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.

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.

N6-methyladenosine modification of the 5' epsilon structure of the HBV pregenome RNA regulates its encapsidation by the viral core protein.

Hepatitis B virus (HBV) contains a partially double-stranded DNA genome. During infection, its replication is mediated by reverse transcription (RT) of an RNA intermediate termed pregenomic RNA (pgRNA) within core particles in the cytoplasm. An epsilon structural element located in the 5' end of the pgRNA primes the RT activity. We have previously identified the N6-methyladenosine (m6A)-modified DRACH motif at 1905 to 1909 nucleotides in the epsilon structure that affects myriad functions of the viral life cycle. In this study, we investigated the functional role of m6A modification of the 5' ε (epsilon) structural element of the HBV pgRNA in the nucleocapsid assembly. Using the m6A site mutant in the HBV 5' epsilon, we present evidence that m6A methylation of 5' epsilon is necessary for its encapsidation. The m6A modification of 5' epsilon increased the efficiency of viral RNA packaging, whereas the m6A of 3' epsilon is dispensable for encapsidation. Similarly, depletion of methyltransferases (METTL3/14) decreased pgRNA and viral DNA levels within the core particles. Furthermore, the m6A modification at 5' epsilon of HBV pgRNA promoted the interaction with core proteins, whereas the 5' epsilon m6A site-mutated pgRNA failed to interact. HBV polymerase interaction with 5' epsilon was independent of m6A modification of 5' epsilon. This study highlights yet another pivotal role of m6A modification in dictating the key events of the HBV life cycle and provides avenues for investigating RNA-protein interactions in various biological processes, including viral RNA genome encapsidation in the context of m6A modification.

Circulating HBV RNA and Hepatitis B Core-Related Antigen Trajectories in Persons With HIV/HBV Coinfection and Hepatitis B Surface Antigen Loss During Tenofovir Therapy.

BACKGROUND: We evaluated long-term trajectories of circulating hepatitis B virus (HBV) RNA and hepatitis B core-related antigen (HBcrAg) in persons with and without hepatitis B surface antigen (HBsAg) loss during tenofovir therapy in the Swiss HIV Cohort Study. METHODS: We included 29 persons with HIV with HBsAg loss and 29 matched persons with HIV without HBsAg loss. We compared HBV RNA and HBcrAg decline and assessed the cumulative proportions with undetectable HBV RNA and HBcrAg levels during tenofovir therapy using Kaplan-Meier estimates. RESULTS: HBsAg loss occurred after a median of 4 years (IQR, 1-8). All participants with HBsAg loss achieved suppressed HBV DNA and undetectable HBV RNA preceding undetectable quantitative HBsAg levels, whereas 79% achieved negative HBcrAg. In comparison, 79% of participants without HBsAg loss achieved undetectable HBV-RNA and 48% negative HBcrAg. After 2 years of tenofovir therapy, an HBV RNA decline ≥1 log10 copies/mL had 100% sensitivity and 36.4% specificity for HBsAg loss, whereas an HBcrAg decline ≥1 log10 U/mL had 91.0% sensitivity and 64.5% specificity. CONCLUSIONS: HBV RNA suppression preceded undetectable quantitative HBsAg levels and had high sensitivity but low specificity for HBsAg loss during tenofovir therapy in persons with HIV. HBcrAg remained detectable in approximately 20% of persons with HBsAg loss and 50% of persons without HBsAg loss.

Quantification of circulating HBV RNA expressed from intrahepatic cccDNA in untreated and NUC treated patients with chronic hepatitis B.

