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Background: Although nucleos(t)ide analogues (NAs) are thought to reduce the risk of hepatitis B virus (HBV)‐related hepatocellular carcinoma (HCC), the effect of NA discontinuation on the prognosis of HBV‐related HCC after hepatectomy is rarely reported. We aimed to investigate the potential for hepatitis B virus e antigen (HBeAg)‐negative HBV‐related HCC patients to discontinue NAs based on preoperative hepatitis B virus surface antigen (HBsAg) status. Methods: This historical cohort study involved 1232 NA‐treated HBeAg‐negative patients who underwent curative hepatectomy for HBV‐related HCC from 2014 to 2019. The recurrence‐free survival (RFS) and overall survival (OS) of patients discontinuing NAs before surgery were compared with those continuing NAs. Propensity score matching (PSM) was used to balance baseline characteristics. Results: Of all enrolled patients, 839 (68.1%) patients continued NAs, and 393 (31.9%) patients discontinued NAs. Continuation of NAs was identified as an independent risk factor for RFS (HR 2.047, 95% CI 1.348–3.109, p < 0.001 before PSM and HR 2.756, 95% CI 1.537–4.942, p < 0.001 after PSM) in HBsAg‐negative patients. Similarly, subgroup survival analyses showed that NA discontinuation was associated with better RFS (p = 0.029 before PSM and p < 0.001 after PSM) and comparable OS (p = 0.935 before PSM and p = 0.115 after PSM) than NA continuation in HBsAg‐negative patients. The interaction between HBsAg status and continuation or discontinuation of NAs was significant (p for interaction <0.001). Conclusions: These findings demonstrate the potential for HBeAg‐negative HBV‐related HCC patients who have achieved HBsAg seroclearance to discontinue NAs under strict monitoring. [ABSTRACT FROM AUTHOR]

Objective To investigate the changes of serum hepatitis B virus surface antigen (HBsAg), hepatitis B virus ribonucleic acid (HBV RNA), γ-glutamyl transpeptidase (GGT), γ-glutamyl transpeptidase to platelet ratio (GPR) levels in patients with different stages of natural history of chronic hepatitis B virus (HBV) infection and the diagnostic value of combined detection in the staging of natural history of chronic HBV infection. Methods A total of 172 patients with chronic HBV infection who were not treated with antiviral therapy in the Third People’ s Hospital of Kunming from November 2020 to December 2023 were selected as the research objects. According to the process of HBV infection, they were divided into HBeAg-positive chronic HBV infection (EPI) group (n = 77), HBeAg-positive CHB (EPH) group (n = 42), HBeAg-negative chronic HBV infection (ENI) group (n = 26), HBeAg-negative CHB (ENH) group (n = 27). The differences of serum HBsAg, HBV RNA, GGT and GPR levels in each group were compared, and the receiver operating characteristic (ROC) curve was drawn to explore the diagnostic value of combined detection of serum HBsAg, HBV RNA, GGT and GPR in EPH and ENH stages in the natural history of chronic HBV infection. Results There were significant differences in serum HBsAg, HBV RNA, GGT and GPR levels among the four groups (P < 0.05). The area under the ROC curve (AUC) of combined detection of serum HBsAg, HBV RNA, GGT and GPR in the diagnosis of EPH and ENH was 0.869 and 0.836, respectively, and the sensitivity and specificity were 76.19%, 59.26% and 84.42%, 96.15%, respectively. Conclusion The combined detection of serum HBsAg, HBV RNA, GGT and GPR has high accuracy and low misdiagnosis rate in the diagnosis of natural history of chronic HBV infection, which is expected to replace the traditional invasive examination. It plays an important clinical significance in the accurate diagnosis and individualized treatment strategy of patients with a natural history of chronic HBV infection. [ABSTRACT FROM AUTHOR]

