Editor’s Note: At the just-concluded Annual Summit of Hepatology, the American Association for the Study of Liver Diseases (AASLD2023), Professor Fengmin Lu’s team from Peking University presented three contributions (1 oral presentation and 2 posters), focusing on the mechanisms related to chronic HBV infection. Their research revealed populations benefiting from interferon therapy in chronic Hepatitis B (CHB) and explored potential new targets for curing. “Hepatology Digest” has specially invited Professor Fengmin Lu to interpret the research findings, their significance and implications for future scientific research or clinical practice, and to envision the prospects for functional cure among the CHB population in our country.
Research One
High TP53BP2 Expression Favors Functional Cure in CHB Patients Treated with Pegylated InterferonTop of Form
72: TP53BP2 IS ASSOCIATED WITH HBsAg CLEARANCE IN PEGINTERFERON-ALFA-TREATED CHRONIC HEPATITIS B
Professor Fengmin Lu delivered an oral presentation.
Top of Form
In patients with CHB undergoing interferon treatment, a small subset experiences HBsAg clearance, achieving the desired goal of functional cure. Identifying crucial host factors essential for CHB cure aids in recognizing patients who can derive maximum benefit from interferon therapy.
In this study, a total of 95 CHB patients treated with Peg-IFNα were included. Among them, 48 achieved HBsAg clearance while 47 did not. A genome-wide association study (GWAS) was conducted on these patients to precisely identify host genes contributing to HBsAg clearance. Subsequently, validation of these findings was performed on another cohort of 81 patients, where 207 individuals were treated with Peg-IFNα and 81 achieved HBsAg clearance. Verification included expression quantitative trait locus (eQTL) analysis and RNA transcriptome sequencing on liver biopsies from interferon-treated patients, alongside in vitro and in vivo experiments related to interferon therapy to validate the functional involvement of candidate genes.
The results revealed a significant association between the single nucleotide polymorphism (SNP) rs7519753-C allele and serum HBsAg clearance in CHB patients treated with Peg-IFNα (P=4.85×10-8, OR=14.47). This association was also observed in the validation cohort, with a significantly higher frequency of the rs7519753-C genotype in the HBsAg clearance group (P=0.008312, OR=1.64). RNA transcriptome sequencing analysis of liver biopsies from interferon-treated patients showed significantly higher TP53BP2 expression in the HBsAg clearance group (P<0.05), and carriers of the rs7519753C allele exhibited significantly elevated liver TP53BP2 expression (P=2.9×10-6). Further experiments demonstrated that under interferon treatment, TP53BP2 enhances the expression of interferon-stimulated genes (ISGs). Mechanistically, TP53BP2 downregulates the expression of the interferon response negative regulator SOCS2 at the transcriptional level, thereby boosting the antiviral activity of the JAK/STAT signaling pathway activated by interferon treatment.
The study findings suggest that the SNP rs7519753-C allele is associated with higher serum HBsAg clearance and increased liver TP53BP2 expression in CHB patients treated with Peg-IFNα. TP53BP2 enhances liver cell response to interferon by suppressing SOCS family gene expression. Hence, TP53BP2 plays a critical role in predicting and enhancing interferon treatment response in CHB patients.
Researchers state:
Researchers emphasize that in the absence of effective curative drugs for CHB, existing interferons exhibit a better HBsAg seroconversion rate compared to nucleos(t)ide analogs. However, when pursuing a cure through interferon treatment, selecting the appropriate patient population remains crucial. Apart from viral factors, the host’s genetic background significantly influences cure outcomes. Additionally, the timing of treatment is pivotal, especially considering the lack of effective means to clear HBV DNA-integrated hepatocytes. Early initiation of antiviral therapy should be considered as an option because initiating treatment early can reduce integration events, and the proliferation of integrated hepatocytes is a significant risk factor for liver cirrhosis and hepatocellular carcinoma.
Research Two
Mutations in the precore region (C region) of the virus enhance viral replication and result in abnormal expression of the core protein, thereby promoting the progression of chronic hepatitis B (CHB).
1518-C: PRECORE MUTATION PROMOTES CHRONIC HEPATITIS B DISEASE PROGRESSION VIA ENHANCED VIRAL REPLICATION AND ABERRANT CORE PROTEIN EXPRESSION
AASLD2023: AASLD Annual Meeting 2023
In patients with chronic HBV infection, mutations in the precore region (PC, G1896A) and the basal core promoter (BCP, A1762T/G1764A) commonly occur naturally. Numerous studies have focused on these mutations’ roles in viral replication and disease progression, yet definitive conclusions remain elusive. Professor Fengmin Lu’s research team conducted an extensive search of Pubmed, Embase, and the Cochrane Library for studies up until June 18, 2022, related to PC mutations and their association with HBV DNA levels, liver cirrhosis, or hepatocellular carcinoma (HCC). They utilized transfection or infection-based cellular and animal models to investigate the impact of singular PC mutations or combined BCP+PC mutations on viral replicability, infectivity, and pathogenicity.
The results from the meta-analysis revealed that among HBeAg-positive patients, serum HBV DNA levels in patients without the G1896A mutation were 0.37 log10 copies/mL higher compared to those with the PC mutation (2.34-fold change). However, among HBeAg-negative patients, those without the G1896A mutation exhibited serum HBV DNA levels 0.87 log10 copies/mL lower than those with the PC mutation (7.41-fold change). Additionally, the PC mutation was closely associated with liver cirrhosis and hepatocellular carcinoma compared to the matched stratified control group without PC mutations.
Subsequently, researchers found that the PC mutation conferred preC RNA with pgRNA-like functionality, enabling the expression of core and P proteins. In transfected cells and mouse models, the PC mutation facilitated HBV replication. Compared to the singular PC mutation, BCP+PC mutations further increased HBV DNA levels. In HepG2-NTCP cells and human liver chimeric mice, both PC and BCP+PC mutations consistently reduced infectivity while enhancing virus DNA replication. RNA sequencing of chimeric mouse liver tissues revealed that the BCP+PC mutations suppressed the host IFN signaling pathway in human liver cells, likely leading to increased cccDNA levels. Crucially, the accumulation of core proteins possibly triggered severe endoplasmic reticulum stress and TNF signaling pathways, causing significant hepatocellular degeneration and cytoplasmic vacuolation in BCP+PC mice livers. The enhanced cell cycle and hippo pathway in these mice’s livers suggest the potential for compensatory hepatocyte proliferation.
Overall, both PC and/or BCP mutations enhance HBV replication in vitro and in vivo. However, the association between PC mutations and higher HBV DNA levels was observed only in HBeAg-negative patients. PC mutations were closely linked to the progressive stages of liver disease in chronic HBV-infected patients. Combined BCP and PC mutations may induce severe hepatocellular damage in human liver-chimeric mice by augmenting viral replication and abnormal core protein expression.
Researchers state:
The high-frequency mutations of A1762T/G1764A in chronic hepatitis B patients have long been observed. In this study, employing advanced long-sequencing techniques, we delved deeper into how A1762T/G1764A affects HBV transcription. We confirmed that A1762T/G1764A not only selectively enhances pgRNA transcription but also suppresses preC mRNA transcription. More importantly, we discovered for the first time that A1762T/G1764A can elevate sub-pgRNA transcription levels, providing a new perspective on how it enhances HBV replication. Furthermore, our research revealed the impact of A1762T/G1764A on the specific binding of HNF1α and HNF4α in the BCP region, offering new directions for a deeper understanding of HBV mechanisms and the development of novel drug strategies.
