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Specific HBsAg mutations are known to hamper HBsAg recognition by neutralizing antibodies thus challenging HBV-vaccination efficacy. Nevertheless, information on their impact and spreading over time is limited. Here, we characterize the circulation of vaccine-escape mutations from 2005 to 2019 and their correlation with virological parameters in a large cohort of patients infected with HBV genotype-D (N = 947), dominant in Europe. Overall, 17.7% of patients harbours ≥1 vaccine-escape mutation with the highest prevalence in subgenotype-D3. Notably, complex profiles (characterized by ≥2 vaccine-escape mutations) are revealed in 3.1% of patients with a prevalence rising from 0.4% in 2005-2009 to 3.0% in 2010-2014 and 5.1% in 2015-2019 (P = 0.007) (OR[95%CI]:11.04[1.42-85.58], P = 0.02, by multivariable-analysis). The presence of complex profiles correlates with lower HBsAg-levels (median[IQR]:40[0-2905]IU/mL for complex profiles vs 2078[115-6037]IU/ml and 1881[410-7622]IU/mL for single or no vaccine-escape mutation [P < 0.02]). Even more, the presence of complex profiles correlates with HBsAg-negativity despite HBV-DNA positivity (HBsAg-negativity in 34.8% with ≥2 vaccine-escape mutations vs 6.7% and 2.3% with a single or no vaccine-escape mutation, P < 0.007). These in-vivo findings are in keeping with our in-vitro results showing the ability of these mutations in hampering HBsAg secretion or HBsAg recognition by diagnostic antibodies. In conclusion, vaccine-escape mutations, single or in complex profiles, circulate in a not negligible fraction of HBV genotype-D infected patients with an increasing temporal trend, suggesting a progressive enrichment in the circulation of variants able to evade humoral responses. This should be considered for a proper clinical interpretation of HBsAg-results and for the development of novel vaccine formulations for prophylactic and therapeutic purposes.

Background and Objectives: There are reports of false qualitative HBsAg results, because of various causes, such as samples with low HBsAg concentrations that may produce false positives. The main aims of this study were to validate the analytical accuracy and to assess the utility of the Elecsys assay compared to that of the qualitative HbsAg assay as a screening test in resolving equivocal qualitative HbsAg results. Materials and Methods: The limit of blank (LoB), the limit of detection (LoD), the limit of quantification (LoQ), and linearity were estimated to validate the analytical accuracy of the Elecsys HBsAg II Quant assay. A total of 449 serum samples showing initial equivocal results (1–50 index) were evaluated by Elecsys HBsAg II Quant and ADVIA Centaur HBsAg II assays. Results: The LoQ of the assay was determined to be 0.050 IU/mL, as provided by the manufacturer. The Kappa agreement between the two assays was almost perfect, at 0.9669, despite seven discordant results. With a specificity of 100% at new cut-off index value ≥5.42, about 78 samples (17%, 78/449) with index value ≥5.42 were interpreted as positives without further duplicate tests, however the remaining 371 samples with index value <5.42 need to be confirmed with additional HBV marker assays. Conclusions: We confirm that the Elecsys HBsAg II Quant assay is accurate and sensitive for HBV infection and recommend it as an alternative confirmatory HBsAg assay for resolving equivocal qualitative HBsAg results.

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

Background Occult hepatitis B virus infection (OBI) carries high risk of transmissibility and pathogenicity. Mutations in the S gene are considered major contributors to OBI development. Our previous study identified the C138R mutation in the S gene of OBI patients. This study aimed to investigate the impact of the C138R mutation on HBsAg function and its mechanisms leading to OBI. Methods Full-length hepatitis B virus (HBV) plasmids (HBV 1.3, genotype B) and mutant plasmids (HBV C138R) were constructed, along with HA-tagged wild-type (sWT) and mutant (sC138R) S protein expression plasmids. The effects of the C138R mutation on HBsAg antigenicity, secretion, and dimer formation were evaluated through in vitro cell transfection and in vivo hydrodynamic tail vein injection in mice. Results The C138R mutation markedly reduced intra- and extracellular HBsAg levels without affecting HBV transcription, replication, or other viral protein expression. HA-tag assays indicated that this reduction resulted from impaired antigenicity. Several commercial ELISA kits results and HA levels in cell supernatants demonstrated that the C138R mutation also impaired HBsAg secretion. Similarly, the C107R mutation significantly decreased both HBsAg antigenicity and secretion. Non-reducing WB assays and Alphafold 3-based structural predictions revealed that the C138R mutation lead to the formation of aberrant HBsAg dimers. These findings were confirmed in the HBV mouse model. Conclusion The C138R mutation contributed to OBI by simultaneously impairing HBsAg antigenicity and secretion. Disruption of the disulfide bond between aa107 and aa138, resulting in the formation of structurally abnormal dimers, represented the primary mechanism underlying HBsAg dysfunction caused by this mutation.

