Hepatocyte Apoptosis: A Novel Mechanism for Targeting Hepatitis B Viral Infection

Posted on: October 30, 2025

Contributed by Abigail Kasberg, PhD

Hepatitis B is a viral infection that is caused by the Hepatitis B virus (HBV). Infection with HBV primarily affects the liver, driving acute and chronic liver disease. Long-term HBV infection can lead to cirrhosis, liver failure, and hepatocellular carcinoma (HCC) (Figure 1). HBV infection is the leading cause of HCC and causes more than 850,000 deaths per year (Shen et al. 2023). Acute liver failure is a rare but severe complication of HBV infection, resulting in the sudden deterioration of liver function (Oketani et al. 2014). When Chronic Hepatitis B (CHB) advances to HCC, the liver undergoes a series of pathological changes through viral gene integration and epigenetic changes, chronic inflammation, persistent immune response, and tissue damage (Shen et al. 2023). Despite the availability of effective vaccines, HBV infection remains a global health concern, contributing to substantial morbidity and mortality worldwide (Shen et al. 2023).

In CHB, the virus persists within hepatocytes as covalently closed circular DNA (cccDNA), creating stable reservoirs that evade immune clearance. HBV cccDNA functions as a mini chromosome, serving as a template for viral mRNA. Conventional antivirals suppress viral replication but rarely eliminate the virus or restore immune control due to the persistence of the cccDNA in the nucleus (Shen et al. 2023). As a result, a wave of investigational therapies are targeting HBV via a different approach: the elimination of HBV-infected hepatocytes through induced apoptosis. Here, we examine the emerging strategies that leverage induced cell death mechanisms to eliminate HBV-infected hepatocytes, highlighting the use of CK18 biomarkers to monitor and quantify this process.

Developing Hepatitis B Treatment Approaches

New therapeutics are in development that explore mechanisms driving apoptosis in HBV-infected hepatocytes, either directly or through immunomodulatory mechanisms to enhance host immune responses that eliminate infected cells. Here’s a sampling of some strategies that have been investigated to study the effects of cell death to clear HBV-infected liver cells:

• CTL-mediated apoptosis of infected hepatocytes
  Apoptosis of infected cells can be a strategy in antiviral defense, however it is important to consider the possibility that apoptosis may also support the release and spread of viruses. HBV-specific cytotoxic T lymphocytes (CTLs) trigger apoptosis in infected hepatocytes of HBV-transgenic mice. However, viral nucleocapsids and DNA intermediates can survive CTL-mediated apoptosis, suggesting that targeting the apoptosis of infected cells may not be effective for complete viral clearance (Pasquetto et al. 2000).
• Induced apoptosis to prevent HBV replication
  Inducing apoptosis through UV-C irradiation and/or anti-CD95 antibody treatment in HBV-producing liver cells significantly reduced the release of mature, infectious HBV virions by 80-90%, instead leading to the release of immature, nonenveloped, and non-infectious viral particles (Arzberger et al. 2010).  These findings suggest that apoptosis is detrimental to the virus, and HBV likely benefits from preventing host cell death to ensure the release of infectious viral progeny (Arzberger et al. 2010). 
• Stimulation of Cytoplasmic Nucleic Acid Sensors
  RNA sensors such as RIG-I recognize viral RNA and trigger antiviral responses through cytokine production and self-induced apoptosis (Gehring et al. 2019). Therapies that stimulate RIG-I, such as modified siRNAs, enhance both antiviral signaling and apoptosis to inhibit HBV replication (Ebert et al. 2011).
• Immune-based vaccines to mobilize immune cells and trigger removal of infected cells.
  An HBx-based therapeutic vaccine acts as a major immunogen, inducing robust systemic HBx-specific T cell responses in HBV carrier mice (Horng et al. 2020). The HBx vaccination drives the mobilization of monocytes into the liver, creating an immune environment that supports effective T cell activity. Together, these vaccine-induced immune responses result in the clearance of HBV-expressing hepatocytes, highlighting HBx as a promising therapeutic target for chronic HBV infection (Horng et al. 2020).
  

CK18: A Biomarker to Investigate Mechanism of Action

As hepatocyte apoptosis gains attention as a therapeutic target, cytokeratin-18 (CK18) has emerged as a valuable biomarker for monitoring hepatocellular death. CK18 is a structural protein abundantly expressed in the liver and, specifically, hepatocytes. When hepatocytes die, intact CK18 and its caspase-cleaved fragment (ccK18) are released into the circulation. Blood levels of ccK18 are indicative of apoptosis, whereas levels of total CK18 (intact and cleaved) are reflective of overall hepatocyte death, encompassing both necrosis and apoptosis. Measuring these proteins – using the Peviva M30® Apoptosense® assay for ccK18 and Peviva M65® assays for total CK18 -provides a noninvasive means to quantify hepatocyte apoptosis and necrosis in the context of liver injury or therapeutic intervention.

