Hepatitis B functional cure: Current and future perspective

Abstract

Chronic hepatitis B (CHB) remains a significant global health challenge, affecting more than 250 million individuals worldwide. A functional cure, defined as the loss of hepatitis B surface antigen (HBsAg) and suppression of hepatitis B virus (HBV) DNA to undetectable levels, represents the optimal therapeutic endpoint for managing CHB. However, the complex pathogenesis of CHB, which includes HBV DNA integration, persistence of covalently closed circular DNA, and impaired immune responses, presents substantial barriers to HBsAg clearance. Current therapies offer limited success in achieving a functional cure, with HBsAg seroclearance occurring in only 3%-5% of patients after 10 years of nucleos(t)ide analogs (NAs) therapy and 8%-14% within 3-5 years of pegylated interferon treatment. To overcome these limitations, novel direct-acting antivirals targeting different stages of the HBV life cycle are being investigated. Additionally, immunomodulatory approaches, including therapeutic vaccines and immune checkpoint inhibitors, are being explored to enhance HBV-specific immune responses. The concept of NAs cessation in carefully selected non-cirrhotic patients may accelerate HBsAg loss, although the risks of hepatic flare and hepatocellular carcinoma necessitate rigorous monitoring. This review provides a comprehensive overview of the current understanding of HBsAg seroclearance in CHB, discussing its clinical significance, therapeutic challenges, and evolving treatment landscape in the pursuit of a functional cure.

Key Words: Hepatitis B; Hepatitis B surface antigen seroclearance; Functional cure; Surface antigen; Virological cure

Core Tip: Achieving a functional cure for chronic hepatitis B, defined as sustained hepatitis B surface antigen loss with undetectable hepatitis B virus (HBV) DNA, remains challenging due to the persistence of covalently closed circular DNA (cccDNA) and immune dysfunction. While standard therapies, such as nucleos(t)ide analogues and pegylated interferon, yield low seroclearance rates, novel therapeutics targeting various steps in the life cycle of HBV-such as cccDNA silencing, RNA degradation, capsid modulation, and immune restoration- show promise. Combinatorial approaches, including small interfering RNA, checkpoint inhibitors, and therapeutic vaccines, are being evaluated. Identifying predictive biomarkers and optimizing the timing for treatment cessation may improve treatment outcomes.

INTRODUCTION

Chronic hepatitis B (CHB) poses a major global health burden, leading to cirrhosis and hepatocellular carcinoma (HCC)[1]. According to the World Health Organization’s hepatitis report for 2024, the global seroprevalence of hepatitis B surface antigen (HBsAg) was 3.8%, with approximately 254 million individuals living with CHB in 2022. Hepatitis B caused an estimated 1.1 million deaths in 2022, primarily from cirrhosis and HCC, which is the second deadliest cancer worldwide[1,2]. The nature of chronic hepatitis B virus (HBV) infection is dynamic, shaped by interactions among the virus, hepatocytes, and host immunity[1,3,4]. While a complete virological cure remains elusive, the loss of HBsAg is considered the optimal therapeutic endpoint. This outcome, considered a functional cure, is defined as sustained undetectable viremia with durable HBsAg loss of at least 24 weeks after the end of therapy, with or without seroconversion to anti-HBsAg antibody[3]. However, a true cure is defined as the elimination of both HBsAg and covalently closed circular DNA (cccDNA)[4]. In recent years, however, achieving a functional cure has become a realistic and meaningful goal in HBV treatment, offering several advantages, including improved survival, reduced HCC risk, and decreased liver-related mortality, comparable to outcomes seen with spontaneous viral clearance[5] (Figure 1). The current standard of care, including nucleos(t)ide analogues (NAs) and immunomodulators like pegylated interferon (PEG-IFN), can induce functional cure, although success rates remain low. However, advances in HBV research and the success of curative therapies for hepatitis C have spurred the development of promising new treatments for CHB, many of which are now in clinical trials. This review aims to provide an overview of current evidence on the functional cure of hepatitis B, limitations of current therapies and an outline of future treatment directions.

Figure 1 Clinical benefits of a functional cure in chronic hepatitis B. A functional cure leads to multiple favorable outcomes, including reduced risk of cirrhosis, hepatocellular carcinoma, and hepatic decompensation. It also enables treatment discontinuation, durable virological response, improved liver histology, reduced transmission risk, and enhanced quality of life. HCC: Hepatocellular carcinoma; HBV: Hepatitis B virus.

MECHANISMS OF HBV PERSISTENCE

The persistence of HBV infection is primarily driven by the stability of cccDNA in hepatocytes and its ability to evade immune clearance[6]. After HBV enters hepatocytes via the sodium taurocholate co-transporting polypeptide (NTCP) receptor, its relaxed circular DNA is transported to the nucleus and repaired by host enzymes to form transcriptionally active cccDNA, which associates with host histones to form a viral minichromosome that serves as a template for viral RNAs, including pregenomic RNA[7,8]. While liver-specific transcription factors and chromatin activating enzymes boost cccDNA-driven gene expression, host restriction complexes, noncoding RNAs, and protein modifiers such as protein arginine methyltransferase-1 inhibit it. This balance governs the long term persistence of HBV[9]. HBV further promotes the persistence of cccDNA by altering cell cycle control via HBV X (HBx)-protein, preventing dilution during mitosis[10,11]. HBV-induced immune dysfunction plays a central role in viral persistence. Kupffer cells and dendritic cells exhibit impaired antigen presentation, diminished interferon (IFN) production, and defective Toll like receptor (TLR) 7/9 signaling[12]. Natural killer (NK) cells show reduced cytotoxic activity and IFN gamma (IFN-γ) secretion, and contribute to the depletion of virus-specific CD8⁺ T cells[12,13]. HBV proteins, such as HBsAg and hepatitis B envelope antigen (HBeAg), inhibit innate immune signaling by blocking pattern recognition receptor (PRR) pathways. The adaptive immune response is also compromised: Both CD8⁺ and CD4⁺ T cells exhibit features of functional exhaustion, characterized by the high expression of inhibitory receptors, mitochondrial dysfunction, and reduced cytokine production[14]. B cells exhibit the expansion of programmed cell death protein-1 (PD-1) atypical memory subsets with impaired antibody responses[15]. Persistent high levels of viral antigens, particularly HBsAg and hepatitis B core antigen (HBcAg), contribute to immune exhaustion and help sustain chronic HBV infection. Notably, there appears to be a bidirectional relationship-while chronic infection drives antigen overproduction, the resulting antigen excess further impairs HBV-specific immune responses, reinforcing viral persistence in a self-perpetuating cycle[13,14]. Regulatory T cells (Tregs) and monocytic myeloid-derived suppressor cells further suppress antiviral immunity through interleukin-10, transforming growth factor-β, and metabolic mediators like arginase-1 and reactive oxygen species[16,17]. In summary, HBV persistence is maintained by stable cccDNA, immune evasion, sustained antigen exposure, and viral interference with host immunity, leading to impaired antiviral responses (Table 1).