OBJECTIVE: A convenient, reproducible biomarker of hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) transcriptional activity is lacking. We measured circulating HBV RNA (cirB-RNA) in untreated and nucleos(t)ide analogues (NUC) treated chronic hepatitis B (CHB) patients to define its correlation with intrahepatic viral markers and HBV core-related antigen (HBcrAg). DESIGN: Paired liver biopsy and serum samples were collected from 122 untreated and 30 NUC-treated CHB patients. We measured cirB-RNA, HBV DNA, hepatitis B surface antigen (HBsAg), HBcrAg and alanine aminotransferase levels. cirB-RNA was quantified using an investigational HBV RNA assay for use on the cobas 6800 system. The test detects a region spanning the HBV canonical polyadenylation site. cccDNA and 3.5 kb RNA in liver tissue were assessed by quantitative PCR and droplet digital PCR. RESULTS: cirB-RNA was detectable in 100% of HBeAg(+) chronic hepatitis (CH), 57% and 14% of HBeAg(-) CH and chronic infection untreated patients and 47% of NUC-treated patients. cirB-RNA undetectability was associated with lower intrahepatic cccDNA transcriptional activity, as well as serum HBcrAg, but no significant differences in HBsAg, in both untreated and treated patients. In untreated HBeAg(-) patients, cirB-RNA correlated with intrahepatic 3.5 kb RNA and cccDNA transcriptional activity, serum HBV DNA and HBcrAg, but not with HBsAg or total cccDNA levels. Combined undetectability of both cirB-RNA and HBcrAg detection in untreated HBeAg(-) patients identified a subgroup with the lowest levels of intrahepatic transcriptionally active cccDNA. CONCLUSION: Our results support the usefulness of quantification of circulating HBV RNA expressed from cccDNA as an indicator of intrahepatic active viral reservoir in both untreated and NUC-treated CHB patients. TRIAL REGISTRATION NUMBER: NCT02602847.

HBcrAg values may predict virological and immunological responses to pegIFN-α in NUC-suppressed HBeAg-negative chronic hepatitis B.

OBJECTIVE: Selected populations of patients with chronic hepatitis B (CHB) may benefit from a combined use of pegylated interferon-alpha (pegIFN-α) and nucleos(t)ides (NUCs). The aim of our study was to assess the immunomodulatory effect of pegIFN-α on T and natural killer (NK) cell responses in NUC-suppressed patients to identify cellular and/or serological parameters to predict better T cell-restoring effect and better control of infection in response to pegIFN-α for a tailored application of IFN-α add-on. DESIGN: 53 HBeAg-negative NUC-treated patients with CHB were randomised at a 1:1 ratio to receive pegIFN-α-2a for 48 weeks, or to continue NUC therapy and then followed up for at least 6 months maintaining NUCs. Serum hepatitis B surface antigen (HBsAg) and hepatitis B core-related antigen (HBcrAg) levels as well as peripheral blood NK cell phenotype and function and HBV-specific T cell responses upon in vitro stimulation with overlapping HBV peptides were measured longitudinally before, during and after pegIFN-α therapy. RESULTS: Two cohorts of pegIFN-α treated patients were identified according to HBsAg decline greater or less than 0.5 log at week 24 post-treatment. PegIFN-α add-on did not significantly improve HBV-specific T cell responses during therapy but elicited a significant multispecific and polyfunctional T cell improvement at week 24 post-pegIFN-α treatment compared with baseline. This improvement was maximal in patients who had a higher drop in serum HBsAg levels and a lower basal HBcrAg values. CONCLUSIONS: PegIFN-α treatment can induce greater functional T cell improvement and HBsAg decline in patients with lower baseline HBcrAg levels. Thus, HBcrAg may represent an easily and reliably applicable parameter to select patients who are more likely to achieve better response to pegIFN-α add-on to virally suppressed patients.

Selective depletion of HBV-infected hepatocytes by class A capsid assembly modulators requires high levels of intrahepatic HBV core protein.

Capsid assembly mediated by hepatitis B virus (HBV) core protein (HBc) is an essential part of the HBV replication cycle, which is the target for different classes of capsid assembly modulators (CAMs). While both CAM-A ("aberrant") and CAM-E ("empty") disrupt nucleocapsid assembly and reduce extracellular HBV DNA, CAM-As can also reduce extracellular HBV surface antigen (HBsAg) by triggering apoptosis of HBV-infected cells in preclinical mouse models. However, there have not been substantial HBsAg declines in chronic hepatitis B (CHB) patients treated with CAM-As to date. To investigate this disconnect, we characterized the antiviral activity of tool CAM compounds in HBV-infected primary human hepatocytes (PHHs), as well as in HBV-infected human liver chimeric mice and mice transduced with adeno-associated virus-HBV. Mechanistic studies in HBV-infected PHH revealed that CAM-A, but not CAM-E, induced a dose-dependent aggregation of HBc in the nucleus which is negatively regulated by the ubiquitin-binding protein p62. We confirmed that CAM-A, but not CAM-E, induced HBc-positive cell death in both mouse models via induction of apoptotic and inflammatory pathways and demonstrated that the degree of HBV-positive cell loss was positively correlated with intrahepatic HBc levels. Importantly, we determined that there is a significantly lower level of HBc per hepatocyte in CHB patient liver biopsies than in either of the HBV mouse models. Taken together, these data confirm that CAM-As have a unique secondary mechanism with the potential to kill HBc-positive hepatocytes. However, this secondary mechanism appears to require higher intrahepatic HBc levels than is typically observed in CHB patients, thereby limiting the therapeutic potential.

