HLA-DR genetic polymorphisms and hepatitis B virus mutations affect the risk of hepatocellular carcinoma in Han Chinese population.

BACKGROUND: Human leucocyte antigen (HLA)-DR plays a crucial role in the immune response against hepatitis B virus (HBV). We aimed to investigate the associations of HLA-DR single nucleotide polymorphisms (SNPs) with the generation of hepatocellular carcinoma (HCC)-related HBV mutations. The effects of HLA-DR SNPs and their interactions with HBV mutations on HCC risks were also determined. METHODS: Five HLA-DR SNPs (rs3135363, rs9268644, rs35445101, rs24755213, and rs984778) were genotyped in 792 healthy controls, 586 chronic hepatitis B (CHB) patients, 536 liver cirrhosis (LC) patients, and 1500 HCC patients using quantitative PCR. Sanger sequencing was used to identify the HBV mutations. Logistic regression model was performed to evaluate the association of HLA-DR SNPs with HCC risk and the frequencies of HCC-related HBV mutations. RESULTS: The variant genotypes at rs3135363, rs9268644, rs35445101, rs24755213, and rs984778 were associated with decreased HCC risks. In genotype C HBV-infected subjects, variant genotypes of these SNPs were associated with decreased frequencies of HCC-related HBV mutations such as C1653T, T1674C/G, G1719T, T1753A/C, A1762T/G1764A, A1846T, G1896A, G1899A, and preS deletion. AG genotype at rs3135363, CA genotype at rs9268644, and AG genotype at rs24755213 reduced the generation of T1753A/C and G1896A in genotype B HBV-infected subjects, respectively. In addition, the interactions of rs3135363, rs9268644, rs24755213 with C1653T, T1753A/C, A1846T, and G1896A decreased the risks of HCC. CONCLUSIONS: HLA-DR genetic polymorphisms might predispose the host to immunoselection of HCC-related HBV mutations and affect the HCC risks possibly through interacting with HBV mutations.

Novel non-HAP class A HBV capsid assembly modulators have distinct in vitro and in vivo profiles.

IMPORTANCE: Chronic hepatitis B is the most important cause of liver cancer worldwide and affects more than 290 million people. Current treatments are mostly suppressive and rarely lead to a cure. Therefore, there is a need for novel and curative drugs that target the host or the causative agent, hepatitis B virus itself. Capsid assembly modulators are an interesting class of antiviral molecules that may one day become part of curative treatment regimens for chronic hepatitis B. Here we explore the characteristics of a particularly interesting subclass of capsid assembly modulators. These so-called non-HAP CAM-As have intriguing properties in cell culture but also clear virus-infected cells from the mouse liver in a gradual and sustained way. We believe they represent a considerable improvement over previously reported molecules and may one day be part of curative treatment combinations for chronic hepatitis B.

Assessment of compensated advanced chronic liver disease based on serum bile acids in chronic hepatitis B patients.

Patients with chronic liver disease progressed to compensated advanced chronic liver disease (cACLD), the risk of liver-related decompensation increased significantly. This study aimed to develop prediction model based on individual bile acid (BA) profiles to identify cACLD. This study prospectively recruited 159 patients with hepatitis B virus (HBV) infection and 60 healthy volunteers undergoing liver stiffness measurement (LSM). With the value of LSM, patients were categorized as three groups: F1 [LSM ≤ 7.0 kilopascals (kPa)], F2 (7.1 < LSM ≤ 8.0 kPa), and cACLD group (LSM ≥ 8.1 kPa). Random forest (RF) and support vector machine (SVM) were applied to develop two classification models to distinguish patients with different degrees of fibrosis. The content of individual BA in the serum increased significantly with the degree of fibrosis, especially glycine-conjugated BA and taurine-conjugated BA. The Marco-Precise, Marco-Recall, and Marco-F1 score of the optimized RF model were all 0.82. For the optimized SVM model, corresponding score were 0.86, 0.84, and 0.85, respectively. RF and SVM models were applied to identify individual BA features that successfully distinguish patients with cACLD caused by HBV. This study provides a new tool for identifying cACLD that can enable clinicians to better manage patients with chronic liver disease.