Table 1 Barriers to achieving functional cure in chronic hepatitis B.

Category	Barrier	Description /mechanism
Viral factors	cccDNA persistence	cccDNA forms a stable nuclear minichromosome, serving as a reservoir for HBV transcription
	High antigen load	Continuous production of HBsAg and HBcAg from cccDNA promotes immune tolerance and T cell exhaustion
	HBV-mediated immune suppression	HBsAg and HBeAg interfere with PRR signaling, hindering innate immune responses via IRF3 and NF-κB inhibition
	HBx-induced cell cycle modulation	HBx protein disrupts hepatocyte proliferation and prevents cccDNA dilution during cell division
Host factors	Epigenetic control of cccDNA	Host enzymes (e.g., HDAC1, Smc5/6) modify cccDNA chromatin, affecting its transcriptional activity
	Effective innate immunity	Kupffer cells, dendritic cells, and monocytes show impaired PRR signaling and reduced IFN production
	NK cell dysfunction	NK cells display reduced cytotoxicity, lower IFN-γ secretion, and contribute to CD8+ T cell depletion
	T cell exhaustion	HBV-specific CD4+ and CD8+ T cells overexpress inhibitory receptors (PD-1, CTLA-4), resulting in functional impairment
	B cell dysfunction	Atypical memory B cells (e.g., PD-1+, FcRL5+) fail to generate effective anti-HBs antibodies
	Expansion of immunosuppressive cells	Regulatory T cells and myeloid-derived suppressor cells suppress antiviral immune responses
Therapeutic gaps	No cccDNA-targeting therapies	Current antivirals suppress replication but do not eliminate or silence cccDNA effectively
	Limited immunotherapeutic tools	PEG-IFN and NAs yield low HBsAg clearance; novel immunotherapies are investigational

HBV: Hepatitis B virus; HBsAg: Hepatitis B surface antigen; HBcAg: Hepatitis B core antigen; HBeAg: Hepatitis B e antigen; cccDNA: Covalently closed circular DNA; HBx: Hepatitis B Virus X protein; PRR: Pattern recognition receptor; IRF3: Interferon regulatory factor 3; HDAC1: Histone deacetylase 1; IFN-γ: Interferon-gamma; PD-1: Programmed cell death protein 1; CTLA-4: Cytotoxic T-lymphocyte-associated protein 4; FcRL5: Fc Receptor–Like 5; NK cells: Natural killer cells; PEG-IFN: Pegylated interferon; NAs: Nucleos(t)ide.

CURRENT EVIDENCE OF HBsAg SEROCLEARANCE

Although HBsAg seroclearance can occur spontaneously, antiviral therapies such as PEG-IFN or NAs are more likely to induce it. Table 2 summarizes published studies on HBsAg seroclearance in CBH patients.

Table 2 Summary of published studies on hepatitis B surface antigen seroclearance (functional cure) in chronic hepatitis B.

Ref.	Year	Study type	n	Cohort and treatment	HBsAg seroclearance rate
Lau et al[36]	2005	RCT	814	HBeAg+ CHB on PEG-IFN α	2.95% at 6 months post-treatment
Marcellin et al[39]	2013	RCT	537	HBeAg- CHB on PEG-IFN α	3% (6 months), 9% (3 years), 12% (5 years)
Bourlière et al[53]	2017	RCT	185	HBeAg- CHB: NA vs NA + PEG-IFN	3.2% vs 7.8% at 96 weeks
Ahn et al[55]	2018	RCT	740	TDF + PEG-IFN vs TDF mono	9.1% vs 0% at 72 weeks
Zhou et al[32]	2019	Meta-analysis	48972	Untreated CHB	1.17% per year
Yeo et al[33]	2019	Meta-analysis	42588	Untreated CHB	1.02% per year
Yip et al[45]	2019	Retrospective	20263	CHB on TDF/ETV	2.1% (mean FU: 4.8 years)
Yeo et al[34]	2020	Prospective cohort	11262	Untreated CHB	1.31% per year
Yang et al[52]	2020	RCT	144	CHB on ETV vs ETV + PEG-IFN	1.8% vs 4.1%
Fonseca et al[56]	2020	Meta-analysis	8719	CHB on IFN+NA vs IFN vs NA (48-130 weeks)	4.8% to 6.5% vs 3.6% to 6.5% vs 0.27% to 0.58%
Song et al[43]	2021	Meta-analysis	1029	IHCs on PEG-IFN	47% at 48 weeks
Hsu et al[47]	2021	Retrospective	4769	CHB on TDF/ETV	0.22% per year
Chan et al[48]	2024	RCT	1298	CHB on TAF	≤ 1.2% at 5 years

HBsAg: Hepatitis B surface antigen; RCT: Randomized controlled trial; HBeAg: Hepatitis B e antigen; CHB: Chronic hepatitis B; PEG-IFN: Pegylated interferon; NA: Nucleos(t)ide analogue; TDF: Tenofovir disoproxil fumarate; ETV: Entecavir; TAF: Tenofovir alafenamide; FU: Follow up; IFN: Interferon; IHCs: Inactive hepatitis B carriers.

