A Dual-domain Engineered Antibody for Efficient HBV Suppression and Immune Responses Restoration.

Chronic hepatitis B (CHB) remains a major public health concern because of the inefficiency of currently approved therapies in clearing the hepatitis B surface antigen (HBsAg). Antibody-based regimens have demonstrated potency regarding virus neutralization and HBsAg clearance. However, high dosages or frequent dosing are required for virologic control. In this study, a dual-domain-engineered anti-hepatitis B virus (HBV) therapeutic antibody 73-DY is developed that exhibits significantly improved efficacy regarding both serum and intrahepatic viral clearance. In HBV-tolerant mice, administration of a single dose of 73-DY at 2 mg kg-1 is sufficient to reduce serum HBsAg by over 3 log10 IU mL-1 and suppress HBsAg to < 100 IU mL-1 for two weeks, demonstrating a dose-lowering advantage of at least tenfold. Furthermore, 10 mg kg-1 of 73-DY sustainably suppressed serum viral levels to undetectable levels for ≈ 2 weeks. Molecular analyses indicate that the improved efficacy exhibited by 73-DY is attributable to the synergy between fragment antigen binding (Fab) and fragment crystallizable (Fc) engineering, which conferred sustained viral suppression and robust viral eradication, respectively. Long-term immunotherapy with reverse chimeric 73-DY facilitated the restoration of anti-HBV immune responses. This study provides a foundation for the development of next-generation antibody-based CHB therapies.

Risk of mother-to-child transmission after amniocentesis in pregnant women with hepatitis B virus: a retrospective cohort study.

BACKGROUND: Amniocentesis is the most widely used invasive prenatal diagnostic sampling technique. However, whether this increases the risk of mother-to-child transmission of infectious diseases remains controversial. OBJECTIVE: This study aimed to determine whether amniocentesis increases the risk of hepatitis B virus infection in infants who received standard prophylaxis, and to assess the related risk factors for mother-to-child transmission in women who underwent amniocentesis during pregnancy. STUDY DESIGN: This retrospective analysis used the clinical data of pregnant women with hepatitis B virus infection at West China Second University Hospital, Sichuan University in 2019. After meeting the inclusion criteria, the participants were divided into 2 groups on the basis of whether they had undergone amniocentesis during pregnancy. The infant hepatitis B virus serologic status was followed 1 to 6 months after completion of immunization. The infant testing positive for hepatitis B surface antigen and negative for Hepatitis B surface antibody indicated mother-to-child transmission of hepatitis B virus. RESULTS: In total, 1764 pregnant women with hepatitis B virus infection were enrolled. Of these, 846 underwent amniocentesis during pregnancy and 918 did not. All offspring received a standardized immunoprophylaxis schedule. The overall mother-to-child transmission rate for hepatitis B virus was 0.6% (5/846) in the amniocentesis group and 0.4% (4/918) in the control group (P=.745). Subgroup analysis showed that the mother-to-child transmission rate in hepatitis B e antigen-positive women was 1.8% (2/111) in the amniocentesis group and 1.0% (2/209) in the control group (P=.612). In women with high viral load, the mother-to-child transmission rate was 1.3% (1/78) vs 0.9% (1/107) (amniocentesis group vs control group; P=1.000). In the amniocentesis group, 31 amniotic fluid specimens had an abnormal appearance (bloody or brown). Univariate analysis showed that the mother-to-child transmission rates of these mothers were statistically higher than those of mothers with pale yellow or transparent amniotic fluid (2/31 vs 3/815; relative risk, 17.527 [3.037-101.151]; P=.012). CONCLUSION: Amniocentesis did not increase the risk of mother-to-child transmission of hepatitis B virus in infants who received a standardized immunoprophylaxis schedule, including those with mothers who were hepatitis B e antigen-positive or had a high viral load. However, the abnormal appearance (bloody or brown) of the amniotic fluid obtained during amniocentesis may indicate increased risk of mother-to-child transmission for hepatitis B virus.

Changing prevalence of chronic hepatitis B virus infection in China between 1973 and 2021: a systematic literature review and meta-analysis of 3740 studies and 231 million people.