Though the world-wide hepatitis B virus (HBV) vaccination program has been well completed for almost thirty years in many nations, almost HBV-related hepatocellular carcinoma (HCC) occurs in unvaccinated middle-aged and elderly adults. Apparently, treating 80% of qualified subjects could decrease HBV-related mortality by 65% in a short period. Nevertheless, globally, only 2.2% of CHB patients undergo antiviral therapy. The HBV markers related to HCC occurrence and prevention are as follows: the HCC risk is the highest at a baseline of HBV DNA of 6–7 log copies/mL, and it is the lowest at a baseline of an HBV DNA level of >8 log copies/mL and ≤4 log copies/mL (parabolic, and not linear pattern). The titer of an HBV core-related antigen (HBcrAg) reflecting the amount of HBV covalently closed circular DNA (ccc DNA) in the liver is related to HCC occurrence. The seroclearance of HBs antigen (HBsAg) is more crucial than HBV DNA negativity for the prevention of HCC. In terms of the secondary prevention of hepatitis B-related HCC involving antiviral therapies with nucleos(t)ide analogues (NAs), unsolved issues include the definition of the immune-tolerant phase; the optimal time for starting antiviral therapies with NAs; the limits of increased aminotransferase (ALT) levels as criteria for therapy in CHB patients; the normalization of ALT levels with NAs and the relation to the risk of HCC; and the relation between serum HBV levels and the risk of HCC. Moreover, the first-line therapy with NAs including entecavir (ETV), tenofovir disoproxil fumarate (TDF), and tenofovir alafenamide (TAF) remains to be clarified. Discussed here, therefore, are the recent findings of HBV markers related to HCC occurrence and prevention, unsolved issues, and the current secondary antiviral therapy for the prevention of HBV-related HCC. [ABSTRACT FROM AUTHOR]

Background: Many efforts are currently focused on functional treatment of the hepatitis B virus (HBV). This can be done by suppressing the secretion of HBV surface antigen (HBsAg). Scientific communities are very interested in natural products in that respect. Objective: Use of root extract of Havachoobe (Onosma dichroanthum BoissI), a Northern Iranian native medical herb, for assessment of its anti-HBsAg secretion activity. Methods: Havachoobe had been bought at a nearby apothecary store. Plant root extract was obtained using a hydroalcoholic process. Cytotoxic activity of the extract was examined on PLC/PRF/5 cells using MTT assay. ELISA has been used to measure HBsAg in the treated cell line supernatants. In addition, real-time PCR analysis was performed to evaluate the expression of HBsAg before and after treatment of Onosma in vitro. Results: The results showed very low root extract cytotoxicity at concentrations under 8 μg/mL. Tissue culture infectious dose 50 was obtained at 63.78 μg/mL. In a dose-dependent and time-dependent manner, a significantly reduced HBsAg secretion was observed at a concentration of 8 ppm at 12 h post-treatment. The real-time PCR result showed relative decreased HBsAg expression at all doses at 12 h post-treatment time. Discussion: In this study, we first reported anti-HBsAg activity on an Iranian herbal medicine. Havachoobe root extract was shown to be able to inhibit HBsAg in a dose-dependent and time-dependent manner. We find the extract exerts its inhibitory effect of HBsAg by targeting transcription of HBsAg. [ABSTRACT FROM AUTHOR]

• Attaching aptamers to agarose through DNA tetrahedral structure to build a novel type of immunosorbent. • This immunosorbent could remove approximately 90% of the HBsAg from human plasma samples. • Better adsorption for samples with high concentration of HBsAg. • The immunosorbents are noncytotoxic. DNA nanostructures are effective intermediates for immobilizing biomolecules because of their high level of controllability, the flexibility to create desired nanostructures through precision "bottom-up" assembly and the convenience to building fine nanostructures. The purpose of this study was to demonstrate the potential use of aptamers bound to agarose through a DNA tetrahedral structure to make a novel type of immunosorbent for the removal of hepatitis B virus surface antigens. Our results show that the proposed immunosorbent exhibits favorable biocompatibility and specificity. Electrophoresis and confocal microscopy were used to confirm the formation of immunosorbents. The ARCHITECT HBsAg assay was performed to quantitatively measure hepatitis B surface antigen. The cytotoxic potential of the immunosorbent was determined by an in vitro viability assay using V79 lung fibroblasts, demonstrating that the immunosorbents are noncytotoxic. The high adsorption capacity of the novel DNA nanostructure-based adsorbents towards hepatitis B surface antigen indicated the potential application of these materials for the treatment of Hepatitis B infection. [ABSTRACT FROM AUTHOR]

