Functional cure for chronic hepatitis B: A state of immune control over cccDNA persistence

Abstract

Functional (clinical) cure is considered a desirable therapeutic endpoint of antiviral therapy for chronic hepatitis B (CHB). Recent studies have detected transcriptionally active covalently closed circular DNA (cccDNA) and integrated hepatitis B virus (HBV) DNA (iDNA) in the liver tissue of patients who achieved functional cure. More than that, a small subset of these patients even showed positive staining for hepatitis B surface antigen (HBsAg) in hepatocytes. Notably, compared to uncured patients, functionally cured patients demonstrate significantly restored HBV-specific immune responses. Based on these findings, it is clear that although cccDNA or iDNA are not completely cleared in certain functionally cured patients, the circulating viral markers are largely undetectable, even with the sensitive measuring methods that are routinely used. This is because, virologically, the residual viral genomic DNA in functionally cured patients is often genetically or epigenetically modified or undergoes post-transcriptional splicing and editing that prevents viral protein expression and productive replication. More importantly, the restored host immune responses further suppress viral protein production and HBV DNA replication to undetectable levels in peripheral blood. Thus, this form of functional cure for CHB might be described more accurately as an “undetectable” state of circulating virologic markers under the control of a restored host immune response against HBV infection. This resembles occult HBV infection to some extent, as replicative forms of HBV DNA—such as relaxed circular DNA and/or cccDNA—persist in the liver, accompanied by a risk of reactivation. As a result, consolidation therapy following serum HBsAg loss and continued follow-up monitoring after discontinuation of treatment become crucial. This review provides a comprehensive overview of the status of CHB patients who achieved functional cure, explains its underlying reasons, and assesses its relevance to clinical treatment and practice.

1. Introduction

Chronic hepatitis B virus (HBV) infection is a major cause of hepatitis B, liver cirrhosis, and hepatocellular carcinoma (HCC)^[1] and remains a significant global public health concern.^[2] As of 2022, approximately 258 million people worldwide continue to live with HBV infection.^[3] China continues to be an intermediate endemic area for HBV infection, posing a substantial threat to public health.^[4] At present, the optimal treatment goal for chronic hepatitis B (CHB) is to achieve a functional cure, which is defined as sustained hepatitis B surface antigen (HBsAg) loss (≥6 months post treatment), with or without HBsAg seroconversion; sustained serum HBV DNA levels below the lower limit of quantification (LLOQ) of highly sensitive assays; hepatitis B e antigen (HBeAg) seroconversion in initially positive patients; alleviation of liver inflammation and improvement in liver histopathology; and a significant reduction in the risk of end-stage liver disease and HBV-related complications.^[1,4,5] However, the cumulative rate of HBsAg clearance with long-term treatment of nucleos(t)ide analogues (NAs), which are the most commonly used in clinical practice, remains as low as 2%–3%,^[6] or only around 10% after a finite course of pegylated interferon-alpha (Peg-IFNα) therapy.^[7] Moreover, although the risk of primary HCC cannot be completely eliminated, patients receiving standard NAs therapy experience a more than 50% reduction in risk.^[8] Furthermore, the risk of developing liver cancer remains relatively high among CHB patients over 50 years of age or with liver cirrhosis even after achieving a functional cure.^[9,10]

Current guidelines regarding a functional cure for CHB do not emphasize the complete eradication of covalently closed circular DNA (cccDNA) from liver tissue, nor do they provide a unified statement on whether integrated HBV DNA (iDNA) and its transcription in the liver tissue of cured patients is completely eliminated.^[4,11] Based on major guidelines and research on intrahepatic virological markers, “cures” for CHB can currently be categorized as follows: (1) Complete (virological) cure, in which all forms of HBV DNA, including integrated DNA, are completely cleared from the liver tissue; (2) Functional cure, in which residual HBV DNA, including cccDNA, may persist in the liver tissue but is suppressed under the selective pressure exerted by the host’s restored antiviral immunity, resulting in very low levels of DNA replication, transcription, and viral protein expression that are undetectable in the bloodstream with current sensitive detection assays.^[4,11,12,13] At least to some extent, this phenomenon resembles the occult hepatitis B virus infection (OBI) observed in some patients who have naturally recovered from HBV infection, in whom replication-competent HBV DNA, including relaxed circular DNA (rcDNA) and/or cccDNA, may still exist in the liver tissue.^[14,15]

