Editor's Note: In the field of chronic HBV infection, the discovery of basic core promoter (BCP) and pre-core (PC) mutations dates back nearly thirty years. However, the potential link between these mutations and the progression of chronic hepatitis B (CHB), as well as their impact on antiviral treatment response, has been a focal point for virologists for a long time. At the 2024 European Association for the Study of the Liver (EASL) Annual Meeting, Professor Fengmin Lu from the Peking University Center for Infectious Diseases shared his team’s latest research findings. Firstly, they revealed the impact of BCP+PC mutant virus infection on hepatocytes, indicating that this mutant virus induces more extensive and severe hepatocyte degeneration and cytoplasmic vacuolization, and suppresses the endogenous IFN-α/γ signaling pathway in human hepatocytes. This discovery may offer a new explanation for the poor response to interferon therapy in some patients. Additionally, Professor Lu delved into the regulation and secretion mechanisms of HBsAg expression in integrated HBV DNA, proposing new strategies for CHB treatment based on these findings. We had an in-depth interview with Professor Lu, and the content is organized here for our readers.Research Progress and Potential Treatment Strategies for BCP and PC Mutations in Chronic HBV Infection
Hepatology Digest: Can you elaborate on the prevalence of BCP and PC mutations in chronic HBV infection and their potential link to disease progression?
Professor Lu: Interest in the pre-core (PC) stop codon mutation and core promoter (BCP) mutations of HBV, which encode the precursor of HBeAg, began with an outbreak of acute hepatitis B in the 1990s at an Israeli hospital due to iatrogenic transmission, resulting in several deaths. Dr. Jack Liang from the NIH in the U.S. conducted pathogenic analysis and traced the source to a CHB patient carrying the PC mutation. Around the same time, Japanese scholar Dr. Omata also reported PC mutations in patients with acute fulminant hepatitis in Japan, linking the mutation to severe hepatitis.
Since PC mutations often occur on the BCP mutation background, the latter also received due attention. Later epidemiological studies showed that both mutations are unfavorable for establishing chronic infection post-exposure. Therefore, we observe that despite the increasing proportion of these mutant strains over time and their dominance in chronic infection, wild-type HBV primarily establishes chronic infection, whether through vertical transmission or adult sexual transmission.
A recent multicenter, multi-ethnic, multi-genotype study of chronic HBV infection showed that approximately 18.4% of patients predominantly (more than 50%) carried PC mutant strains, 24.2% carried BCP mutants, and 11.0% carried both PC and BCP mutations. Only 46.3% predominantly carried wild-type HBV. With the disappearance of HBeAg and/or seroconversion, the proportion of mutant strains as the dominant virus in chronic infection patients rises to 71.1%. Similarly, with age, the proportion of mutant strains increases significantly, reaching 55.8% and 75.2% in HBeAg-positive and negative patients over 40 years old, respectively. This suggests these mutations may play a role in maintaining chronic infection.
Consistent with this, our recent meta-analysis shows that PC mutations are associated with higher serum HBV DNA levels in HBeAg-negative chronic HBV carriers, but not in HBeAg-positive patients. Further stratified analysis found that PC mutations are associated with chronic hepatitis, indicating they may promote disease progression by exacerbating liver damage. The meta-analysis indeed shows a significant association between PC mutations and the occurrence of cirrhosis and HCC, and antiviral treatment can reduce the risk of cirrhosis related to PC mutations. Furthermore, the pathogenicity of PC mutations may not differ significantly among different genotypes.
Hepatology Digest: What key genes and molecular pathways related to BCP+PC mutations did you discover in your transcriptome analysis? How do these findings help us better understand the pathogenesis of HBV infection?
Professor Lu: The experimental mice used in this study were human-liver-chimeric mice created by transplanting primary human hepatocytes into the livers of immunodeficient mice, resulting in a co-existing state of human and residual mouse hepatocytes. It is generally believed that liver damage is caused by HBV-specific immune clearance of infected hepatocytes, but our H&E staining of liver tissues from these immunodeficient human-liver-chimeric mice revealed that, compared to wild-type (WT) virus, BCP+PC mutant virus infection induces more extensive and severe hepatocyte degeneration and cytoplasmic vacuolization, indicating that BCP+PC mutant virus infection causes more severe damage to human hepatocytes in chimeric mice.
Differential expression profiling, clustering, and functional analysis of sequencing results from human hepatocytes infected with WT and mutant viruses revealed that ER stress (ERS) and TNF signaling pathways are activated in human hepatocytes infected with BCP+PC mutant strains. RT-qPCR and 8-OHdG histochemical staining showed significantly elevated mRNA levels of GRP78, XBP1, ATF4, CHOP, GADD34, ERO1A, and ERO1B in human hepatocytes infected with BCP+PC mutant viruses, and 8-OHdG-positive cells were present in liver tissues from BCP+PC group mice, but rarely detected in WT group mice. This confirms that mutant strains cause stronger ERS and ROS formation in human hepatocytes. Enrichment analysis also shows that BCP and PC mutations jointly suppress the endogenous IFNα/γ signaling pathway in human hepatocytes, suggesting this could be why cccDNA with BCP and PC mutations responds poorly to IFNα treatment.
