Drug development for chronic hepatitis B functional cure: Recent progress

Abstract

Chronic hepatitis B virus (HBV) infection affects approximately 254 million individuals globally, contributing to significant morbidity and mortality due to HBV-related liver failure and cirrhosis, which result in millions of fatalities each year. Although approved antiviral nucleos(t)ide analogues can effectively suppress HBV replication, their ability to reduce hepatitis B surface antigen (HBsAg) levels in plasma remains limited. The clinical application of the immunomodulator interferon-alpha is restricted by concerns regarding its safety and the severity of associated adverse reactions, rendering long-term administration challenging. Therefore, current drug development efforts for chronic hepatitis B aim to achieve a functional cure, which is defined as HBsAg serological clearance and sustained suppression of HBV DNA. This review discusses recent advancements in novel direct-acting therapeutic strategies for the treatment of chronic hepatitis B by focusing on the progresses in HBV entry inhibitors, monoclonal antibodies, RNA interferences, and other agents that directly target the virus. Furthermore, we discuss the development of immunomodulatory therapies, including TLR-7/8 agonists, immune checkpoint inhibitors, and therapeutic vaccines. In the end, we conclude by highlighting the importance of the rational combination-strategy design to improve the functional cure rate of HBV.

Core Tip: As we know, chronic hepatitis B virus (HBV) infection can lead to liver failure and cirrhosis, resulting in significantly increased morbidity and mortality rates. Current antiviral nucleos(t)ide analogues suppress HBV replication but are limited in reducing hepatitis B surface antigen (HBsAg) levels. Interferon alpha, an immunomodulator, is restricted by safety concerns and adverse reactions. New drug development aims for a functional cure, defined as HBsAg clearance and sustained HBV DNA suppression. This review highlights recent advancements in novel therapies targeting HBV, including HBsAg entry inhibitors, monoclonal antibodies, transcription inhibitors, and immunomodulatory agents like TLR-7/8 agonists, immune checkpoint inhibitors, and therapeutic vaccines.

INTRODUCTION

Chronic hepatitis B virus (HBV) infection represents a significant global public health challenge. According to the World Health Organization, an estimated 254 million individuals are living with chronic hepatitis B in 2022, with approximately 1.1 million deaths attributed to HBV-related complications such as cirrhosis and hepatocellular carcinoma (primary liver cancer)[1]. The challenges in achieving a cure for HBV infection are attributable to the virological and immunological features of chronic HBV infection, including the persistence of covalently closed circular DNA (cccDNA) in the nuclei of infected hepatocytes as a viral transcription template; the accumulation of viral integration events throughout the long-term infection that contributes to hepatitis B surface antigen (HBsAg) production; and the high viral antigen load that drives the exhaustion of HBV-specific immunity[2]. As current available antiviral therapies are rarely effective in treating HBV, a group of novel antiviral drugs with distinct mechanisms are being developed, which can be categorized into the following two classes: Direct-acting antivirals that further block viral replication and lower the viral antigen load, and immunomodulators that boost immunity and restore anti-HBV immune surveillance.

THE HBV REPLICATION PROCESS

HBV is a partially double-stranded DNA virus belonging to the Hepadnaviridae family, has a structure composed of an envelope surrounding a nucleocapsid that houses the relaxed circular DNA (rcDNA) genome. The virus specifically infects hepatocytes, initiating a complex replication cycle. The process begins with the interaction between the Pre-S1 glycoprotein on the viral envelope and the sodium taurocholate co-transporting polypeptide (NTCP) receptor on the hepatocyte surface, which facilitates the entry of the viral nucleocapsid into the cell. After entry, the rcDNA is transported to the nucleus, where it is converted into cccDNA, serving as a stable template for the transcription of pre-genomic RNA (pgRNA) and messenger RNA (mRNA), which are essential for viral replication and protein synthesis. The pgRNA undergoes reverse transcription, generating new rcDNA molecules while simultaneously encoding core proteins and polymerase, which are required for the assembly of new nucleocapsids. Concurrently, the mRNA directs the production of other viral proteins. The mature nucleocapsids acquire viral envelope proteins to form mature virions that are released from the cell. A subset of nucleocapsids returns to the nucleus, where rcDNA is reconverted into cccDNA, perpetuating the intracellular reservoir of viral DNA. This recycling mechanism enables the persistence of HBV within hepatocytes, even in the absence of new infections. Moreover, HBV DNA can integrate into the host genome, contributing to the development and progression of chronic infection[3].