Therefore, to elucidate in detail the actual manifestations and essence of functional cure, this review first describes the fact that viral genomes persist in the liver tissue of many functionally cured patients. Subsequently, the underlying reasons are thoroughly explored from multiple angles, including immunology, virological factors and the inherent limitations of antiviral drugs. Finally, the potential implications of this phenomenon for clinical treatment and management are discussed, and reasonable recommendations are proposed to further improve the functional cure rate and prognosis of patients with CHB.

2. Functional cure: undetectable circulating viral markers under host immune pressure

2.1. Some cases of functional cure resemble the OBI

In fact, OBI originates from the concept of transfusion safety. It is defined as the presence of replication-competent HBV DNA (specifically cccDNA) in the livers of individuals who test negative for serum HBsAg using current sensitive detection assays, regardless of whether HBV DNA is detectable in their serum. OBI primarily results from the resolution of acute self-limited infection, including acute hepatitis B, and the spontaneous resolution of chronic infection.^[16] Although some functionally cured patients should be considered to have OBI due to the presence of transcriptionally active cccDNA, they are distinct from OBI patients in several ways (Table 1): In OBI patients, HBV DNA may be intermittently detectable in peripheral blood (typically <200 IU/mL), whereas in functionally cured patients, HBV DNA is consistently undetectable. A possible explanation is that in such OBI cases, cccDNA is occasionally transcriptionally active, which leads to HBV DNA being detectable in peripheral blood at low levels. By contrast, functionally cured patients may have lower copy numbers of intrahepatic cccDNA in a state of transcriptional silence^[17]; functionally cured patients may exhibit a more complete state of immune reconstitution compared to those with OBI, in whom transcriptionally active cccDNA may occasionally exist, resulting in intermittent low-level detection of serum HBV DNA. However, we must emphasize that they share a common characteristic: Functionally cured patients also remain at risk of HBV reactivation under immunosuppressive therapy (especially anti-CD20 monoclonal antibody treatment) because cccDNA may persist in the liver.^[18] Therefore, prophylactic direct antivirals should be given to these cured patients while they are receiving treatments that may cause immunosuppression due to the possible persistence of cccDNA in liver tissue.

Table 1.

Characteristics of complete and functional cure for chronic hepatitis B and occult hepatitis B virus infection^{[4,9,10,16,17]}

Characteristics	Complete cure	Functional cure	Occult HBV infection
HBV DNA	–	<LLOQ	–; or with occasionally positive, usually < 200 IU/mL
HBsAg	–	< 0.05 IU/mL	<0.05 IU/mL
Anti-HBc	+	+	+/–a
Anti-HBs	+/–	+/–	+/–
Liver rc/cccDNA	–	+/–	+
Specific immune status against HBV	Restored	Restored	Partially restored; or uncertain for those with intermittent HBV DNA positivity
Risk of HCC	–	Significantly decreased, except in patients with cirrhosis or HBsAg loss at >50 years of age	A noticeable association may still exist in patients with occasionally positive HBV DNA
Risk of blood transmission	–	– (cannot be completely excluded)	+ (absent if cccDNA transcription is completely silenced)
Risk of organ transplant transmission	–	+ (if liver tissue cccDNA is positive)	+
Risk of reactivation	–	+ (if liver tissue cccDNA is positive)	+
Antiviral therapy	–	Recommended in patients with cirrhosis	Recommended if >30 years of age and with occasional HBV DNA positivity b
Prophylactic antiviral treatment c	–	+ (if liver tissue cccDNA is positive)	+

^aThe symbol +/– indicates the presence or absence. ^bGuidelines for the prevention and treatment of chronic hepatitis B (version 2022)^[4]. ^cWhen individuals are undergoing immunosuppressive therapy.

HBV: Hepatitis B virus; HBsAg: Hepatitis B surface antigen; Anti-HBc: Antibody to hepatitis B core antigen; Anti-HBs: Antibody to HBsAg; rcDNA; Relaxed circular DNA; cccDNA: Covalently closed circular DNA; HCC: Hepatocellular carcinoma; LLOQ: Lower limit of quantification.