Abstract: A critical challenge in producing an antibody‐based assay with the highest reproducibility and sensitivity is the strategy to immobilize antibodies to solid phase. To date, numerous methods of antibody immobilization were reported but each was subjected to its advantages and limitations. The current study proposes a new potential antibody binding protein, the human neonatal fragment crystallizable (Fc) receptor. This protein has shown its high affinity to the Fc of antibody either in vivo or in vitro. Human neonatal Fc receptor is a heterodimer constructed by p51 α‐heavy chain and β2‐microglobulin light chain; however, the binding sites toward the antibody are located in the p51 α‐heavy chain. Hence, vector cloning and recombinant protein expression were carried out to express the p51 α‐heavy chain of the human neonatal Fc receptor (hFcRn‐α). The recombinant protein expressed, hFcRn‐α, was adopted to pin rabbit IgG against hepatitis B virus surface antigen to a solid phase. A sandwich enzyme‐linked immunosorbent assay was further developed to evaluate the efficiency of hFcRn‐α‐directed immobilization in antigen detection. The result was compared with the conventional physical adsorption method. The findings demonstrated that human neonatal Fc receptor was efficient in pinning antibodies and generating higher signals compared with the physical adsorption of antibody. [ABSTRACT FROM AUTHOR]

Herein, we present a novel sandwich fluorescence enzyme linked immunosorbent assay (ELISA) for highly sensitive detection of Hepatitis B virus surface antigen (HBsAg) based on glucose oxidase (GOx)-induced fluorescence quenching of mercaptopropionic acid-modified CdTe quantum dots (MPA-QDs). In this system, hydrogen peroxide (H 2 O 2 ) sensitive MPA-QDs was used as a signal output, and glucose oxidase (GOx) was used as label which can generate H 2 O 2 via catalytic oxidation of glucose. The proposed method showed dynamic linear detection of HBsAg both in the range of 47 pg mL −1 ~ 380 pg mL −1 and 0.75 ng mL −1 ~ 12.12 ng mL −1 . The detection limit of the proposed fluorescence ELISA was 1.16 pg mL −1 , which was approximately 430-fold lower than that of horseradish peroxidase (HRP)-based conventional ELISA. The average recoveries for HBsAg-spiked serum samples ranged from 98.0% to 126.8% with the relative standard derivation below 10%, thus indicating acceptable precision and high reproducibility of the proposed fluorescence ELISA for HBsAg detection. Additionally, the developed method showed no false positive results analyzing 35 real HBsAg-negative serum samples, and exhibited excellent agreement (R 2 =0.9907) with a commercial time-resolved fluorescence immunoassay (TRFIA) kit for detecting 31 HBsAg-positive serum samples. In summary, the proposed method based on fluorescence quenching of H 2 O 2 sensitive QDs is considerably to be an excellent biodetection platform with ultrahigh sensitivity, good accuracy and excellent reliability. [ABSTRACT FROM AUTHOR]

Abstract: A sensitive electrochemical immunosensor for Hepatitis B virus surface antigen (HBsAg) detection was fabricated based on hemin/G‐quadruplex interlaced onto Fe3O4‐AuNPs or hemin ‐amino‐reduced graphene oxide nanocomposite (H‐amino‐rGO‐Au). G‐quadruplex DNAzyme, which is composed of hemin and guanine‐rich nucleic acid, is an effective signal amplified tool for its outstanding peroxidase activity and Fe3O4‐AuNPs or (H‐amino‐rGO‐Au) nanocomposites with quasi‐enzyme activity provide appropriate support for the immobilization of hemin/G‐quadruplex. The target protein was sandwiched between the primary antibody immobilized on the GO and secondary antibody immobilized on the Fe3O4‐AuNPs or (H‐amino‐rGO‐Au) nanocomposites and glutaraldehyde was used as linking agent for the immobilization of primary antibody on the surface of GO. Both Fe3O4‐AuNPs and H‐amino‐rGO‐Au nanocomposite and also hemin/G‐quadruplex can cooperate the electrocatalytic reduction of H2O2 in the presence of methylene blue as mediator. The proposed immunosensor has a wide linear dynamic range of 0.1 pg/ml to 300 pg/ml with a detection limit of 60 fg/ml when Fe3O4‐AuNPs was used for immobilization of hemin/G‐quadruplex, while the dynamic range and DL were 0. 1–1000 pg/mL and 10 fg/mL, respectively in the presence of H‐amino‐rGO‐ Au nanocomposite as platform for immobilizing of hemin/G‐quadruplex. The proposed immunosensor was also used for analysis of HBsAg in spiked human serum samples with satisfactory results. [ABSTRACT FROM AUTHOR]