CURRENT SITUATION OF CHRONIC HEPATITIS B TREATMENT

The treatment of chronic hepatitis B relies on two main classes of drugs approved by the United States FDA, the European Medicines Agency, and regulatory bodies in many Asian countries: Immunomodulators and nucleos(t)ide analog antiviral drugs as listed in Table 1. The approved immunomodulators include conventional interferon alpha-2α (IFN-2α) and pegylated IFN-2α (PEG-IFN-2α)[4]. The nucleos(t)ide analog family has three drugs used as first-line therapy, namely, entecavir, tenofovir disoproxil fumarate (TDF), and tenofovir alafenamide. Adefovir dipivoxil, lamivudine, and telbivudine are less ideal in terms of potency, resistance, and long-term safety profiles.

Table 1.

Current available drugs for the treatment of chronic hepatitis B

Types of drugs	Name	Antiviral mechanism	Limitations/comments
Immunomodulators	IFN-2α and PEG-IFN-2α	Inhibit HBV replication Activate antiviral immune responses	Poor tolerability; Anti-PEG antibody mediated immune clearance[4]
Nucleoside analogue	ETV	HBV DNA polymerase inhibitor	High potency and low resistance
Nucleotide analogue	TDF	HBV DNA polymerase inhibitor	Potent, but with kidney and bone toxicity concern in long-term use
Nucleotide analogue	TAF	HBV DNA polymerase inhibitor	TDF analog with improved renal and bone safety profiles
Nucleotide analogue	ADV	HBV DNA polymerase inhibitor	Less potent, nephrotoxicity
Nucleoside analogue	3TC	HBV DNA polymerase inhibitor	Less potent, high rate of resistance
Nucleoside analogue	LdT	HBV DNA polymerase inhibitor	High rate of resistance

HBV: Hepatitis B virus; ETV: Entecavir; TAF: Tenofovir alafenamide; ADV: Adefovir dipivoxil; 3TC: Lamivudine; LdT: Telbivudine; TDF: Tenofovir disoproxil fumarate; IFN-2α: Interferon alpha-2α; PEG-IFN-2α: Pegylated IFN-2α.

A hallmark of chronic HBV infection is the presence of an enormous number of subviral particles composed of hepatitis B surface antigen (HBsAg) in the plasma that outnumber complete virions by a ratio of 10000-100000: 1[5]. This excess of subviral particles interfere with the function of innate and adaptive immune cells, impeding the clearance of circulating viruses and infected hepatocytes. Thus, hepatitis B treatment goals are categorized into functional and complete cure, whereby functional cure is defined as HBsAg clearance or levels below 0.05 IU/mL, undetectable HBV DNA or levels below 10 IU/mL, with or without HBsAg seroconversion[6], the complete cure is considered as the total eradication of HBV DNA, including cccDNA and integrated HBV DNA, from the liver and serum[7]. However, the development of therapies targeting cccDNA remains a significant unmet need, making complete cure currently unattainable.

Nucleos(t)ide analog therapy is effective in suppressing HBV replication but demonstrates limited efficacy in reducing serum HBsAg, with an annual HBsAg clearance rate of approximately 1%[8]. Combination therapy using PEG-IFN-2α and TDF achieves higher rates of HBsAg seroclearance, with approximately 10% of patients achieving this outcome. However, the use of interferon (IFN) is frequently associated with severe adverse effects[9]. It is worth noting that discontinuation of nucleos(t)ide analogs has recently been suggested as a novel treatment strategy to enhance the rate of HBsAg loss by allowing immune reconstitution. Although the STOP-NUC and FINITE trials that enrolled HBsAg-negative patients found that stopping nucleos(t)ide analogs substantially increased the rate of HBsAg loss (10.1% vs 0%; 19% vs 0%, respectively), data from other trials do not suggest such favorable outcomes. A significant proportion of patients suffer clinical relapse and hepatic flares after stopping treatment[10-14]. This discrepancy suggests that multiple factors such as patient ethnicity and baseline HBsAg levels may impact clinical outcomes and strongly argues that treatment discontinuation should be carefully considered based on patient qualifications and that the patients should be subsequently monitored for potential relapses and hepatic flares[15].

Taken together, the limited cure rate with currently available treatment options underscores the urgent needs for the development of new, safe, and effective drugs for the treatment of chronic hepatitis B.

NEW THERAPEUTIC TARGETS FOR HEPATITIS B

New therapeutic strategies aim to achieve long-lasting viral suppression with shorter treatment durations while also restoring the body’s antiviral immune response[16,17], and are divided into two main categories: Direct-acting antiviral agents and immunomodulators (Figure 1)[18].

Figure 1.