OBJECTIVE: China concentrates a large part of the global burden of HBV infection, playing a pivotal role in achieving the WHO 2030 global hepatitis elimination target. METHODS: We searched for studies reporting HBV surface antigen (HBsAg) seroprevalence in five databases until January 2023. Eligible data were pooled using a generalised linear mixed model with random effects to obtain summary HBsAg seroprevalence. Linear regression was used to estimate annual percentage change (APC) and HBsAg prevalence in 2021. RESULTS: 3740 studies, including 231 million subjects, were meta-analysed. HBsAg seroprevalence for the general population decreased from 9.6% (95% CI 8.4 to 10.9%) in 1973-1984 to 3.0% (95% CI 2.1 to 3.9%) in 2021 (APC=-3.77; p<0.0001). Decreases were more pronounced in children <5 years (APC=-7.72; p<0.0001) and 5-18 years (-7.58; p<0.0001), than in people aged 19-59 years (-2.44; p<0.0001), whereas HBsAg seroprevalence increased in persons ≥60 years (2.84; p=0.0007). Significant decreases were observed in all six major Chinese regions, in both men (APC=-3.90; p<0.0001) and women (-1.82; p<0.0001) and in high-risk populations. An estimated 43.3 million (95% uncertainty interval 30.7-55.9) persons remained infected with HBV in China in 2021 (3.0%), with notable heterogeneity by region (<1.5% in North China to>6% in Taiwan and Hong Kong) and age (0.3%, 1.0%, 4.7% and 5.6% for <5 years, 5-18 years, 19-59 years and ≥60 years, respectively). CONCLUSIONS: China has experienced remarkable decreases in HBV infection over the last four decades, but variations in HBsAg prevalence persist in subpopulations. Ongoing prevention of HBV transmission is needed to meet HBV elimination targets by 2030. TRIAL REGISTRATION NUMBER: PROSPERO (CRD42021284217).

ARCHITECT HBsAg Next assay is positioned better to resolve and refine challenging weak reactive clinical samples.

BACKGROUND: Ultrasensitive HBsAg assays are replacing the previous versions. Unlike the sensitivity, the specificity, and its positioning to resolve weak-reactives (WR) are not studied. We investigated the ability of ARCHITECT HBsAg-Next (HBsAg-Nx) assay to resolve WR and sought its clinical validation and correlation with confirmatory/reflex testing. METHODS: Among 99,761 samples between Jan 2022 - 2023, 248 reactive samples in HBsAg-Qual-II were compared with HBsAg-Nx assay. Sufficient samples were further subjected to neutralization (n = 108) and reflex (anti-HBc total/anti-HBs antibody) testing. RESULTS: Out of 248 initial reactive samples in HBsAg-Qual-II, 180 (72.58%) were repeat reactive, and 68 (27.42%) were negative, whereas in HBsAg-Nx, 89 (35.89%) were reactive and 159 (64.11%) were negative (p<0.0001). Comparing the results of two assays (Qual-II/Next), 57.67% (n = 143) were concordant (++/-) and 105 (42.33%) were discordant (p = 0.0025). Testing of HBsAg-Qual-II + & HBsAg-Nx - samples revealed that 85.71% (n = 90) were anti-HBc total negative and 98.08% (n = 51) were not neutralized as well as significant proportion (89%) had no clinical correlation. The proportion of samples neutralized was significantly different between ≤5 S/Co (26.59%) and >5 S/Co (71.42%) (p = 0.0002). All samples (n = 26) with enhanced reactivity in HBsAg-Nx were effectively neutralized, while samples with no increase in reactivity, 89% (n = 72) failed neutralization (p=<0.001). CONCLUSIONS: HBsAg-Nx assay is positioned better to resolve and refine challenging WR samples than Qual-II which correlated well with confirmatory/reflex tests and clinical disease. This superior internal benchmarking significantly reduced the cost and quantum of retesting, confirmatory/reflex testing in the diagnosis of HBV infection.

A multicenter randomized-controlled trial of nucleos(t)ide analogue cessation in HBeAg-negative chronic hepatitis B.