Low Hepatitis B Core-Related Antigen Levels Correlate Higher Spontaneous Seroclearance of Hepatitis B Surface Antigen in Chronic Hepatitis B Patients With High Hepatitis B Surface Antigen Levels.

BACKGROUND & AIMS: Seroclearance of hepatitis B surface antigen (HBsAg) indicates functional cure for hepatitis B virus (HBV) infection. Low HBsAg levels can predict HBsAg seroclearance over time. However, little is known about the association between hepatitis B core-related antigen (HBcrAg) levels and spontaneous seroclearance of HBsAg. METHODS: We conducted a retrospective cohort study including 2614 treatment-naïve patients with chronic HBV infection who received long-term follow-up at the National Taiwan University Hospital. The primary end point was spontaneous HBsAg seroclearance. We aimed to explore whether HBcrAg levels could predict HBsAg seroclearance, especially for patients with HBsAg levels >1000 IU/mL. RESULTS: There were 465 patients who cleared HBsAg with 32,414.72 person-years of follow-up, with a mean clearance rate of 1.43% per year. We found that lower HBcrAg levels at baseline were associated with an increased likelihood of HBsAg seroclearance (log rank P < .001). When restricting the study population to 1539 patients with HBsAg levels >1000 IU/mL, only HBcrAg <10,000 U/mL (vs ≥100,000 U/mL) served as an independent viral predictor for HBsAg seroclearance, with adjusted hazard ratio of 1.95 (95% CI, 1.16-3.27). In contrast to the late decline of HBsAg levels (5-9 years before HBsAg seroclearance), HBcrAg levels became undetectable 10-14 years before HBsAg seroclearance. This finding was confirmed by the different annual HBsAg seroclearance rates in the first and second decades of follow-up (0.97% vs 3.75%; P < .001) in patients achieving undetectable HBcrAg levels. CONCLUSIONS: Lower serum HBcrAg levels were associated with increased probability of HBsAg seroclearance over time. In patients with HBsAg levels >1000 IU/mL, clearing HBcrAg may serve as an early biomarker for HBsAg seroclearance.

Incidences of new hepatitis B infection and anti-hepatitis B core-negative occult hepatitis B infection among Japanese blood donors in relation to anti-hepatitis B surface antigen levels.

A transfusion-transmitted hepatitis B virus (HBV) infection caused by blood only positive for anti-hepatitis B surface antigen (anti-HBs) was reported. Occult HBV infection (OBI) with sole anti-HBs among blood donors is an issue. The incidence of HBV infection among repeat blood donors was investigated with a detailed HBV infection phase, focusing on the influence of anti-HBs level. This study followed 3 435 653 donors for HBV DNA conversion over 4 years and 9 months. Infection phase was determined based on marker changes over DNA conversion. This study identified 115 hepatitis B surface antigen (HBsAg) conversions, 72 DNA-only conversions, and 15 DNA plus anti-hepatitis B core (anti-HBc) conversions among donors all negative for HBV DNA, HBsAg, and anti-HBc. Total incidence was 2.38/100 000 person-years (PY). None of these 202 new HBV infections arose in the group with anti-HBs titer ≥ 10 mIU/mL. In total, 30 anti-HBc-negative OBIs were identified (incidence; 0.35/100 000 PY); 7 showed typical secondary anti-HBs response, and 23 showed stable anti-HBc and anti-HBs levels at DNA conversion. The HBV infection-protective ability of anti-HBs ≥ 10 mIU/mL was reinforced. In addition to new infections, the blood donor population includes anti-HBc-positive- and negative OBI with immune reactions or abortive HBV infection.