HBV transcription and translation persist despite viral suppression in HBV-HIV co-infected patients on antiretroviral therapy.

BACKGROUND AND AIMS: Liver injury may persist in patients with HBV receiving antiviral therapy who have ongoing transcription and translation. We sought to assess ongoing HBV transcription by serum HBV RNA, translation by serum hepatitis B core related antigen (HBcrAg), and their associations with hepatic HBsAg and HBcAg staining in patients coinfected with HBV and HIV. METHODS: This is a cross-sectional study of 110 adults coinfected with HBV and HIV who underwent clinical assessment and liver biopsy. Immunohistochemistry (IHC) was performed for HBsAg and HBcAg. Viral biomarkers included quantitative HBsAg, HBV RNA, and HBcrAg. RESULTS: Participants' median age was 49 years (male, 93%; Black, 51%; HBeAg+, 65%), with suppressed HBV DNA (79%) and undetectable HIV RNA (77%) on dually active antiretroviral therapy. Overall, HBV RNA and HBcrAg were quantifiable in 81% and 83%, respectively (96% and 100% in HBeAg+, respectively). HBcAg staining was detected in 60% and HBsAg in 79%. Higher HBV RNA was associated with higher HBcAg and HBsAg IHC grades (both p < 0.0001). The HBsAg membranous staining pattern was significantly associated with higher HBV-RNA and HBcrAg levels. CONCLUSION: HBcAg and HBsAg IHC staining persisted despite viral suppression, and IHC grades and staining patterns correlated with markers of transcription (HBV RNA) and translation (HBcrAg). These data indicate that apparent HBV suppression is associated with residual transcription and translation that could contribute to liver pathology. Additional antiviral strategies directed to HBV protein expression may be useful to ameliorate liver injury.

Efficacy and Safety of Bepirovirsen in Chronic Hepatitis B Infection.

BACKGROUND: Bepirovirsen is an antisense oligonucleotide that targets all hepatitis B virus (HBV) messenger RNAs and acts to decrease levels of viral proteins. METHODS: We conducted a phase 2b, randomized, investigator-unblinded trial involving participants with chronic HBV infection who were receiving or not receiving nucleoside or nucleotide analogue (NA) therapy. Participants were randomly assigned (in a 3:3:3:1 ratio) to receive weekly subcutaneous injections of bepirovirsen at a dose of 300 mg for 24 weeks (group 1), bepirovirsen at a dose of 300 mg for 12 weeks then 150 mg for 12 weeks (group 2), bepirovirsen at a dose of 300 mg for 12 weeks then placebo for 12 weeks (group 3), or placebo for 12 weeks then bepirovirsen at a dose of 300 mg for 12 weeks (group 4). Groups 1, 2, and 3 received loading doses of bepirovirsen. The composite primary outcome was a hepatitis B surface antigen (HBsAg) level below the limit of detection and an HBV DNA level below the limit of quantification maintained for 24 weeks after the planned end of bepirovirsen treatment, without newly initiated antiviral medication. RESULTS: The intention-to-treat population comprised 457 participants (227 receiving NA therapy and 230 not receiving NA therapy). Among those receiving NA therapy, a primary-outcome event occurred in 6 participants (9%; 95% credible interval, 0 to 31) in group 1, in 6 (9%; 95% credible interval, 0 to 43) in group 2, in 2 (3%; 95% credible interval, 0 to 16) in group 3, and 0 (0%; post hoc credible interval, 0 to 8) in group 4. Among participants not receiving NA therapy, a primary-outcome event occurred in 7 participants (10%; 95% credible interval, 0 to 38), 4 (6%; 95% credible interval, 0 to 25), 1 (1%; post hoc credible interval, 0 to 6), and 0 (0%; post hoc credible interval, 0 to 8), respectively. During weeks 1 through 12, adverse events, including injection-site reactions, pyrexia, fatigue, and increased alanine aminotransferase levels, were more common with bepirovirsen (groups 1, 2, and 3) than with placebo (group 4). CONCLUSIONS: In this phase 2b trial, bepirovirsen at a dose of 300 mg per week for 24 weeks resulted in sustained HBsAg and HBV DNA loss in 9 to 10% of participants with chronic HBV infection. Larger and longer trials are required to assess the efficacy and safety of bepirovirsen. (Funded by GSK; B-Clear ClinicalTrials.gov number, NCT04449029.).