BACKGROUND & AIMS: Nucleos(t)ide analogues (NUCs) are the standard and mostly lifelong treatment for chronic HBeAg-negative hepatitis B, as functional cure (loss of HBsAg) is rarely achieved. Discontinuation of NUC treatment may lead to functional cure; however, to date, the evidence for this has been based on small or non-randomized clinical trials. The STOP-NUC trial was designed with the aim of increasing the HBsAg loss rate using a NUC treatment interruption approach. METHODS: In this multicenter, randomized-controlled trial, 166 HBeAg-negative patients with chronic hepatitis B on continuous long-term NUC treatment, with HBV DNA <172 IU/ml (1,000 copies/ml) for ≥4 years, were randomized to either stop (Arm A) or continue NUC treatment (Arm B) for a 96-week observation period. In total, 158 patients were available for final analysis, 79 per arm. The primary endpoint was sustained HBsAg loss up to week 96. RESULTS: Our study met its primary objective by demonstrating HBsAg loss in eight patients (10.1%, 95% CI 4.8%-19.5%) in Arm A and in no patient in Arm B (p = 0.006). Among patients with baseline HBsAg levels <1,000 IU/ml, seven (28%) achieved HBsAg loss. In Arm A, re-therapy was initiated in 11 (13.9%) patients, whereas 32 (40.5%) patients achieved sustained remission. A decrease of HBsAg >1 log IU/ml was observed in 16 patients (20.3%) in Arm A and in one patient (1.3%) in Arm B. No serious adverse events related to treatment cessation occurred. CONCLUSIONS: Cessation of NUC treatment was associated with a significantly higher rate of HBsAg loss than continued NUC treatment, which was largely restricted to patients with end of treatment HBsAg levels <1,000 IU/ml. IMPACT AND IMPLICATIONS: As HBeAg-negative patients with chronic hepatitis B on nucleos(t)ide analogues (NUCs) rarely achieve functional cure, treatment is almost always lifelong. The STOP-NUC trial was conducted to investigate whether discontinuing long-term NUC treatment can increase the cure rate. We found that some patients achieved functional cure after stopping NUCs, which was especially pronounced in patients with HBsAg levels <1,000 at the end of NUC treatment, and that many did not need to resume therapy. The results of the Stop-NUC trial provide evidence for the concept of stopping NUC treatment as a therapeutic option that can induce functional cure.

Hepatitis delta: Epidemiology to recent advances in therapeutic agents.

Hepatitis D virus (HDV) was first described in 1977 and is dependent on the presence of hepatitis B surface antigen (HBsAg) for its entry into cells and on the human host for replication. Due to the envelopment with the hepatitis B virus (HBV) envelope, early phases of HDV entry resemble HBV infection. Unlike HBV, HDV activates innate immune responses. The global prevalence of HDV is estimated to be about 5% of HBsAg positive individuals. However, recent studies have described a wide range of prevalence between 12 to 72 million individuals. Infection can occur as super-infection or co-infection. The diagnosis of active HDV infection involves screening with anti HDV antibodies followed by quantitative PCR testing for HDV RNA in those who are HBsAg positive. The diagnostic studies have evolved over the years improving the validity and reliability of the tests performed. HDV infection is considered the most severe form of viral hepatitis and the HDV genotype may influence the disease course. There are eight major HDV genotypes with prevalence varying by geographic region. HDV treatment has been challenging as HDV strongly depends on the host cell for replication and provides few, if any viral targets. Better understanding of HDV virology has led to the development of several therapeutic agents currently being studied in different phase II and III clinical trials. There is increasing promise of effective therapies that will ameliorate the course of this devastating disease.

Toxoplasma gondii, HBV, and HCV co-infection and their correlation with CD4 cells among Iranian HIV-positive patients.

INTRODUCTION: Human immunodeficiency virus (HIV/AIDS) infected patients have a higher risk of opportunistic infections (OIs) depending on their immunological status, especially CD4 + cell count. Toxoplasma gondii, hepatitis C virus (HCV), and hepatitis B virus (HBV) are important OIs among Human Immunodeficiency Virus (HIV)/Acquired Immune Deficiency Syndrome (AIDS) patients. However, little is known about co-infection of these pathogens among HIV-infected individuals and their correlation with the patient's CD4 + cell count. Hence, this study aimed to investigate the serological and molecular status of T. gondii infection among HIV-infected individuals who had co-infection with HBV and HCV infections. METHODS: A total of 100 HIV/AIDS patients in two cities in the southwest of Iran was tested for T. gondii Immunoglobulin G (IgG) and Immunoglobulin M (IgM) antibodies as well as DNA detection by polymerase chain reaction (PCR) targeting the RE gene. HBV and HCV were detected by hepatitis B surface antigen (HBsAg) test, hepatitis C antibody (HCV Ab) test, and Real-Time PCR. The number of CD4 + cell counts was determined by Flow cytometry. RESULTS: Anti-T. gondii IgG was positive in 22% of the patients, but anti-T. gondii IgM and PCR were negative in all samples. HBV and HCV were positive in 8% and 33% of the patients, respectively. Co-infections were as followed: HIV + HCV (16%), HIV + HCV + T. gondii (11%), HIV + T. gondii (5%), HIV + HBV (1%), HIV + HBV + T. gondii (1%), HIV + HBV + HCV (1%), and HIV + HBV + HCV + T. gondii (5%). A significant decline in CD4 + cell counts was found in such co-infection groups (HIV + T. gondii, HIV + HCV + T. gondii, and HIV + HBV + HCV + T. gondii) compared with the HIV mono-infection group. CONCLUSIONS: Our study showed that co-infections of T. gondii, HCV, and HBV were common among HIV-infected patients and co-infections had a negative correlation with CD4 + cell counts of the patients.

