The significance of detecting HBV pgRNA and HBcrAg in HBV patients treated with NAs.

The value of detecting hepatitis B virus (HBV), pregenomic RNA (pgRNA), and hepatitis B core-related antigen (HBcrAg), both separately and jointly, in the management of HBV patients undergoing treatment with Nucleotide Analog was investigated. A total of 149 HBV patients who were being treated with Nucleotide Analog were enrolled in this study. The quantitative levels of HBV pgRNA and HBcrAg in the sera of these patients were determined, aiming to comprehend their replication levels and expression during the course of antiviral therapy. The patients were separated into 3 groups based on treatment duration: treatment time ≤ 12 months, treatment time ranging from 12 months to <60 months, and treatment time ≥ 60 months. Significantly different levels of HBcrAg and HBV pgRNA were observed among 3 groups (P < .05). In the group of patients with positive hepatitis B e antigen, both HBcrAg and pgRNA levels were higher compared to the group with negative hepatitis B e antigen, and this difference between the 2 groups was found to be statistically significant. Stratified analysis based on levels of hepatitis B surface antigen (HBsAg) revealed that the group with HBsAg levels < 100 IU/mL had lower levels of both HBcrAg and pgRNA compared to the group with HBsAg levels ≥ 100 IU/mL (P < .001). Following antiviral therapy, various degrees of transcription of covalently closed circular DNA continue to exist within the liver of HBV patients. The levels of serum HBcrAg and HBV pgRNA vary among patients with different treatment durations, indicating their efficacy in evaluating disease conditions during antiviral therapy.

Was antiviral prophylaxis necessary after kidney transplantation utilizing HBcAb+ donors? A systematic review and meta-analysis.

BACKGROUND: Current guidelines lack consensus on whether antiviral prophylaxes should be administered after kidney transplantation from HBcAb+ donors. This systematic review and meta-analysis aimed to evaluate the incidence and risk factors of de novo HBV (DNH) infection, as well as graft and patient survival. METHODS: We searched PubMed, Embase, and the Cochrane Library up to December 31, 2023. We included relevant studies that assessed clinical outcomes following transplantation utilizing HBcAb+ kidneys. Summary measures of effect and 95% confidence intervals (CI) for prevalence, risk factors, as well as graft and patient survival were estimated using random-effects meta-analysis. RESULTS: Thirteen studies were included for the final analysis. The DNH incidence was at 0.36% (9/2516) with low heterogeneity (I2 = 6%). HBsAb+ recipients (OR: 0.78, 95%CI: 0.25-2.38), HBcAb+ recipients (OR: 3.11, 95%CI: 0.91-10.66, P = 0.071), and recipients not receiving any antiviral prophylaxis (OR: 1.26, 95%CI: 0.15-10.58) were not associated with higher DNH risk. Specifically, HBsAb-/HBcAb+ recipients had the highest DNH incidence (4.65%), followed by HBsAb-/HBcAb- (0.49%), HBsAb+/HBcAb- recipients (0.45%), and HBsAb+/HBcAb+ (0%). Furthermore, recipients receiving HBcAb+ kidneys had comparable graft survival (HR: 1.06, 95%CI: 0.94-1.19, P = 0.55) and patient survival (HR:1.16, 95%CI: 0.98-1.38, P = 0.090) compared with recipients receiving HBcAb- kidneys. CONCLUSION: Kidney transplantation utilizing HBcAb+ kidneys contributed to comparable graft and patient survival with an extremely low risk of HBV transmission. Antiviral prophylaxes may only be administered in HBsAb-/HBcAb+ recipients.

The downregulation of Tapasin in dendritic cell regulates CD8+ T cell autophagy to hamper hepatitis B viral clearance in the induced pluripotent stem cell-derived hepatocyte organoid.