Research studies measuring ccK18 and CK18 highlight their potential as noninvasive indicators of HBV-related liver injury and disease activity.

• Histological Correlation:
  Serum ccK18 levels positively correlated with histological activity index (HAI) scores in both asymptomatic HBV carriers and subjects with CHB. Notably, ccK18 levels were significantly higher in CHB subjects compared to asymptomatic carriers (Balkan et al., 2017). These findings position ccK18 as a promising noninvasive biomarker of liver disease activity in HBV infection.
• Indicator of Fibrosis and Inflammation Severity:
  ccK18 levels showed significant correlation with liver inflammation and fibrosis in HBV-infected subjects (Swiderska et al., 2017). Importantly, ccK18 demonstrated sensitivity and specificity in discriminating fibrosis severity and inflammation, even in cases where the standard biomarker ALT remained within normal limits.
• Prognostic value for HBV-related ACLF:
  In another study, both ccK18 and CK18 were elevated in CHB subjects and in those with CHB-related acute-on-chronic liver failure (ACLF), compared to healthy controls (Zheng et al., 2014). These results support an association between CK18 biomarkers, hepatocyte apoptosis/necrosis, and disease severity. Moreover, the M30®/M65® ratio correlated with outcomes in ACLF, suggesting potential prognostic value for monitoring progression of HBV-related liver deterioration (Zheng et al., 2014).

Preclinical Use: Drug Candidate Screening & Mechanistic Studies

Evaluating hepatocyte apoptosis early in the research & development process is beneficial for evaluating the impact of new Hepatitis B drug candidates on HBV-infected hepatocytes. By incorporating ccK18 measurements into preclinical research and clinical trials, researchers can:

• Distinguish apoptosis-driven liver injury from other forms of liver damage.
• Correlate ccK18 levels with virologic and immunologic outcomes.
• Optimize dosing of investigational pro-apoptotic therapies to reduce off-target toxicity.

This can be studied by treating HBV-infected cell lines or liver organoids with investigational drugs, followed by measuring changes in apoptotic cell death using biomarkers such as ccK18. This is important for multiple reasons:

1. Antiviral drugs aim to reduce ongoing hepatocyte apoptosis by suppressing the virus. Measurements of ccK18 could be leveraged to verify if a drug candidate effects baseline apoptosis caused by HBV.
2. Investigational therapies may include mechanisms of action that target and induce cell death of HBV-infected hepatocytes as a means of eradicating infection. M30^® and M65^® assays can be used to detect increases in cell death in treated cell cultures, providing measurable evidence of the drug’s ability to target and eliminate HBV-infected cells.
3. Toxicology studies of novel compounds assess unintended hepatocyte cell death and liver damage caused by investigational drugs. ccK18 and CK18 biomarkers have shown efficacy in detecting hepatoxicity in preclinical studies of drug-induced cell death and toxicity studies (Antoine et al. 2009; Cummings et al. 2008).

Clinical Trial Use: Cell Death Biomarker

Hepatitis B clinical trials are increasingly incorporating biomarkers of hepatocyte apoptosis to monitor treatment response, guide subject stratification, and evaluate the physiological effects of investigational drugs on the liver. Therapies that suppress HBV replication typically reduce hepatocyte apoptosis (Figure 2). In contrast, some therapies may intentionally induce apoptosis in HBV-infected hepatocytes to eliminate infected cells, while others could cause unintended hepatotoxicity, resulting in increased cell death (Figure 2). Monitoring these different outcomes is critical for assessing efficacy and safety in HBV clinical trials.

Subject Stratification:

As stated above, ccK18 and CK18  have shown promise as a indicators of CHB disease activity (Zheng et al., 2014; Swiderska et al., 2017). These findings highlight the potential of CK18 and ccK18 to be leveraged as subject stratification biomarkers in clinical trials. By quantifying apoptosis versus necrosis, ccK18 and CK18 measurements may help identify subjects more likely to recover versus those at higher risk of decompensation, enabling more targeted enrollment, stratification, monitoring, and decision-making. Further research is warranted to validate the predictive value of ccK18 and CK18 in these populations.

Response to Treatment Biomarker:

HBV therapeutic developers can benefit from incorporating ccK18 and CK18 biomarkers into drug development programs. For therapies such as vaccines, T-cell therapies, or toll-like receptor agonists, the M30^® Apoptosense assay can quantify immune-mediated killing of infected hepatocytes in clinical trials.