The replication of hepatitis B virus (HBV) may be modulated by a variety of cell signaling pathways, including the phosphatidylinositol 3‑kinase (PI3K)‑RAC‑α serine/threonine‑protein kinase (AKT)‑serine/threonine‑protein kinase mTOR (mTOR) pathway. The aim of the present study was to determine the regulatory effects of this pathway on the infection and replication of HBV. The results indicated that the HBV entry process may activate the AKT pathway, as demonstrated by AKT phosphorylation in HBV natural infection. However, inhibition of AKT phosphorylation by short‑term treatment with AKT inhibitors was unable to block HBV entry, which suggested that AKT activation induced by HBV infection is not essential for viral entry process. Prolonged treatment with PI3K‑AKT‑mTOR pathway inhibitors markedly promoted HBV replication in HBV replicating and natural infection models. The PI3K‑AKT‑mTOR pathway was therefore identified to be a negative regulator of HBV replication. These inhibitors enhanced the replication and transcription of HBV in an HBx‑dependent way. The results additionally indicated that a PI3K inhibitor, Ly294002, inhibited the secretion of the small surface antigen of HBV in a PI3K‑AKT‑independent manner. The inhibitor Ly294002 may be used as a tool for the drug development of surface antigen secretion inhibitors. [ABSTRACT FROM AUTHOR]

Background: Hepatitis B is the world's most common serious liver infection. Infection by hepatitis B virus during pregnancy has high rate of vertical transmission and adverse effect on both the mother and child. Data on seroprevalence and associated factors among pregnant women in Dawuro or surrounding was scarce. Therefore, the aim of this study was to assess prevalence of hepatitis B virus and factors associated among pregnant women in Dawuro Zone. Methods: Institution based cross-sectional study was conducted from 1st April to May 31, 2015 in Dawuro zone. Venous blood was collected, plasma was separated and hepatitis B virus surface antigen was screened using rapid test. Logistic regression was employed with 95% CI and p value of <0.05 was used as statistically significant. Results: Data were collected on 289 women. The overall prevalence from this study was (p=3.5%, 95% CI 1.4-5.6%) multivariate analysis using logistic regression showed multiple sexual partner (AOR=6.923; 95% CI 1.685-28.441), and abortion history (AOR = 4.975; 95% CI 1.21-20.456), were significantly associated with hepatitis B virus surface antigen (HBsAg) infection. Conclusion: This seroprevalence was categorized as intermediate endemicity according to WHO classification criteria. Health education on sexual transmission of hepatitis B virus and inclusion of screening hepatitis b virus as routine antenatal care service is recommended. [ABSTRACT FROM AUTHOR]

Here we prepared an electrochemical immunosensor employing Au sheet as working electrode, Fe 3 O 4 magnetic nanoparticles (MNPs) as supporting matrix and hemin/G-quadruplex DNAzyme as signal amplifier for determination of hepatitis B virus surface antigen (HBsAg). First, the primary antibody of HBs (Ab 1 ) was immobilized on the surface of the carboxyl-modified MNPs. Then, the assembly of antibody and alkylthiol/G-quadruplex DNA/hemin on gold nanoparticles was used as bio-bar-coded nanoparticle probe. Protein target was sandwiched between the primary antibody of HBs (Ab 1 ) immobilized on the MNPs and hemin bio-bar-coded AuNPs probe labeled antibody (Ab 2 ). Hemin/G-quadruplex structure as HRP mimicking-DNAzyme significantly improved the catalytic reduction of H 2 O 2 by oxidation of methylene blue (MB). Square wave voltammetry signals of MB provided quantitative measurements of HBsAg with a linear concentration range of 0.3–1000 pgmL −1 and detection limit of 0.19 pgmL −1 . Due to efficient catalytic activity of HRP mimicking-DNAzyme, the proposed immunosensor exhibited high sensitivity and it holds great promise for clinical application and provides a new platform for immunosensor development and fast disease diagnosis. [ABSTRACT FROM AUTHOR]

