea and the Philippines) and besifovir (nucleotide approved in South Korea) [31]. Although clevudine was approved as an antiviral agent for HBV without significant toxicity during the six-month clinical trial, longer therapy (14 months) was found to cause reversible mitochondrial myopathy [32]. This nucleoside analog was one of the few drugs that seemed to have an impact on HBV cccDNA in a woodchuck model (Tennant, personal communication).

Thymosin alpha-1 (Zadaxin) is an immune modulator, administered subcutaneously with minimal side effects approved as monotherapy for chronic hepatitis B in Asian countries [33]. The activity is via an enhancement of T cell differentiation and maturation and is especially effective in settings where there is a reduction in T cell number and/or function [33].

5.5. Drugs in the Pipeline

There are several novel antiviral agents being developed for chronic hepatitis B. The drugs can be divided according to their strategies to eradicate chronic HBV infection (Table 5.2) [34]:

1. Virologic (direct-acting agents or DAAs)

2. Host immune approaches (indirect-acting agents or immune therapy)

Table 5.2.

Classes of antiviral agents in the pipeline for HBV

Direct-acting antiviral agents (DAAs)

Capsid assembly inhibitors or modulators (CAM)

Entry inhibitor

Small interfering RNA (siRNA)

Nucleic acid polymers (NAPs)

HBsAg inhibitors

s-Antigen transport inhibiting oligonucleotide polymers (STOPs)

Antisense molecules

Nucleoside analogs

Indirect-acting agents (immune therapy)

Therapeutic vaccines

Innate immune stimulation

TLR-8 agonist

TLR-7 agonist

Host acting pathway

Apoptosis inducer

Cyclophilin inhibitor

Gene editing

Gene-editing CRISPR/Cas 9

Gene-editing ARCUS platform

Other mechanisms

Monoclonal antibody

FXR agonist

Host targeting antisense (LNA)

PD-L1

Cell immunotherapy

MicroRNA

Nucleic acid-directed HBV cell killing

5.5.1. Direct-Acting Antiviral Agents (DAAs)

Virologic antiviral agents or DAAs are new therapies that could directly target HBV replication steps without killing infected cells [35]. Nucleoside analogs target the viral reverse transcriptase enzyme, thus inhibiting HBV replication. Several nucleoside analogs are approved for chronic hepatitis B treatment as mentioned above, but because they require long-term use and do not completely clear HBV from hepatocytes, new DAAs are being developed, and next we will discuss different strategies used.

5.5.1.1. Capsid Assembly Effectors or Modulators (CAM)

The HBV nucleocapsid plays an essential role in the viral replication cycle that includes HBV genome packaging, reverse transcription, intracellular trafficking of relaxed circular DNA (rcDNA) into the nucleus, and maintenance of chronic infection. Capsid assembly modulators (CAM) are characterized by two types (Table 5.3 and Fig. 5.3): (1) class I or heteroarylpyrimidines (HAP) are core protein allosteric modulators (CpAM) that upon binding to HBV capsids promote their misassembled to aberrant non-capsid core polymers, and (2) class II or phenylpropanamides (PP), sulfamoylbenzamides (SBA), or derivatives are capsid assembly modulators that upon binding to the capsid form normal but empty nonfunctional capsids devoid of pgRNA/rcDNA.

Table 5.3.

Clinical status of capsid assembly effectors or modulators

Drug/class	Route	HBV DNA Log reductiona	Company	Clinical trial phase	ClinicalTrials.gov identifier
Morphothiadin (GLS4)/I	Oral	2.3	HEC Pharma, PR China	2	NCT03638076/NCT04147208/NCT04147208
JNJ 56136379/II	Oral	2.9	Janssen, Ireland	2	NCT02662712
ABI-H0731/II	Oral	3.9	Assembly Biosciences, USA	2	NCT03109730/NTC03780543/NTC03577171/NTC03576066/NTC04454567/NTC02908191
ABI-H2158/II	Oral	2.3	Assembly Biosciences, USA	2	NCT04398134
QL-007/	Oral	-	Qilu, PR China	2	NCT04157257/NCT04157699
RG7907 (RO7049389)/I	Oral	3–5	Roche, Switzerland	1	NCT02952924
EDP-514/II	Oral	> 4.0b	Enanta Pharma, USA	1	NCT04470388
ABI-H3733/II	Oral	5.0c	Assembly Biosciences, USA	1	NCT04271592
ZM-H1505R/pyrazole	Oral		ZhiMeng Biopharma, PR China	1	NCT04220801
ALG-000184/II	Oral	5.0b	Aligos Therapeutics, USA/Emory University	1	NCT04536337
GLP-26/II	Oral	1–3b	Emory University, Aligos Therapeutics	Preclinical	n/a
CB-HBV-001/pyrazole	Oral	12c	ZhiMeng Biopharma, PR China	Preclinical	n/a

N/A not applicable

^aHBV DNA Log₁₀ IU/ml in vivo (data obtained from clinical trials)

^bLog₁₀ decrease in HBV DNA (data obtained from mice models)

^cHBV DNA, EC₅₀ (nM) in vitro

Fig. 5.3.

