Class A capsid assembly modulator apoptotic elimination of hepatocytes with high HBV core antigen level in vivo is dependent on de novo core protein translation.

Capsid assembly is critical in the hepatitis B virus (HBV) life cycle, mediated by the viral core protein. Capsid assembly is the target for new anti-viral therapeutics known as capsid assembly modulators (CAMs) of which the CAM-aberrant (CAM-A) class induces aberrant shaped core protein structures and leads to hepatocyte cell death. This study aimed to identify the mechanism of action of CAM-A modulators leading to HBV-infected hepatocyte elimination where CAM-A-mediated hepatitis B surface antigen (HBsAg) reduction was evaluated in a stable HBV replicating cell line and in AAV-HBV-transduced C57BL/6, C57BL/6 SCID, and HBV-infected chimeric mice with humanized livers. Results showed that in vivo treatment with CAM-A modulators induced pronounced reductions in hepatitis B e antigen (HBeAg) and HBsAg, associated with a transient alanine amino transferase (ALT) increase. Both HBsAg and HBeAg reductions and ALT increase were delayed in C57BL/6 SCID and chimeric mice, suggesting that adaptive immune responses may indirectly contribute. However, CD8+ T cell depletion in transduced wild-type mice did not impact antigen reduction, indicating that CD8+ T cell responses are not essential. Transient ALT elevation in AAV-HBV-transduced mice coincided with a transient increase in endoplasmic reticulum stress and apoptosis markers, followed by detection of a proliferation marker. Microarray data revealed antigen presentation pathway (major histocompatibility complex class I molecules) upregulation, overlapping with the apoptosis. Combination treatment with HBV-specific siRNA demonstrated that CAM-A-mediated HBsAg reduction is dependent on de novo core protein translation. To conclude, CAM-A treatment eradicates HBV-infected hepatocytes with high core protein levels through the induction of apoptosis, which can be a promising approach as part of a regimen to achieve functional cure. IMPORTANCE: Treatment with hepatitis B virus (HBV) capsid assembly modulators that induce the formation of aberrant HBV core protein structures (CAM-A) leads to programmed cell death, apoptosis, of HBV-infected hepatocytes and subsequent reduction of HBV antigens, which differentiates CAM-A from other CAMs. The effect is dependent on the de novo synthesis and high levels of core protein.

Activation of AIM2 by hepatitis B virus results in antiviral immunity that suppresses hepatitis C virus during coinfection.

IMPORTANCE: Clinical data suggest that Hepatitis C virus (HCV) levels are generally lower in Hepatitis B virus (HBV) co-infected patients, but the mechanism is unknown. Here, we show that HBV, but not HCV, activated absent in melanoma-2. This in turn results in inflammasome-mediated cleavage of pro-IL-18, leading to an innate immune activation cascade that results in increased interferon-γ, suppressing both viruses.

Immune response and treatment targets of chronic hepatitis B virus infection: innate and adaptive immunity.

Chronic hepatitis B virus (HBV) infection is a major global public health risk that threatens human life and health, although the number of vaccinated people has increased. The clinical outcome of HBV infection depends on the complex interplay between viral replication and the host immune response. Innate immunity plays an important role in the early stages of the disease but retains no long-term immune memory. However, HBV evades detection by the host innate immune system through stealth. Therefore, adaptive immunity involving T and B cells is crucial for controlling and clearing HBV infections that lead to liver inflammation and damage. The persistence of HBV leads to immune tolerance owing to immune cell dysfunction, T cell exhaustion, and an increase in suppressor cells and cytokines. Although significant progress has been made in HBV treatment in recent years, the balance between immune tolerance, immune activation, inflammation, and fibrosis in chronic hepatitis B remains unknown, making a functional cure difficult to achieve. Therefore, this review focuses on the important cells involved in the innate and adaptive immunity of chronic hepatitis B that target the host immune system and identifies treatment strategies.