Background and aim To prospectively evaluate the efficacy of vaccine alone compared with vaccine plus HBIG for preventing HBV transmission in neonates of HBsAg (+)/HBeAg (−) mothers. Methods Combined immunization is currently recommended for neonates of HBsAg (+) mothers in China. As a result, a randomized design is infeasible due to ethical reasons. In practice, Guangxi Zhuang Autonomous Region and Jiangsu Province implement vaccine alone and vaccine plus HBIG strategies for neonates born to HBsAg (+)/HBeAg (−) mothers, respectively. We alternatively enrolled neonates of HBsAg (+)/HBeAg (−) mothers from these two regions. Three doses of a recombinant yeast-derived hepatitis B vaccine were given at 0, 1 and 6 months with or without HBIG at birth. Results At 7 months, sera were collected from 132 neonates in Guangxi Zhuang Autonomous Region and 752 neonates in Jiangsu Province. Baseline characteristics of both mothers and neonates were comparable in the two regions. No differences were revealed regarding the occurrence of perinatal HBV transmission with or without HBIG at birth [0.1% (1/752) vs. 0.0% (0/132), p = 1.000]. The anti-HBs response rates were 97.7% (129/132) and 98.5% (740/751) for the neonates with vaccine alone and with HBIG ( p = 0.758), respectively. Vaccine alone induced a significantly higher anti-HBs GMC as compared to vaccine plus HBIG at 7 months of age (1555.3 mIU/mL vs. 654.9 mIU/mL, p < 0.0001). At 12 months of age, protective levels of anti-HBs remained in 97.4% (596/612) and 98.3% (118/120) of the neonates receiving and not receiving HBIG, respectively ( p = 0.771). The neonates receiving combined prophylaxis had a markedly lower anti-HBs GMC (210.7 mIU/mL vs. 297.0 mIU/mL, p = 0.011). Horizontal HBV transmission occurred in none of the successfully immunized neonates for both compared groups at 12 months of age. Conclusions Vaccine alone may be enough for preventing HBV transmission in neonates of HBsAg (+)/HBeAg (−) mothers. [ABSTRACT FROM AUTHOR]

In this paper, a novel time-resolved fluorescence immunoassay (TRFIA) is described that allows the simultaneous quantitative detection of hepatitis B virus surface antigen (HBsAg) and hepatitis B e antigen (HBeAg) in human serum to aid the diagnosis and monitoring of hepatitis B virus infection. The proposed method was developed based on a two-step sandwich immunoassay protocol in which monoclonal antibodies against HBsAg and HBeAg were co-coated in 96 microtitration wells, then tracer polyclonal antibodies against HBsAg labeled with samarium and tracer monoclonal antibodies against HBeAg labeled with europium chelates were used for detection. The detection range was 0.1-150 IU/mL for HBsAg and 0.5-160 PEIU/mL for HBeAg, and the detection limits were 0.03 IU/L and 0.09 PEIU/ml, respectively. The intra- and inter-assay coefficients of variation were below 8 % for both virus antigens. The dilution linearity and accuracy of the assay were satisfactory. No statistically significant differences were observed in sensitivity or specificity for the serum samples between the dual-label TRFIA and a commercial single-label TRFIA. These results demonstrate that an effective, reliable and convenient HBsAg/HBeAg dual-label TRFIA was successfully developed that may be clinically applicable for blood screening to monitor the course of hepatitis B virus infection and predict treatment responses. [ABSTRACT FROM AUTHOR]

Objective: To determine the frequency of hepatitis B virus surface antigen (HBsAg) and hepatitis C virus (HCV) antibodies in children with hematological disorders having multiple transfusions. Methodology: This descriptive cross-sectional study was conducted in the Department of Pediatrics, Lady Reading Hospital, Peshawar from December 10, 2016 to June 10, 2017. A total of 230 children with different hematological disorders who received two or more blood transfusions during the past two years were included in the study. Each child was screened for HBsAg and anti-HCV antibodies using 3rd generation commercial ELISA micro-plate kits (DIA.PRO, ITALY) according to the manufacturer instructions. Results: Out of 230, 122 (53%) patients were males and 108 (47%) were females. Their mean age was 10 ±10.86 years. In this study, 23% of patients were positive for anti-HCV antibodies, while 7% of patients were positive for HBsAg. Out of 53 patients positive for hepatitis C antibodies, 55% were males. Similarly, out of 16 patients positive for HBsAg, 56% were males. Conclusion: The frequency of anti-HCV antibodies was 23% and HBsAg was positive in 7% of all children who received multiple transfusions in the past two years. [ABSTRACT FROM AUTHOR]