The difference in the hepatocyte membrane receptor NTCP between humans and mice prevents mouse hepatocytes from being infected by human HBV. The extensive damage and death of human hepatocytes in mice infected with BCP+PC mutant viruses suggest compensatory proliferation of residual mouse hepatocytes in the chimeric liver tissue. Consistent with this, analysis based on the mouse genome reference confirms significant activation of the cell cycle and Hippo signaling pathways in liver tissues from mice infected with BCP+PC mutant strains, indicating that human hepatocyte damage induces compensatory proliferation of mouse hepatocytes.
Hepatology Digest: Based on this study, what potential therapeutic strategies do you see for addressing BCP+PC mutations in chronic HBV infection? How should these strategies be developed further in future research and clinical trials?
Professor Lu: Mutant viruses have a stronger ability to maintain the cccDNA pool. This hypothesis aligns somewhat with previous clinical findings: compared to baseline HBeAg-positive patients, HBeAg-negative patients achieve undetectable HBV DNA levels more quickly with nucleos(t)ide analogs (NAs) treatment but are more prone to virological rebound after discontinuation. Additionally, we can even anticipate that HBeAg-negative patients (predominantly wild-type strains) who seroconvert to HBeAg-negative status during NAs treatment might achieve better treatment outcomes and clinical cure compared to naturally occurring HBeAg-negative patients dominated by mutant strains. This suggests we should initiate antiviral therapy early in patients to reduce potential mutations and accumulation, thereby slowing disease progression.
It is important to emphasize that there have been some reports on the poor response of PC/BCP mutant viruses to IFN therapy, but whether PC/BCP mutations affect the efficacy of NA treatment and whether they can guide antiviral therapy requires further clinical observation. This is feasible in the current era of NA-dominated treatment, and such studies can be easily conducted. Additionally, future prospective cohort studies could evaluate the impact of sustained infection with mutant viruses on liver cancer immunotherapy, guided by HBV mutation detection.
In summary, from a clinical practice perspective, detecting PC mutations has the potential to predict disease progression and antiviral treatment response and may guide the clinical management of chronic HBV infection in the future, especially in East Asia and Southeast Asia, where HBV genotypes B and C are prevalent.
Mechanisms of HBsAg Secretion from Integrated HBV DNA and New Strategies for CHB Treatment
Hepatology Digest: Your other study found that HBsAg expressed by integrated HBV DNA is inconsistent in composition and secretion with that expressed by cccDNA. Could you explain the biological significance and clinical importance of this phenomenon?
Professor Lu: Achieving “functional cure” for CHB remains a significant challenge. One major reason is HBV DNA integration, which hinders functional cure. To uncover the unique characteristics of HBsAg from integrated HBV DNA and provide new insights for achieving functional cure, our team conducted stratified analysis of clinical cohorts, using animal and cell models to simulate the expression and secretion characteristics of integrated HBV DNA.
Through semi-quantitative analysis of intrahepatic HBsAg protein levels in 563 untreated patients, we found that although serum HBsAg levels were significantly lower in HBeAg-negative patients compared to HBeAg-positive patients, intrahepatic HBsAg protein levels did not significantly decrease. Further studies showed this might be due to the relatively higher proportion of 2.4 kb HBV RNA in integrated HBV DNA, leading to overexpression of large surface antigen (L-HBsAg), affecting HBsAg secretion efficiency.
Mechanistic analysis revealed this change results from the loss of the core promoter in integrated HBV DNA, causing the Enhancer I (EnhI) to reorient towards the preS1 (SP1) promoter through a loss-of-scan mechanism. This uneven activation of SP1 and SP2 promoters results in L-HBsAg overexpression and intracellular retention, affecting HBsAg secretion.
Hepatology Digest: Based on this study, what insights can we draw? How should future CHB treatment strategies address the HBsAg produced by integrated HBV DNA?
Professor Lu: The expression and secretion of HBsAg from integrated HBV DNA are not fully consistent with serum HBsAg levels, indicating that besides cccDNA, integrated HBV DNA expression and secretion are critical factors making CHB difficult to cure.
Based on this finding, we propose new strategies for future CHB treatment. Firstly, the timing of treatment is crucial for preparing for future CHB cure. For example, antiviral treatment should be initiated during the immune tolerance phase to prevent integration and reduce liver inflammation, stopping the clonal expansion and active expression of HBsAg in hepatocytes carrying integrated HBV DNA, laying the groundwork for future clinical cure. Secondly, RNA interference therapy is currently known to effectively suppress HBsAg expression from integrated HBV DNA. Prolonged treatment to continuously reduce HBsAg expression might help restore cytotoxic T lymphocyte (CTL) function. This treatment approach may bring new hope for CHB cure.
In conclusion, this study reveals the expression and secretion mechanisms of HBsAg from integrated HBV DNA and their potential impact on CHB treatment strategies. This discovery not only provides new perspectives on the pathogenesis of CHB but also offers new approaches for future CHB treatment. With further research on integrated HBV DNA and the development of novel treatments, we have reason to believe that curing CHB will no longer be an unattainable dream.