Guidance on treatment endpoints and study design for clinical trials aiming to achieve cure in chronic hepatitis B and D: Report from the 2022 AASLD-EASL HBV-HDV Treatment Endpoints Conference.

Representatives from academia, industry, regulatory agencies, and patient advocacy groups convened under the American Association for the Study of Liver Diseases (AASLD) and the European Association for the Study of the Liver (EASL) in June 2022 with the primary goal of achieving consensus on chronic HBV and HDV treatment endpoints to guide clinical trials aiming to "cure" HBV and HDV. Conference participants reached an agreement on some key points. The preferred primary endpoint for phase II/III trials evaluating finite treatments for chronic hepatitis B (CHB) is a "functional" cure, defined as sustained HBsAg loss and HBV DNA less than the lower limit of quantitation (LLOQ) 24 weeks off-treatment. An alternate endpoint would be "partial cure" defined as sustained HBsAg level < 100 IU/mL and HBV DNA < LLOQ 24 weeks off-treatment. Clinical trials should initially focus on patients with HBeAg positive or negative CHB, who are treatment-naive or virally suppressed on nucleos(t)ide analogs. Hepatitis flares may occur during curative therapy and should be promptly investigated and outcomes reported. HBsAg loss would be the preferred endpoint for chronic hepatitis D, but HDV RNA < LLOQ 24 weeks off-treatment is a suitable alternate primary endpoint of phase II/III trials assessing finite strategies. For trials assessing maintenance therapy, the primary endpoint should be HDV RNA < LLOQ assessed at on-treatment week 48. An alternate endpoint would be ≥2 log reduction in HDV RNA combined with normalization of alanine aminotransferase level. Suitable candidates for phase II/III trials would be treatment-naiive or experienced patients with quantifiable HDV RNA. Novel biomarkers (hepatitis B core-related antigen [HBcrAg] and HBV RNA) remain exploratory, while nucleos(t)ide analogs and pegylated interferon still have a role in combination with novel agents. Importantly, patient input is encouraged early on in drug development under the FDA/EMA patient-focused drug development programs.

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.

Clinical performance of Circulating HBV RNA and iTACT-HBcrAg Assays in HBeAg-negative and HBsAg-cleared Chronic Hepatitis B Patients.

Hepatitis B virus (HBV) RNA and hepatitis B core-related antigen (HBcrAg) have been reported to reflect the transcriptional activity of covalently closed circular HBV DNA. We retrospectively investigated the proportions of quantifiable serum HBV RNA and immunoassay for total antigen including complex via pretreatment-hepatitis B core-related antigen (iTACT-HBcrAg) in chronic hepatitis B patients negative for hepatitis B e antigen (HBeAg) and/or with hepatitis B surface antigen (HBsAg) seroclearance. This study included 246 HBeAg-negative HBV-infected patients, who comprised 13 with liver cirrhosis (LC, the LC group), 118 chronic hepatitis (CH, the CH group), and 115 inactive carriers (IC, the IC group), and 44 patients with HBsAg seroclearance. iTACT-HBcrAg and HBV RNA levels were determined using stored serum samples. Higher proportions of the patients had quantifiable iTACT-HBcrAg than HBV RNA in all groups of HBeAg-negative patients (iTACT-HBcrAg: 84.6%, 90.7%, 35.7%, HBV RNA: 23.1%, 26.3%, 14.8%, for the LC, CH, IC groups). With HBsAg seroclearance (HBsAg <0.05 IU/mL), the proportions of quantifiable samples for HBV RNA were also lower than iTACT-HBcrAg (0% for HBV RNA). Thus, iTACT-HBcrAg was more often detectable than circulating HBV RNA in this study population. Further long-term prospective evaluation of iTACT-HBcrAg is desirable for its utilization in clinical practice.

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.

Predictive role of early treatment dynamics of HBV RNA and HBcrAg for HBeAg seroconversion in children with chronic hepatitis B.