Tapasin, a crucial molecular chaperone involved viral antigen processing and presentation, plays an important role in antivirus immunity. However, its impact on T cell differentiation in the context of virus clearance remains unclear. In this study, we employed induced pluripotent stem cells to differentiate into hepatocyte-like cell, which were subsequently inserted to the inverted colloidal crystal scaffolds, thus establishing a hepatocyte organoid (HO). By inoculating hepatitis B virus (HBV) particles in the system, we successfully engineered a robust in vitro HBV infection model for at least 3 weeks. Furthermore, we aimed to explore the effects of lentivirus-mediated short hairpin RNA (shRNA) targeting human Tapasin on the differentiation and antiviral function of CD8+ T cells. Specifically, we transfected dendritic cells (DCs) with Tapasin-shRNA and cocultured with T cells. The results demonstrated that Tapasin-shRNA transfected DCs effectively suppressed T cell proliferation and impeded HBV-specific cytotoxic T lymphocyte responses. Our investigation also revealed the role of mTOR pathway activation in reducing autophagy activity within CD8+ T cells. Expressions of autophagy-related proteins, beclin-1, LC3II/LC3I were decreased and PI3K/AKT/mTOR activity was increased in Tapasin-shRNA group. Collectively, our findings elucidate that shRNA targeting the Tapasin gene within DCs inhibits T cell differentiation by reducing autophagy activity to hamper viral clearance in the HBV-infected HO.

Clinical implications of hepatitis B virus core antigen-mediated immunopathologic T cell responses in chronic hepatitis B.

Hepatitis B virus (HBV) infection significantly impacts Asian populations. The influences of continuous HBV antigen and inflammatory stimulation to T cells in chronic hepatitis B (CHB) remain unclear. In this study, we first conducted bioinformatics analysis to assess T-cell signaling pathways in CHB patients. In a Taiwanese cohort, we examined the phenotypic features of HBVcore -specific T cells and their correlation with clinical parameters. We used core protein overlapping peptides from the Taiwan prevalent genotype B HBV to investigate the antiviral response and the functional implication of HBV-specific T cells. In line with Taiwanese dominant HLA-alleles, we also evaluated ex vivo HBVcore -specific T cells by pMHC-tetramers targeting epitopes within HBV core protein. Compared to healthy subjects, we disclosed CD8 T cells from CHB patients had higher activation marker CD38 levels but showed an upregulation in the inhibitory receptor PD-1. Our parallel study showed HBV-specific CD8 T cells were more activated with greater PD-1 expression than CMV-specific subset and bulk CD8 T cells. Moreover, our longitudinal study demonstrated a correlation between the PD-1 fluctuation pattern of HBVcore -specific CD8 T cells and liver inflammation in CHB patients. Our research reveals the HBV core antigen-mediated immunopathologic profile of CD8 T cells in chronic HBV infection. Our findings suggest the PD-1 levels of HBVcore -specific CD8 T cells can be used as a valuable indicator of personal immune response for clinical application in hepatitis management.

Breakthrough infection by hepatitis B virus in a vaccinated blood donor: An emerging threat for transfusion safety in low-endemic countries?

We present the case of a breakthrough infection by hepatitis B virus (HBV), intending to warn about the challenge that HBV represents for transfusion safety. Virological markers for HBV infection were assayed during a blood donor screening by detection of HBsAg, anti-HBc, and viral nucleic acid (HBV DNA) by a nucleic acid test (NAT). Additionally, samples were analyzed for detection of immunoglobulin M anti-HBc, HBeAg, anti-HBe, and anti-HBs. A first-time donor repeatedly tested positive for HBV DNA by NAT and nonreactive for HBV-serological markers of infection. He stated having completed the anti-HBV vaccination schedule; thus, study of anti-Hbs resulted in reactive at protective level (18 mIU/mL). The donor denied clinical symptoms of hepatitis and remained healthy during the follow-up period. 95 days postdonation, NAT was negative, seroconversion of anti-HBc ab was detected, and a significant increase in anti-HBs concentration was measured (>1000 mIU/mL). This is the first case of HBV-breakthrough infection reported in Argentina and to our knowledge, this potential threat to transfusion safety is novel in an HBV low-endemic region with high coverage of HBV vaccination. The occurrence of breakthrough infections challenges the current protocols for the identification of HBV-infected subjects, could be a source of silent HBV transmission.