Therefore, for such functional cures, we should carefully consider several factors, including the residual abundance of cccDNA and iDNA in the liver, their transcriptional activity, and the functional status of the restored HBV-specific immune response. By evaluating these factors, we can better understand the true state of functional cure in CHB. This also offers insights into its underlying mechanisms from both the pathogen and the host immune perspective, which is essential for optimizing existing treatment strategies, enhancing antiviral efficacy, increasing the rate of functional cure, and improving patients’ prognosis through appropriate post-treatment monitoring.

2.2. The presence of viral genomes in most functionally cured patients

It is well established that there are two distinct forms of HBV DNA in hepatocytes. The first is cccDNA, which exists as a small chromosomal form in the nucleus of infected hepatocytes and is repaired from rcDNA. This episomal cccDNA transcribes all viral mRNA and serves as the primary template for progeny virus production. The second form, double-stranded linear DNA, can integrate into the host genome to form iDNA.^[6] Importantly, iDNA constitutes the predominant source of serum HBsAg in both HBeAg-negative and HBeAg-positive patients who have undergone prolonged NAs therapy.^{[19,20,21,22]}

Recent studies have shown that only a subset of patients meeting current functional cure criteria achieve complete viral DNA eradication. Notably, 68.1% of functionally cured CHB patients exhibited persistent cccDNA detection in hepatic tissue upon biopsy.^[13] This result suggests that in most “cured” patients with sustained serum HBsAg loss, replication-competent HBV DNA, including rcDNA and/or cccDNA, may persist in liver tissue.^[14,23] Furthermore, 72.2% of these patients showed positive HBV RNA detection in liver tissue, with roughly 19%–25.5% of functional cured patients demonstrating persistent intrahepatic HBsAg expression.^[13,20]

Consistent with this, a recent study conducted in China that applied spatial transcriptomics found that cccDNA-derived transcripts could be detected in the liver tissue of cured patients.^[12] It is worth noting that, due to the limitations of liver biopsy techniques, the sampled tissue may not fully represent the overall virological status of the entire liver. Following a functional cure, the proportion of individuals who still harbor intrahepatic cccDNA, its transcripts, and viral proteins may be higher than previously assumed. In addition, since most functionally cured patients are monitored using noninvasive assessments to evaluate treatment outcomes, it is difficult to accurately determine the true intrahepatic virological status of these individuals.

Some studies have even suggested that cccDNA in the liver tissue of cured patients retains transcriptional activity for the 3.5 kb HBV RNA, similar to that observed in untreated patients, indicating that cccDNA in these functionally cured patients may still sustain viral replicative potential and maintain a minimal cccDNA pool through intracellular replenishment.^[24,25] These reports indicate that in some functionally cured patients, cccDNA may persist and retain transcriptional activity; however, due to the restored HBV-specific immune response post-treatment, the production of viral DNA and the expression of viral proteins are suppressed below the LLOQ of the current sensitive assay.

As we know, achieving a functional cure, with sustained HBsAg loss as the key criterion, is accompanied by well restoration of the patients’ anti-HBV immune response. For example, cured patients often demonstrate significantly higher levels of HBsAg-specific and HBV polymerase (Pol)-specific cytotoxic T lymphocytes (CTLs) in peripheral blood than uncured patients.^[24] Such a robust host immune response can suppress the transcription and replication of residual viruses, keeping the circulating blood viral markers below the sensitivity threshold of detection assays. These findings also provide a mechanistic explanation for the spontaneous clearance of serum HBsAg after acute and chronic HBV infections as well as the virological relapse observed in functionally cured CHB patients receiving immunosuppressive therapy. For example, among B-cell non-Hodgkin lymphoma patients who are HBsAg-negative and hepatitis B core antibody-positive, over 10% develop HBV reactivation while undergoing B-cell depletion therapy.^[18] Therefore, it is crucial to emphasize that even when patients achieve serum HBsAg loss, the risk of HBV reactivation persists, and regular surveillance and monitoring are still necessary.