Background A multivariate predictive model was constructed using baseline and 12-week clinical data to evaluate the rate of clearance of hepatitis B surface antigen (HBsAg) at the 48-week mark in patients diagnosed with chronic hepatitis B who are receiving treatment with pegylated interferon α (PEG-INFα). Methods The study cohort comprised CHB patients who received pegylated interferon treatment at Mengchao Hepatobiliary Hospital, Fujian Medical University, between January 2019 and April 2024. Predictor variables were identified (LASSO), followed by multivariate analysis and logistic regression analysis. Subsequently, predictive models were developed via logistic regression, random forest (RF), gradient boosting decision tree (GBDT), extreme gradient boosting (XGBoost), and support vector machine (SVM) algorithms. The efficacy of these models was assessed through various performance metrics, including the area under the receiver operating characteristic curve (AUC), sensitivity, specificity, and F1 score. Results This study included a total of 224 individuals diagnosed with chronic hepatitis B. The variables baseline log 2 (HBsAg), gender, age, neutrophil count at week 12, HBsAg decline rate at week 12, and HBcAb at week 12 were closely associated with functional cure and were included in the predictive model. In the validation term, the logistic regression model had an AUC of 0.858, which was better than that of the other machine learning models (AUC = 0.858,F1 = 0.753). Consequently, this model was selected for the development of the predictive tool. Conclusions The combined use of the baseline log 2 (HBsAg) value, HBsAg decline rate at week 12, gender, neutrophil count at week 12, and age can serve as a foundational predicting model for anticipating the clearance of HBsAg in individuals with chronic hepatitis B who are receiving PEG-INFα therapy.

We aimed to establish the optimal value of the alanine aminotransferase (ALT)/quantitative HBsAg (qHBsAg) ratio and qHBsAg level at end of treatment (EOT) to predict HBsAg seroclearance following cessation of entecavir versus tenofovir disoproxil fumarate (TDF) in HBeAg-negative with chronic hepatitis B (CHB) patients. This retrospective cohort study included 715 HBeAg-negative patients without cirrhosis who had previously received entecavir (n=438) or TDF (n=277). The cumulative incidences of HBsAg seroclearance at 5 and 10 years after cessation of entecavir or TDF were 13.0% and 33.8%, respectively. Both a qHBsAg level <100 IU/mL and an ALT/qHBsAg ratio ≥0.2 at EOT were independent predictors of HBsAg seroclearance (p <0.001). A qHBsAg of 100 IU/mL had significantly lower Akaike information criterion (AIC) and Bayesian information criterion (BIC) values than an ALT/qHBsAg ratio of 0.2 at EOT (AIC: 1273 vs. 1305, BIC: 1276 vs. 1308, both p <0.001). Additionally, patients who discontinued entecavir therapy had a higher rate of HBsAg seroclearance than patients who discontinued TDF therapy, regardless of qHBsAg ≥100 IU/mL (p=0.027) or ALT/qHBsAg ratio <0.2 (p=0.028). The optimal values for predicting HBsAg seroclearance were 300 and 100 IU/mL for qHBsAg and 0.1 and 0.2 for the ALT/qHBsAg ratio after stopping entecavir versus TDF, respectively. A qHBsAg level <100 IU/mL at EOT is better predictor of HBsAg seroclearance than an ALT/qHBsAg ratio ≥0.2. Compared to TDF, entecavir was associated with higher rates of HBsAg seroclearance in patients with higher qHBsAg level or a lower ALT/qHBsAg ratio at EOT.