Spontaneous clearance

Multiple cohort studies from Asian and European countries reported variable annual spontaneous HBsAg seroclearance rates in CHB patients. In Asian cohorts, seroclearance rates ranged from 0.12%-2.38% per year[18-23], while Western cohorts showed rates of 0.54%-1.98% per year[24-31]. Among cirrhotic patients, the annual clearance rate was 0.8% in European patients and 1.5% in Taiwanese patients[23,30]. A meta-analysis by Zhou et al[32] (48972 CHB patients, 352381 person-years) reported a pooled clearance rate of 1.17%. This was higher in HBeAg-negative than HBeAg-positive patients, and in those aged ≥ 40 years (1.7%) compared to < 40 years (1.1%). Inactive carriers experienced a rate of 1.24%, while cirrhotic patients showed rates of 0.8%-1.4%. Another meta-analysis of 34 studies by Yeo et al[33] found a 1.02% annual clearance rate among untreated patients. Yeo et al[34] studied a large longitudinal cohort of 11264 patients, reporting a 1.31% clearance rate, with higher rates in men; those aged > 55 years; those with HBV genotype C, HBV DNA ≤ 2000 IU/mL, quantitative HBsAg (qHBsAg) ≤ 100 IU/mL, and HBeAg-negative status; and among community-based populations. In summary, annual spontaneous HBsAg seroclearance rates in CHB patients show wide variation across studies, shaped by multiple viral and host determinants. Differences in geography, population profiles, and study design likely contribute to this heterogeneity, while limited data from regions outside Asia and Europe restrict broader generalizability.

Treatment induced HBsAg seroclearance

PEG-IFN: PEG-IFN has demonstrated modest efficacy in achieving HBsAg clearance. In a multicenter randomized controlled trial (RCT), 266 HBeAg-positive patients treated with PEG-IFN-α2b for 52 weeks reported a 7% HBsAg loss rate at one year, with no additional benefit from combining lamivudine[35]. Another study reported a 2.95% HBsAg seroconversion rate with PEG-IFN-α2a in HBeAg-positive patients[36]. In HBeAg-negative CHB, a multicenter trial showed 3% HBsAg loss at 6 months, increasing to 9% at year 3 and 12% at year 5[37-39]. A retrospective study in 233 HBeAg-negative patients reported cumulative HBsAg loss rates of 4.7%, 9.4%, and 14.2% at 3, 5, and 10 years, respectively[40]. Although 48 weeks of PEG-IFN-α2a is standard, extended therapy improves viral suppression, alanine transaminase (ALT) normalization, and HBsAg clearance. A meta-analysis of four studies (350 patients) found significantly higher clearance rates with extended therapy at the end of treatment and at 24 and 48 weeks post-treatment (OR = 2.45, 3.17, and 5.02; P ≤ 0.02)[41]. Another meta-analysis of 9 studies (545 patients) reported a pooled HBsAg loss rate of 11%, similar to HBeAg-positive (14%) and HBeAg-negative (10%) groups, with no significant difference between monotherapy and NAs combination[42]. Patients with inactive HBsAg carriers achieved a high HBsAg clearance rate after PEG-IFN treatment. In a meta-analysis of 11 studies consisting of 1029 inactive CHB patients, the overall HBsAg clearance rate was 47% (31%-64%), with a conversion rate of 26% (15%-38%) after 48 weeks of PEG-IFN treatment[43]. To summarize, PEG-IFN remains a viable option for a subset of CHB patients aiming for a functional cure, especially those with favorable predictors. However, its limited efficacy, slow response, and tolerability issues underscore the need for more effective and safer treatment strategies.

NAs: NAs are effective in suppressing HBV replication and reducing disease progression, but they rarely achieve HBsAg clearance. Long-term studies indicate that only 2%-5% of patients achieve HBsAg loss after up to 10 years of continuous NA therapy[44]. Large retrospective analyses report clearance rates of 2.1% over 4.8 years[45], 4.58% over 6 years[46], and 2.11% at 10 years[47]. Even in tenofovir alafenamide (TAF)-treated cohorts, HBsAg loss remained ≤ 1.2% over 5 years[48]. Across studies, clearance rates consistently ranged from 1.4% to 5.1%, underscoring the limited capacity of NAs to achieve a functional cure[45,46,49-51]. The consistently low HBsAg clearance rates with NAs, even after a decade of therapy, reflect their inability to eliminate cccDNA or restore effective immune control-highlighting the need for novel agents targeting functional cure.

PEG-IFN plus NAs: Individual trials have shown modest but improved HBsAg clearance with combination therapy using PEG-IFN and NAs compared to monotherapy. In a study by Yang et al[52], the addition of PEG-IFN to entecavir (ETV) resulted in a 4.1% clearance rate, compared to 1.8% with ETV alone. Similarly, Bourlière et al[53] reported 7.8% clearance with NAs + PEG-IFN compared to 3.2% with NAs monotherapy. The highest efficacy was observed with tenofovir disoproxil fumarate (TDF) plus PEG-IFN for 48 weeks, resulting in a 10.4% clearance rate at week 120[54,55]. A meta-analysis comparing combination therapy to IFN alone showed only a modest difference in HBsAg loss (4.8% vs 3.6%)[56]. However, compared to NAs monotherapy, combination therapy was significantly superior (6.55% vs 0.58%), particularly when PEG-IFN was administered at standard doses for ≥ 48 weeks[56]. Among various regimens, switch-to-IFN strategies yielded the highest end-of-treatment HBsAg loss, although this benefit diminished over time. Sensitivity analyses revealed that clearance peaked at one year post-treatment, followed by a gradual decline[56]. In summary, while combination therapy with PEG-IFN and NAs offers a modest enhancement in HBsAg clearance, especially with prolonged treatment and appropriate patient selection, its limited long-term durability and tolerability challenges emphasize the need for more effective and better-tolerated therapeutic options.