3. Causes of intrahepatic HBV genomes in functionally cured patients

3.1. The antiviral immune function is well restored in functionally cured patients

In patients with CHB, both innate and virus-specific adaptive immune responses are typically characterized by profound functional impairment and atypical exhaustion.^[24,26] By contrast, individuals who achieve a functional cure often exhibit substantial restoration of both innate and adaptive immune functions. Enhanced innate immunity is evidenced by the activation of liver-resident natural killer (NK) cells, increased antigen-presenting capacity of specific Kupffer cells, and the recruitment of marginal neutrophils into hepatic tissue.^[24]

With regard to adaptive immune responses, functionally cured patients demonstrate markedly improved differentiation and antibody-secreting capacity of HBsAg-specific B cells compared with uncured patients. Notably, these patients frequently harbor immunoglobulin G⁺ classical memory B cells and plasmablasts as well as increased frequencies and functional enhancement of HBsAg-specific B cells.^[27,28] However, the differentiation and antibody-secreting function of hepatitis B core antigen (HBcAg)-specific B cells do not appear to be significantly restored after a functional cure,^[28] suggesting that humoral immunity targeting HBsAg may play a more critical role in achieving a functional cure.

Furthermore, HBV-specific T-cell responses are often reconstituted in functionally cured individuals. This is evidenced by a notable increase in both the number and functionality of HBV-specific T cells,^[29] with CD4⁺ T cells playing a pivotal role.^[30] Interestingly, a subset of CD4-CTLs has also been identified in these individuals.^[24] Consistent with this, the proportion of exhausted CD8⁺ T cells is significantly reduced, and the frequency of FOXP3⁺ regulatory T cells is markedly decreased. Moreover, compared to non-cured individuals, functionally cured patients often possess a higher proportion of HBsAg- and Pol-specific CTLs,^[24] highlighting the critical role of these virus-specific T-cell responses in controlling HBV infection. Finally, it has recently been found that hepatocytes expressing major histocompatibility complex class II molecules are present in functionally cured patients and that this population of cells may interact with LR-NK and CD4-CTL cells.^[24]

Importantly, recent studies have suggested that in CHB patients, virus-specific T cells are more functionally impaired than terminally exhausted.^[26] Therefore, it is reasonable to propose the use of agents such as IL-2 and 4-1BB agonists as immunomodulatory therapies to restore T-cell functionality.^[31,32] Such direct activation of functionally impaired T cells might be more effective than the use of immune checkpoint inhibitors (e.g., PD-1 inhibitors) in this context.

3.2. Transcriptional control of HBV cccDNA and iDNA by the host Immune System

As previously mentioned, functional cure, defined primarily as the sustained undetectability of viral markers in circulation, does not exclude the presence of cccDNA and iDNA in liver tissue. However, suppose these forms of DNA are not transcriptionally silenced to prevent the expression of viral proteins, how do hepatocytes harboring transcriptionally active cccDNA and iDNA evade host immune surveillance, allowing patients to attain a functionally cured state? To address this, we discuss the status of cccDNA and iDNA in the liver tissue of patients with a functional cure.

As the source of HBV replication and transcription, cccDNA in infected hepatocytes exists in a chromatin-like structure wrapped around nucleosomes, forming minichromosomes.^[33] Consequently, cccDNA adheres to the transcriptional regulatory mechanisms of the host gene, including epigenetic modifications.^[34] For example, the structural maintenance of chromosome 5/6 complex broadly suppresses cccDNA transcriptional activity,^[35,36] while histone methylation and acetylation of cccDNA can either enhance or inhibit the transcriptional activity of its promoters (Figure 1).^[37] These mechanisms provide an alternative explanation for the presence of cccDNA in up to 68.1% of patients who have achieved a functional cure (defined as sustained HBsAg loss) with Peg-IFNα based therapy.^[13] Specifically, selective suppression or silencing of the SP1/2 promoter activity in cccDNA could enable infected hepatocytes to evade clearance by HBsAg-specific CTLs.^[25]

Figure 1.