Forkhead O Transcription Factor 4 Restricts HBV Covalently Closed Circular DNA Transcription and HBV Replication through Genetic Downregulation of Hepatocyte Nuclear Factor 4 Alpha and Epigenetic Suppression of Covalently Closed Circular DNA via Interacting with Promyelocytic Leukemia Protein.

Nuclear located hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) remains the key obstacle to cure chronic hepatitis B (CHB). In our previous investigation, it was found that FoxO4 could inhibit HBV core promoter activity through downregulating the expression of HNF4α. However, the exact mechanisms whereby FoxO4 inhibits HBV replication, especially its effect on cccDNA, remain unclear. Here, our data further revealed that FoxO4 could effectively inhibit cccDNA mediated transcription and HBV replication without affecting cccDNA level. Mechanistic study showed that FoxO4 could cause epigenetic suppression of cccDNA. Although FoxO4-mediated downregulation of HNF4α contributed to inhibiting HBV core promoter activity, it had little effect on cccDNA epigenetic regulation. Further, it was found that FoxO4 could colocalize within promyelocytic leukemia protein (PML) nuclear bodies and interact with PML. Of note, PML was revealed to be critical for FoxO4-mediated inhibition of cccDNA epigenetic modification and of the following cccDNA transcription and HBV replication. Furthermore, FoxO4 was found to be downregulated in HBV-infected hepatocytes and human liver tissues, and it was negatively correlated with cccDNA transcriptional activity in CHB patients. Together, these findings highlight the role of FoxO4 in suppressing cccDNA transcription and HBV replication via genetic downregulation of HNF4α and epigenetic suppression of cccDNA through interacting with PML. Targeting FoxO4 may present as a new therapeutic strategy against chronic HBV infection. IMPORTANCE HBV cccDNA is a determining factor for viral persistence and the main obstacle for a cure of chronic hepatitis B. Strategies that target cccDNA directly are therefore of great importance in controlling persistent HBV infection. In present investigation, we found that FoxO4 could efficiently suppress cccDNA transcription and HBV replication without affecting the level of cccDNA itself. Further, our data revealed that FoxO4 might inhibit cccDNA function via a two-part mechanism: one is to epigenetically suppress cccDNA transcription via interacting with PML, and the other is to inhibit HBV core promoter activity via the genetic downregulation of HNF4α. Of note, HBV might dampen the expression of FoxO4 for its own persistent infection. We propose that manipulation of FoxO4 may present as a potential therapeutic strategy against chronic HBV infection.

Risk of hepatocellular carcinoma in antiviral treatment-naïve chronic hepatitis B patients treated with entecavir or tenofovir disoproxil fumarate: a network meta-analysis.