In severe HBV infection, progression to acute liver failure increases mortality and may necessitate transplantation, with hepatocyte death as a key feature (Cao et al., 2019; Zheng et al., 2014). Monitoring treatment response in real time is therefore critical. Preliminary research evidence supports CK18 as a useful biomarker. In HBV-associated acute liver failure subjects treated with entecavir, serum CK18 levels dropped significantly within the first week, reflecting reduced hepatocyte death and providing an early indication of therapeutic efficacy (Jochum et al., 2009).

Physiological Impacts of investigational Drug:

In Hepatitis B clinical trials, longitudinal monitoring of ccK18 and CK18 biomarkers provides insight into the physiological impact of investigational therapies. A decline in ccK18 may reflect reduced hepatocyte apoptosis and mitigation of liver injury, whereas rising CK18 levels could signal ongoing damage or potential hepatotoxicity. Incorporating ccK18 and CK18 biomarkers alongside virological endpoints provides drug developers with a more complete view of treatment effects, enabling early detection of both beneficial and adverse physiological effects.

Nucleos(t)ide analogue antivirals reduce necroinflammation and support hepatocyte recovery during CHB treatment. In a clinical study, CHB subjects receiving these antivirals exhibited significant decreases in serum ccK18 and CK18 levels, paralleling declines in viral load (Farnik et al. 2011). These findings suggest that effective antiviral treatment alleviates liver injury, likely by limiting hepatocyte death and allowing the liver to begin healing from HBV-mediated damage, though the precise mechanisms warrant further investigation.

Pharmaceutical Hepatitis B Pipeline

The development of novel therapies for Hepatitis B remains an active area of investigation. Multiple investigational drugs in the pipeline either directly induce hepatocyte apoptosis or indirectly influence hepatocyte cell death (Table 2).

Investigational Drug	Developer	Mechanism of Action	Target	Clinical Trial	Potential Effect on Hepatocyte Cell Death
Hepatocyte Death Modulators
APG-1387	Ascentage Pharma	SMAC mimetic	HBV-infected hepatocytes	Phase 2 NCT04568265	Directly induces apoptosis of infected hepatocytes
Antivirals
Bepirovirsen (GSK3228836)	GSK	Antisense oligonucleotide; reduces HBV RNA and surface antigen	HBV RNA	Phase 2 NCT04449029Phase 3NCT05630807 NCT05630820	Indirect reduction of apoptosis (via viral suppression)
CRISPR-Cas13 mRNA Therapy	VCCC Alliance	RNA-targeting CRISPR to degrade HBV RNA	HBV RNA	Preclinical	Potentially induces apoptosis of infected hepatocytes
RG6346 (RO7445482; Xalnesiran)	Dicerna/Roche	siRNA that targets and silences HBV transcripts	HBV mRNA	Phase 2 NCT04225715	Indirectly promotes apoptosis via immune clearance
Tune-401	Tune Therapeutics	Epigenetic silencing of HBV gene expression	HBV DNA &   HBV cccDNA	Phase 1b NCT06671093	Indirectly affects apoptosis via reduced viral activity
Immune Modulators
GS-9620 (Vesatolimod)	Gilead Sciences	TLR7 agonist; induce antiviral response	HBV-infected hepatocytes	Phase 2 NCT02166047	May induce apoptosis via immune activation
GS-9688 (Selgantolimod)	Gilead Sciences	TLR8 agonist; induce pro-inflammatory and immunomodulatory cytokines	HBV-infected hepatocytes	Phase 2  NCT03491553	May induce apoptosis via immune activation
HB-400 (GS-2829; GS-6779)	Gilead / HOOKIPA Pharma	Arenavirus-based vaccine; induce immune response against Chronic Hepatitis B	HBV-infected hepatocytes	Phase 1 NCT05770895	May induce clearance of HBV-infected cells via immune stimulation

Advancing HBV Therapies with Cell Death Insight

From early-stage screening in the lab to late-stage clinical trials, ccK18 and CK18 biomarkers are being leveraged in valuable ways to support HBV drug development. ccK18 and CK18 biomarkers enable researchers to track cell death in HBV-infected hepatocytes with precision, whether to detect liver injury in response to a novel toxic compound before it goes too far, or to verify that an immune-based therapy is performing as expected by eliminating infected cells. Importantly, these apoptosis and cell death measurements tie into the larger goals of HBV treatment to prevent liver failure and cancer. By monitoring and modulating hepatocyte death, companies can ensure their drug candidates are working toward viral clearance and liver healing, rather than uncontrolled cell death. Together, this research paves the way for broader use of ccK18 and CK18 biomarkers among HBV research programs, fostering smarter trial designs, robust biomarker-supported endpoints, and ultimately, better outcomes for individuals with HBV infection.