Newer agents: Several newer antiviral agents are under clinical investigation for their potential to enhance HBsAg seroclearance by targeting different stages of the viral replication cycle. Bepirovirsen, an antisense oligonucleotide (ASO) designed to degrade HBV mRNA and pregenomic RNA, has shown promising initial efficacy. In phase 2 trials, it achieved HBsAg seroclearance in approximately 26%-29% of patients after 24 weeks of monotherapy or in combination with NAs. However, the durability of this response is limited; only 12%-14% of patients maintained HBsAg loss 24 weeks after treatment discontinuation, underscoring the challenge of achieving a sustained off-treatment response[57]. These findings highlight that while ASOs can effectively suppress antigen production, they may not be sufficient to achieve a functional cure without concurrent immune restoration. Xalnesiran, a small interfering RNA (siRNA) targeting the S gene of HBV, represents another novel approach aimed at reducing HBsAg levels by promoting RNA degradation. In a recent phase 2 study, when combined with NAs and either PEG-IFN-α2a or ruzotolimod (a TLR 7 agonist), Xalnesiran achieved HBsAg seroclearance rates ranging from 3% to 23%, with higher efficacy noted in the PEG-IFN combination group[58]. Despite these encouraging short-term outcomes, post-treatment follow-up revealed a decline in response rates, mirroring the limited durability seen with bepirovirsen. These observations suggest that while RNA-targeting therapies can induce significant reductions in viral antigenemia, their long-term effectiveness may require combinatorial strategies, including immunomodulatory agents, to sustain virological control.

To summarize, the data suggest that while spontaneous or NAs-induced functional cure remains uncommon, the strategic use of PEG-IFN-especially in combination with NAs-offers a more promising route to achieving HBsAg loss. PEG-IFN plus NAs consistently outperforms monotherapy, with seroclearance rates of up to 12% at 5 years compared to < 2% for NAs alone. Notably, inactive HBsAg carriers show high response rates (up to 47%), highlighting a potential target group.

PREDICTORS OF HBsAg CLEARANCE

HBsAg seroclearance is influenced by both host and viral factors (Table 3). Spontaneous clearance is more likely in older males, those with HBeAg-negative status, and genotype C infection, while high HBV DNA (> 20000 IU/mL) and qHBsAg ≥ 1000 IU/mL reduce the likelihood of clearance[59]. In PEG-IFN treated patients, favorable predictors include younger age, female sex, genotype A, combination therapy, low baseline HBsAg/HBV DNA, early HBsAg decline, and ALT elevation at week 12[60]. In NAs treated patients, predictors of functional cure include early HBeAg seroconversion, initiation of high-barrier NAs before seroconversion, and HBsAg < 1000 IU/mL at 18 months post-seroconversion[61]. In HBeAg-negative CHB, older age, shorter consolidation, and baseline/end-of-treatment HBsAg ≥ 1000 IU/mL increase relapse risk[62], while HBsAg < 100 IU/mL and or an end-treatment ALT/qHBsAg ratio ≥ 0.2 strongly predict clearance[63]. A low HBsAg glycan isomer level (≤ 3.5 Log ng/mL) is associated with a fivefold higher 10 year clearance probability[64].

Table 3 Predictors of hepatitis B surface antigen clearance in chronic hepatitis B.

Category	Subcategory	Predictor variables
Spontaneous clearance	Host factors	Male sex; older age (hazard ratio 1.16-1.21)
	Viral factors	HBeAg-negative status; genotype C infection; lower HBV DNA (≤ 20000 IU/mL); low quantitative HBsAg (≤ 1000 IU/mL)
PEG-IFN therapy	Host factors	Younger age; female sex; ALT elevation at week 12
	Treatment factors	Combination therapy (PEG-IFN + NAs) more effective than PEG-IFN alone
	Viral factors	Genotype A; low baseline HBsAg and HBV DNA; early HBsAg decline (at week 12 or 24)
NAs therapy	Treatment factors	Early HBeAg seroconversion (before age 18); initiating high-barrier NA before seroconversion
	Viral factors	Low HBsAg (< 1000 IU/mL) at 18 months post-seroconversion
NA discontinuation in HBeAg-negative CHB	Treatment factors	End-of-treatment HBsAg < 100 IU/mL; ALT/qHBsAg ratio ≥ 0.2
	Viral factor	Low HBsAg glycan isomer (≤ 3.5 Log ng/mL) associated with 5-fold higher 10-year clearance
Prediction models	Nomogram	BMI, HBeAg status, qHBsAg, HBV DNA (C-index 0.91)
	HepBLOSS-1 & HepBLOSS-2	Age, sex, HBsAg, HBV DNA; AUROC 0.81-0.89; > 50% 10-year clearance in high-risk groups
	HBRN-SQuARe	Sex, age, race, qHBsAg; AUROC 0.99 (1 year), 0.95 (3 years) in untreated HBeAg-negative CHB

HBsAg: Hepatitis B surface antigen; HBV: Hepatitis B virus; HBeAg: Hepatitis B e antigen; qHBsAg: Quantitative hepatitis B surface antigen; PEG-IFN: Pegylated interferon; NAs: Nucleos(t)ide analogues; ALT: Alanine aminotransferase; BMI: Body mass index; CHB: Chronic hepatitis B; AUROC: Area under the receiver operating characteristic curve; C-index: Concordance index; HepBLOSS: Hepatitis B Long-term Outcome Scoring System; HBRN-SQuARe: Hepatitis B Research Network-Surface Quantitative Antigen Risk estimator.