Chemical structures of prototypical capsid assembly effectors or modulators (CAM)

Both classes of HBV capsid effectors can interfere with several steps of HBV replication cycle including pre- and post-capsid formation, prevention of capsid assembly, perturbation of capsid integrity of incoming virus particles, entry of HBV capsid and core particles into the cell nucleus, pregenomic RNA encapsidation, and consequently its reverse transcription. All these changes in the HBV replication cycle may ultimately prime inhibition of cccDNA formation and/or amplification (Fig. 5.1).

There are five capsid effectors in phase 2 clinical trials. Morphothiadin (GLS4) is a class I HAP compound developed from Bay41–4109 that has shown potent in vitro inhibition of HBV DNA replication; nevertheless, in vivo studies with health volunteers have shown that GLS4 needs an extra-booster (ritonavir) to increase its plasma concentration and achieve effective antiviral activity in humans [36]. Two CpAM ABI-H0731 and ABI-H2158 are in phase 2 clinical trials. ABI-H0731 has shown a decline in HBV RNA that correlated with HBV DNA decline in a 4-week therapy [37], and several phase 2 clinical trials are being conducted with this compound in combination with nucleoside analogs, including entecavir or tenofovir. ABI-H2158 has shown in vivo decline of HBV DNA and pgRNA by ~2 log₁₀ IU/ml and is in phase 2 clinical trial in combination with entecavir [38]. JNJ56136379 is an inducer of empty nonfunctional HBV capsids (CAM-N) that was well tolerated by healthy volunteers in phase 1 and has shown reduced HBV DNA and RNA levels; in a 4-week phase 1b monotherapy study, baseline polymorphisms or enrichment of substitutions did not show an impact on virological response, though the emergence of resistance to longer treatments are underway in phase 2 studies [39]. QL-007 (Qilu, PR China) is in phase 2 clinical trials with entecavir or tenofovir for both safety and efficacy evaluation (Table 5.3).

Four capsid effectors are in phase 1 clinical trial including RG7907, EDP-514, ABI-H3733, and ZM-H1505R. RG7907 (RO7049389), a class I CpAM, reduced both HBV DNA and RNA levels at the end of 28-days treatment, with favorable PK profiles [40]. EDP-514 is a class II core inhibitor that has shown to prevent de novo formation of cccDNA in human primary hepatocytes, and it is in phase 1a/1b study with healthy volunteers [41]. ABI-H3733 is a class II capsid inhibitor that has shown to be a potent inhibitor of HBV DNA (EC₅₀ = 5 nM) and cccDNA formation (EC₅₀ = 125 nM) in vitro [42]. ZM-H1505R is a new pyrazole compound that inhibits HBV DNA replication by inhibiting pgRNA encapsidation and cccDNA formation.

Three main capsid effectors are in preclinical studies: GLP-26, ALG-000184, and CB-HBV-001. GLP-26 (Emory University) is a novel class II CAM, with a unique glyoxamidopyrrolo backbone. It showed substantial in vitro effects in HBV DNA replication and HBe antigen with low nanomolar ranges (EC₅₀ = 3 nM for both markers), with >1 log reduction in cccDNA, and no apparent cytotoxicity. Sustained decreases in HBeAg and HBsAg levels were also observed in HBV-infected humanized mouse model treated with GLP-26 in combination with entecavir up to 3 months after drug cessation [43–46]. ALG-000184 (Aligos Therapeutics/Emory University) is the prodrug of ALG-001075, another potent class II CAM that has shown picomolar activity in vitro and substantial effects in HBV DNA replication in mouse model, with no apparent signs of toxicity and markedly improved solubility [47]. This drug is now entering phase 1a/1b clinical trial in New Zealand, Hong Kong and Republic of Moldova. CB-HBV-001 is a new oxazolidinone, pyrazole capsid inhibitor that is being evaluated in preclinical trials (AASLD 2018).