Isolated Hepatitis B Core Antibody Positivity and Long-Term Liver-Related Mortality in Korea: A Cohort Study.

INTRODUCTION: Whether isolated hepatitis B core antibody (anti-HBc) positivity is a risk factor for long-term liver-related outcomes in hepatitis B virus (HBV)-endemic areas remains unclear. We aimed to investigate liver-related and liver cancer mortality of isolated anti-HBc positivity in Korean adults. METHODS: A cohort study comprised 609,299 Korean adults who underwent hepatitis B serologic markers, as a part of health examination. Liver-related and liver cancer mortality were determined using the National Death Records. RESULTS: During a median follow-up of 9.0 years (interquartile range, 5.5-13.7 years), 554 liver-related deaths were identified (liver-related mortality, 9.6 cases per 10 5 person-years). The prevalence of isolated anti-HBc positivity was 3.8% (n = 23,399) and was age-dependent. After adjustment for age, sex, and other confounders, hazard ratios (95% confidence interval) for liver-related mortality in isolated anti-HBc-positive and hepatitis B surface antigen-positive subjects compared with HBV-unexposed subjects were 1.69 (1.22-2.33) and 27.02 (21.45-34.04), respectively. These associations were pronounced in the analyses using liver cancer mortality as an outcome. Among isolated anti-HBc-positive patients, the risks of liver-related and liver cancer mortality were significantly higher in those with high fibrosis-4 scores compared with patients unexposed to HBV with the multivariable-adjusted hazard ratios (95% confidence interval) of 15.59 (9.21-26.37) and 72.66 (36.96-142.86), respectively. DISCUSSION: In this cohort of Korean adults, isolated anti-HBc positivity was associated with an increased risk of liver-related and liver cancer mortality, especially when accompanied by a high fibrosis score. Isolated anti-HBc positivity may be an independent risk factor for liver-related outcomes, especially in high-endemic areas.

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.

Long-term Hepatitis B Surface Antigen Profile and Seroclearance after Severe Acute Flares of Chronic Hepatitis B.

Background/Aims: Hepatitis B surface antigen (HBsAg) seroclearance remains uncommon in chronic hepatitis B (CHB) infection. During acute flares of CHB (AFOCHB), alanine aminotransferase elevation reflects a mounting immune response toward viral clearance. We hypothesized that severe AFOCHB is associated with a greater quantitative HBsAg (qHBsAg) decline and HBsAg seroclearance rate. Methods: A total of 75 patients with severe AFOCHB with alanine aminotransferase 10× the upper limit of normal were matched to a control group by age and sex in a 1:2 ratio. qHBsAg levels were measured at the time of flare and annually (for both cases and controls) until the last follow-up. Results: The median follow-up times for patients with severe AFOCHB and controls were 8.8 and 10.5 years, respectively. The cumulative rate of HBsAg seroclearance was higher in the severe AFOCHB group than in the control group (11.8% vs 5.0%, p=0.04) despite the former group having a trend of a higher baseline median qHBsAg (3,127 IU/mL vs 1,178 IU/mL, p=0.076). Compared with the control group, the severe AFOCHB group had a greater annual qHBsAg reduction (-242.4 IU/mL/yr vs -47.3 IU/mL/yr, p=0.002). Increasing age (p=0.049), lower baseline qHBsAg (p=0.002), and severe AFOCHB (p=0.014) were independently associated with HBsAg seroclearance. However, the cumulative rate of hepatocellular carcinoma was significantly higher in the severe AFOCHB group than in the control group (15.8% vs 1.9%, p<0.001). Conclusions: Severe AFOCHB was associated with a greater incidence of HBsAg seroclearance and qHBsAg decline. However, it was associated with a higher incidence of hepatocellular carcinoma.

Interleukin-37 suppresses the cytotoxicity of hepatitis B virus peptides-induced CD8+ T cells in patients with acute hepatitis B.