As the field of HBV therapy moves toward curative strategies, hepatocyte apoptosis is becoming a cornerstone of drug development. Whether directly induced via SMAC mimetics or indirectly through immune activation, the goal is to eliminate infected cells while preserving overall liver function. ccK18 offers a promising research tool to monitor these effects, enabling precision in drug development.

Further Reading:

Peviva M30® Apoptosense® for ccK18 and Peviva M65® assays for total CK18 are labeled for Research Use Only (RUO) and are not intended for clinical diagnostic use. Any mention of clinical utility in this reference list is not supported by the manufacturer or distributor.

Antoine, Daniel J., et al. “High-Mobility Group Box-1 Protein and Keratin-18, Circulating Serum Proteins Informative of Acetaminophen-Induced Necrosis and Apoptosis in Vivo.” Toxicological Sciences: An Official Journal of the Society of Toxicology, vol. 112, no. 2, Dec. 2009, pp. 521–31. PubMed, https://doi.org/10.1093/toxsci/kfp235.

Arzberger, Silke, et al. “Apoptosis of Hepatitis B Virus-Infected Hepatocytes Prevents Release of Infectious Virus.” Journal of Virology, vol. 84, no. 22, Nov. 2010, pp. 11994–2001. DOI.org (Crossref), https://doi.org/10.1128/JVI.00653-10.

Balkan, Ayhan, et al. “Relationship between Liver Injury and Serum Cytokeratin 18 Levels in Asymptomatic Hepatitis B Virus Carriers and in Patients with Chronic Hepatitis B Infection.” Arab Journal of Gastroenterology: The Official Publication of the Pan-Arab Association of Gastroenterology, vol. 18, no. 2, Jun. 2017, pp. 98–103. PubMed, https://doi.org/10.1016/j.ajg.2017.05.008.

Cao, Zhujun, et al. “Serum Keratin-18 Fragments as Cell Death Biomarker in Association with Disease Progression and Prognosis in Hepatitis B Virus-Related Cirrhosis.” Journal of Viral Hepatitis, vol. 26, no. 7, Jul. 2019, pp. 835–45. PubMed, https://doi.org/10.1111/jvh.13100.

Cummings, Jeffrey, et al. “Preclinical Evaluation of M30 and M65 ELISAs as Biomarkers of Drug Induced Tumor Cell Death and Antitumor Activity.” Molecular Cancer Therapeutics, vol. 7, no. 3, Mar. 2008, pp. 455–63. DOI.org (Crossref), https://doi.org/10.1158/1535-7163.MCT-07-2136.

Ebert, Gregor, et al. “5’ Triphosphorylated Small Interfering RNAs Control Replication of Hepatitis B Virus and Induce an Interferon Response in Human Liver Cells and Mice.” Gastroenterology, vol. 141, no. 2, Aug. 2011, pp. 696–706, 706.e1-3. PubMed, https://doi.org/10.1053/j.gastro.2011.05.001.

Farnik, Harald, et al. “Nucleos(t)Ide Analogue Treatment Reduces Apoptotic Activity in Patients with Chronic Hepatitis B.” Journal of Clinical Virology, vol. 52, no. 3, Nov. 2011, pp. 204–09. DOI.org (Crossref), https://doi.org/10.1016/j.jcv.2011.08.009.

Horng, Jau-Hau, et al. “HBV X Protein-Based Therapeutic Vaccine Accelerates Viral Antigen Clearance by Mobilizing Monocyte Infiltration into the Liver in HBV Carrier Mice.” Journal of Biomedical Science, vol. 27, no. 1, May 2020, p. 70. PubMed, https://doi.org/10.1186/s12929-020-00662-x.

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Pasquetto, Valerie, et al. “Intracellular Hepatitis B Virus Nucleocapsids Survive Cytotoxic T-Lymphocyte-Induced Apoptosis.” Journal of Virology, vol. 74, no. 20, Oct. 2000, pp. 9792–96. DOI.org (Crossref), https://doi.org/10.1128/JVI.74.20.9792-9796.2000.

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Zheng, Su-Jun. “Prognostic Value of M30/M65 for Outcome of Hepatitis B Virus-Related Acute-on-Chronic Liver Failure.” World Journal of Gastroenterology, vol. 20, no. 9, 2014, p. 2403. DOI.org (Crossref), https://doi.org/10.3748/wjg.v20.i9.2403.