Multifaceted Defects in Monocytes in Different Phases of Chronic Hepatitis B Virus Infection: Lack of Restoration after Antiviral Therapy.

Monocytes play an important role in the control of microbial infection, but monocyte biology during chronic hepatitis B virus (HBV) infection (CHI) remains inadequately studied. We investigated the frequency, phenotype, and functions of monocyte subsets in different phases of CHI, namely, immune tolerance (IT), hepatitis B early antigen (HBeAg)-positive/HBeAg-negative chronic hepatitis B (EP-/EN-CHB, respectively), and inactive carrier (IC), identified factors responsible for their functional alterations, and determined the impact of antiviral therapy on these cells. Flow cytometric analysis indicated that HLA-DR+ CD14++ CD16- classical monocytes were significantly reduced while HLA-DR+ CD14++ CD16+ intermediate and HLA-DR+ CD14+ CD16++ nonclassical monocytes were expanded in IT and EP-/EN-CHB compared with those in IC and healthy controls (HC). In comparison to IC/HC, monocytes in IT and CHB exhibited diminished expression of Toll-like receptor 2 (TLR-2)/TLR-4/TLR-9 and cytokines interleukin-12 (IL-12)/tumor necrosis factor alpha (TNF-α)/IL-6 but produced higher levels of IL-10/transforming growth factor ß (TGF-ß). Further, monocytes in CHB/IT showed impaired phagocytosis and oxidative response relative to those in IC/HC. In vitro assays indicated that high titers of hepatitis B surface antigen (HBsAg) present in IT/CHB and of IL-4 in CHB triggered the functional defects in monocytes via induction of ß-catenin. Additionally, monocyte-derived M1 macrophages of CHB/IT produced fewer proinflammatory and more anti-inflammatory cytokines than those of IC/HC, while in CHB/IT, the monocytes skewed the differentiation of CD4+ T cells more toward regulatory T cells and a Th2 phenotype. Moreover, monocytes in CHB and IT overexpressed chemokine receptor CCR2, which coincided with increased intrahepatic accumulation of ß-catenin+ CD14+ cells. One year of tenofovir therapy failed to normalize monocyte functions or reduce serum HBsAg/IL-4 levels. Taken together, monocytes are functionally perturbed mostly in IT and EP-/EN-CHB phases. Targeting intramonocytic ß-catenin or reducing HBsAg/IL-4 levels might restore monocyte function and facilitate viral clearance. IMPORTANCE Chronic HBV infection (CHI) is a major cause of end-stage liver disease for which pharmacological treatments currently available are inadequate. Chronically HBV-infected patients fail to mount an efficient immune response to the virus, impeding viral clearance and recovery from hepatitis. Monocytes represent a central part of innate immunity, but a comprehensive understanding on monocyte involvement in CHI is still lacking. We here report a multitude of defects in monocytes in chronically HBV-infected patients that include alteration in subset distribution, Toll-like receptor expression, cytokine production, phagocytic activity, oxidative response, migratory ability, polarization of monocyte-derived macrophages, and monocyte-T-cell interaction. We demonstrated that high levels of hepatitis B virus surface antigen and IL-4 potentiate these defects in monocytes via ß-catenin induction while therapy with the nucleotide analog tenofovir fails to restore monocyte function. Our findings add to the continuing effort to devise new immunotherapeutic strategies that could reverse the immune defects in CHI.

In situ analysis of hepatitis B virus (HBV) antigen and DNA in HBV-induced hepatocellular carcinoma.

AIMS: Hepatitis B Virus (HBV) infection is the major risk factor for hepatocellular carcinoma (HCC) in East Asia. Here we aimed to further investigate the abundance of viral antigen and DNA within HBV-related HCC and surrounding tissues at histological level. METHOD: In addition to routine histopathology, in situ hybridization (ISH) of HBV DNA and immunohistochemistry (IHC) of HBsAg were performed in tissues from 131 HBsAg-positive HCC patients undergoing liver resection. Serum α-fetoprotein together with basic biochemical and immunological parameter was also measured. RESULTS: Overall, the ISH of HBV DNA and IHC of HBsAg showed 31.3% and 92.9% positive rate respectively (p < 0.0001). The level of correlation between these two markers was much more significant in tumor (p < 0.0001) than in tumor-surrounding tissue (p = 0.01). HBsAg exhibited a much higher positive rate in tumor-adjacent tissue than in tumor tissue (86.6% versus 29.9%, p < 0.0001) with significantly different staining pattern. By contrast, the positive rate of HBV DNA ISH was comparable in tumor and surrounding tissue (17.6% versus 22.9%, p = 0.36). Yet the HBV DNA signal in tumor tissue showed predominant nuclear localization (87.0%) whereas staining pattern in adjacent tissue was mixed (43.3% nuclear localization, p = 0.0015). Finally, no significant association between intra-tumor HBV DNA/HBsAg positivity and major histological markers (microvascular invasion, tumor differentiation, etc.) or recurrence after surgery was observed. CONCLUSIONS: These data confirmed the largely integrated state of HBV DNA, weaker expression and altered localization of surface antigen in tumor compared with surrounding tissue. The strikingly different prevalence and localization of HBsAg and HBV DNA reflected the complex and heterogeneous mechanisms leading to HBV-induced tumorigenesis.