Some epigenetic modifications of cccDNA. cccDNA: Covalently closed circular DNA; Smc5/6: Structural maintenance of chromosome 5/6; HBx: Hepatitis B virus X protein; DDB1: Damaged DNA binding protein 1; CRL4: Cullin-RING ligase 4. (Created with Biorender.com)

Moreover, cccDNA can also evade host immune attacks through post-transcriptional modifications involving alternative splicing of mRNA.^[38,39] In such cases, while cccDNA retains some transcriptional activity, the splicing variants inhibit the effective translation and expression of viral proteins, with one of the main HBV splicing sites located at 489nt.^[40,41] Consequently, the host immune system cannot effectively clear these infected cells, leading to undetectable levels of serum HBV DNA and HBsAg despite positive HBV RNA detection in liver tissue. In addition, these splicing variants cannot support the replication of progeny viral DNA. For example, the predominant splice variant Sp1 disrupts the translational integrity of the Pol open reading frame, thereby preventing the completion of the HBV replication lifecycle.^[42] Finally, it is important to emphasize that such epigenetic suppression of viral promoters or post-transcription splicing editing is reversible, which means that hepatocytes harboring transcriptionally active cccDNA or iDNA with the potent capability of reexpressing viral proteins and HBV DNA replication can persist in the liver tissue of these “cured” patients.

A study conducted by Gao et al.^[13] revealed that approximately 72% of patients who achieved a functional cure still exhibited persistent HBV transcription in liver tissue, with iDNA being the primary source of transcription. Additionally, 25.5% of functionally cured patients still showed detectable HBsAg expression in liver tissue,^[13] indicating that iDNA within the liver tissue of functionally cured patients can still undergo transcription and translation.

Two categories of proteins derived from iDNA are present in these liver tissues. The first type comprises viral-host fusion proteins. The expression of HBsAg-host fusion proteins have been detected in HCC cell lines, such as Hep3B, HuH-1, and PLC/PRF/5.^[43,44] Clinically, multiple HBV-host chimeric transcripts are detected in HBeAg-negative CHB patients.^[19] Consistent with this observation, Wen et al.^[45] reported the sustained presence of HBs-FN1 transcripts in CHB patients receiving long-term antiviral therapy, further supporting this viewpoint. Most importantly, a recent study also confirmed the presence of HBV-host chimeric transcripts in the liver tissue of cured patients.^[12]

The second type is the HBsAg protein, but its secretion is impaired and retained intracellularly in dominant.^[20] This occurs because the core promoter (CP) of iDNA is deleted, which enables the enhancer I to selectively activate the SP1 and SP2 promoters, with a stronger effect on SP1 than on SP2. As a result, large HBsAg expression predominates, leading to reduced secretion efficiency of HBsAg derived from iDNA in HBeAg-negative patients.^[20] This finding provides a reasonable explanation for the phenomenon in HBeAg-negative patients, whereby liver tissue shows abundant HBsAg-positive hepatocytes while serum HBsAg levels remain much lower than in HBeAg-positive patients. Based on this, it is reasonable to ask whether reduced circulating HBsAg levels still allow effective stimulation of the immune system to generate HBsAg-specific cellular immunity and whether this may hinder the achievement of a functional cure.^[46] Of course, another possibility for undetectable serum HBsAg is that serum HBsAg has not been truly cleared, but instead, undergoes mutations in the HBV S region that affect the antigenicity of HBsAg and allow it to escape detection by most detection kits. This represents a “false” HBsAg clearance due to mutation.^[47] However, with the continual development and improvement of detection kits, such instances of false-negative results will decrease.

In summary, under sustained host immune surveillance, infected hepatocytes expressing/secreting viral antigens would be immediately identified and eliminated, which provides an explanation for the absence of serum viral protein detection. In other words, the “functional cure” achieved through the maintenance of viral protein expression silence and viral replication suppression under host immune pressure resembles a “whack-a-mole” game between the host immune system and the residual virus (Figure 2). Thus, unsurprisingly, a substantial proportion of functionally cured patients remain at risk of viral rebound and clinical recurrence. Therefore, it is necessary to continue consolidation therapy for at least 24 weeks after HBsAg seroclearance and to conduct regular follow-up monitoring after treatment cessation, as the active transcription of viral DNA is “eternal” , at least to some extent.

Figure 2.