Overview of chronic hepatitis B drug therapy targets[18]. This diagram illustrates the life cycle of hepatitis B virus (HBV), including viral entry, uncoating, formation of relaxed circular DNA, formation of covalently closed circular DNA, integration of genomic DNA, transcription, and translation. It also describes various therapeutic strategies targeting different stages of the HBV life cycle, including entry inhibitors, immunomodulators, and inhibitors that directly target viral DNA and RNA. This figure was created by BioRender.com (Supplementary material). cccDNA: Covalently closed circular DNA; rcDNA: Relaxed circular DNA; ASO: Antisense oligonucleotide; HBsAg: Hepatitis B surface antigen; CAM: Capsid modulators; mRNA: Messenger RNA; pgRNA: Pre-genomic RNA.

Direct-acting HBV antivirals

HBV replication involves several key steps, such as viral entry, transcription, translation, DNA synthesis, assembly, and virion secretion. Direct-acting antiviral drugs target these processes to block viral entry, disrupt RNA transcription, prevent nucleocapsid assembly, inhibit DNA synthesis, or stop the secretion of HBsAg.

HBV entry inhibitors: HBV enters hepatocytes by interacting with the Pre-S1 glycoprotein on the viral envelope and the sodium taurocholate cotransporting polypeptide (NTCP) receptor[19]. Bulevirtide, formerly called Myrcludex B, is a linear lipopeptide derived from HBV PreS1 region that potently blocks this interaction by competing with the virus for NTCP binding, thereby preventing viral entry[20,21]. This drug was approved by the European Union in 2020 for treating chronic HBV/hepatitis delta virus (HDV) co-infection, and clinical studies have shown that bulevirtide monotherapy or combination with PEG-IFN-α for 48 weeks significantly reduces HDV RNA levels and HDV infected hepatocytes, and even leads to HBsAg dropping by more than 1 Log IU/mL[22-24]. Bulevirtide is also being investigated in clinical trials for chronic hepatitis B. The success of bulevirtide in treating HDV infections largely relies on HDV being an RNA virus with a much higher rate of spread and re-infection than HBV, making it more vulnerable to blocking entry. However, it is still unclear whether preventing new HBV infections by bulevirtide in patients with previously established chronic infections can significantly improve treatment outcomes. Nevertheless, considering the recent discovery in HBV-infected mice with humanized liver that a new HBV infection is necessary to reactivate previously silenced cccDNA, blocking new infections by bulevirtide shows potential especially in combination therapies[25].

HBV monoclonal antibodies: HBV-neutralizing monoclonal antibodies target the virus’s envelope proteins to block viral entry into hepatocytes, reduce circulating HBsAg levels and activate immune cells through Fc-mediated mechanisms[26]. Several monoclonal antibodies are currently under investigation in Phase II clinical trials, including lenvervimab (GC1102), HH-003, and tobevimab (VIR3434) (Table 2).

Table 2.