Distinct clinical and genetic features of hepatitis B virus-associated follicular lymphoma in Chinese patients.

Hepatitis B virus (HBV) infection has been associated with an increased risk for B-cell lymphomas. We previously showed that 20% of diffuse large B-cell lymphoma (DLBCL) patients from China, an endemic area of HBV infection, have chronic HBV infection (surface antigen-positive, HBsAg+) and are characterized by distinct clinical and genetic features. Here, we showed that 24% of follicular lymphoma (FL) Chinese patients are HBsAg+. Compared with the HBsAg- FL patients, HBsAg+ patients are younger, have a higher histological grade at diagnosis, and have a higher incidence of disease progression within 24 months. Moreover, by sequencing the genomes of 109 FL tumors, we observed enhanced mutagenesis and distinct genetic profile in HBsAg+ FLs, with a unique set of preferentially mutated genes (TNFAIP3, FAS, HIST1H1C, KLF2, TP53, PIM1, TMSB4X, DUSP2, TAGAP, LYN, and SETD2) but lack of the hallmark of HBsAg- FLs (ie, IGH/BCL2 translocations and CREBBP mutations). Transcriptomic analyses further showed that HBsAg+ FLs displayed gene-expression signatures resembling the activated B-cell-like subtype of diffuse large B-cell lymphoma, involving IRF4-targeted genes and NF-κB/MYD88 signaling pathways. Finally, we identified an increased infiltration of CD8+ memory T cells, CD4+ Th1 cells, and M1 macrophages and higher T-cell exhaustion gene signature in HBsAg+ FL samples. Taken together, we present new genetic/epigenetic evidence that links chronic HBV infection to B-cell lymphomagenesis, and HBV-associated FL is likely to have a distinct cell-of-origin and represent as a separate subtype of FL. Targetable genetic/epigenetic alterations identified in tumors and their associated tumor microenvironment may provide potential novel therapeutic approaches for this subgroup of patients.

[Comparison of clinicopathological features for indications of different types of antiviral therapy in chronic hepatitis B].

Objective: To compare and analyze the clinicopathological features and significance for indications of different types of antiviral therapy in chronic hepatitis B (CHB). Methods: Clinical data of 861 CHB cases who received liver biopsy, had hepatitis B virus (HBV) DNA-positive (> 30 IU/ml) and met the indications for antiviral therapy from January 2014 to December 2019 were included. Liver pathological changes and their correlation with clinical characteristics were compared and analyzed. According to different data, t-test, analysis of variance, nonparametric test, χ2 test, Ridit and logistic regression analysis were used for statistical analysis. Results: Most of the cases (72.24%) had remarkable pathological damage. The degree of liver fibrosis was higher in the normal than the abnormal group (P<0.001). 17.54% cases had hepatic steatosis. The vast majority of cases (97.33%) had positive hepatitis B surface antigen (HBsAg), while only 50.87% had positive hepatitis B core antigen (HBcAg). The positive correlation factors affecting the severity of liver histopathology were alkaline phosphatase level, while the negative correlation factors were positive HBcAg staining, albumin and platelet level. The degree of liver inflammation and fibrosis had statistically significant differences with different HBcAg staining levels (χ2=44.142 and 102.386, respectively; P<0.001), and the severity was more apparent in the negative group. Conclusion: There exist differences in clinicopathological features for indications of different types of antiviral therapy in patients with CHB. Liver function test range is inconsistent with degrees of hepatic histological severity. The positive and intensity of liver tissue HBcAg staining, and albumin and alanine aminotransferase levels have negative correlation with disease severity.

Incidence and prediction of HBsAg seroclearance in a prospective multi-ethnic HBeAg-negative chronic hepatitis B cohort.