Interleukin-37 (IL-37) is a newly identified anti-inflammatory cytokine, owning immunosuppressive activity in infectious diseases. The aim of this study was to investigate the regulatory function of IL-37 on CD8+ T cells during hepatitis B virus (HBV) infection. Eighteen acute hepatitis B (AHB) patients, thirty-nine chronic hepatitis B (CHB) patients, and twenty controls were enrolled. IL-37 concentration was measured by ELISA. IL-37 receptor subunits expressions on CD8+ T cells were assessed by flow cytometry. Purified CD8+ T cells were stimulated with HBV peptides and recombinant IL-37. Perforin and granzyme B secretion was investigated by ELISPOT. Programmed death-1 (PD-1) and cytotoxic T-lymphocyte associated protein-4 (CTLA-4) mRNA expressions were semi-quantified by real-time PCR. CD8+ T cell cytotoxicity was assessed in direct contact and indirect contact coculture with HepG2.2.15 cells. Plasma IL-37 level was down-regulated and negatively correlated with aminotransferase levels in AHB patients. There were no significant differences of IL-37 receptor subunits among AHB patients, CHB patients, and controls. Exogenous IL-37 stimulation suppressed HBV peptides-induced perforin and granzyme B secretion by CD8+ T cells in AHB patients, but not in CHB patients. Exogenous IL-37 stimulation did not affect proinflammatory cytokines secretion as well as PD-1/CTLA-4 mRNA expressions in CD8+ T cells in AHB and CHB patients. Exogenous IL-37 stimulation dampened HBV peptide-induced CD8+ T cell cytotoxicity in a cell-to-cell contact manner. The current data indicated that acute HBV infection might induce down-regulation of IL-37, which might be associated with enhanced CD8+ T cell cytotoxicity and liver damage.

Interleukin-33 mediates both immune-related and non-immune-related inhibitory effects against hepatitis B virus.

Chronic infection by hepatitis B virus (HBV) is associated with high risks of liver fibrosis, cirrhosis and hepatocellular carcinoma. HBV covalently closed circular DNA (cccDNA) in the nucleus of infected hepatocyte serves as transcription template. Neither natural resolution of acute infection nor current treatment options for chronic infection are believed to cause cccDNA clearance. We previously showed that injection of IL-33-expressing plasmid facilitated clearance of intrahepatic HBV DNA in a mouse model of HBV persistence. In this work, HBV-targeting therapeutic effects of IL-33 were further explored. Murine IL-33 delivered by recombinant adeno-associated virus (AAV-mIL-33) induced clearance of both serum HBV markers and intrahepatic HBV DNA in two mouse models of HBV persistence based on replicon plasmid and recombinant cccDNA (rcccDNA) respectively. Clearance was associated with serum ALT elevations and liver infiltrations by CD4+ and CD8+ T cells, indicating IL-33-induced cellular immune responses against HBV-harboring cells. Adoptive transfer of splenocytes from AAV-mIL-33-cured mice was indeed sufficient to engender similar clearance in recipient mice. In vitro, intracellular, instead of extracellular, IL-33 was mainly responsible for repressing viral transcription, protein production and genome replication in Huh7 cells transfected with HBV replicon or rcccDNA. IL-33 was shown to be recruited onto rcccDNA minichromosome accompanied by loss of transcriptional activation epigenetic marks. Finally, transfection of IL-33 into HBV-infected HepG2/NTCP cells resulted in reduced transcription, antigen expression and genome replication, suggesting repression of canonical cccDNA. These data demonstrated diverse inhibitory effects on HBV and HBV-infected cells mediated by IL-33, and suggest IL-33 as an interesting therapeutic candidate.

Barriers Against Hepatitis B Vaccination in High-Risk Adults: A Cross-Sectional Study.