Current investigational drugs for the treatment of chronic hepatitis B

Types of drugs	Mechanism of action	Representative drugs	Global clinical R&D phase	Efficacy
HBV entry inhibitors	Acts on NTCP to inhibit virus entry into cells	Bulevirtide	Clinical phase II	Profound HDV RNA reduction but minimal HBsAg reduction
HBV monoclonal antibodies	Binding to HBV virus indicates that HBsAg binds and neutralizes the virus	Lenvervimab	Clinical phase II	HBsAg decreased after each administration but rebounded rapidly; 6 out of 27 patients had HBsAg loss when combined with siRNA and IFN-α
		Tobevibart (VIR-3434)	Clinical phase II
		HH-003	Clinical phase II	N/A on HBsAg loss rate; Efficacious against HDV
		BJT-778	Clinical phase II	N/A for HBsAg loss rate; Efficacious against HDV
RNA interference (siRNA)	Small interfering RNA targeted degradation of HBV mRNA	Daplusiran + tomligisiran (JNJ-3989)	Clinical phase II	Mean HBsAg reduction of 1.89 Log10 IU/mL after a 48-week therapy
		Elebsiran (VIR-2218)	Clinical phase II	11 out of 69 patients had HBsAg loss after different regimens of VIR-2218 plus IFN-α
		Imdusiran (AB-729)	Clinical phase II	4 out of 12 patients had HBsAg loss after 48 weeks Imdusiran plus 24 weeks IFN-α and ongoing nucleos(t)ide analogs
		RBD1016	Clinical phase I	HBsAg decline of 1.26 Log10 IU/mL by week 12 after two 3 mg/kg dosing
		Xalnesiran (RG6346)	Clinical phase II	30% HBsAg loss by Xalnesiran 200 mg and IFN-α combination at the end of 48-week treatment
RNA interference (ASO)	Targeted degradation of HBV mRNA by antisense oligonucleotides	Bepirovirsen	Clinical phase III	9%-10% patients had HBsAg loss after 300 mg weekly for 24 weeks
		AHB-137	Clinical phase II	62% patients had HBsAg loss after 12-week 300 mg dosing (interim results)
Inhibitor of capsid assembly	By affecting nucleocapsid formation, it reduces virus formation and cccDNA internal recruitment	JNJ-6379	Clinical phase I	HBV DNA reduction after a 28-day therapy, no HBsAg loss
		Morphothiadine (GLS4)	Clinical phase III	HBV DNA and RNA suppression. HBsAg decline but no HBsAg loss
		Canocapavir	Clinical phase II	HBV DNA and pregenomic RNA reduction after a 28-day therapy
		EDP-514	Clinical phase I	HBV DNA and RNA reduction in treatment-naïve and viral suppressed patients after a 28-day therapy
		ALG-000184	Clinical phase I	HBV DNA and RNA reduction, and modest HBsAg reduction after a 28-day monotherapy or ETV combination
HBsAg secretion inhibitors	Nucleic acid polymers interfere with the release of subviral particles by interacting with chaperone proteins, such as DNAJB12, and increase the intracellular degradation of subviral particles	REP 2139	Clinical phase II	14 of 40 patients had HBsAg loss after 48 weeks of combination therapy with TDF and peg-IFN
		REP 2165	Clinical phase II	14 of 40 patients had HBsAg loss after 48 weeks of combination therapy with TDF and peg-IFN
Direct-acting cccDNA drugs	Gene editing technology was used to knock out cccDNA	PBGENE-HBV	Clinical phase I	Reduction in HBsAg in 2 out of 3 patients after the first administration at 0.2 mg/kg dose
TLR-7 agonist	TLR-7 is activated to activate innate immune cells	Vesatolimod (GS-9620)	Clinical phase II	No patient has HBsAg loss after 12-week therapy with TDF combination
		TQ-A3334	Clinical phase II	N/A
TLR-8 agonist	TLR-8 is activated to activate innate immune cells	Selgantolimod	Clinical phase II	2 out of 39 achieved HBsAg loss after 24-week therapy
		HRS9950	Clinical phase II	N/A
		TQA3810	Clinical phase II	N/A
Immune checkpoint inhibitors	Anti-PD-1 antibody, by inhibiting PD-1, activates immune cells	Nivolumab	Clinical phase II	1 out of 12 patients had HBsAg loss after 12-week therapy
		Serplulimab	Clinical phase II	N/A
		Sintilimab	Clinical phase I	N/A
	Anti-PD-L1 antibody, by inhibiting PD-L1, activates immune cells	Envafolimab	Clinical phase II	3 out of 33 patients had HBsAg loss after 24-wekk therapy
Therapeutic vaccines	Enhanced T- and B-cell immunity	VBI-2601 (BRII-179)	Clinical phase II	A 9 doses regimen with HBV siRNA induced anti-HBs antibody (16/40) but no HBsAg loss
		VVX001	Clinical phase II	N/A
		CVI-HBV-002	Clinical phase II	2 out 18 patients had HBsAg loss after ChAdOx1-HBV on Day 0 and MVA-HBV on Day 28 (VTP-300)

HBV: Hepatitis B virus; HBsAg: Hepatitis B surface antigen; siRNA: Small interfering RNA; mRNA: Messenger RNA; ASO: Antisense oligonucleotide; TDF: Tenofovir disoproxil fumarate; IFN-2α: Interferon alpha-2α; PEG-IFN-2α: Pegylated IFN-2α; cccDNA: Covalently closed circular DNA; HDV: Hepatitis delta virus.

Lenvervimab has demonstrated modest efficacy in clearing HBsAg. In clinical trials, a 12.5% HBsAg clearance rate was observed in the 80000 IU dose group, while the 240000 IU dose group achieved a 22.2% clearance rate after one month of treatment. However, HBsAg levels rebounded rapidly following the cessation of therapy[27]. HH-003 is a fully human monoclonal antibody that specifically targets the pre-S1 region of the HBV envelope protein. By inhibiting the binding of the pre-S1 region to the NTCP receptor, HH-003 effectively prevents HBV entry into hepatocytes. A single-center, open-label Phase IIb clinical trial showed that treatment with HH-003 for 24 weeks in patients with chronic hepatitis B and co-existing chronic hepatitis D resulted in a 2.18 Log10 IU/mL reduction in HDV RNA levels[28]. VIR3434, another fully human monoclonal antibody, targets the antigenic loop shared by large, medium, and small HBsAg proteins. This antibody has been engineered to enhance its half-life through LS mutation in Fc region to increase its affinity for Fc-γ receptors (Fc-γRIIa and Fc-γRIIIA). These modifications improve its ability to activate immune cells. VIR3434 mediates its therapeutic effects via three mechanisms: (1) Neutralizing HBV to prevent its entry into hepatocytes; (2) Enhancing antigen presentation and stimulating T-cell responses, producing a vaccine-like effect; and (3) Neutralizing and facilitating HBsAg and HBV clearance through antibody-dependent cellular phagocytosis[29]. Clinical trials on VIR3434 have reported significant reductions in plasma HBsAg levels, with most patients experiencing decreases greater than 1 Log10 IU/mL within 1-3 days of administration[30]. Furthermore, the combination of VIR3434 with the small interfering RNA (siRNA) drug VIR-2218 which targets all HBV transcripts achieved an average reduction in HBsAg levels exceeding 2.5 Log10 IU/mL, with most patients reaching HBsAg levels below 10 IU/mL[31].