5.5.1.2. Entry Inhibitors

HBV enters the cell by attaching the receptor binding region of pre-S1 to the NTCP receptor at the membrane of the hepatocyte [48] (Fig. 5.1). Bulevirtide (Myrcludex B) binds irreversibly to NTCP inhibiting the HBV entry into the hepatocyte [49]. This drug is administered subcutaneously and is being studied for chronic hepatitis B and delta in phase 2b with or without peginterferon (PEG-IFN) alfa-2a (Table 5.4) [49]. Preliminary results showed that 12/30 (40%) of individuals treated with bulevirtide plus PEG-IFN for 48 weeks had alanine aminotransferase (ALT) normalization and HDV RNA negative. In the follow-up of 24 weeks of treatment, 4 out 15 individuals treated with 2 mg bulevirtide plus PEG-IFN had undetectable HBsAg, and three out four had HBsAg seroconversion [50]. Bulevirtide was well tolerated, with some drug-related adverse events primarily caused by an increase in total bile salts [50]. This is explained because the drug binding to NTCP prevents infection but also inhibits hepatic bile salt uptake leading to the transiently elevated bile salt level [51].

Table 5.4.

Entry Inhibitor in development

Drug	Route	Dose	Company	Clinical trial phase	ClinicalTrials.gov identifier
Bulevirtide (myrcludex B)	Parenteral	2–5 mg SC qd 48 weeks	Hepatera, Russia with MYR GmbH, Germany	2b	NCT02888106

5.5.1.3. Small Interfering RNA (siRNA)

RNA interference (RNAi) is the mechanism through which double-stranded RNAs silence cognate genes (Fig. 5.1). It is characterized by the presence of RNAs about 22 nucleotides homologous to the gene that is being suppressed. Dicer is the cellular nuclease that cleaves double-stranded RNAs and can produce putative guide RNAs or small interfering RNA (siRNA) [52]. After the sense strand is removed and the antisense strand is loaded on the RNA-induced silence complex (RISC), it hybridizes to a complementary region of a target mRNA, which results in its degradation [53]. This phenomenon provides effective agents for inhibiting infectious, metabolic, cancer, and genetic diseases [53]. A critical issue in the development of siRNA-based drugs is to avoid toxicity such as (1) immunogenic reactions to dsRNA (2′-O-methyl base modifications have largely avoided this issue), (2) toxicity of excipients (work continues on developing potent and nontoxic nanoparticles), (3) unintended RNAi activity (avoided by detailed screening target sites against human genome sequences), and (4) on target RNAi activity in nontarget tissues (selection of highly diseased selective genes and delivery routes which reduce accumulation in nontarget tissues) [54]. Previous studies showed that siRNA could significantly inhibit HBV transcripts and cccDNA in vitro in HepG2 cells and in vivo in mice [53, 55, 56]. Currently, several siRNAs are being evaluated in preclinical and phases 1, 1/2, and 2 clinical trials shown in Table 5.5. VIR-2218 has shown dose-dependent HBsAg reductions (mean decline of 1.0 log₁₀) in HBeAg negative or positive patients virally suppressed on nucleos (t)ide analogs without significant fibrosis [58]. Another siRNA drug, JNJ-3989 (ARO-HBV) that is in a phase 2a study, has demonstrated a ≥log₁₀ reduction in HBsAg at nadir was achieved in 98% of patients [59]. In total, 15/38 (39%) of patients who were responders throughout the study were sustained responder at day 392 [59].

Table 5.5.

Small interfering RNA (siRNA) drugs in development

Drug	Route	Company	Clinical trial phase	ClinicalTrials.gov identifier
VIR-2218 (ALN-HBV02)	Parenteral (SC)	Alnylam and Vir Biotech, USA	2	NCT03672188
JNJ-3989 (ARO-HBV)	Parenteral (SC)	Arrowhead Pharma with Janssen, USA	2a	NCT03365947
RG6346 (DCR HBVS)	Parenteral (SC)	Roche, Switzerland	2/1	NCT03772249
AB-729 [57]	Parenteral (SC)	Arbutus Biopharma, USA	1	NA

NA not available

5.5.1.4. Nucleic Acid Polymers (NAPs)

NAPs are phosphorothioate oligonucleotides (PS-ONs) that inhibit HBV via a post-entry mechanism blocking the assembly/release of HBV subviral particles (Fig. 5.1). The universal model for NAP pharmacology is based on the interaction of the amphipathic protein domain and the hydrophobic side of NAPs, preventing the conformational changes in the target or its interaction with other amphipathic helices [60]. In this class of antivirals, there are the HBsAg inhibitors and the STOPs (s-antigen transport inhibiting oligonucleotide polymers).