BACKGROUND: The hepatitis B vaccination has been strongly recommended by regulatory bodies. However, there are great discrepan- cies between routine practices and the recommendations of regulatory agencies in many countries. We aimed to identify the barriers against Hepatitis B Vaccination (HBV) for high-risk patients by comparing the awareness, attitude, and knowledge among vaccinated and unvaccinated patients. METHODS: A 34-item questionnaire was applied to 156 patients, consisting of renal transplant recipients, allogeneic hematopoietic stem cell transplant recipients, and patients with chronic hepatitis C. Multiple logistic regression analysis was employed to identify indepen- dent predictors for patients receiving the hepatitis B virus vaccination. RESULTS: The multiple logistic regression analysis revealed that the independent risk factors against the HBV vaccination were a require- ment of a separate appointment for hepatitis B virus vaccination (aOR: 3.35, 95% CI, 1.18-9.47), and fear of severe side effects that can be related with hepatitis B virus vaccination (aOR: 3.67, 95% CI, 1.18-9.47). However, taking a recommendation for hepatitis B virus vaccination at least once from a health care provider (aOR: 0.04, 95% CI, 0.01-0.11), and having a health insurance (aOR: 0.09, 95% CI, 0.01-0.55) were independent protective factors for being vaccinated. In further analysis among patients with at least a single dose of vaccine, the lack of recommendation from a health care provider for hepatitis B virus vaccination and the absence of a healthcare pro- vider who is responsible for monitoring the completion of the 3-dose vaccination were identified as independent risk factors for failure to complete the 3-dose hepatitis B virus vaccination. CONCLUSION: In high-risk adults, the barriers against hepatitis B virus vaccination should be handled by a comprehensive action plan to achieve the WHO 2030 hepatitis elimination target.

Role of Innate Immune Regulatory Genes, FOXP3 and FOS in Chronic Hepatitis B Infection.

Persistence of hepatitis B virus (HBV) infection leading to chronic infection and its sequalae is responsible for over half a million deaths worldwide. The reason for persistence of chronic hepatitis B (CHB) infection is still not clearly understood. An attempt was made to understand the role of immune regulatory genes in CHB in comparison to spontaneously cleared HBV infection. Relative gene expression of 26 genes involved in innate immunity were studied using Real-Time Polymerase Chain Reaction Array. A total of 679 subjects from three different geographical regions of Northeast India (Assam, Arunachal Pradesh, and Tripura) were included in this case-control study. The cases were subdivided into CHB cases with HBeAg(+)(72), CHB with HBeAg(-)(278), spontaneously cleared controls (88), and healthy controls (228). Overall, 28.3% of the subjects had previous exposure with HBV, while 28.6% had protective antibodies IgG/IgM against HBV. There was a statistically higher number of CHB in men (66.4%) compared to women (33.6%) (p = 0.0001). Proto-oncogene FOS has been found to be moderately upregulated in CHB with HBeAg +ve (2.3-fold) and significantly upregulated (4.1-fold upregulation) in hepatocellular carcinoma. Further, FOXP3 was found to be significantly upregulated (3.0-fold, p = 0.01) in CHB with HBeAg (+) compared to spontaneously cleared HBV infection. In conclusion, CHB with HBeAg positivity was found to have disrupted immune response with upregulation of FOS and FOXP3. Thus, early induction of HBeAg seroconversion with interferon-based therapy or oral nucleos(t)ide analogs along with FOS inhibitors can have important clinical implications in the management of CHB and preventing cirrhosis and HCC.

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.

Hepatitis B Flare in Hepatitis B e Antigen-Negative Patients: A Complicated Cascade of Innate and Adaptive Immune Responses.