Overall, HBV monoclonal antibodies are predicted to block viral entry, clear HBsAg, and enhance immune control. However, similar to bulevirtide, monotherapy with HBV monoclonal antibodies is unlikely to cure HBV infections and is further limited by potential viral rebound after treatment discontinuation. Therefore, proper drug combinations with additional directacting antivirals or immunomodulators are necessary to improve treatment outcomes.

RNA interference: RNA interference agents, including siRNA and antisense oligonucleotides (ASO), are designed to reduce viral antigen burden by targeting viral transcripts as well as block HBV replication by limiting replication templates and factors. siRNA is a double-stranded RNA molecule that recognizes HBV transcripts to trigger RNA-induced silencing complex and Argonaute 2 mediated viral RNA degradation and translation repression. Distinct from siRNA, ASO is single-stranded DNA oligo that binds complementary HBV RNA to promote viral RNA degradation by recruiting RNA-degrading enzyme RNaseH. Overall, facilitated by liver targeted delivery and chemical modification to extend the half-life, RNA interference agents have demonstrated exceptional efficiency and durability in lowering HBsAg.

Currently, several siRNA drugs, including JNJ-3989, AB-729, RG-6346, and VIR-2218, are being investigated in Phase II clinical trials. JNJ-3989 is a combination of two siRNA molecules (JNJ-3976 and JNJ-3924 in a 2: 1 ratio) designed to target the viral RNAs encoding HBV surface antigens as well as all viral transcripts including the HBx mRNAs, with N-acetylgalactosamine (GalNAc) conjugation for liver-targeting delivery. The Phase IIb REEF 1 trial (CNT-03982186) evaluated its efficacy in patients randomized into three groups: (1) Nucleoside analog (NA) monotherapy; (2) NA with JNJ-3989 at doses of 40, 100, or 200 mg every four weeks; and (3) NA with JNJ-3989 (100 mg every four weeks) combined with JNJ-6379, a core-capsid assembly regulator. The primary clinical endpoint was defined as alanine aminotransferase levels < 3 times the upper limit of normal, HBV DNA below the lower limit of quantification, and HBsAg < 10 IU/mL after 48 weeks of treatment. The results demonstrated a dose-dependent reduction in HBsAg levels with JNJ-3989, with mean reductions of 1.5 Log10 IU/mL (40 mg), 2.1 Log10 IU/mL (100 mg), and 2.6 Log10 IU/mL (200 mg). At 48 weeks, 19% of patients receiving the 200 mg dose achieved the primary clinical endpoint. The reduction in HBsAg levels in the group treated with the combination of JNJ-3989 and JNJ-6379 was 1.8 Log10 IU/mL, which was lower than the reduction achieved with JNJ-3989 monotherapy, indicating that JNJ-6379 may not contribute significantly to the anti-HBsAg therapeutic effect[32].

In the REEF 2 trial (NCT04129554), 130 HBeAg-negative patients with chronic hepatitis B were randomized to receive either NA plus JNJ-3989 (200 mg every 4 weeks) with JNJ-6379 (250 mg daily) or NA with placebo for 48 weeks, followed by treatment discontinuation. The primary endpoint was serum clearance of HBsAg at week 72 (24 weeks after treatment cessation). At week 48, the combination therapy group achieved a mean HBsAg reduction of 1.89 Log10 IU/mL. Although no patients met the primary endpoint after drug withdrawal, 67.1% of the combination therapy group maintained HBsAg levels below 100 IU/mL, and 57.9% showed sustained reductions at the 24-week follow-up[33].

VIR-2218 is also a GalNAc-conjugated siRNA therapeutic targeting conserved regions within the HBx coding sequence. In patients with chronic hepatitis B, the treatment regimen involved administering 200 mg every four weeks for six doses, followed by a 40-week observation period after treatment cessation. At the end of the six-dose treatment, the mean reduction in HBsAg levels was 1.96 Log10 IU/mL. After the 40-week post-treatment observation period, the mean HBsAg reduction remained at 1.61 Log10 IU/mL[34]. When VIR-2218 was combined with IFN-α, the HBsAg seroconversion rate reached 15.9% (11 out of 69 patients), which was significantly higher than that with IFN-α or VIR-2218 monotherapy[35]. This promising result indicates that HBsAg reduction by siRNA could potentially create a conducive environment in the liver to improve treatment outcomes by concurrent or subsequent immune modulators including IFN-α.