Background Previous studies by our group and others have demonstrated that pegylated interferon (PEG-IFN) therapy results in a relatively high rate of clinical cure in inactive hepatitis B surface antigen carriers (IHCs). Emerging evidence suggests that B lymphocytes play a pivotal role in HBsAg clearance. This study aimed to evaluate the efficacy of PEG-IFN in IHCs, investigate dynamic changes in global (non-HBV-specific) B-cell subset frequencies and their association with HBsAg clearance, and characterize the immunological profiles of B cells in CHB patients who achieved a functional cure. Methods A total of 458 inactive IHCs who were enrolled at Beijing You’an Hospital, Capital Medical University, between January 2008 and February 2023 were included in this study. All participants received once-weekly subcutaneous injections of PEG-IFNα-2b, and clinical outcomes were retrospectively analyzed in the entire cohort. B-cell phenotyping was performed in a subset of 36 patients (21 with HBsAg clearance and 15 without) to assess the frequencies of PD-1⁺ and IgG⁺ B-cell subsets, including total B cells, plasmablasts, naïve B cells, immature B cells, and memory B cells. These assessments were conducted at baseline and at weeks 12 and 24 of treatment. Results Clinical cure rates were 15.98% at week 24 and 28.27% at week 48 of PEG-IFN therapy. At week 24, the frequency of PD-1⁺ total B cells was significantly lower in the C group than in the NC group (0.63% vs. 1.61%, P  = 0.037). The proportion of IgG⁺ plasmablasts was significantly higher in the C group compared to the NC group (10% vs. 5.5%, P  = 0.016). Conclusion IHCs who achieved HBsAg clearance under PEG-IFN therapy had lower PD-1⁺ total B-cell frequencies and relatively higher proportions of IgG⁺ plasmablasts than those without clearance. These findings show limited observational differences in PD-1⁺ B-cell and IgG⁺ plasmablast frequencies between groups, which may be associated with clinical cure, but the biological implications remain uncertain and warrant confirmation in larger mechanistic studies.

Background and Objectives: This study aims to estimate the analytical performance of the Sysmex HISCL HBsAg assay and to assess the analytical correlation with the Roche Elecsys HBsAg II quant assay with clinical samples and the WHO International Standard (IS). Materials and Methods: The intra-assay precision, linearity, assay limitation, accuracy, and comparative evaluation of the HISCL HBsAg assay were estimated. Results: Extrapolating from the plot of the average total allowable error versus the reference value, an accuracy goal of 20% would be achieved around a limit of quantification (LoQ) of 0.014867 IU/mL. The percentage of biases for each level of the WHO IS measured by the two assays were less than 15%, except for the WHO 3rd IS, for which the HISCL HBsAg assay achieved a percentage of bias of 33%. In the comparative evaluation, Passing–Bablok regression analysis did not reveal any significant deviation from linearity between the two assays (y = −48.6998 + 1.9206x; p = 0.79 by the CUSUM test for linearity). The mean difference of the quantitative HBsAg level between the two assays was 1762.5 IU/mL in the Bland–Altman plot. Conclusions: The HISCL HBsAg assay, with a highly sensitive LoQ of 0.03 IU/mL, showed similar analytical performance in HBsAg quantification to the Elecsys HBsAg II quant assay and may be helpful in obtaining better diagnoses and therapeutic strategies for treating HBV infections.