HBsAg Inhibitors

Aside from the ability of HBsAg to sequester anti-HBs from the blood system, HBsAg has direct immunoinhibitory action against both innate and adaptive immune responses (Fig. 5.1). HBsAg loss is infrequently achieved with the current therapy; therefore, antivirals targeting the inhibition of HBsAg are being developed. NAPs have the ability to interact with hydrophobic surfaces of proteins and have emerged as the first therapy to be able to achieve rapid HBsAg loss [61].

REP 2139 is a phosphorothioate oligodeoxyribonucleotide (PS-ONs) with the sequence (dAdC)₂₀ (Table 5.6 and Fig. 5.4) [62]. Clinical studies of REP 2139 in combination with thymosin or PEG-IFN was well tolerated and resulted in liver flares (without liver dysfunctions) following initial reductions of serum HBsAg and HBV DNA [62]. REP 2165 is a version of REP 2139 which has been shown preclinically to retain antiviral activity with lower accumulation in the liver. The results of phase 2 randomized trial showed that addition of NAPs to tenofovir and PEG-IFN increased functional cure after therapy without altering tolerability [63].

Table 5.6.

HBsAg inhibitors in development

Drug	Route	Company	Clinical trial phase	ClinicalTrials.gov identifier
REP 2139	Parenteral	Replicor, Canada	2	NCT02726789
REP 2165	Parenteral	Replicor, Canada	2	NCT02565719

Fig. 5.4.

Chemical structures of HBsAg inhibitor (a), nucleoside analog (b), innate immune stimulation (c), cellular inhibitor of apoptosis proteins (cIAPs) (d), and cyclophilin inhibitor (e)

STOPs (s-Antigen Transport Inhibiting Oligonucleotide Polymers)

STOPs are oligonucleotide aptamers (protein binding) comprised of a repeating poly AC sequence (Fig. 5.1). STOPs share the structural similarity with NAPs but contain several novel chemical features. STOPs can reduce HBsAg secretion by affecting protein trafficking from the infected cell resulting in its degradation [64]. In HepG2.2.15 cells, ALG-10133 reduced HBsAg secretion in nanomolar range and with synergistic effects when combined with class II CAMs [65]. ALG-10133 has been selected as the lead candidate, starting clinical trials on 2020 with projected human efficacious dose of 30–75 mg delivered SC weekly (Table 5.7) [66].

Table 5.7.

STOPs (s-antigen transport inhibiting oligonucleotide polymers) in development

Drug	EC50 μM	Route	Company	Clinical trial phase	ClinicalTrials.gov identifier
ALG-10133	0.0032	Parenteral (SC)	Aligos Therapeutics, USA	1	NCT04485663

EC₅₀ median effective concentration to inhibit HBV DNA replication

5.5.1.5. Antisense Molecules

Antisense oligonucleotides (ASO) (Table 5.8.) are small single-stranded nucleic acid sequences that bind with high selectivity to their target RNAs. This triggers degradation via an RNAse H-dependent pathway [68]. GSK 3228836 is a 2′-O-methoxyethyl free ASO currently in development for the treatment of chronic hepatitis B. It has been tested as a subcutaneous injection in doses up to 120 mg, and no safety concerns were identified [69]. GSK3389404 (GSK404) is a second-generation ASO that showed an acceptable safety profile [70]. GSK3389404 presented platelet dose-dependent declines that plateaued on treatment and started to recover after dose completion [70, 71]. RO7062931 is an N-acetylgalactosamine (GalNAc) conjugated single-stranded oligonucleotide (SSO) with locked nucleic acid (LNA) that is complementary to messenger RNAs (mRNAs) of the HBV genome [72, 73]. Gal-Nac conjugation should reduce ASO renal and platelet toxicities. It was well tolerated in healthy volunteers. Phase 1 studies showed a mean nadir of HBsAg of −0.5 log₁₀ IU/mL, with treatment emergent ALT elevations with transient concurrent HBsAg decline (0.6–0.8 log₁₀ IU/mL) with no changes in liver function [74].

Table 5.8.