This study aimed to assess the predictive capacity of emerging serological markers, serum HBV RNA and HBcrAg, for HBeAg seroconversion in children with HBeAg-positive chronic hepatitis B (CHB). Treatment-naïve HBeAg-positive CHB children who admitted to the Liver Disease Center of Hunan Children's Hospital between April 2021 and September 2022 and received treatment with the combined entecavir and interferon-alpha treatment were recruited. Serum HBV RNA and HBcrAg were measured at baseline and Weeks 12, 24, and 48 of treatment. Our study showed that serum HBV RNA (HR = 0.71, 95% CI: 0.56-0.91, p = 0.006), HBcrAg (HR = 0.60, 95% CI: 0.43-0.84, p = 0.003), and HBsAg (HR = 0.49, 95%CI: 0.36-0.69, p < 0.001) at Week 12 were independent predictors of HBeAg seroconversion. ROC curve analysis presented that serum HBV RNA decline value (ΔHBV RNA) at Week 36 and HBcrAg decline value (ΔHBcrAg) at Week 12 (AUC = 0.871, p = 0.003 and AUC = 0.810, p = 0.003, respectively) could effectively predict HBeAg seroconversion. Furthermore, the optimal critical values were determined and the children with ΔHBV RNA > 3.759 log10 copies/mL at Week 36 or ΔHBcrAg >0.350 log10 U/mL at Week 12 more likely to achieve HBeAg seroconversion. The serum HBV RNA and HBcrAg provide new insights into the treatment of CHB in children. Early assessment of serum HBV RNA and HBcrAg during treatment can assist clinical decision-making and optimize individualized therapeutic approaches.

Use of HBV RNA and to predict change in serological status and disease activity in CHB.

BACKGROUND AND AIMS: Predicting changes in disease activity and serological endpoints is necessary for the management of patients with chronic hepatitis B (CHB). We examined whether HBV RNA and hepatitis B core-related antigen (HBcrAg), two specialized virological markers proposed to reflect the activity of covalently closed circular DNA, may improve the ability to predict not sustained inactive carrier phase, spontaneous alanine aminotransferase (ALT) flare, HBeAg loss, and HBsAg loss. APPROACH AND RESULTS: Among eligible participants enrolled in the North American Hepatitis B Research Network Adult Cohort Study, we evaluated demographic, clinical, and virologic characteristics, including HBV RNA and HBcrAg, to predict not sustained inactive carrier phase, ALT flare, HBeAg loss, and HBsAg loss through a series of Cox proportional hazard or logistic regression models, controlling for antiviral therapy use. Among the study population, 54/103 participants experienced not sustained inactive carrier phase, 41/1006 had a spontaneous ALT flare, 83/250 lost HBeAg, and 54/1127 lost HBsAg. HBV RNA or HBcrAg were predictive of all 4 events. However, their addition to models of the readily available host (age, sex, race/ethnicity), clinical (ALT, use of antiviral therapy), and viral factors (HBV DNA), which had acceptable-excellent accuracy (e.g., AUC = 0.72 for ALT flare, 0.92 for HBeAg loss, and 0.91 for HBsAg loss), provided only small improvements in predictive ability. CONCLUSION: Given the high predictive ability of readily available markers, HBcrAg and HBV RNA have a limited role in improving the prediction of key serologic and clinical events in patients with CHB.

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.

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.

Hepatitis B screening in hematology patients receiving intravenous immunoglobulin.

BACKGROUND: Transient positivity for hepatitis B core antibody (Anti-HBc) following intravenous immunoglobulin (IVIG) and subcutaneous immunoglobulin exposure is a well-described phenomenon. The aim of this study was to retrospectively review Hepatitis B viral screening practices in IVIG recipients in a hematology specific cohort at a single center. METHODS: Electronic databases were analyzed to identify all hematology patients who received IVIG from September 2022 to March 2022 at a single Irish center (n = 43). The proportion of patients that had a baseline anti-HBc tested prior to IVIG receipt was assessed as well as the proportion of patients that developed a transiently positive anti-HBc following IVIG exposure. Data were also collected relating to signal cut-off ratios in patients with detectable anti-HBc post-IVIG. RESULTS: 58.1% of patients had at least one serological hepatitis B viral test sent prior to IVIG exposure. Anti-HBc was the least common serological investigation performed prior to IVIG exposure (21% of recipients). A positive or equivocal "low level antibody" was identified in 15% of recipients and this was proven to be transient in all cases. CONCLUSION: The minority of hematology patients had a baseline anti-HBc assessed prior to IVIG exposure. All patients in this study had the potential to require further immunosuppressive therapies, which could be limited by a misleading anti-HBc result. We therefore advocate for baseline anti-HBc testing to be performed prior to IVIG exposure in hematology patients and for cautious interpretation of anti-HBc results taking into account signal cut-off ratios post-IVIG exposure.