In addition to siRNA therapeutics, several ASO drugs, including bepirovirsen and AHB-137, are under clinical investigation. Bepirovirsen is currently in the Phase III development stage. Results from its Phase IIb trial showed that patients receiving 300 mg weekly for 24 weeks, followed by a 24-week drug-free observation period, achieved HBsAg seroclearance rates of 9%-10%[36]. It should be noted that the immune-modulation function of bepirovirsen may be partially responsible for its efficacy. Interestingly, a recent study comparing 24-week bepirovirsen vs 12-week bepirovirsen treatment followed by 24-week PEG-IFN-α administration in virus-suppressed patients found that longer duration of antigen reduction before the sequential addition of PEG-IFN-α could reduce HBsAg relapse rates[37]. This finding further implies that lower antigen load by RNA interference is associated with better treatment outcomes upon the subsequent addition of immunomodulators.

Capsid assembly modulators: Capsid assembly is an essential step in the HBV life cycle, involving core proteins as building blocks to form a capsid which houses the reverse transcription of pgRNA by HBV polymerase, with direct connections with viral particle assembly and amplification of cccDNA. Core protein allosteric modulators (CpAMs) disrupt this process by inducing the formation of unstable capsids (class I) or empty capsids, which then reduce viral particle production and impair the nuclear trafficking of rcDNA-containing capsids, limiting the accumulation of cccDNA within hepatocytes[38,39]. Monotherapy with CpAMs has shown reductions in serum HBV DNA and RNA levels; however, HBsAg levels remained largely unaffected after 12 weeks of treatment. In a Phase Ib clinical trial, JNJ-6379 significantly reduced HBV DNA levels after 28 days of continuous treatment, although no changes in HBsAg levels were observed[40]. Similarly, investigational class II CpAMs, such as EDP-514 and ALG-000184, are currently in Phase I trials but have not demonstrated significant effects on HBsAg levels. It is not completely surprising that CpAMs are limited in their ability in reducing HBsAg, given their primary mechanism of action is blocking HBV DNA replication. However, cell culture studies have revealed that certain CpAMs can disrupt nucleocapsid disassembly in normal HBV during entry, thereby reducing cccDNA formation. It remains to be elucidated whether more potent CpAMs can provide such benefits in a clinical setting. Of note, being another HBV DNA replication inhibitor, orally available potent CpAMs with high resistant barrier may be potential alternatives as nucleos(t)ide analogs if early treatment is to be initiated for chronic HBV infections[41,42].

HBsAg secretion inhibitors: Nucleic acid polymers (NAPs) are single-stranded thiophosphate oligonucleotides that inhibit the release of subviral particles to enhance the intracellular degradation of subviral particles without affecting HBV RNA levels, Dane particles, or the production of HBc/HBeAg[43,44]. While its precise mechanism of action is still debatable, it seems to involve host chaperone proteins such as DNAJB12[45]. REP 2139 and REP 2165 are notable NAPs under investigation. A Phase II clinical trial demonstrated that combining REP 2139/REP 2165 with TDF and pegylated PEG-IFN effectively reduced serum HBsAg levels over 24 weeks, with 60% of patients achieving HBsAg levels below 0.05 IU/mL. At 48 weeks post-treatment cessation, 35% of patients (14 out of 40) achieved complete serum HBsAg clearance[46]. While the clinical data appear promising, larger-sized clinical trials are warranted to gain deeper insights into its efficacy, safety, and mechanism of action.

Direct-acting cccDNA drugs: Drugs that can eradicate cccDNA need to be developed to completely cure chronic hepatitis B. However, cccDNA removal from the already infected hepatocytes poses a significant challenge due to its chromatinized nature, making targeting structurally difficult when using the currently available small-molecule drugs. To date, no drug targeting cccDNA has entered clinical trials. Experimentally, a high-throughput screening study of 846000 compounds in HBV-infected primary human hepatocytes has led to the identification of ccc_R08, which reduces cccDNA in a dose-dependent manner[47,48]. Additional studies on this compound and a deeper understanding of HBV cccDNA biology are necessary to develop more potent compounds and design novel approaches to specifically reduce cccDNA in HBV-infected livers. In addition to the use of small-molecule inhibitors, in vitro and in vivo studies have shown that the CRISPR/Cas system can significantly reduce cccDNA levels in the liver based on the unique sequence of HBV, which is distinct from the chromosomal DNA of the host[49-51]. Despite the potential of the CRISPR/Cas system as a cccDNA-targeting tool, several challenges persist, including issues related to delivery efficiency and the risk of off-target effects[52]. An alternative strategy for eradicating cccDNA is by the transcriptional silencing of cccDNA. Accordingly, several in vitro studies, including the CRISPR baseediting approach and HBV X protein-disruption approach, have shown promising results[53-55]. While these results are preliminary, specifically silencing cccDNA by novel virological and immunological strategies represents an exciting research direction with immense translational potential.