Antisense molecules in development

Drug	Route	Company	Clinical trial phase	ClinicalTrials.gov identifier
GSK 3228836 /IONIS-HBVRx/ISIS505358	Parenteral (SC) [67]	Ionis with GSK, USA	2a	NCT04449029/NCT02981602
GSK3389404/Ionis-HBV-LRx	Parenteral (SC)	Ionis with GSK	2a	NCT02647281
RO7062931	Parenteral (SC)	Roche	1a	NCT03505190/NCT03038113

5.5.1.6. Nucleoside Analogs

ATI-2173 is a novel phosphoramidate prodrug of clevudine in preclinical studies for chronic hepatitis B (Table 5.9 and Fig. 5.4) [67]. Long-term use of clevudine was found to exhibit reversible skeletal myopathy in a small group of individuals and therefore subsequently discontinued from development. ATI-2173 was designed by modifying clevudine that bypasses the first phosphorylation step where the 5′-monophosphate is converted to the active 5′-triphosphate in the liver [67]. ATI-2173 activity was decreased by 25 viral polymerase mutations associated with entecavir, lamivudine, and adefovir resistance, but not capsid inhibitor resistance mutations [67]. It has been claimed that this compound could behave as a non-nucleoside antiviral agent.

Table 5.9.

Nucleoside analog in development

Drug	EC50 μM	Route	Company	Clinical trial phase	ClinicalTrials.gov identifier
ATI-2173	0.0013	Oral	Antios Therapeutics, USA	1	NCT04248426

EC₅₀ median effective concentration to inhibit HBV DNA replication

5.5.1.7. RNAseH Inhibitors

RNAseH is one of the two enzymatically active domains on HBV polymerase and destroys the HBV RNA after it has been copied into DNA by the reverse transcriptase [75]. RNAseH is a potential target for antiviral drugs, and over 150 RNAseH inhibitors are divided in four compound classes: (1) α-hydroxytropolones (αHT), (2) N-hydroxyisoquinolinediones (HID), (3) N-hydroxypyridinediones (HPD), and (4) and N-hydroxynapthyridinones [76–81]. Novel amide αHT were studied with EC₅₀ values from 0.31 to 54 μM [79]. Studies in chimeric mouse showed that an HPD and an αHT suppressed HBV replication to up to 1.4 log₁₀ after two weeks of treatment followed by a rebound in the viral titers [82].

5.5.2. Indirectly Acting Antiviral Agents (Immune Therapy)

Specific immune therapy can maintain the HBV replication under control of a functional host antiviral response [9] (Fig. 5.1). An example of approved immune therapy for chronic hepatitis B is interferon alpha (pegylated or not). Pegylated interferon alpha alone or in combination therapy can achieve sustained off-treatment control but in only a small portion of individuals [26].

Therapeutic restoration of protective immunity is a strategy that can be considered to achieve the functional cure of HBV [83]. Several approaches are being considered such as therapeutic vaccines, innate immune stimulation (TLR-8 and TLR-7 agonists), host acting pathway (apoptosis inducer and cyclophilin inhibitor), gene editing, and many other mechanisms.

5.5.2.1. Therapeutic Vaccines

There is a renewed interest in therapeutic vaccines with the development of novel formulations, suitable immunization routes for designed adequate antigens, and adjuvant strategies (Table 5.10). In addition, it is important to consider adequate strategies, including combination therapy with other antivirals, either concomitant or sequential strategies.

Table 5.10.

Therapeutic vaccines in development

Drug	Platform	Route	Company	Clinical trial phase	ClinicalTrials.gov Identifier
NASVAC (ABX203)	HBs and HBc antigen mixed with carboxyl vinyl polymer [84]	Nasal	CIGB, Cuba	3	NCT01374308/NCT02249988
HepTcell (FP-02.2)	Synthetic HBV-derived peptides formulated with IC31®, a TLR9based adjuvant [85]	IM [86]	Altimmune, USA	2	NCT02496897
AIC 649	Parapoxvirus (iPPVO) [87]	IV	AiCuris, Germany	1
JNJ 64300535 (HB-110)	Plasmids encoding HBsAg, HBcAg, and IL12 [88]	IM Electroporation	Janssen, Ireland/Ichor Medical Genexine, USA	1	NCT03463369
TG1050	Ad5 which encodes truncated HBV core, POL, and two small domains from the ENV [89]	SC	Transgene, France	1	NCT04168333/NCT02909023/NCT04168333
MVA-VLP-HBV	Modified vaccinia Ankara-virus-like particle-hepatitis B virus [90]		GeoVax, USA	Preclinical	n/a
Chimigen HBV	Recombinant chimeric fusion protein comprising hepatitis B virus (HBV) S1 and S2 surface antigen fragments, core antigen, and a murine monoclonal antibody heavy chain fragment (Fc) [91]		Akshaya, Canada	Preclinical	n/a
TherVacB	HBsAg, HBcAg, and a boost using a modified vaccinia virus Ankara (MVA) vector [92]		Helmholtz Zentrum Muenchen, Germany	Preclinical	n/a
3xT2A and Mix2A	VLVs expressing polymerase (Pol), core (HBcAg), and MHBs [93]		CaroGen, USA	Preclinical	n/a
HBV	TheraT® and VaxWave® investigational arenavirus-based immunization technologies [94]		HOOKIPA Pharma, Austria, with Gilead	Preclinical	n/a
VBI-2601 (BRII-179)	Recombinant, protein-based immunotherapeutic [95]		VBI Vaccines, USA	1b/2a