Several prediction models have shown high accuracy. A nomogram using body mass index, HBeAg status, qHBsAg, and HBV DNA achieved a C-index of 0.913, validated externally (C-index 0.886)[65]. Additionally, HepBLOSS-1 (HBV DNA, age, sex, HBsAg) and HepBLOSS-2 (excluding sex) predicted 5-, 10-, and 15-year seroclearance in untreated HBeAg-negative patients, with an area under the receiver operating characteristics (AUROC) curve of 0.81-0.89 and > 50% 10-year clearance in high scorers[66]. Recently, the HBRN-SQuARe model, incorporating sex, ∆qHBsAg, age, and race, predicted HBsAg loss with AUROC of 0.99 and 0.95 at 1 and 3 years, respectively, in a multiethnic cohort of 1240 patients[67]. In summary, HBsAg clearance is influenced by multiple host and viral factors. Predictors vary by treatment type, with early seroconversion and HBsAg decline being favorable.

THE WAY FORWARD IN ACHIEVING FUNCTIONAL CURE

The development of curative therapy for hepatitis C virus (HCV) stands in sharp contrast to the ongoing challenge of curing HBV infection, primarily due to fundamental differences in viral biology and persistence mechanisms. Direct-acting antivirals have revolutionized HCV treatment by targeting key viral enzymes involved in replication. Since HCV does not integrate into the host genome or establish a stable nuclear reservoir, viral eradication is achievable in over 95% of treated patients, resulting in a true virological cure. In contrast, curing CHB requires addressing cccDNA persistence and restoring effective immune responses[3,5]. While NAs effectively suppress viral replication, they do not eliminate cccDNA. PEG-IFN can target cccDNA to a limited extent but achieves only modest HBsAg clearance, with its efficacy constrained by immune evasion, hepatic flares, and poor tolerability, restricting its broader application[68-70]. To overcome these limitations, novel therapies targeting multiple stages of the HBV life cycle, including viral entry, replication, and assembly, as well as cccDNA disruption and immune stimulation, are being developed[71,72]. These innovative strategies represent a paradigm shift from viral suppression to functional cure. While most of the evidence on newer molecules comes from preclinical studies (in vitro or animal models), some agents have progressed to early-phase clinical trials, including phase 1 and phase 2. Figure 2 illustrates the HBV life cycle within hepatocytes and highlights current and emerging therapeutic intervention points.

Figure 2 This schematic illustrates key steps in the hepatitis B virus life cycle and corresponding therapeutic intervention points. The virus enters hepatocytes, forms Covalently closed circular DNA (cccDNA) in the nucleus, and undergoes transcription, translation, encapsidation, and release. Potential therapeutic targets include entry inhibition (e.g., Myrcludex B), cccDNA silencing (e.g., clustered regularly interspaced short palindromic repeats-Cas), post-transcriptional suppression (e.g., small interfering RNAs, antisense oligonucleotides), polymerase inhibition (e.g., tenofovir alafenamide, tenofovir disoproxil fumarate, entecavir), capsid assembly modulation (e.g., core protein allosteric modulators), release inhibition (e.g., nucleic acid polymers), and immune modulation strategies (e.g., therapeutic vaccines and checkpoint inhibitors). These interventions aim to disrupt viral persistence and promote functional cure. HBV: Hepatitis B virus; cccDNA: Covalently closed circular DNA; siRNA: Small interfering RNA; ASO: Antisense oligonucleotide; TAF: Tenofovir alafenamide; TDF: Tenofovir disoproxil fumarate; ETV: Entecavir; CpAM: Core protein allosteric modulator; NAP: Nucleic acid polymer; NTCP: Sodium taurocholate co-transporting polypeptide.

Therapies targeting cccDNA

Targeting cccDNA represents a key strategy in the pursuit of a functional cure; however, therapeutic progress is limited by an incomplete understanding of the formation, stability, and regulation of cccDNA. While approaches to silence or eliminate cccDNA are advancing, they remain largely experimental. Endonuclease-based genome editing tools like zinc finger nucleases, transcription activator-like effector nucleases, and clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated proteins (Cas) systems have shown potential in disrupting cccDNA in vitro[72]. Among these, CRISPR-Cas9 has demonstrated the ability to cleave more than 90% of HBV DNA. However, a small proportion (approximately 7%) may undergo functional repair, necessitating the use of multiplexed guide RNAs to ensure complete inactivation of viral genes[73]. Key barriers to the clinical translation of this novel approach include hepatocellular delivery efficiency, off target risks to the host genome, and the cleavage of chromosomally integrated HBV DNA and DNA recombination. Despite these concerns, endonuclease-mediated mutagenesis remains the only experimentally proven method for permanently inactivating cccDNA in tissue. Transcriptional regulation of cccDNA involves both viral factors (e.g., HBx, HBcAg) and host pathways. A key mechanism includes HBx-mediated degradation of the structural maintenance of chromosomes 5/6 (SMC5/6), which otherwise represses HBV transcription. Nitazoxanide, an antiparasitic agent, has been shown to preserve SMC5/6 and inhibit HBV replication, including cccDNA activity. In a pilot study of 9 CHB patients treated with nitazoxanide for 48 weeks, 89% achieved undetectable HBV DNA, HBeAg loss occurred in 2 patients, and HBsAg clearance in 33%, though larger studies are needed to validate these findings[74,75]. Pevonedistat, a ubiquitin-like protein inhibitor, suppresses HBV cccDNA transcription by inhibiting the HBx protein-mediated degradation of the SMC5/6 complex[76]. Epigenetic modifiers offer another promising strategy by reversing pro-viral chromatin states. Agents such as AGK2 and GS-5801 (a lysine demethylase inhibitor) have shown antiviral activity in preclinical models, but clinical efficacy remains limited[77]. Lastly, host apolipoprotein B mRNA-editing catalytic polypeptide-like 3 (APOBEC3) family deaminases can induce cccDNA degradation following IFN stimulation, although the clearance is partial and variable[78]. Thus, both epigenetic modifiers and APOBEC3 based approaches offer novel and mechanistically distinct strategies for cccDNA silencing or degradation, but are currently limited by efficacy, delivery, and safety challenges.