High end-of-treatment hepatitis B core-related antigen levels predict hepatitis flare after stopping nucleot(s)ide analogue therapy.

BACKGROUND AND AIMS: Accurate biomarkers to predict outcomes following discontinuation of nucleos(t)ide analogue (NA) therapy are needed. We evaluated serum hepatitis B core-related antigen (HBcrAg) level as a biomarker for predicting outcomes after NA discontinuation. METHODS: Patients with HBeAg-negative chronic hepatitis B (CHB) without cirrhosis were enrolled in a prospective trial evaluating clinical outcomes until 96 weeks after NA discontinuation. End of treatment (EOT) and off-treatment levels of serum HBcrAg, HBsAg, HBV RNA and HBV DNA were used to predict key clinical outcomes including hepatitis flare (ALT ≥5 × ULN and HBV DNA > 2000 IU/mL). The SCALE-B score was calculated for the purposes of model validation. RESULTS: HBcrAg was tested amongst 65 participants. The median age was 54 years, 54% were male and 83% were Asian. HBcrAg was detectable in 86% patients. HBcrAg level ≥4 log U/mL at EOT was predictive of hepatitis flare [8/10 (80%) vs. 17/55 (31%), p = .001]. The presence of either HBcrAg ≥4 log U/mL or detectable HBV RNA at EOT predicted for both biochemical relapse and hepatitis flare. The SCALE-B model at EOT predicted for virological relapse, biochemical relapse, hepatitis flare and HBsAg loss in this cohort. An increase in the serum HBcrAg level off-treatment was also associated with hepatitis flare. No participant with EOT HBcrAg level ≥4 log U/mL achieved HBsAg loss. CONCLUSIONS: High levels of serum HBcrAg predict for hepatitis flare after stopping NA therapy and low likelihood of HBsAg loss at week 96. People with high levels of serum HBcrAg are not suitable candidates for NA discontinuation.

What is the benefit of prophylaxis to prevent HBV reactivation in HBsAg-negative anti-HBc-positive patients? Meta-analysis and decision curve analysis.

BACKGROUND AND AIMS: Patients with overt or occult hepatitis B virus (HBV) infection receiving immunosuppressive treatments have a wide risk of HBV reactivation (HBVr). We performed meta-analysis with decision curve analyses (DCA) to estimate the risk of HBVr in HBsAg-negative anti-HBc-positive patients naïve to nucleos(t)ide analogues (NAs) receiving immunosuppressive treatments. APPROACH AND RESULTS: Studies were identified through literature search until October 2022. Pooled estimates were obtained using random-effects model. Subgroup analyses were performed according to underlying disease and immunosuppressive treatments. DCA was used to identify the threshold probability associated with the net benefit of antiviral prophylaxis in HBsAg-negative anti-HBc-positive patients. We selected 68 studies (40 retrospective and 28 prospective), including 8034 patients with HBsAg negative anti-HBc positive. HBVr was 4% (95% CI 3%-6%) in HBsAg-negative anti-HBc-positive patients, with a significantly high heterogeneity (I2 69%; p < .01). The number-needed-to-treat (NNT) by DCA ranged from 8 to 24 for chemotherapy plus rituximab, from 12 to 24 for targeted therapies in cancer patients and from 13 to 39 for immune-mediated diseases. Net benefit was small for monoclonal antibodies. CONCLUSIONS: Our DCA in HBsAg-negative anti-HBc-positive patients provided evidence that NA prophylaxis is strongly recommended in patients treated with chemotherapy combined with rituximab and could be appropriate in patients with cancer treated with targeted therapies and in patients with immune-mediated diseases. Finally, in patients with cancer treated with monoclonal antibodies or with chemotherapy without rituximab, the net benefit is even lower.