ID intradermal, SC subcutaneous, Ad adenovirus, POL polymerase, ENV envelope, VLV virus-like vesicles, N/A not applicable

5.5.2.2. Innate Immune Stimulation

The host immune responses to HBV determine if the individuals will clear (functional cure) or fail to clear the virus (chronic hepatitis B). Toll-like receptor (TLR) family and its functions are one way to modulate the immunological host responses [96]. TLR8 and TLR7 are endosomal TLRs members with a high degree of sequence and function similarity. They recognize pathogen-associated molecular patterns (viral single-stranded RNA fragments) and trigger innate and adaptive immune responses[96, 97]. Agonist ligands of Toll-like receptors 7 and 8 have immune-stimulating activity allowing to intervene several diseases and to be valuable vaccine adjuvant candidates [96].

Selgantolimod (formerly GS-9688) is a small molecular agonist of Toll-like receptor 8 (TLR8) [98]. It sustained reduced intrahepatic RNA and DNA of woodchuck hepatitis virus (WHV) in animal model. With a finite, short duration treatment, the serum WHsAg level reduced with half of animals with levels below the limit of detection [97]. Selgantolimod is an oral drug under phase 2 clinical trial (Table 5.11 and Fig. 5.4). RO7020531 (RG7854) is an oral prodrug of a TLR-7 agonist in phase 2 clinical trial (Table 5.11). Carboxylesterase (mainly CES2) and oxidation by aldehyde oxidase converts RO702053 into the active metabolite RO7011785 [99]. Preclinical data showed that a combination of HBV locked nucleic acid antisense oligonucleotide (HBV-LNA ASO) with RO7020531 reduced HBsAg and HBV DNA with delayed rebound off-treatment in mice [100].

Table 5.11.

Innate immune stimulation in development

Drug	Class	Route	Company	Clinical trial phase	ClinicalTrials.gov identifier
GS9688 (Selgantolimod)	TLR-8 agonist	Oral	Gilead Sciences, USA	2	NCT03491553/NCT03615066
RO7020531 (RG7854)	TLR-7 agonist	Oral	Roche, Switzerland	2	NCT02956850/NCT03530917/NCT04225715

5.5.2.3. Host Acting Pathway

Cellular inhibitor of apoptosis proteins (cIAPs) impairs clearance of hepatitis B virus (HBV) infection by preventing TNF-mediated killing/death of infected cells. Animal studies showed that drug inhibitors of cIAPs were able to reduce serum HBV DNA, hepatitis B surface, and core antigens [101]. APG-1387 is an apoptosis inducer; it is a second mitochondria-derived activator of caspase (SMAC) mimetic, and it targets inhibitors of apoptosis proteins (IAPs) [102]. Currently, APG-1387 is under clinical trial phase 1 study for chronic hepatitis B (Table 5.9 and Fig. 5.4).

CRV-431 is an oral cyclophilin inhibitor, non-immunosuppressive analog of cyclosporine A. CRV 431 is a small molecule under clinical development for the treatment of liver diseases including fibrosis and hepatocellular carcinoma [103]. Preclinical studies showed antiviral activity against hepatitis B reducing HBV DNA and HBsAg levels in transgenic mice and a phase 1 is ongoing (Table 5.12 and Fig. 5.4) [104].

Table 5.12.