De novo hepatitis B infection following liver transplantation with core antibody positive grafts: The role of surface antibody status in guiding long-term prophylaxis.

Liver transplantation (LT) with hepatitis B core antibody (anti-HBc) positive grafts to hepatitis B surface-antigen (HBsAg) negative recipients is safe and has likely contributed to improvements in organ access over the years. The incidence of de novo hepatitis B infection (HBV) in these instances is low with appropriate prophylaxis and is affected by recipient immunologic status. There is debate as to whether hepatitis B surface antibody (anti-HBs) positivity may safely inform prophylaxis discontinuation post-LT. In this retrospective study of all hepatitis B surface antigen (HBsAg) negative recipients of anti-HBc positive organs at three large academic centers between January 2014 and December 2019, nine LT recipients discontinued prophylaxis after developing anti-HBs antibodies 1 year or later post-LT. Three of the nine patients (33%) developed de novo HBV, defined by positive HBsAg or hepatitis B virus (HBV) DNA, during the study period. The remaining six patients had no evidence of HBV infection after a mean follow-up of 37 months. The patients without de novo HBV had higher anti-HBs titers at the time of prophylaxis discontinuation and were less likely to have negative anti-HBs at the time of transplant or negative anti-HBc at any time point. These results suggest that quantitative anti-HBs titer thresholds rather than qualitative anti-HBs positivity at 1 year or later after LT should be used to identify patients at decreased risk of de novo infection and help guide prophylaxis duration.

Biomarkers of transfusion transmitted occult hepatitis B virus infection: Where are we and what next?

Blood transfusion is a vital procedure, where transfusion-transmitted infection of hepatitis B virus (HBV) remains an important issue, especially from blood donors with occult hepatitis B virus infection (OBI). Occult hepatitis B virus infection is a complex entity to detect using surrogate blood biomarkers for intrahepatic viral transcriptional activity, requiring a continually refined battery of tests utilised for screening. This review aims to critically evaluate the latest advances in the current blood biomarkers to guide the identification of OBI donors and discuss novel HBV markers that could be introduced in future diagnostic practice. Challenges in detecting low HBV surface antigen levels, mutants, and complexes necessitate ultrasensitive multivalent dissociation assays, whilst HBV DNA testing requires improved sensitivity but worsens inaccessibility. Anti-core antibody assays defer almost all potentially infectious donations but have low specificity, and titres of anti-surface antibodies that prevent infectivity are poorly defined with suboptimal sensitivity. The challenges associated with these traditional blood HBV markers create an urgent need for alternative biomarkers that would help us better understand the OBI. Emerging viral biomarkers, such as pre-genomic RNA and HBV core-related antigen, immunological HBV biomarkers of T-cell reactivity and cytokine levels, and host biomarkers of microRNA and human leucocyte antigen molecules, present potential advances to gauge intrahepatic activity more accurately. Further studies on these markers may uncover an optimal diagnostic algorithm for OBI using quantification of various novel and traditional blood HBV markers. Addressing critical knowledge gaps identified in this review would decrease the residual risk of transfusion-transmitted HBV infection without compromising the sustainability of blood supplies.

[Clinical significance and research progress of quantitative hepatitis B virus core antibody measurement].

Hepatitis B virus core antibodies are specific antibodies produced after viral infection that appear early and last for a long time, and its levels in serum are measured by the double-antigen sandwich chemiluminescent microparticle immunoassay method, which has higher sensitivity and specificity, providing new clinical indicators for hepatitis B patients diagnosis, treatment, and drug withdrawal management. This article reviews the clinical significance and research progress of quantitative hepatitis B core antibody measurement and expounds on its research applications and prospects in clinical practice.