Functional cure resembles a “whack-a-mole” game, where host immune pressure strongly suppresses viral replication and protein expression. After a significant reduction in viral load, host HBV-specific immunity is restored. Under the surveillance of restored host immunity, residual viral DNA transcription and translation activities become effectively suppressed, and as a consequence, viral replication persists in a sustained suppression state through host-mediated regulatory mechanisms. cccDNA: Covalently closed circular DNA; iDNA: Integrated HBV DNA; HBV: Hepatitis B virus; HBsAg: Hepatitis B surface antigen; CTL: Cytotoxic T lymphocytes. (Created with Biorender.com)

3.3. Limitations of current therapies in achieving a functional cure for CHB

In addition to the aforementioned mechanisms of immune escape, such as epigenetic modifications silencing viral promoters and post-transcriptional splicing variants, current antiviral agents are inherently limited in achieving complete viral eradication. However, they provide a foundation for achieving a functional cure. For example, the commonly used clinical treatment strategy involves using NAs to reduce viral load, followed by sequential Peg-IFNα therapy in patients with low HBsAg levels to achieve a functional cure.^[48,49] Nevertheless, both drugs have some shortcomings that partially explain why most functional cures result in undetectable serum virological markers under immune surveillance rather than sterilizing viral clearance.

NAs do not directly target cccDNA or iDNA. Their antiviral mechanism involves metabolic conversion into structural analogues of deoxynucleotides, which competitively inhibit HBV polymerase reverse transcriptase activity by binding to its catalytic site. Alternatively, they can be incorporated into the negative strand of HBV during reverse transcription, terminating its elongation and blocking rcDNA synthesis from pregenome RNA (pgRNA), exerting antiviral effects.^[50] However, because NAs function by competitively inhibiting viral polymerase, it is theoretically impossible for them to achieve complete viral suppression.^[51]

Peg-IFNα primarily activates the host antiviral immune response.^[52] However, HBV splicing variants prevent the effective translation and expression of viral proteins, rendering Peg-IFNα incapable of mediating immune clearance against these transcriptionally silenced or spliced transcripts. Additionally, studies have reported that Peg-IFNα can upregulate PD-L1 expression in hepatocytes, induce T-cell apoptosis,^[53,54] and diminish CD8⁺ T-cell populations,^[55] which also hinders the immune system from completely clearing HBV infection. These factors collectively provide a theoretical basis for the maintenance of extremely low cccDNA levels under current antiviral therapies.

4. The implications of the functional cure concept for therapeutic development and clinical practice

It is widely accepted that viral load reduction is essential to alleviate immune exhaustion and suppression in hosts chronically exposed to elevated viral antigen levels.^[56] The “undetectable” virological markers in circulation in functionally cured patients indicate that although the HBV in the body has not been completely eradicated, it is suppressed by the host immune system at an extremely low level. This phenomenon suggests that achieving a functional cure requires both viral load reduction and the restoration and enhancement of the host antiviral immune response. Therefore, the primary goal of a functional cure is to reduce the viral load. In short, to enable more patients to achieve a functional cure in the future, it is essential to develop more potent and faster-acting drugs that directly target and inhibit the transcription of cccDNA and iDNA as well as viral replication. In this regard, RNA interference (RNAi) and antisense oligonucleotides (ASO), which can simultaneously target viral transcripts from both cccDNA and iDNA, hold great promise.^[57,58,59] Blocking all viral protein expression would lead to undetectable circulating HBV DNA and the loss of HBsAg. However, because of the persistence of cccDNA, this phenomenon should not be overinterpreted. As expected, rapid virologic rebound and relapse inevitably occur after discontinuation of treatment.^[60,61]

Clustered regularly interspaced short palindromic repeats (CRISPR)-based gene editing targeting cccDNA and iDNA has the potential to eliminate all HBV genomes.^[62,63] However, safety concerns and effective delivery remain regarding the application of such gene editing technologies,^[64] though we have developed a blue-light inducible system to enhance the intracellular delivery efficiency of CRISPR-associated protein 9/guide RNA in infected hepatocytes.^[65] Thus, further extensive research is required to fully address and resolve these issues before clinical implementation.