Immunomodulators

Chronic HBV infection suppresses both innate and adaptive immunity, indicating that immune-related targets could play a pivotal role in combination therapies. Persistent HBV infection has been associated with the exhaustion and depletion of HBV-specific T cells, potentially driven by continuous exposure to high level of HBsAg[56]. Additionally, inadequate activation of the innate immune system and the presence of an immunotolerant liver microenvironment hinder the clearance of HBV[57]. Therefore, strategies to enhance the body’s immune response to HBV are essential for virus elimination and achieving a cure for chronic hepatitis B. We herein discuss several classes of HBV immune modulation approaches that are under clinical investigation.

TLR-7/TLR-8 agonists: TLRs are essential components of the innate immune system as they can recognize pathogen-associated molecular patterns. TLR-7 is primarily expressed in plasmacytoid dendritic cells, B cells, and macrophages. Upon ligand binding, TLR-7 induces the secretion of type I IFNs, tissue necrosis factor-α (TNF-α), and chemokines such as CXCL10, and enhances antigen presentation, T-cell activation, and plasma cell differentiation[58,59]. Conversely, TLR-8 is predominantly expressed in macrophages and myeloid dendritic cells, contributing to the secretion of pro-inflammatory cytokines[60]. Several TLR-7 and TLR-8 agonists are being investigated in clinical trials, however, current reports suggest that their efficacy in reducing HBsAg levels is limited. For instance, Vesatolimod, a TLR-7 agonist, showed that it can induce ISG15 expression but did not significantly increase plasma IFN-α levels or reduce HBsAg levels in Phase II trials, failing to meet its primary endpoint[61,62]. Another TLR-7 agonist, TQ-A3334, is undergoing Phase II clinical trials, with Phase I data confirming its potential safety and tolerability in healthy adults[63].

Selgantolimod (GS-9688), a TLR-8 agonist, stimulates the secretion of cytokines such as p40, IL-6, IL-12, TNF-α and IFN-γ to enhance NK cell function and suppress myeloid-derived suppressor cells[64]. In a Phase II trial, patients with chronic hepatitis B on nucleos(t)ide analogs received weekly doses of 3 mg, 1.5 mg, or placebo for 24 weeks and were followed for 24 weeks after Selgantolimod treatment. Results revealed that 26% (10/39) of patients experienced a reduction in HBsAg levels (> 0.1 Log10 IU/mL) after 24 weeks of Selgantolimod administration, and 5% (2/39) achieved HBsAg loss[65]. These data suggest that TLR8 agonist exhibit modest but durable clinical benefits for chronic HBV infection and may constitute a component of future combination therapies.

Immune checkpoint inhibitors: The increasing clinical use of immune checkpoint inhibitors in treating malignant tumors has prompted research into their potential role in HBV infection. The PD-1/PD-L1 signaling pathway is known to regulate T-cell proliferation, activation, and cytokine release[66]. Chronic hepatitis B is associated with elevated expression levels of PD-1 on CD8+ T cells. In vitro studies have shown that blocking the PD-1 pathway enhances IFN-γ secretion by peripheral blood mononuclear cells and promotes cell proliferation[67]. Several PD-1/PD-L1 inhibitors are currently in Phase II clinical trials for chronic hepatitis B, including nivolumab, serplulimab, and envafolimab. In a preliminary trial, a single dose of 0.3 mg/kg nivolumab (a PD-1 antibody) administered over 12 weeks resulted in a mean HBsAg reduction of 0.3 Log10 IU/mL, with serological clearance of HBsAg achieved in only one patient (1 out of 12) accompanied with a minor alanine aminotransferase flare and increased peripheral HBsAg-specific T cells[68]. Envafolimab, an anti-PD-L1 antibody, was administered to 33 patients at a dose of 1 mg/kg every two weeks for 24 weeks to evaluate changes in serum HBsAg levels. Post-treatment results showed an HBsAg reduction of > 0.5 Log10 IU/mL in seven patients, with three patients achieving serological clearance of HBsAg[69,70]. While immune checkpoint inhibitors demonstrate some efficacy in chronic hepatitis B, their impact on HBsAg decline remains limited. A recent discovery in a mouse model of HBV suggested that the PD-1/PD-L1 axis may not be the primary inhibitory signaling pathway accountable for dysfunctional HBV-specific T cells. Instead, activation of the co-stimulatory factor, 4-1BB, which is expressed on the surface of T cells, rejuvenates dysfunctional HBV-specific CD8+ T cells[71]. Therefore, combination therapies incorporating broader immune-signaling modulators and other treatment modalities may improve treatment outcomes, thereby warranting further investigation.