Capsid assembly inhibitors

Capsid assembly inhibitors are promising adjuncts to NAs in CHB, aiming for deeper viral suppression to aid immune function restoration[79,80]. These agents target the HBV core protein, a key component in the viral replication cycle, and are primarily categorized into two types based on their mechanism of action. The first group includes core protein allosteric modulators.

Such as GLS4 and RO7049389, which induce the formation of defective or misassembled capsids that are incapable of supporting viral replication. The second group includes capsid assembly modulators such as AT-130, NVR-3778, and JNJ-6379, which promote the assembly of empty capsids lacking viral genomic material[71,79,80]. Both classes effectively inhibit the encapsidation of pregenomic RNA, thereby reducing the production of HBV DNA particles. More importantly, they prevent the recycling of nucleocapsids back into the nucleus, a key step in the replenishment of cccDNA during successive infection cycles. By blocking this step, capsid assembly inhibitors help to limit the persistence of HBV within hepatocytes. Clinical studies evaluating RO7049389 and the combination of NVR-3778 with PEG-IFN have demonstrated enhanced antiviral activity, with significant reductions in HBV DNA and RNA levels; however, these interventions did not produce a meaningful decline in HBsAg levels[81,82]. These findings suggest that while capsid assembly inhibitors are effective at disrupting viral replication and cccDNA amplification, their impact on key functional cure markers such as HBsAg remains limited.

Targeting viral gene expression

Persistent high levels of HBV antigens contribute to immune dysfunction and chronicity in hepatitis B[13,14,17]. Therapeutic strategies aimed at reducing antigen expression by degrading viral RNA are gaining traction as potential avenues to restore immune control. Direct degradation of viral mRNA offers a route to reduce antigen levels. RNA interference (RNAi), particularly siRNAs, targets overlapping HBV transcripts, suppressing the production of HBsAg, HBeAg, and HBcAg. While early siRNAs like ARC-520 showed limited efficacy in HBeAg-negative and NAs-experienced patients due to integrated HBV DNA, next-generation agents have improved outcomes. JNJ-3989 achieved > 1 Log IU/mL HBsAg reduction in 98% of patients, with durable responses in more than half[83,84]. AB-729 also demonstrated potent and sustained HBsAg decline in NAs-suppressed patients, alongside improved HBV-specific T cell responses and modest ALT flares[85-87]. Similarly, VIR-2218 targeting HBx showed > 1 Log IU/mL HBsAg reduction in 71% of patients, though sustained responses were seen in only 20%. However, its combination with PEG-IFN enhanced HBsAg suppression compared to monotherapy[88]. Collectively, these findings highlight the potential of targeting HBV gene expression, either at the transcriptional or post-transcriptional level, to lower antigen load, enhance immune restoration, and support efforts toward functional cure.

HBsAg release inhibitors

HBsAg is secreted in massive quantities as non-infectious subviral particles (SVPs), making up more than 99.9% of circulating HBV particles. Inhibiting its release may reduce antigen load and promote immune reactivation[89]. Nucleic acid polymers (NAPs) developed by Replicor Inc, such as RNA-based REP-2139 and REP-2165, and DNA-based REP-2055 and REP-2031, block SVP assembly or secretion. In a trial, REP-2139 followed by PEG-IFN achieved sustained HBsAg loss and anti-HBs seroconversion in 42% of HBV/hepatitis D virus (HDV) co-infected patients, lasting more than a year[90]. However, REP-2139 was associated with heavy metal-like toxicity due to accumulation in plasma and liver, prompting the development of REP-2165, which demonstrated comparable efficacy with reduced hepatic build-up[91]. A phase 2 study combining NAPs with TDF and PEG-IFN showed enhanced efficacy: 60% of patients achieved HBsAg ≤ 0.05 IU/mL, and 35% maintained a functional cure 48 weeks post-treatment[92]. Notably, over 90% of patients experienced ALT flares, especially those who lost HBsAg, indicating that immune-mediated responses contributed to viral control. To summarize, NAPs significantly reduce circulating HBsAg by blocking SVP release, thereby promoting immune reactivation and enabling functional cure in a subset of patients. Their integration into combination therapies holds promise, though future refinements in safety and broader validation are essential to enhance their clinical utility.

HBV entry inhibitors

The identification of NTCP as the entry receptor for HBV and HDV has enabled novel therapeutic strategies aimed at preventing the entry of the virus and hepatocyte reinfection particularly relevant in HBV/HDV co-infection, where HDV relies on HBV surface proteins and shares the NTCP receptor. As HDV is replication defective and requires HBsAg for assembly and propagation, the loss of HBsAg in the context of HBV infection signifies not only a functional cure of HBV but also facilitates the suppression or potential eradication of HDV. This dual therapeutic advantage underscores the significance of achieving HBsAg clearance in co-infected patients, making it a critical therapeutic goal[93]. Myrcludex B (MyrB; bulevirtide), a myristoylated peptide derived from the pre-S1 domain of large HBsAg, is a key entry inhibitor. While MyrB monotherapy has been shown to reduce HDV RNA, it has a limited effect on HBsAg levels[94,95]. In an open-label phase 2 study, combining MyrB with PEG-IFN led to HBsAg loss in 27% of patients at 24 weeks, compared to 0% in monotherapy arms[95]. However, MyrB use is associated with elevated bile salts, a predictable outcome of NTCP inhibition. Additional entry inhibitors, such as ezetimibe and cyclosporin derivatives, are under investigation and may act independently of NTCP[96,97].

Immune modulation therapies

Spontaneous HBV clearance highlights the importance of a robust innate and adaptive immune response, making immunomodulatory strategies a promising avenue for restoring HBV-specific immunity[71]. PRRs such as TLRs and retinoic acid-inducible gene (RIG)-I detect HBV and trigger antiviral cytokines, promoting NK cell activity and adaptive immune restoration. Pharmacologic activation of these pathways is under exploration.