Kinetics and Value of Hepatitis B Core-Related Antigen in Patients with Chronic Hepatitis B Virus Infection during Antiviral Treatment.

BACKGROUND: The hepatitis B core-related antigen (HBcrAg) correlates with HBV DNA in patients with chronic HBV infection without antiviral treatment. Its utility in monitoring patients during and after the cessation of nucleos(t)ide analog (NA) treatment is unknown. METHODS: The levels of HBcrAg were longitudinally determined in two cohorts of chronic HBV-infected patients with (A) newly started NA treatment or (B) after NA cessation during a median follow up (FU) of 60 months or 48 weeks, respectively. The correlation of HBcrAg and HBV DNA and the predictive value for HBeAg seroconversion and HBsAg loss were evaluated. RESULTS: Fifty-six patients with newly-started NA treatment and 22 patients with NA cessation were identified. HBcrAg and HBV DNA strongly correlated before NA treatment (r = 0.77, p < 0.0001) and at virological relapse (0.66, p = 0.0063). At the individual level, the discrepant kinetics of HBcrAg and HBV DNA became evident. During NA treatment, 33% (6/18) and 9% (5/56) of patients showed HBeAg seroconversion or HBsAg loss/HBsAg < 100 IU/mL, respectively. Low levels of HBcrAg were associated with these endpoints. CONCLUSION: HBcrAg levels before antiviral treatment help to identify patients with chances of HBsAg loss or HBeAg seroconversion. However, its utility in replacing quantitative HBV DNA to evaluate treatment efficacy or virological relapse off-treatment is limited.

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.

A case-control study of risk factors for incident hepatitis B virus infection in South African blood donors.

OBJECTIVES: Hepatitis B virus (HBV) infection remains a global health problem. Risk factors for HBV infection are usually assessed in prevalent rather than incident infections. To identify demographic and behavioral risks associated with incident HBV among South African blood donors. METHODS: A case-control study was performed between November 2014 and January 2018. Cases were blood donors testing positive for HBV DNA with or without hepatitis B surface antigen but negative for antibody to hepatitis B core antigen. Participants completed an audio computer-assisted structured interview on exposures during the previous 6 months. Sex-specific multivariable logistic regression yielded independent associations between risks and HBV infection. RESULTS: 56 females and 37 males with incident HBV were compared to 438 female and 439 male controls, respectively. For females, risk factors were accepting money or goods for sex, using agents to prepare one's anus prior to anal sex, penetrating injury, non-Black race, and lower educational status. Men reporting homosexual or bisexual orientation or sex with other men, previous injury, referral for HBV testing, or lack of medical insurance were at increased risk. For both sexes, having more than two male sexual partners increased risk. CONCLUSIONS: Sexual behaviors predominated over parenteral exposures as risks for incident HBV in both female and male blood donors.

PreS1BP mediates inhibition of Hepatitis B virus replication by promoting HBx protein degradation.