Chronic hepatitis B virus (HBV) infection is a dynamic process involving interactions among HBV, hepatocytes, and the host immune system. The natural course of chronic hepatitis B (CHB) is divided into four chronological phases, including the hepatitis B e antigen (HBeAg)-positive and HBeAg-negative phases. During HBV flare, alanine aminotransferase (ALT) levels abruptly rise to >5× the upper limit of normal; this is thought to occur due to the immune response against an upsurge in serum HBV DNA and antigen levels. Hepatitis flares may occur spontaneously, during or after antiviral therapy, or upon immunosuppression or chemotherapy in both HBeAg-positive and HBeAg-negative patients. The clinical spectrum of HBV flares varies from asymptomatic to hepatic decompensation or failure. HBeAg seroconversion with ≥ 1 year of consolidation therapy is accepted as an endpoint of oral antiviral therapy in HBeAg-positive patients, but recommendations for treating HBeAg-negative patients differ. Thus, the management of HBeAg-negative patients has attracted increasing interest. In the current review, we summarize various types of HBV flares and the associated complex cascade of innate and adaptive immune responses, with a focus on HBeAg-negative CHB patients. Hopefully, this review will provide insight into immunopathogenesis to improve the management of HBV flares in HBeAg-negative CHB patients.

Effectiveness of Hepatitis B Vaccination Campaign in Italy: Towards the Control of HBV Infection for the First Time in a European Country.

BACKGROUND: In 1991, a mass immunization campaign against the hepatitis B virus (HBV) for children and teenagers was introduced in Italy. This study evaluated the impact of the immunization campaign on the incidence and modes of HBV transmission. METHOD: Acute HBV cases of viral hepatitis were reported to the National Surveillance System (SEIEVA). Hepatitis A cases reported to the same system were used as controls to calculate the adjusted odds ratios and the population attributable risk for potential risk factors. RESULTS: The incidence of acute HBV declined from 5.0 in 1990 to 0.4 in 2019 per 100,000 population. The fall was almost total in people targeted by the campaign: in 2019, zero cases (100% reduction) in the age-group 0-14 years and 0.1 cases per 100,000 population (99.4% reduction) in the age-group 15-24 years were reported. In the decade 2010-2019, nearly one-fifth (19.3%) of cases occurred in foreigners. Intravenous drug use is no longer a risk factor (OR = 0.7; 95% CI = 0.5-1.02). Beauty treatments, risky sexual exposure, and household contact with an HBsAg carrier were found to be independent predictors of acute hepatitis B. CONCLUSIONS: The HB vaccination campaign proved effective in minimising acute HBV in Italy. Control of the infection is close to being reached for the first time in Europe.

Community-Based Screening for Hepatitis B and C Infectivity Using Two Quantitative Antigens to Identify Endemic Townships.

Screening and linkage to care are essential to achieve viral hepatitis elimination before 2030. The accurate identification of endemic areas is important for controlling diseases with geographic aggregation. Viral activity drives prognosis of chronic hepatitis B and hepatitis C virus infection. This screening was conducted in Chiayi County from 2018-2019. All residents aged 30 years or older were invited to participate in quantitative HBsAg (qHBsAg) and HCV Ag screening. Among the 4010 participants (male:female = 1630:2380), the prevalence of qHBsAg and HCV Ag was 9.9% (396/4010) and 4.1% (163/4010), respectively. High-prevalence townships were identified, three for qHBsAg > 15% and two for HCV Ag > 10%. The age-specific prevalence of qHBsAg was distributed in an inverse U-shape with a peak (16.0%, 68/424) for subjects in their 40 s; for HCV, prevalence increased with age. Concentrations of qHBsAg < 200 IU/mL were found in 54% (214/396) of carriers. The rate of oral antiviral treatment for HCV was 75.5% (114/151), with subjects younger than 75 years tending to undergo treatment (85.6% vs. 57.4%, p < 0.001). QHBsAg and HCV Ag core antigens can reflect the concentration of the viral load, which serves as a feasible screening tool. Using quantitative antigen screening for hepatitis B and C in community-based screening, two hyperendemic townships were identified from an endemic county.