BACKGROUND AND AIMS: Achieving HBsAg loss is an important landmark in the natural history of chronic hepatitis B (CHB). A more personalized approach to prediction of HBsAg loss is relevant in counseling patients. This study sought to develop and validate a prediction model for HBsAg loss based on quantitative HBsAg levels (qHBsAg) and other baseline characteristics. METHODS: The Hepatitis B Research Network (HBRN) is a prospective cohort including 1240 untreated HBeAg-negative patients (1150 adults, 90 children) with median follow-up of 5.5 years. Incidence rates of HBsAg loss and hepatitis B surface antibody (anti-HBs) acquisition were determined, and a predictor score of HBsAg loss using readily available variables was developed and externally validated. RESULTS: Crude incidence rates of HBsAg loss and anti-HBs acquisition were 1.6 and 1.1 per 100 person-years (PY); 67 achieved sustained HBsAg loss for an incidence rate of 1.2 per 100 PY. Increased HBsAg loss was significantly associated with older age, non-Asian race, HBV phenotype (inactive CHB vs. others), HBV genotype A, lower HBV-DNA levels, and lower and greater change in qHBsAg. The HBRN-SQuARe (sex,∆quantHBsAg, age, race) score predicted HBsAg loss over time with area under the receiver operating characteristic curve (AUROC) (95% CIs) at 1 and 3 years of 0.99 (95% CI: 0.987-1.00) and 0.95 (95% CI 0.91-1.00), respectively. In validation in another cohort of 1253 HBeAg-negative patients with median follow-up of 3.1 years, HBRN SQuARe predicted HBsAg loss at 1 and 3 years with AUROC values of 0.99 (0.98-1.00) and 0.88 (0.77-0.99), respectively. CONCLUSION: HBsAg loss in predominantly untreated patients with HBeAg-negative CHB can be accurately predicted over a 3-year horizon using a simple validated score (HBRN SQuARe). This prognostication tool can be used to support patient care and counseling.

Hepatitis B virus infection and the risk of gastrointestinal cancers among Chinese population: A prospective cohort study.

Our study aims to explore the relationship between chronic hepatitis B virus (HBV) infection and the risk of gastrointestinal (GI) cancers including liver, gastric, gallbladder or extrahepatic bile duct, pancreatic, small intestine, esophageal and colorectal cancer in the Kailuan Cohort study. We prospectively examined the relationship between HBV infection and new-onset GI cancers among 93 402 participants. Cox proportional hazards regression models, subgroup analyses and competing risk analyses were used to evaluate the association between HBV infection and the risk of new-onset GI cancers. During a median follow-up of 13.02 years, 1791 incident GI cancer cases were diagnosed. Compared to HBsAg seronegative participants, a significant positive association between HBV infection and GI cancers was observed in the multivariate-adjusted models (HR 5.59, 95% CI: 4.84-6.45). In the site-specific analyses, participants with HBsAg seropositive exhibited an increased risk of liver cancer (HR = 21.56, 95% CI: 17.32-26.85), gallbladder or extrahepatic bile duct cancer (HR = 14.89, 95% CI: 10.36-21.41), colorectal cancer (HR = 1.75, 95% CI: 1.15-2.96) and pancreatic cancer (HR = 1.86, 95% CI: 1.10-3.99). After taking death as the competing risk event, the associations of HBV infection with the risk of these cancers were attenuated but remained significant both in the cause-specific hazards models, the subdistribution proportional hazards models and sensitivity analyses. Our study suggests that HBV infection is associated with the elevated risk of liver cancer and extrahepatic cancer including gallbladder or extrahepatic bile duct, pancreatic and colorectal cancer among adults in Northern China.

Diagnostic Accuracy and Validity of Serological and Molecular Tests for Hepatitis B and C.

INTRODUCTION: Hepatitis B and C viruses are one of the leading causes of health problems in the world and early diagnosis and treatment of them are very important. Thereby, this study aimed to evaluate the validity and reliability of usable diagnostic tests for the detection of hepatitis B and C viruses in the clinical setting and to compare them with each other. MATERIALS AND METHODS: In this review article, we have searched major online databases, including PubMed and EMBASE. 42 retrieved articles were published between January 2000 and January 2020, which are summarized in this review. RESULTS: Immunoassay approaches are general techniques for the identification of pathogenic agents, among which ELISA is the gold standard for the detection of HBsAg. While serological techniques are not conclusive, molecular assays are really important because of the high sensitivity to detect chronic hepatitis B without HBeAg, in which viral loads are very low. Biosensors have more elevated selectivity and sensitivity and faster responses compared to other methods. CONCLUSION: This study suggests that all of the molecular, serological, and biotechnological assays have advantages and disadvantages for diagnosing hepatitis B and C viruses which are dependent on the condition, so we should choose one of them in regards to the time, cost, and laboratory equipment along with the clinical symptoms.