BACKGROUND: Long-term antiviral treatments are associated with a significantly lower hepatocellular carcinoma (HCC) incidence in chronic hepatitis B (CHB) patients by reducing HBV DNA concentrations. However, it is still controversial whether antiviral strategies affect HCC development in antiviral treatment-naïve CHB patients. This study aimed to estimate the incidence of HCC in antiviral treatment-naïve CHB patients who were treated with Entecavir (ETV) and Tenofovir Disoproxil Fumarate (TDF) and compare the efficacy of two treatment regimens in HCC reduction. METHODS: The PubMed, Embase, China National Knowledge Infrastructure, and Wanfang databases were systematically searched until June 24, 2021. The pooled incidence and 95% confidence interval of HCC were calculated by the Freeman-Tukey double arcsine transformation method. The efficacies of ETV and TDF treatments in HCC reduction were compared through a network meta-analysis. RESULTS: A total of 27 studies were identified as eligible for this systematic review. The incidence densities in the ETV and TDF treatment groups were 2.78 (95% CI: 2.21-3.40) and 2.59 (95% CI: 1.51-3.96) per 100 persons-year among patients with preexisting cirrhosis and 0.49 (95% CI: 0.32-0.68) and 0.30 (95% CI: 0.06-0.70) per 100 persons-year among patients without preexisting cirrhosis. As the proportion of CHB patients with preexisting cirrhosis increased, the incidence density of HCC also increased gradually. Compared with other Nucleos(t)ide analogs (NAs) treatments, ETV and TDF treatments significantly lowered the risk of HCC, with hazard ratios (HRs) of 0.60 (95% CI: 0.40-0.90) and 0.56 (95% CI: 0.35-0.89), respectively. However, there was no difference in the incidence density of HCC between ETV and TDF treatments (HR = 0.92, 95% CI: 0.71-1.20) regardless of preexisting cirrhosis. CONCLUSION: ETV and TDF treatments were associated with significantly lower risks of HCC than other NAs treatments. However, no difference was observed between ETV and TDF treatments in the risk of HCC development regardless of preexisting cirrhosis among treatment-naïve CHB patients.

Superiority of tenofovir alafenamide fumarate over entecavir for serum HBsAg level reduction in patients with chronic HBV infection: A 144-week outcome study after switching of the nucleos(t)ide analog.

BACKGROUND: To evaluate the long-term efficacy of switching of the nucleos(t)ide analog used for treatment from entecavir (ETV) to tenofovir alafenamide fumarate (TAF) in patients with chronic HBV infection. METHODS: A total of 103 patients with serum HBsAg levels of ≥100 IU/mL who had received ETV were enrolled. The nucleos(t)ide analog used for the treatment was switched from ETV to TAF, and the changes in serum HBsAg levels during the 144-week period before and after the drug switching were compared in 74 patients who had received ETV at least for 192 weeks. RESULTS: Significant decreases of serum HBsAg levels were observed during both the ETV and the TAF administration period, although the degree of reduction was greater during the latter period than during the former period (P<0.001). Significant decreases of serum HBsAg levels were seen in both patients with genotype B HBV infection and genotype C HBV infection, irrespective of the serum HBsAg and HBcrAg levels at the time of the drug switching. CONCLUSION: Switching of the nucleos(t)ide analog used for treatment from ETV to TAF merits consideration in patients with chronic HBV infection, since the extent of reduction of the serum HBsAg level was greater during the TAF treatment period than during the ETV treatment period.

Approaches for Detection of Hepatitis B Virus Pre-S Gene Deletions and Pre-S Deleted Proteins and Their Application in Prediction of Higher Risk of Hepatocellular Carcinoma Development and Recurrence.

Hepatocellular carcinoma (HCC) is among the most common and lethal human cancers worldwide and is closely associated with chronic hepatitis B virus (HBV) infection. Pre-S deleted proteins are naturally occurring mutant forms of HBV large surface proteins that are expressed by HBV surface genes harboring deletion mutations over the pre-S gene segments. It has been well demonstrated that HBV pre-S deleted proteins function as important oncoproteins, which promote malignant phenotypes of hepatocytes through the activation of multiple oncogenic signaling pathways and result in HCC formation. The oncogenic signaling pathways activated by pre-S deleted proteins have been verified as potential therapeutic targets for the prevention of HCC development. Moreover, the presence of pre-S gene deletions and the expression of pre-S deleted proteins in the blood and liver tissues of HBV-infected patients have been evaluated as valuable biomarkers for predicting a higher risk of HCC development and recurrence after curative surgical resection. Therefore, the precise detection of pre-S gene deletions and pre-S deleted proteins holds great promise as regards identifying the patients at higher risk of HCC development and recurrence, thus aiding in more timely and better treatments to improve their survival. This review summarizes the major approaches used for the detection of pre-S gene deletions and pre-S deleted proteins, including the approaches based on Sanger DNA sequencing, pre-S gene chips, next-generation sequencing and immunohistochemistry staining, and it highlights their important applications in the prediction of higher risks of HCC development and recurrence.