An in silico study to unveil potential drugs and vaccine chimera for HBV capsid assembly protein: combined molecular docking and dynamics simulation approach.

Humans are a major reservoir of the hepatitis B virus (HBV), therefore promising treatment and control vaccination strategies are needed to eradicate the virus. Though promising drugs and vaccines are available against HBV, still efforts are required to enrich the therapy options. Herein, the HBV assembly protein was explored to identify novel targets for future use against HBV. Computer-aided drug designing and immune-informatics techniques were employed for the identification of putative inhibitors and vaccine ensemble against HBV using capsid assembly protein. The identified drug molecule binds with high affinity to the active pocket of the protein, and several epitopes are scanned in the protein sequence. The drug molecule, besides being a good putative inhibitor, has acceptable drug-like properties. A multi-epitope vaccine is also constructed to overcome the limitations of weakly immunogenic epitopes. In contrast to the MHC II level, the set of predicted epitopes has been recognized to interact with significant numbers of HLA alleles of MHC I. Selected epitopes are extremely virulent, antigenic, nontoxic, nonallergic, have suitable affinity to bind with the prevailing DRB*0101 allele, and also spectacle 86% mediocre population coverage. A multi-epitope peptide-based vaccine chimera having 73 amino acids was designed. It emerged as substantially immunogenic, thermally stable, robust in producing cellular as well as humoral immune responses, and had competent physicochemical properties to analyze in vitro and in vivo studies. The capsid assembly protein is a in more stable nature in the presence of the drug molecule compared to the TLR3 receptor in the vaccine presence. These particulars were confirmed by exposing the docked molecules to absolute and relative binding free energy approaches of MMGBSA/PBSA. The purpose to investigate the interactions between the vaccine and a representative TLR3 immune receptor can reveal the intermolecular affinity and possible presentation mechanism of the vaccine by TLR3 to the host immune system. It was revealed that the vaccine is showing a very good affinity of binding for the TLR3 and forming a network of hydrophobic and hydrophilic interactions. Overall, the findings of this study are promising and might be useful for further experimental validations.

[Sero-epidemiological characteristics of the hepatitis D virus infection among hepatitis B virus infected-patients at a single center in Xinjiang region].

Objective: To investigate the sero-epidemiological characteristics of the hepatitis D virus (HDV) infection among hepatitis B virus (HBV)-infected patients in Xinjiang region. Methods: A single-center cross-sectional analysis method was used to select 264 cases of hepatitis B virus infection who were hospitalized in the Center for Infectious Diseases and Liver Diseases of the First Affiliated Hospital of Xinjiang Medical University from August 2021 to January 2022. All patients were tested for HDV Ag, HDV IgM, HDV IgG, and HDV RNA. The infection status of hepatitis D virus was analyzed by grouping according to their clinical type, HBV viral load, and HBsAg level. A paired t-test was used for data with measurement data conforming to normal distribution. A paired rank sum test was used for data that did not conform to normal distribution before and after treatment. Results: A total of 36 cases (13.64%) and 26 cases (9.85%) were positive for HDV serological markers and HDV RNA. According to clinical type grouping, the positive rates of HDV serum markers in patients with chronic hepatitis B, hepatitis B-related cirrhosis, liver cancer, and liver failure were 13.46%, 12.43%, and 20.83%, respectively, and there was no statistically significant difference among the three groups (χ2=0.86, P=0.649). The positive rates of HDV RNA were 11.54%, 8.11%, and 20.83%, respectively, and there was no statistically significant difference among the three groups (χ2=4.015, P=0.134). According to HBV viral load grouping, the positive rates of HDV serum markers among patients with viral loads <20, 20-2 000, and >2 000 IU/ml were 17.15%, 7.81%, and 6.67%, respectively, and the difference was not statistically significant among the three groups (χ2=4.846, P=0.089). The positive rates of HDV RNA were 9.47%, 10.94%, and 10%, respectively, and the difference was not statistically significant among the three groups (χ2=0.113, P=0.945). According to HBsAg level grouping, the positive rates of HDV serum markers in HBsAg<0.05, 0.05~250, and >250 IU/ml were 14.29%, 16.67%, and 10.85%, respectively, and there was no statistically significance between the three groups (χ2=1.745, P=0.418). The positive rates of HDV RNA were 4.76%, 8.77%, and 11.63%, respectively, and there was no statistically significant difference among the three groups (χ2=1.221, P=0.543). Clinical outcome, disease course, HBV DNA, serological markers of viral hepatitis, routine blood test, biochemical indicators, coagulation function, and other laboratory indicators were compared between HDV serum marker and/or nucleic acid positive and negative patients, and there was no statistically significant difference (P>0.05). Conclusion: The positive rate of HDV serological markers and HDV RNA is 13.64% and 9.85%, respectively, at a single center in the Xinjiang region, and there is still a high HDV infection rate among the HBV-infected patients with low levels of viral load and HBsAg.