PRR agonists: RO7020531, a TLR7 agonist, demonstrated good safety in healthy individuals and is currently under investigation in combination with capsid inhibitors in CHB patients[98]. Vesatolimod (GS-9620), another TLR7 agonist, showed strong antiviral activity in animal models, but its efficacy in humans was limited-likely due to lower dosing strategies[99]. Selgantolimob (GS-9688), a TLR8 agonist, produced modest outcomes in CHB patients, with no patient achieving > 1 Log decline in HBsAg, and only 6% achieving a ≥ 0.5 Log reduction. However, when combined with TAF, GS-9688 resulted in a > 0.3 Log IU/mL reduction in HBsAg[100]. RIG-I, a cytoplasmic sensor of double-stranded RNA, recognizes the epsilon encapsidation signal in HBV pregenomic RNA, triggering the production of interferon lambda (IFN-λ), which inhibit HBV replication and activate innate immune responses[101,102]. Additionally, RIG-I disrupts the interaction between the epsilon signal and HBV polymerase, further suppressing viral replication. The RIG-I agonist SB 9200 (Inarigrivir) demonstrated dose-dependent reductions in HBV DNA and RNA, with a 0.5 Log HBsAg decline observed in 22% of patients. However, the drug was discontinued due to severe adverse events, including hepatocellular dysfunction and one death reported in the phase 2 CATALYST trial[71]. Recent studies also suggest that PEG-IFN may restore PRR expression, supporting the rationale for combination strategies in CHB treatment[103,104].

Immune checkpoint inhibitors: Immune checkpoint inhibitors (ICIs) have garnered attention as potential immunotherapeutic agents in CHB, inspired by their growing use in cancer therapy. Immune checkpoint receptors such as PD-1, cytotoxic T-lymphocyte antigen-4, and T-cell immunoglobulin and mucin-domain containing-3 contribute to HBV-specific T cell exhaustion. In CHB patients, elevated PD-1 expression on HBV-specific CD8⁺ T cells correlates with higher viral loads and HBeAg levels[105]. Multiple PD-1/programmed death-ligand 1 (PD-L1) inhibitors, including nivolumab, serplulimab, and envafolimab, are currently in phase II trials[106,107]. In a phase Ib trial, nivolumab (0.3 mg/kg), with or without GS-4774 (therapeutic vaccine), induced modest HBsAg declines in HBeAg-negative CHB patients[106]. In a recent Phase IIb study, subcutaneous administration of ASC22 (envafolimab), a humanized, single-domain PD-L1 antibody, at 1.0 mg/kg every two weeks for 24 weeks was found to be safe and well-tolerated in virally suppressed CHB patients receiving NAs. Treatment induced a decline in HBsAg levels, particularly in patients with baseline HBsAg ≤ 100 IU/mL. Notably, 3 out of 10 patients in the 1.0 mg/kg cohort with baseline HBsAg ≤ 100 IU/mL achieved on-treatment HBsAg loss[107]. Additionally, a cohort study involving cancer patients demonstrated functional cure in 9.1% of those with HCC and 6.8% of non-HCC cancer patients treated with ICIs[108]. Among patients with baseline qHBsAg levels < 100 IU/mL, HBsAg clearance rates reached 13.0% at 12 months and 38.4% at 24 months, highlighting the potential of ICIs in achieving a functional cure for HBV[108]. Accordingly, while PD-1/PD-L1 blockade exhibits immunological efficacy and leads to a moderate decrease in HBsAg among individuals with virologic suppression, therapeutic outcomes are markedly influenced by baseline antigen levels. Multiple studies have demonstrated that patients presenting with low initial HBsAg concentrations (< 100 IU/mL) derive the most pronounced benefit, highlighting the critical role of antigen load reduction as a potential prerequisite for effective ICI therapy.

IFN-λ: IFN-λ is a complex and highly regulated host innate immune defense system that can impact HBV replication and cccDNA[109]. It offers antiviral effects similar to IFN-α but with reduced systemic toxicity due to its limited receptor distribution[110]. In the LIRA-B2a trial, PEG-IFN-λ had similar on-treatment HBeAg seroconversion rates but failed non inferiority at 24 weeks post-treatment. However, combination therapy, ETV followed by ETV plus PEG-IFN-λ (LIRA-B2b trial) improved immune profiles and preserved CD8⁺ T cell function, showing promise for future use[111].

Therapeutic vaccines: Despite the availability of an effective prophylactic vaccine, no therapeutic vaccine for CHB has yet been approved. Therapeutic vaccines aim to restore impaired HBV specific T cell responses and induce de novo adaptive immunity. Several candidates such as TG1050, VTP-300, GS-4774, and BRII-179 are currently in phase II clinical trials, with encouraging results in preclinical models[112-115]. In a phase II trial by Lok et al[112], GS-4774 a heat-inactivated yeast-based vaccine expressing HBsAg, HBcAg, and HBx failed to induce HBsAg decline or clearance at weeks 24 or 48. However, when combined with TDF, it boosted IFN-γ, TNF-α, and IL-2 production by HBV specific CD8+ T cells[113]. BRII-179 (VBI-2601), a protein-based vaccine expressing all three envelope proteins, was evaluated in a phase 2a trial in combination with the siRNA agent elebsiran (BRII-835), with or without IFN-α. While siRNA monotherapy alone yielded limited immune recovery, adding BRII-179 Led to the generation of durable neutralising anti-HBs antibodies in approximately 40% of participants, along with expansion of IL-2-producing Pre-S1/Pre-S2-specific CD4⁺ T cells. Although no group achieved sustained HBsAg loss, this data confirm that deep antigen reduction can “unmask” HBV-specific humoral immunity when paired with a broad-spectrum vaccine[114]. VTP-300, a vector-based vaccine, achieved a 0.7-1.4 Log₁₀ IU/mL reduction in HBsAg among patients with baseline HBsAg < 50 IU/mL, with two participants reaching undetectable levels, suggesting potential synergy when combined with siRNA[115]. A key challenge across these platforms is the generation of immune responses against the vaccine vector itself, which may blunt HBV specific immunity and limit dosing frequency. A heterologous prime-boost strategy may help overcome this hurdle. In conclusion, current evidence suggests that vaccines alone demonstrate limited efficacy in achieving HBsAg loss; nevertheless, they consistently enhance HBV specific immune responses when used in conjunction with antigen reducing agents such as siRNA. The implementation of heterologous prime-boost strategies or the incorporation of RNA targeting therapies appears to offer increased potential for achieving clinically significant outcomes.