Evaluation of dried blood spots for hepatitis B and D serology and nucleic acid testing.

Areas with the highest burden of hepatitis B virus (HBV) infection are often low-middle-income countries with limited access to diagnosis due to isolation, affordability, and/or feasibility. Dried blood spots (DBSs) provide an alternative for remote areas where collection and transportation of serum is impractical. In this study, the application of DBS for serological and molecular detection of HBV and hepatitis D virus (HDV) was evaluated. Hepatitis B surface antigen was detected in 87 of 91 (95.6%) DBS. Seventeen of 21 (81%) had detectable HBeAg and 52 of 71 (73.2%) were anti-HBe positive. Anti-HD was detectable in 11 of 12 (91.6%) spiked control DBS after an initial failure to detect in patient DBS. HBV DNA was detected from 50 of 70 (71.4%) DBS with serum loads greater than 200 IU/mL in an in-house assay and 18 of 24 (75%) DBS with loads exceeding 389 IU/mL in a commercial assay. Using linear regression, HBV DNA loads from DBS were able to predict serum loads in 46 of 50 (92%) samples to within 1 log of actual serum load. HDV RNA was detected in 42 of 47 (89%) DBS with serum levels greater than 7200 IU/mL. DBSs are recommended for diagnosis of HBV, monitoring, and detection of high loads in pregnant women where peripheral blood testing remains unfeasible. Detection of HDV RNA from DBS may prove useful in endemic areas.

Role of anti-HBs in functional cure of HBeAg+ chronic hepatitis B patients infected with HBV genotype A.

BACKGROUND & AIMS: HBsAg-specific antibody responses are difficult to detect during chronic hepatitis B infection (CHB) and are often overlooked. The aim of this study was to examine whether anti-HBs may be involved in functional cure (FC) by profiling anti-HBs responses in patients with CHB using a panel of specific assays. METHODS: Longitudinal serum samples were obtained from 25 patients with CHB who were infected with HBV genotype A and were undergoing nucleos(t)ide analogue (NA) treatment: 14 achieved FC while 11 remained infected (non-FC). Anti-HBs immune complexes (HBsAg-IC), FcγRIIIa dimer binding, epitope specificity and neutralisation efficacy were measured. RESULTS: HBsAg-IC peaks were detected prior to HBsAg loss in 10/14 FC patients. These HBsAg-IC peaks overlapped with either an alanine aminotransferase (ALT) flare (8/10 patients), or a rise in ALT (2/10 patients). HBsAg-IC peaks were detected in 7/11 non-FC patients, but were not associated with an ALT flare. FCγRIIIa binding was detected in 9/14 FC patients, independent from detection of overlapping HBsAg-IC/ALT peaks. FC patients had stable HBsAg epitope occupancy across the study, whereas non-FC patients had a reduction in HBsAg epitope occupancy within the first 12-24 weeks of NA treatment. Convalescent sera from FC patients recognised more HBsAg epitopes and neutralised HBV infection more potently than anti-HBs derived from vaccinees. Neutralisation potency appeared to increase post-HBsAg loss in 4/5 FC patients examined. CONCLUSIONS: Using these assays, we confirm that anti-HBs responses are present and fluctuate over time in this cohort of patients with HBeAg+ CHB, who were infected with HBV genotype A and treated with NAs. Key anti-HBs profiles associated with either FC or failure to achieve FC were also identified, suggesting a role for anti-HBs responses in FC. LAY SUMMARY: Using a panel of assays to characterise hepatitis B surface antibody (anti-HBs) responses in a group of patients with chronic hepatitis B, we identified anti-HBs profiles associated with either functional cure, or failure to achieve functional cure. Functional cure was associated with immune complex peaks which overlapped with alanine aminotransferase flares. Conversely, in those who did not achieve functional cure, immune complex peaks were present, but were not associated with alanine aminotransferase flares, and a decline in anti-HBs diversity was observed early during treatment.

Ubiquitinated Hepatitis D Antigen-Loaded Microvesicles Induce a Potent Specific Cellular Immune Response to Inhibit HDV Replication in Vivo.