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.

Development, Production, and Characterization of Hepatitis B Subviral Envelope Particles as a Third-Generation Vaccine.

Vaccination still represents the most efficient and inexpensive strategy in the control of hepatitis B virus (HBV) infection. However, about 10% of the population vaccinated with the current S yeast-derived vaccine fail to induce an adequate immune response. Our group has developed a new-generation hepatitis B vaccine candidate composed by the three surface proteins of the HBV. Here we describe the methods to develop and characterize a stable CHO-K1 recombinant cell line able to produce and secrete hepatitis B subviral envelope particles (HBV-SVPs) containing L and M glycoproteins in addition to S glycoprotein. In addition, Western blot and immunogold electron microscopy techniques to evaluate the size, morphology, and composition of the particles are explained. Finally, immunization protocols are described in order to study the immunogenicity of HBV-SVPs and the ability of the antibodies triggered by these particles to recognize the binding site of HBV with the hepatocyte.

Ethanol attenuates presentation of cytotoxic T-lymphocyte epitopes on hepatocytes of HBV-infected humanized mice.

BACKGROUND AND AIMS: Approximately 3.5% of the global population is chronically infected with Hepatitis B Virus (HBV), which puts them at high risk of end-stage liver disease, with the risk of persistent infection potentiated by alcohol consumption. However, the mechanisms underlying the effects of alcohol on HBV persistence remain unclear. Here, we aimed to establish in vivo/ex vivo evidence that alcohol suppresses HBV peptides-major histocompatibility complex (MHC) class I antigen display on primary human hepatocytes (PHH), which diminishes the recognition and clearance of HBV-infected hepatocytes by cytotoxic T-lymphocytes (CTLs). METHODS: We used fumarylacetoacetate hydrolase (Fah)-/-, Rag2-/-, common cytokine receptor gamma chain knock-out (FRG-KO) humanized mice transplanted with human leukocyte antigen-A2 (HLA-A2)-positive hepatocytes. The mice were HBV-infected and fed control and alcohol diets. Isolated hepatocytes were exposed ex vivo to HBV 18-27-HLA-A2-restricted CTLs to quantify cytotoxicity. For mechanistic studies, we measured proteasome activities, unfolded protein response (UPR), and endoplasmic reticulum (ER) stress in hepatocytes from HBV-infected humanized mouse livers. RESULTS AND CONCLUSIONS: We found that alcohol feeding attenuated HBV core 18-27-HLA-A2 complex presentation on infected hepatocytes due to the suppression of proteasome function and ER stress induction, which diminished both the processing of HBV peptides and trafficking of HBV-MHC class I complexes to the hepatocyte surface. This alcohol-mediated decrease in MHC class I-restricted antigen presentation of the CTL epitope on target hepatocytes reduced the CTL-specific elimination of infected cells, potentially leading to HBV-infection persistence, which promotes end-stage liver disease outcomes.