To summarize, Immune modulation therapies in CHB-including PRR agonists, ICIs, IFN-λ, and therapeutic vaccines aim to restore HBV specific immunity. While some agents show immunologic activity, most have demonstrated limited HBsAg reduction, with future success likely dependent on optimized combinations and improved patient selection.

Combination therapies: Given the distinct mechanisms of action of NAs and PEG-IFN, combination strategies might enhance treatment outcomes. These include de-novo (simultaneous initiation), add-on (adding PEG-IFN to ongoing NAs therapy), and switch-to approaches (transitioning from NAs to PEG-IFN). Emerging data suggest that sequential strategies, particularly switch-to or add-on after achieving viral suppression and low antigenemia, may improve response rates, especially in patients who have already achieved HBeAg loss on NAs[116,117]. However, further research is needed to optimize these combinations, with factors like timing, baseline HBsAg levels, and virological suppression likely playing key roles in success. Combinatorial therapies of novel agents are being actively explored to enhance functional cure rates in CHB. In a phase IIa trial, combining the RNAi agent JNJ-3989 with NAs, with or without the capsid modulator JNJ-6379, resulted in ≥ 1 Log IU/mL HBsAg reduction in all patients receiving triple therapy-regardless of HBeAg status. Higher doses of JNJ-3989 (100-400 mg) yielded greater responses, while reducing the interval between doses did not confer additional benefit. Impressively, 38% of patients maintained significant HBsAg suppression nearly a year after treatment, underscoring the potential durability of response from combination regimens[83].

CONCEPT OF NAs CESSATION AND HBsAg LOSS

NAs are the cornerstone of CHB treatment, yet decisions regarding therapy cessation remain complex[118]. Discontinuation is generally advised in patients achieving HBeAg seroconversion or HBsAg loss[119]. For non-cirrhotic HBeAg positive patients, all major guidelines support stopping NAs after seroconversion and at least one year of virological suppression[5,120], with the Asian Pacific Association for the Study of the Liver (APASL) recommending a longer 3-year consolidation to reduce relapse risk[121]. However, guidelines diverge significantly for HBeAg-negative CHB. Since HBsAg decline is gradual, lifelong therapy is often needed. However, the 2008 APASL guideline proposed stopping therapy after at least two years of undetectable HBV DNA, confirmed thrice over 18 months. Supporting this, Hadziyannis et al[122] reported 39% HBsAg loss in 33 patients after stopping adefovir. Similarly, the FINITE RCT by Berg et al[123] showed 19% HBsAg loss post-tenofovir discontinuation. Based on such evidence, the 2017 European Association for the Study of the Liver guideline recommended stopping NAs in non-cirrhotic HBeAg-negative patients after three years of sustained suppression[124].

HBsAg loss post-therapy may stem from restored immune function control, including revived CD8+ T cell function and cytokine upregulation[125]. HBsAg level, especially its decline during therapy and low levels at cessation, strongly predicts HBsAg loss. In a study of 1075 HBeAg-negative patients, HBsAg seroclearance rose from 0.15% annually on therapy to 1.78% annually post-therapy, reaching 13% at 6 years. Predictors included early HBV DNA suppression, HBsAg decline > 1 Log₁₀, end-of-treatment HBsAg < 100 IU/mL, and no retreatment despite relapse[126]. The RETRACT-B study (n = 1552) found cumulative HBsAg loss rates of 3.2% at 12 months and 13% at 48 months post-treatment, with a higher likelihood in patients with end of treatment HBsAg < 100 IU/mL (HR 22.5). HBsAg loss exceeded 30% at 48 months in Whites individuals with < 1000 IU/mL and Asians with < 100 IU/mL at cessation; others had < 10% probability[127]. In the CREATE study (n = 572), 4.2% achieved HBsAg loss, with non-Asians faring better (15% vs 1.5%). Lower levels of HBcrAg (< 2 Log U/mL) and HBsAg (< 100 IU/mL) predicted favorable outcomes[128].

Key concerns with cessation of NAs include ALT flares, decompensation, and HCC. In RETRACT-B study, hepatic decompensation occurred in 1.22% (4.3% in cirrhotics) study subjects, and HCC in 2.2% with cirrhosis vs 0.7% without[127]. Another study (n = 691) reported comparable HCC rates post-cessation and during therapy, with clinical relapse in 60.6%[126]. Smaller studies reported clinical relapse in 57.9%-86.8% within 6months of tenofovir withdrawal[129], and virological and clinical relapse rates of 70% and 43.6% respectively[130]. Based on these findings, treatment cessation may enhance the chance of functional cure and is generally safe in non-cirrhotic patients when supported by rigorous follow-up.

CONCLUSION

Hepatitis B treatment has evolved significantly over the past few decades, yet finding a functional cure still presents daunting obstacles. The incorporation of cccDNA into the host genome and the modulation of HBV specific immunity remain major obstacles in antiviral therapy approaches. Research on promising medications is progressing rapidly, but may take time before they are translated into clinical settings. Furthermore, clinical trials of several emerging drugs have shown that monotherapy is not as effective as intended. Therefore, multi-target and multi-drug combination therapy that includes a mix of PEG-IFN, NAs, or other drugs constitutes a promising strategy to achieve functional cure in a large proportion of CHB patients.