Current Trend in Antiviral Therapy for Chronic Hepatitis B.

Since active hepatitis B virus (HBV) replication is the key driver of hepatic necroinflammation and disease progression, the treatment aim of chronic hepatitis B (CHB) is to suppress HBV replication permanently to prevent hepatic decompensation, liver cirrhosis and/or hepatocellular carcinoma and prolong survival. Currently, pegylated interferon (Peg-IFN), entecavir (ETV), tenofovir disoproxil fumarate (TDF) and tenofovir alafenamide (TAF) are the first-line drugs of choice. Peg-IFN therapy has been used rarely due to its subcutaneous injection and significant side effect profile. Once daily oral ETV, TDF and TAF can suppress HBV DNA profoundly but have no direct action on cccDNA of the HBV-infected hepatocytes, hence continuing long-term therapy is usually needed to maintain HBV suppression, but the ultimate goal of HBsAg loss was rarely achieved (10 year 2%). In addition, long-term NUC therapy comes with several concerns such as increasing cost, medication adherence and loss-to-follow-up. Studies, mainly from Taiwan, have shown that finite NUCs therapy of two to three years in HBeAg-negative patients is feasible, safe and has a great benefit of much increasing HBsAg loss rate up to 30%/5 year. These have led an emerging paradigm shift to finite NUC therapy in HBeAg-negative patients globally. However, off-NUC relapse with hepatitis B flares may occur and have a risk of decompensation or even life-threatening outcomes. Therefore, proper monitoring, assessment, and retreatment decisions are crucial to ensure safety. Ideally, retreatment should be not too late to ensure safety and also not too early to allow further immune response for further HBsAg decline toward HBsAg loss. Assessment using combined HBsAg/ALT kinetics during hepatitis flare is better than biochemical markers alone to make a right retreatment decision. The strategy of finite NUC therapy has set a benchmark of high HBsAg loss rate to be achieved by the new anti-HBV drugs which are under preclinical or early phase study.

Crosstalk between Hepatitis B Virus and the 3D Genome Structure.

Viruses that transcribe their DNA within the nucleus have to adapt to the existing cellular mechanisms that govern transcriptional regulation. Recent technological breakthroughs have highlighted the highly hierarchical organization of the cellular genome and its role in the regulation of gene expression. This review provides an updated overview on the current knowledge on how the hepatitis B virus interacts with the cellular 3D genome and its consequences on viral and cellular gene expression. We also briefly discuss the strategies developed by other DNA viruses to co-opt and sometimes subvert cellular genome spatial organization.

Serum miR-192-5p levels predict the efficacy of pegylated interferon therapy for chronic hepatitis B.

We examined the association between serum miRNA (-192-5p, -122-3p, -320a and -6126-5p) levels and the efficacy of pegylated interferon (Peg-IFN) monotherapy for chronic hepatitis B (CHB) patients. We enrolled 61 CHB patients treated with Peg-IFNα-2a weekly for 48 weeks, of whom 12 had a virological response (VR) and 49 did not VR (non-VR). A VR was defined as HBV DNA < 2,000 IU/ml, hepatitis B e antigen (HBeAg)-negative, and nucleos(t)ide analogue free at 48 weeks after the end of treatment. The non-VR group showed a significantly higher HBeAg-positivity rate, ALT, HBV DNA, and serum miR-192-5p levels at baseline (P = 0.024, P = 0.020, P = 0.007, P = 0.021, respectively). Serum miR-192-5p levels at 24-weeks after the start of treatment were also significantly higher in the non-VR than the VR group (P = 0.011). Multivariate logistic regression analysis for predicting VR showed that miR-192-5p level at baseline was an independent factor (Odds 4.5, P = 0.041). Serum miR-192-5p levels were significantly correlated with the levels of HBV DNA, hepatitis B core-related antigen, and hepatitis B surface antigen (r = 0.484, 0.384 and 0.759, respectively). The serum miR-192-5p level was useful as a biomarker for the therapeutic efficacy of Peg-IFN in CHB treatment.