Among the advantaged population with clinical cure of chronic hepatitis B, chronic inactive hepatitis B virus carriers (IHCs) and nucleoside analog-experienced patients have similar serological manifestations. This study established non-interferon-treated groups as controls to compare the efficacy of pegylated interferon α-2b (Peg-IFNα-2b) in achieving clinical cure between IHCs and nucleoside analog (NA)-experienced patients. A total of 270 patients were enrolled in this observational study. The IHC cohort comprised 55 patients who received Peg-IFNα-2b (Peg-IFN group), and the other 70 patients did not receive any antiviral treatment (untreated group). Patients treated with NAs were divided into two groups: one group (70 patients) receiving NA add-on Peg-IFNα-2b therapy regimen (NA add-on Peg-IFN group) and another group (75 patients) receiving continuous NA monotherapy (NA group). The primary endpoints were hepatitis B surface antigen (HBsAg) clearance and HBsAg seroconversion at 48 weeks and 72 weeks. At 48 weeks, 65.5% (36/55) and 52.9% (37/70) patients achieved HBsAg clearance in the Peg-IFN group and NA add-on Peg-IFN group, respectively (p = 0.156). HBsAg seroconversion was achieved in 47.3% (26/55) of the Peg-IFN group and 34.3% (24/70) of the NA add-on Peg-IFN group (p = 0.141). At the follow-up of 72 weeks, 36 patients in the Peg-IFN group achieved HBsAg loss (65.5%, 36/55), and 33 patients in the NA add-on Peg-IFN group achieved HBsAg clearance (47.1%, 33/70), which were significantly higher than in the Peg-IFN group (p = 0.041). The HBsAg seroconversion rates in the Peg-IFN group and NA add-on Peg-IFN group at 72 weeks were 45.5% (25/55) and 32.9% (23/70), respectively (p = 0.151). No patient achieved HBsAg clearance or seroconversion in the NA group and untreated group. Furthermore, the receiver operating characteristic curve showed baseline HBsAg< 72 IU/mL, and the decline of HBsAg of more than 80% and 98% from baseline to 12 and 24 weeks provided good predictions for HBsAg clearance. Meanwhile, 77% of patients with baseline HBsAg< 100 IU/mL achieved a clinical cure at 48 weeks. Peg-IFNα-2b results in a high rate of HBsAg clearance and seroconversion in both IHCs and NA-experienced patients, especially for those patients who have HBsAg below 100 IU/mL.

Previous studies have suggested the potential of PD-1/PD-L1 inhibitors in the treatment of chronic HBV infection. However, since phase III clinical trials have not yet been announced, additional clinical insights may be obtained by observing changes in serum hepatitis B surface antigen (HBsAg) and HBV-DNA levels in cancer patients undergoing PD-1 inhibitor therapy. To explore the effects of PD-1 inhibitor combinational therapy on serum HBsAg and HBV-DNA levels, investigate the incidence of HBsAg loss, HBV reactivation (HBVr), and immune-related adverse events (irAEs), and identify the risk factors associated with significant HBsAg fluctuations and HBVr. A retrospective study including 1195 HBsAg-positive cancer patients who received PD-1 inhibitors between July 2019 and June 2023 was conducted, and 180 patients were enrolled in this study. Serum HBsAg levels before and after PD-1 inhibitor administration were compared across different subgroups. The Pearson χ or Fisher exact test was performed to investigate the relationships between categorical variables. Univariable and multivariable analysis were performed to identify the risk factors associated with significant HBsAg fluctuations and HBVr. With the concurrent use of antiviral agents, serum HBsAg levels decreased (Z=-3.966, P < 0.0001) in 129 patients and increased (t=-2.047, P=0.043) in 51 patients. Additionally, 7 patients (3.89%) achieved serum HBsAg loss. Virus replication was suppressed in most of the enrolled patients. When divided patients into different subgroups, significant HBsAg decreases after PD-1 inhibitor administration were discovered in lower baseline HBsAg group (Z=-2.277, P=0.023), HBeAg-seronegative group (Z=-2.200, P=0.028), non-irAEs occurrence group (Z=-2.007, P=0.045) and liver cancer group (Z=-1.987, P=0.047). Of note, 11 patients and 36 patients experienced HBVr (6.11%) and irAEs (20%), respectively, which could lead to discontinuation or delayed use of PD-1 inhibitors. After multivariable analysis, HBeAg-seropositive (OR, 7.236 [95% CI, 1.757-29.793], P=0.01) and the occurrence of irAEs (OR, 4.077 [95% CI, 1.252-13.273], P=0.02) were identified as the independent risk factors for significant HBsAg increase, the occurrence of irAEs (OR, 5.560 [95% CI, 1.252-13.273], P=0.01) was identified as the only independent risk factor for HBVr. PD-1 inhibitors combined with nucleos(t)ide analogues (NAs) may exert therapeutic potential for chronic HBV infection in cancer patients. However, attention also should be paid to the risk of significant elevation in HBsAg levels, HBVr, and irAEs associated with PD-1 inhibitor combinational therapy.