Several recent studies have demonstrated that the use of RNAi could effectively reduce viral load and possibly serve as a foundational treatment in combination with peg-IFNα as immune modulators, which could increase functional cure rates in CHB patients.^[66,67] As for residual silenced cccDNA, it is unresponsive to direct antiviral drugs or immune modulators such as interferons.^[68] Therefore, restoring and establishing a robust host immune response against HBV is critical to preventing viral rebound and clinical relapse.

The phenomenon of “undetectable” virological markers in circulation in functionally cured patients suggests that, in the selection of treatment strategies, we should advocate for future antiviral therapies that may involve NAs, RNAi, and capsid inhibitors to reduce viral load and deplete the cccDNA pool, followed by or combined with Peg-IFNα therapy, therapeutic vaccines, and other immunomodulatory drugs. By optimizing drug combinations in cocktail therapies, we can facilitate functional cures for HBV.^[60,61]

In terms of current clinical treatment strategies, some studies have shown that consolidation of antiviral therapy for 12–24 weeks or longer after serum HBsAg loss to further facilitate immune reconstitution beyond viral suppression, thereby lowering the risk of relapse after discontinuation.^[69,70,71] Additionally, evidence suggests that higher levels of protective antibodies at the time of discontinuation are associated with a lower rate of HBsAg seroreversion and virological relapse.^[71,72] Therefore, appropriately extending the duration of interferon therapy and combining it with therapeutic vaccines^[73] may promote the emergence of protective antibodies, increase their levels, and reduce the risk of HBsAg reversion and viral relapse after discontinuation of treatment. Furthermore, for patients who have achieved a functional cure, structured post-treatment surveillance should include dynamic monitoring of virological biomarkers coupled with longitudinal evaluation of HBV-specific immune responses (e.g., quantitative hepatitis B surface antibody titers and levels of HBcAg-, Pol-, and HBsAg-specific T-cell response).^[24,74] Notably, although HBV-related complications, including HCC, are significantly reduced in functionally cured patients, a subset of individuals—particularly those with cirrhosis or those who achieve a functional cure after the age of 50—may still harbor iDNA and transcriptionally inactive cccDNA in the liver. Consequently, lifelong HCC surveillance is warranted due to the persistent risk of HBV-related hepatocarcinogenesis.^[9,10] Specifically, regular monitoring of virological markers, liver function indicators, and serum tumor markers (such as alpha-fetoprotein and protein induced by vitamin K absence or antagonist-II^[75]), as well as liver ultrasound, should be conducted. When necessary, contrast-enhanced computed tomography or contrast-enhanced magnetic resonance imaging may be performed with precise timing of image acquisition phases to avoid missed diagnoses.^[4]

5. Summary and outlook

In summary, for most CHB patients, functional cure fundamentally constitutes a state of virologically undetectable circulation mediated by reconstituted host immune control. Achieving this balance requires the comprehensive suppression of HBV replication and related viral protein expression alongside the enhancement of host antiviral immunity, both of which are indispensable. Consequently, the strategic integration of direct-acting antivirals with immunomodulators, complemented by therapeutic regimen optimization, may increase functional cure rates. The development of new drugs, such as RNAi/ASO, offers new hope for a functional cure for CHB. However, it is important to note that the rapid decline or even disappearance of serum HBsAg induced by these therapies differs significantly from the declination observed during natural disease progression or under prolonged NAs treatment,^[76] and this difference must be carefully considered. Lastly, we would like to emphasize that most patients who achieve a functional cure still face the risk of HBV reactivation. Therefore, consolidation therapy after HBsAg loss and protocolized long-term surveillance are essential.

Author Contributions

Kunyu Zhao, Lili Wu, and Yu Wu contributed to the conception and drafting of the manuscript; Kunyu Zhao, Lili Wu, Qianqian Jiang, Mengmeng Zhu and Suzhen Jiang were responsible for the design and revision of the manuscript; Chao Zhang formulated the writing strategy and made revisions to the manuscript; Fengmin Lu formulated the writing strategy, guided the preparation and revision of the manuscript. All authors read and approved the final manuscript.

Conflicts of Interest

None.

Funding Statement

The work was supported by Sanming Project of Medicine in Shenzhen (SZSM202311032).