Host-acting pathway

Drug	Class	Route	Company	Clinical trial phase	ClinicalTrials.gov identifier
APG-1387	Apoptosis inducer	IV	Ascentage Pharma, PR China	1	NCT03585322
CRV-431	Cyclophilin inhibitor	Oral [103]	Hepion, USA (formerly ContraVir)	1	NCT03596697

IV intravenous

5.5.2.4. Gene Editing

Clustered regularly interspaced short palindrome repeats (CRISPR)/Cas9-based antiviral strategy is one of the most versatile gene-editing tools, discovered as a bacterial adaptive immune system [105]. The CRISPR/Cas9 system can specifically destruct HBV genomes in vitro and in vivo, mediating specific cleavage of cccDNA [106–108] (Fig. 5.1). Several optimal targets in HBV genome have been described, such as the surface and polymerase overlap region; the YMDD RT motif and the HBV enhancer I, II, X protein; and pre-core regions with high efficacy [109]. However, CRISPR/Cas system inevitably targets integrated HBV DNA and induces double-strand breaks (DSBs) of host genome, raising concerns of genome instability and carcinogenicity [108, 110]. To avoid DSBs of the host genome, recently it was described a permanently Cas9-mediated base editors that effectively introduced nonsense mutations that generated premature stop codons of surface gene in both integrated and cccDNA reducing HBsAg secretion [110]. EBT107 is a gene-editing CRISPR/Cas 9 drug that uses a duplex gRNA excision knockout as a candidate for HBV in preclinical studies (Table 5.13) [111].

Table 5.13.

Gene editing

Drug	Class	Company	Clinical trial phase
EBT107	Gene-editing CRISPR/Cas 9	Excision Bio, USA	Preclinical
ARCUS nucleases	Gene-editing ARCUS platform	Precision Biosciences, USA	Preclinical

IV intravenous

ARCUS genome-editing technology is another platform of gene editing being developed for chronic hepatitis B [112]. The ARCUS technology is based on the properties of a naturally occurring gene-editing enzyme – the homing endonuclease I-CreI—and reduces the risk of additional off-target DNA edits [113].

5.5.2.5. Other Mechanisms

Recombinant hepatitis B human monoclonal antibody:

Lenvervimab (GC1102) is a recombinant hepatitis B human monoclonal antibody expected to improve sustained virological response reducing HBsAg levels in individuals with chronic hepatitis B infection [114]. It is under study for HBV-related liver transplant recipients (Table 5.14).

Table 5.14.

Drugs with other mechanisms in development

Drug	Route	Mechanisms	Company	Clinical trial phase	ClinicalTrials.gov identifier
Lenvervimab (GC1102)	IV	Monoclonal antibody	Green Cross, South Korea	2a	NCT03801798/NCT02304315
Vonafexor (EYP001)	Oral	FXR agonist	Enyo Pharma, France	1	NCT04365933
ASC22 (KN035)	SC	PD-L1 pathway	Ascletis Pharma, PR China	2a	NCT04465890
LTCR-H2–1	IV	T cell immunotherapy	Lion TCR, Singapore	1	NCT04745403

IV intravenous, SC subcutaneous, N/A not applicable

Farnesoid X receptor (FXR) agonist:

HBV enters the hepatocyte by binding to NTCP, the genome of which contains two active farnesoid X receptor (FXR)α response elements that participate in HBV transcriptional activity [115]. In vitro studies showed that FXR agonists inhibited viral mRNA, DNA, and protein production and reduced the cccDNA pool size [115]. Vonafexor (EYP001) is a farnesoid X receptor (FXR) agonist with anti-HBV effects [116, 117]. It is under study in combination with PEG-IFN, nucleoside analogs in double or triple therapy (Table 5.14).

PD-L1 pathway:

The programmed cell death protein 1 (PD-1)/programmed death-ligand 1(PD-L1) pathway is a key immune checkpoint regulator that controls the induction and maintenance of immune tolerance in chronic hepatitis B infection [118]. ASC22 (KN035) is a novel fusion anti-PDL1 antibody being studied for the treatment of solid tumors and in clinical trials for chronic hepatitis B phase 2a (Table 5.14).

T cell immunotherapy:

LTCR-H2–1 (Table 5.14) is a preclinical drug that boosts adaptive immune response through T cell receptor (TCR) gene transfer [119]. It is engineered to target virus-derived peptides presented on MHC class I on the surface of virus-infected cells. This technology is based on leukapheresis to isolate white blood cells, followed by T cell expansion; HBV targeting TCR are introduced into the activated T cells by viral transduction or electroporation, and then after phenotypic and functional validation, the TCR-engineered T cells are infused back into the individual [120].