BACKGROUND: PreS1-binding protein (PreS1BP), recognized as a nucleolar protein and tumor suppressor, influences the replication of various viruses, including vesicular stomatitis virus (VSV) and herpes simplex virus type 1 (HSV-1). Its role in hepatitis B virus (HBV) replication and the underlying mechanisms, however, remain elusive. METHODS: We investigated PreS1BP expression levels in an HBV-replicating cell and animal model and analyzed the impact of its overexpression on viral replication metrics. HBV DNA, covalently closed circular DNA (cccDNA), hepatitis B surface antigen (HBsAg), hepatitis B core antigen (HBcAg), and HBV RNA levels were assessed in HBV-expressing stable cell lines under varying PreS1BP conditions. Furthermore, co-immunoprecipitation and ubiquitination assays were used to detect PreS1BP- hepatitis B virus X protein (HBx) interactions and HBx stability modulated by PreS1BP. RESULTS: Our study revealed a marked decrease in PreS1BP expression in the presence of active HBV replication. Functional assays showed that PreS1BP overexpression significantly inhibited HBV replication and transcription, evidenced by the reduction in HBV DNA, cccDNA, HBsAg, HBcAg, and HBV RNA levels. At the molecular level, PreS1BP facilitated the degradation of HBx in a dose-dependent fashion, whereas siRNA-mediated knockdown of PreS1BP led to an increase in HBx levels. Subsequent investigations uncovered that PreS1BP accelerated HBx protein degradation via K63-linked ubiquitination in a ubiquitin-proteasome system-dependent manner. Co-immunoprecipitation assays further established that PreS1BP enhances the recruitment of the proteasome 20S subunit alpha 3 (PSMA3) for interaction with HBx, thereby fostering its degradation. CONCLUSIONS: These findings unveil a previously unidentified mechanism wherein PreS1BP mediates HBx protein degradation through the ubiquitin-proteasome system, consequentially inhibiting HBV replication. This insight positions PreS1BP as a promising therapeutic target for future HBV interventions. Further studies are warranted to explore the clinical applicability of modulating PreS1BP in HBV therapy.

ENPP1 inhibits the transcription activity of the hepatitis B virus pregenomic promoter by upregulating the acetylation of LMNB1.

Current therapies for hepatitis B virus (HBV) infection can slow disease progression but cannot cure the infection, as it is difficult to eliminate or permanently silence HBV covalently closed circular DNA (cccDNA). The interaction between host factors and cccDNA is essential for their formation, stability, and transcriptional activity. Here, we focused on the regulatory role of the host factor ENPP1 and its interacting transcription factor LMNB1 in HBV replication and transcription to better understand the network of host factors that regulate HBV, which may facilitate the development of new antiviral drugs. Overexpression of ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) in Huh7 cells decreased HBV pregenomic RNA (pgRNA) and hepatitis B core antigen (HBcAg) expression levels, whereas knockdown of ENPP1 increased them. A series of HBV promoter and mutant plasmids were constructed, and a luciferase reporter assay showed that overexpression of ENPP1 caused inhibition of the HBV promoter and its mutants. A DNA pull-down assay showed that lamin B1 (LMNB1), but not ENPP1, interacts directly with the HBV enhancer II/ basic core promoter (EnhII/BCP). ZDOCK and PyMOL software were used to predict the interaction of ENPP1 with LMNB1. Overexpression of LMNB1 inhibited the activity of the HBV promoter and its mutant. The acetylation levels at the amino acids 111K, 261K, and 483K of LMNB1 were reduced compared to the control, and an LMNB1 acetylation mutant containing 111R, 261Q, 261R, 483Q, and 483R showed increased promoter activity. In summary, ENPP1 together with LMNB1 increased the acetylation level at 111K and 261K, and LMNB1 inhibited the activity of HBV promoter and downregulated the expression of pregenomic RNA and HBcAg. Our follow-up studies will investigate the expression, clinical significance, and relevance of ENPP1 and LMNB1 in HBV patient tissues, explore the effect of LMNB1 on post-transcriptional progression, and examine whether ENPP1 can reduce cccDNA levels in the nucleus.

Suggested blood donor deferral strategy regarding hepatitis B infections in China.