Hepatitis D is the most severe form of human viral hepatitis and currently lacks an efficient therapy. Dendritic cell-derived exosomes (Dexs) have been found to induce immune responses capable of eliminating viruses. However, the therapeutic potential of antigen-loaded exosomes in hepatitis D is still unknown. Recently, we designed exosomes loaded with ubiquitinated hepatitis delta virus (HDV) small delta antigen (Ub-S-HDAg) and then treated mice bearing replicating HDV with these exosomes to explore their antiviral effect and mechanism. Mature dendritic cell-derived exosomes (mDexs) were loaded with Ub-S-HDAg and their antivirus function was evaluated in mice with HDV viremia. Furthermore, the proportion of CD8+ cells, the ratio of Th1/Th2 cells, the postimmunization levels of cytokines were explored, and the Janus kinases (JAK)/signal transducer and activator of transcription (STAT) pathway was evaluated with a JAK2 inhibitor AG490. In Ub-S-HDAg-Dexs group, the HDV RNA viral load was significantly decreased compared with other groups by CD8+ cell enrichment and an increase Th1/Th2 cell ratio. Furthermore, lymphocyte infiltration was increased, while the HDAg level was decreased in mouse liver tissue. However, there were no significant differences in HBV surface antigen (HBsAg), alanine aminotransferase (ALT), or aspartate aminotransferase (AST) levels among the groups. Moreover, p-JAK2, p-STAT1, p-STAT4, STAT1, and STAT4 expression was increased in Ub-S-HDAg-Dexs group. In conclusion, Ub-S-HDAg-Dexs might be a potential immunotherapeutic agent for eradicating HDV by inducing specific cellular immune response via the JAK/STAT pathway. IMPORTANCE Hepatitis D is the most severe viral hepatitis with accelerating the process of liver cirrhosis and increasing the risk of hepatocellular carcinoma. However, there are no effective antiviral drugs. Exosomes derived from mature dendritic cells are used not only as immunomodulators, but also as biological carriers to deliver antigens to induce robust immune response. Based on these properties, exosomes could be used as a biological immunotherapy by enhancing adaptive immune response to inhibit hepatitis D virus replication. Our research may provide a new therapeutic strategy to eradicate HDV in the future.

Silicon Nanowire Field-Effect Transistor as Label-Free Detection of Hepatitis B Virus Proteins with Opposite Net Charges.

The prevalence of hepatitis B virus (HBV) is a global healthcare threat, particularly chronic hepatitis B (CHB) that might lead to hepatocellular carcinoma (HCC) should not be neglected. Although many types of HBV diagnosis detection methods are available, some technical challenges, such as the high cost or lack of practical feasibility, need to be overcome. In this study, the polycrystalline silicon nanowire field-effect transistors (pSiNWFETs) were fabricated through commercial process technology and then chemically functionalized for sensing hepatitis B virus surface antigen (HBsAg) and hepatitis B virus X protein (HBx) at the femto-molar level. These two proteins have been suggested to be related to the HCC development, while the former is also the hallmark for HBV diagnosis, and the latter is an RNA-binding protein. Interestingly, these two proteins carried opposite net charges, which could serve as complementary candidates for evaluating the charge-based sensing mechanism in the pSiNWFET. The measurements on the threshold voltage shifts of pSiNWFETs showed a consistent correspondence to the polarity of the charges on the proteins studied. We believe that this report can pave the way towards developing an approachable tool for biomedical applications.

Evaluation of the in situ assay for HBV DNA: An observational real-world study in chronic hepatitis B.

ABSTRACT: The visualization of intrahepatic hepatitis B virus (HBV) DNA by in situ hybridization (ISH) has uncovered some interesting aspects of HBV life cycle at the single-cell level. In the current study, we intend to evaluate the reliability and robustness of this assay in the real-world clinical scenario and its relationship with currently available clinical biomarkers in chronic hepatitis B (CHB) patients.In this cross-sectional study, 94 CHB patients and 10 patients with non-HBV related liver diseases were enrolled. Liver biopsies and routine histopathology analysis were performed. Intrahepatic HBV DNA and viral antigens (HBsAg and HBcAg) were detected by ISH and immunohistochemistry (IHC), respectively. The basic biochemical and virological parameters such as alanine transaminase, serum HBV DNA, and serum HBsAg were measured.The HBV DNA-ISH assay showed 55.8% (53/94 cases) positive rate in CHB patients, no false positive was found in non-HBV related hepatitis. The IHC of HBsAg and HBcAg showed a positive rate of 94.7% (89/94 cases) and 19.5% (17/87 cases), respectively. Quantification of HBV DNA-ISH signal showed a significant correlation with serum HBV DNA (rs = 0.6223, P < .0001). In addition, the staining pattern of HBV DNA in situ in the context of collagen deposition informed the histopathological progression of chronic liver disease.The application of this ISH assay in evaluating intrahepatic viral replication in real-world CHB patients showed favorable performance. It can be a complementation to conventional liver histopathology examination and IHC detection of viral antigens. This methodology provides an intuitive assessment of virological and pathological state of CHB patients, and further supports clinical diagnosis and management.