Tunable Duplex Semiquantitative Detection of Nucleic Acids with a Visual Lateral Flow Immunoassay Readout.

Quantitative nucleic acid amplification testing (NAAT) is a key enabling technology for infectious disease management, especially in instances where viral load informs therapeutic decisions. Inadequate access to quantitative NAATs remains a challenge to the successful deployment of antiretroviral therapy (ART) regimens for patients with chronic hepatitis B virus (CHB) in low resourced settings (LRS). Current field-deployable NAATs are generally qualitative (yes/no) rather than quantitative in nature, making them ill-suited for viral load monitoring programs for CHB patients. Here, we report the development of a proof-of-concept molecular diagnostic test, the semiquantitative ligation and amplification (SQLA) assay, which achieves semiquantitative detection of input target DNA at two independently tunable detection thresholds with a simple visual readout. The SQLA assay utilizes a duplex competitive thermophilic helicase-dependent amplification (tHDA) chemistry and can be performed in under 1 h.

Soluble programmed cell death-1 predicts hepatocellular carcinoma development during nucleoside analogue treatment.

Soluble immune checkpoint molecules are emerging novel mediators of immune regulation. However, it is unclear whether soluble immune checkpoint proteins affect the development of hepatocellular carcinoma (HCC) during nucleos(t)ide analogue (NA) treatment in patients with chronic hepatitis B virus infection. This study included 122 NA-naïve patients who received NA therapy. We assessed the associations of clinical factors, including soluble immune checkpoint proteins, with HCC development during NA treatment. The baseline serum concentrations of 16 soluble immune checkpoint proteins were measured using multiplexed fluorescent bead-based immunoassay. In total, 13 patients developed HCC during the follow-up period (median duration, 4.3 years). Of the 16 proteins, soluble inducible T-cell co-stimulator (≥ 164.71 pg/mL; p = 0.014), soluble programmed cell death-1 (sPD-1) (≤ 447.27 pg/mL; p = 0.031), soluble CD40 (≤ 493.68 pg/mL; p = 0.032), and soluble herpes virus entry mediator (≤ 2470.83 pg/mL; p = 0.038) were significantly associated with HCC development (log-rank test). In multivariate analysis, an sPD-1 level ≤ 447.27 pg/mL (p = 0.014; hazard ratio [HR], 4.537) and α-fetoprotein level ≥ 6.4 ng/mL (p = 0.040; HR, 5.524) were independently and significantly associated with HCC development. Pre-treatment sPD-1 is a novel predictive biomarker for HCC development during NA treatment.

Novel X gene point mutations in chronic hepatitis B and HBV related cirrhotic patients.

INTRODUCTION: HBx is a multifunctional modulator viral protein with key roles in various biological processes such as signal transduction, transcription, proliferation, and cell apoptosis. Also, HBx has an important role in the progression of cirrhosis and hepatocellular carcinoma (HCC). This study aimed to determine mutations in X gene, enhancer II (EnhII), and basal core promoter (BCP) of genotype D of Hepatitis B Virus (HBV) in cirrhotic and chronic HBV patients. MATERIAL AND METHODS: This cross-sectional study was performed on 68 cases with chronic HBV (cHBV) and 50 cases with HBV related cirrhosis. Serum samples were obtained for genomic DNA extraction. Semi-nested PCR was used to amplify the HBx region. Point mutations in the HBx region were detected by sequencing. RESULT: Novel mutations were detected, including C1491G, C1500T, G1613T, and G1658T in the N-terminal of the X gene. The frequency of C1481T/G1479A, T1498C, C1500T, G1512A, A1635T, C1678T, A1727T, and A1762T/ G1764A/ C1773T was significantly higher in cirrhotic patients compared to chronically HBV infected ones. A higher rate of A1635T, C1678T, A1727T, A1762T, G1764A, and C1773T was observed in cirrhotic patients. CONCLUSION: Our findings showed that the frequency of mutations in the basal-core promoter, enhancer II, and regulatory region of the HBx gene was more seen in cirrhotic patients than in chronic HBV cases. Novel mutations were detected in the HBx gene, causing amino acid substitutions; however, the clinical impact of these novel mutations is yet to be cleared.