BACKGROUND: Hepatitis B virus (HBV) reactivity in individual immunologic and nucleic acid tests (NAT) tests does not represent the true infectious status of the blood donor. This study discusses the use of confirmatory tests to determine when deferral of blood donors is appropriate. METHODS: HBsAg or HBV NAT reactive samples were confirmed via a neutralisation test. All the HBsAg reactive but neutralisation test negative samples were subjected to further anti-HBc testing. The receiver operating characteristic curve was used to obtain the best threshold value using signal-to-cut-off ratios of two HBsAg enzyme-linked immunosorbent assay reagents. RESULTS: Of the 780 HBV reactive samples collected, there were 467 HBsAg reactive but HBV DNA negative samples, of which 65 (13.92%) and 402 (86.08%) were neutralisation test positive and negative, respectively. Of the 402, 91 samples (30% of tested samples) were anti-HBc reactive. HBV DNA positive specimens negative by virus neutralisation were >80% HBcAg positive. A screening strategy was proposed for Chinese blood collection agencies. CONCLUSION: These findings suggest that adopting a screening algorithm for deferring HBV reactive blood donors based on HBsAg and NAT testing followed with HBsAg S/CO consideration and HBcAg testing can be both safe and feasible in China.

Seroreactivity of antibodies to hepatitis B core antigen among hepatitis B surface antigen-screened negative blood donors and its implications for blood safety in a resource-constrained country.

BACKGROUND AND OBJECTIVES: Transfusion-related hepatitis B infections have been reduced significantly with the implementation of blood screening using both serology and nucleic acid amplification technology (NAT) in developed countries. However, in resource-constrained countries, where NAT is inaccessible, the risk persists from early acute and occult cases. This study aimed to determine the antibodies to hepatitis B core antigen (anti-HBc) reactive rate among hepatitis B surface antigen (HBsAg)-screened negative blood donors and its impact on blood safety in the Philippines. MATERIALS AND METHODS: A total of 1602 HBsAg-negative samples, randomly collected from nine leading blood service facilities representative of each region in the Philippines, were tested for anti-HBc immunoglobulin M (IgM), Total and antibodies to HBsAg (anti-HBs) using the Architect i2000SR Immunoassay Analyser (Abbott Laboratories, IL). Anti-HBc IgM and/or Total repeat reactive were further tested for hepatitis B virus (HBV) NAT using the Cobas TaqScreen MPX v2.0 (Roche Diagnostics, Basel). RESULTS: Overall, 19.16% HBsAg-negative samples (n = 307/1602) were reactive for either anti-HBc IgM or Total or a combination of both, of which 1.3% (n = 4/307) had detectable HBV-DNA and 80.5% (n = 247/307) were anti-HBs positive. About the anti-HBs titres, 30.27% (n = 485/1602) were positive (≥10 IU/L) with 55.67% (n = 270/485) having titres ≥100 IU/L. Anti-HBs-only-positive samples were 14.85% (n = 238/1602). CONCLUSION: We observed a high anti-HBc reactive rate (19.16%) with 3.7% anti-HBc-only reactive (anti-HBs negative) and 1.3% HBV-DNA positive. This warrants the need to reconsider existing screening practices to improve blood safety in the country.

A novel phthalazinone derivative as a capsid assembly modulator inhibits hepatitis B virus expression.

Development of new anti-hepatitis B virus (HBV) drugs that target viral capsid assembly is a very active research field. We identify a novel phthalazinone derivative, compound 5832, as a potent HBV inhibitor. In this study, we intend to elaborate the antiviral effect and mechanism of 5832 against HBV in vitro and in vivo. Compound 5832 treatment induces the formation of genome-free empty capsid by interfering with the core protein assembly domain, which significantly decreases the extracellular and intracellular HBV DNA. In the AAV-HBV transduced mouse model, 5832 suppresses serum HBV DNA after 4-week treatment, and decreases HBsAg and HBeAg levels. 5832 treatment also reduces intrahepatic HBV RNA, DNA and HBcAg levels. During the follow-up period after treatment withdrawal, serum antigen levels demonstrated no increase. We demonstrate 5832 treatment could active apoptotic signaling by elevating the expression of death receptor 5 (DR5), which participated in corresponding HBcAg-positive hepatocyte eradication. Phthalazinone derivative 5832 may serve as a promising anti-HBV drug candidate to improve the treatment options for chronic HBV infection.