Therapeutic vaccines: Despite having a highly effective preventive vaccine for HBV, there is currently no approved therapeutic vaccine for HBV. The primary goal of therapeutic vaccine development is to activate HBV-specific B and T cells to restore adaptive immunity. In this regard, several therapeutic vaccines, including TG1050, VTP-300, GS-4774 and BRII-179, are undergoing Phase II clinical trials.

TG1050 is an adenovirus (Ad5)-based vaccine that encodes multiple HBV antigens, including polymerase, HBcAg, and HBsAg domains. Preclinical studies in murine models have shown that TG1050 induces both immunogenic and antiviral effects, promoting the activation and proliferation of HBV-specific T cells[72]. However, its clinical impact on HBsAg levels in patients with chronic hepatitis B has been minimal, with most patients experiencing reductions of less than 0.2 Log10 IU/mL[73]. VTP-300 is a therapeutic vaccine derived from a chimpanzee adenovirus vector (ChAdOx1 HBV) combined with a Modified Vaccinia Ankara Enhanced (MVA-HBV) vaccine. These encode polymerase, core, and S antigens from a genotype C HBV sequence. In a Phase Ib/IIa clinical trial, patients received an intramuscular injection of ChAdOx1 HBV on day 0 and MVA-HBV on day 28. The results showed HBsAg reductions of 0.7, 0.7, and 1.4 Log10 IU/mL in 3 of the 18 participants at two months post-treatment, with 2 patients achieving durable undetectable HBsAg level. However, these patients had baseline HBsAg levels below 50 IU/mL before the therapy[74]. It remains to be further elucidated whether the combination with HBsAg-reducing agents, such as siRNA, to lower baseline HBsAg levels could further increase the functional cure rate.

BRII-179 (VBI-2601) is a protein-based recombinant HBV immunotherapy expressing Pre-S1, Pre-S2, and S HBV surface antigens. In a recent phase 2a study, 40% (16/40) of patients with chronic hepatitis B treated with nine 4-weekly doses of HBV-targeted siRNA elebsiran (BRII835) in combination with the therapeutic vaccine BRII-179 developed robust anti-HBs antibodies, whereas none of the patients in the siRNA-alone arm showed the presence of antiHBs antibodies despite a 1.2-2.6-fold log reduction in serum HBsAg levels[75]. Overall, this vaccine enhanced HBV-specific B cells and PreS1/S2-specific CD4+ T cells but not Sspecific CD8+ T cells. Another striking finding in this trial was that although neutralizing anti-HBs antibodies induced by BRII-179 contributed to HBsAg reduction, it did not lead to HBsAg seroconversion (no HBsAg loss). Concurrent treatment with IFN-α also failed to provide additional clinical benefits. This study once again highlights the complex interplay between chronic HBV infections and host immunity and implies that efficient reactivation of both humoral and cellular adaptive immunities against HBV may be needed for a durable functional cure.

CONCLUSION

The use of approved nucleos(t)ide analogues and IFN-α in the treatment of chronic hepatitis B has been associated with low functional cure rates and a limited range of treatment options. These limitations are primarily attributed to the persistent nature of HBV cccDNA and ineffective immune control over HBV-infected cells. To overcome these challenges, novel therapeutic approaches with diverse mechanisms of action are currently under development and urgently needed, targeting a functional cure for patients with chronic hepatitis B. Results from multiple clinical trials indicate that monotherapy may be insufficient to achieve the serological clearance of HBsAg. Thus, future treatment directions should focus on multidrug combinations including siRNA or ASO drugs with immunomodulators on the backbone of nucleos(t)ide analogs or other potent orally available antivirals such as capsid modulators[16]. An important factor that can likely impact the response rate is the selection and time sequence of combination therapies. A stepwise treatment scheme is proposed, which suggests commencing with viral DNA suppression, sustaining the downregulation of viral antigens, and then boosting antiviral immune responses of the host to shift the balance from chronic infection to immune control of HBV[76]. Another key factor is the starting HBsAg level, as multiple clinical studies have convincingly shown better rates of HBsAg loss in patients with lower baseline serum HBsAg levels. Therefore, sequential treatment with agents to first durably reduce the HBsAg load before initiating immunomodulators may enhance the functional cure rate. Finally, to establish a more effective and rational treatment strategy for chronic hepatitis B and to meet the goal of functional cure, larger cohort studies are essential to clarify both the efficacy and safety of these emerging therapeutic options.