Hepatitis B Virus Small Envelope Protein Promotes Hepatocellular Carcinoma Angiogenesis via Endoplasmic Reticulum Stress Signaling To Upregulate the Expression of Vascular Endothelial Growth Factor A.

Hepatocellular carcinoma (HCC) is a hypervascular tumor, and accumulating evidence has indicated that stimulation of angiogenesis by hepatitis B virus (HBV) may contribute to HCC malignancy. The small protein of hepatitis B virus surface antigen (HBsAg), SHBs, is the most abundant HBV protein and has a close clinical association with HCC; however, whether SHBs contributes to HCC angiogenesis remains unknown. This study reports that the forced expression of SHBs in HCC cells promoted xenograft tumor growth and increased the microvessel density (MVD) within the tumors. Consistently, HBsAg was also positively correlated with MVD counts in HCC patients' specimens. The conditioned media from the SHBs-transfected HCC cells increased the capillary tube formation and migration of human umbilical vein endothelial cells (HUVECs). Intriguingly, the overexpression of SHBs increased vascular endothelial growth factor A (VEGFA) expression at both the mRNA and protein levels. Higher VEGFA expression levels were also observed in xenograft tumors transplanted with SHBs-expressing HCC cells and in HBsAg-positive HCC tumor tissues than in their negative controls. As expected, in the culture supernatants, the secretion of VEGFA was also significantly enhanced from HCC cells expressing SHBs, which promoted HUVEC migration and vessel formation. Furthermore, all three unfolded protein response (UPR) sensors, inositol-requiring enzyme 1α (IRE1α), protein kinase RNA-like endoplasmic reticulum (ER) kinase (PERK), and activating transcription factor 6 (ATF6), associated with ER stress were found to be activated in SHBs-expressing cells and correlated with VEGFA protein expression and secretion. Taken together, these results suggest an important role of SHBs in HCC angiogenesis and may highlight a potential target for preventive and therapeutic intervention for HBV-related HCC and its malignant progression. IMPORTANCE Chronic hepatitis B virus infection is one of the important risk factors for the development and progression of hepatocellular carcinoma (HCC). HCC is characteristic of hypervascularization even at early phases of the disease due to the overexpression of angiogenic factors like vascular endothelial growth factor A (VEGFA). However, a detailed mechanism of HBV-induced angiogenesis remains to be established. In this study, we demonstrate for the first time that the most abundant HBV protein, i.e., small surface antigen (SHBs), can enhance the angiogenic capacity of HCC cells by the upregulation of VEGFA expression both in vitro and in vivo. Mechanistically, SHBs induced endoplasmic reticulum (ER) stress, which consequently activated unfolded protein response (UPR) signaling to increase VEGFA expression and secretion. This study suggests that SHBs plays an important proangiogenic role in HBV-associated HCC and may represent a potential target for antiangiogenic therapy in HCC.

Hepatitis B Virus Infection and Extra-Hepatic Manifestations: A Systemic Disease.

People living with hepatitis B virus (HBV) chronic infection are exposed to high rates of liver complications including end-stage liver disease and hepatocellular carcinoma. Extrahepatic manifestations of HBV infection have long been underestimated. Several of these extrahepatic syndromes have been well described, including systemic vasculitides, glomerulonephritis, and cutaneous manifestations. Other manifestations have been more recently described such as hematological malignancies and neurological diseases. These extrahepatic manifestations are associated with significant morbidity and mortality. Although not completely understood, underlying mechanisms include HBV-induced local and systemic inflammation. Suppression of HBV replication usually improves extrahepatic manifestations. This review will discuss how HBV induces inflammation and the extrahepatic manifestations of HBV infection to guide clinical management.