5.6. Conclusions

Currently, nucleoside analogs and peginterferon are available for chronic hepatitis B treatment and are quite effective and safe. They can prevent progression of disease, but even persons treated with these drugs can develop hepatocellular carcinoma. The treatments can achieve inhibition of HBV replication; however, few individuals achieve “functional cure” status (HBsAg clearance with or without surface antibody). Several novel drugs are in the pipeline for treatment and elimination of chronic hepatitis B. The drugs are at different stages of development from preclinical to phase 2 clinical trials, and some of them are considered for combination strategies. These drugs will be instrumental for a sustained HBV DNA undetectability with sustained clearance of HBsAg and for preventing liver cancer. Elimination of cccDNA and integrated HBV DNA will be key to eradicate chronic hepatitis B infection. Currently, there are numerous drugs that have the potential to cure HBV, but most do not have the necessary potency to clear all cccDNA. We now know that the half-life of cccDNA (several months and not decades) is shorter than was previously reported [121]. Thus, it may be possible to eliminate cccDNA in approximately 1 year with more potent agents or more likely a combined modality (e.g., capsid effector plus STOPs). As expounded above, a great number of approaches are being tried to eliminate HBV, and it is clear that we are beginning to turn the tide.

Key Points.

• Current treatments do not completely clear HBV from hepatocytes leading to the establishment of lifetime chronic infection.

• Novel anti-HBV therapies targeting different steps of HBV replication cycle with the potential of curing individuals chronically infected are needed.

• Elimination of cccDNA from the nuclei of hepatocytes and clearance of HBV surface antigen (HBsAg) from blood are crucial to achieving a functional and complete cure.

• Drug-drug combinations synergistically targeting key steps of HBV replication cycle and immunomodulators boosting the host immune response may lead to a functional cure.

• Novel strategies including CRISPR and siRNA technologies which can inactivate persistent HBV cccDNA and also target integrated viral DNA may eliminate HBV from chronically infected human hepatocytes.

Abbreviations

Ad

Adenovirus

ALT

Alanine aminotransferase

APOBEC

Apolipoprotein B mRNA editing catalytic polypeptide-like

ARCUS

Gene-editing platform

ASO

Antisense oligonucleotide

CAM

Capsid assembly effectors or modulators

CAS

CRISPR associated

cccDNA

Covalently closed circular DNA

CES

Carboxylesterase

cIAPS

Cellular inhibitor of apoptosis proteins

CpAM

Core protein allosteric modulators

CRISPR

Clustered regularly interspaced short palindrome repeats

DAAs

Direct-acting agents

DS

Double stranded

DSBs

Double-strand breaks

EC₅₀

Median effective concentration to inhibit HBV DNA replication

ENV

Envelope

FXR

Farnesoid X receptor

GalNAc

N-acetylgalactosamine

GLS4

Morphothiadin

HAP

Heteroarylpyrimidines

HBcAg

HBV core antigen

HBeAg

HBV e antigen

HBsAg

HBV surface antigen

HBV

Hepatitis B virus

HID

N-hydroxyisoquinolinediones

HPD

N-hydroxypyridinediones

IAPs

Inhibitors of apoptosis proteins

ID

Intradermal

IM

Intramuscular

IV

Intravenous

L

HBV large surface protein

LNA

Locked nucleic acid

M

HBV middle surface protein

MHC

Major histocompatibility complex

mRNA

Messenger RNA

n/a

Not applicable

NAPs

Nucleic acid polymers

nM

Nanomolar

NTCP

Sodium taurocholate cotransporting polypeptide

PD-1

Programed cell death protein 1

PD-L1

Programed death ligand protein 1

PEG-IFN

Peginterferon

pgRNA

Pregenomic RNA

PK

Pharmacokinetics

POL

Polymerase

PP

Phenylpropanamides

PS-ONs

Phosphorothioate oligonucleotides

rcDNA

Relaxed circular DNA

RIG-I

Retinoic acid-inducible gene-I

RISC

RNA-induced silence complex

RNAi

RNA interference

RT

Reverse transcriptase

S

HBV small surface protein

SBA

Sulfamoylbenzamides

SC

Subcutaneous

siRNA

Small interfering RNA

SMAC

Second mitochondria-derived activator of caspases

SSO

Single-stranded oligonucleotide

STOPs

s-Antigen transport inhibiting oligonucleotide polymers

TCR

T cell receptor

TLR

Toll-like receptor

TNF

Tumor necrosis factor

VLV

Virus-like vesicles

WHsAg

WHV surface antigen

WHV

Woodchuck hepatitis virus

YMDD RT

Tyrosine, methionine, aspartate, motif aspartate reverse transcriptase motif

αHT

α-hydroxytropolones

μM

Micromolar