• Tenofovir alafenamide (TAF)-containing antiretroviral therapy effectively suppressed lamivudine-resistant hepatitis B virus (HBV) in people living with human immunodeficiency virus (HIV). • The decrease in hepatitis B virus surface antigen (HBsAg) level was minimal, and HBsAg loss occurred rarely (2.2%) after switching to a TAF-containing regimen. • Hepatitis B virus envelope antigen seroconversion occurred more frequently in patients with HIV co-infected with lamivudine-susceptible HBV than in those co-infected with lamivudine-resistant HBV. • Switching from a tenofovir-disoproxil-fumarate- to a TAF-containing regimen led to improved proteinuria and bone density in patients co-infected with HIV and HBV. Data on the effectiveness of tenofovir alafenamide (TAF) against lamivudine-resistant (LAM-R) hepatitis B virus (HBV) among patients co-infected with human immunodeficiency virus (HIV) and HBV are limited. Between April and December 2018, HIV-positive patients co-infected with LAM-R or lamivudine-susceptible (LAM-S) HBV who switched from tenofovir-disoproxil-fumarate-containing antiretroviral therapy (ART) to TAF-containing ART were followed for 96 weeks. Plasma HBV and HIV loads, HBV serological markers, and liver function before and after the switch were analysed. In total, 182 patients co-infected with HIV and HBV were included in this study: 45 with LAM-R HBV and 137 with LAM-S HBV. At baseline, 28.9% and 7.4% of patients in the LAM-R and LAM-S groups, respectively, tested positive for hepatitis B virus envelope antigen (HBeAg) (P <0.001), and the respective percentages of patients who had achieved plasma HBV DNA <20 IU/mL were 95.5% and 97.1%. At weeks 48 and 96, 100% and 94.9% of patients in the LAM-R group, respectively, and 97.1% and 95.6% of patients in the LAM-S group, respectively, maintained plasma HBV DNA <20 IU/mL. Lamivudine resistance of HBV and baseline hepatitis B virus surface antigen (HBsAg) level were associated with HBsAg decrement at week 96 at a degree of 0.25 log 10 IU/mL [95% confidence interval (CI) 0.059–0.246] and 0.22 log 10 IU/mL (per 1-log 10 IU/mL increase, 95% CI 0.018–0.101), respectively. At week 96, 2.2% (4/182) of patients had HBsAg loss; no patients in the LAM-R group and 25.0% (2/8) of patients in the LAM-S group had HBeAg seroconversion. Switching to TAF-containing regimens maintained high rates of HBV viral suppression in patients co-infected with either LAM-R or LAM-S HBV. The decrease in HBsAg was minimal, and HBsAg seroconversion occurred infrequently. [ABSTRACT FROM AUTHOR]

Immunogenicity and safety of different adjuvants combined with a model antigen (HBsAg) were compared. Healthy HBV-naïve adults were randomized to receive HBs adjuvanted with alum or Adjuvant Systems AS01B, AS01E, AS03A or AS04 at Days 0 and 30. Different frequencies of HBs-specific CD4 + T cells 14 days post dose 2 but similar polyfunctionality profiles were induced by the different adjuvants with frequencies significantly higher in the AS01B and AS01E groups than in the other groups. Antibody concentrations 30 days post-dose 2 were significantly higher in AS01B, AS01E and AS03A than in other groups. Limited correlations were observed between HBs-specific CD4 + T cell and antibody responses. Injection site pain was the most common solicited local symptom and was more frequent in AS groups than in alum group. Different adjuvants formulated with the same antigen induced different adaptive immune responses and reactogenicity patterns in healthy naïve adults. The results summary for this study (GSK study number 112115 – NCT# NCT00805389 ) is available on the GSK Clinical Study Register and can be accessed at www.gsk-clinicalstudyregister.com . [ABSTRACT FROM AUTHOR]

The aim of the present study was to investigate the in vitro effects of hepatitis B virus surface antigen (HBsAg) on the immune function of human monocyte-derived dendritic cells (MD-DCs), and the moderating role of T cell immunoglobulin and mucin domain-containing molecule-3 (Tim-3) signaling molecule. The monocytes, obtained from healthy adult peripheral blood, were incubated with recombinant human granulocyte-macrophage colony-stimulating factor and interleukin (IL)-4 to induce DCs. DC-associated cell markers were detected using flow cytometry. MD-DCs were treated with HBsAg (5 μg/ml) in vitro for 48 h and subsequently, cell markers, lymphocyte stimulatory capacity, signaling protein and downstream cytokines were assessed. In addition, a Tim-3 monoclonal antibody was used to inhibit the Tim-3 signaling pathway, and subsequently the immune responses of MD-DCs to HBsAg stimulation were determined using the aforementioned method. The cell phenotype expressions of MD-DCs were all significantly increased with cluster of differentiation (CD)11c at 70.09±0.57%, human leukocyte antigen-DR at 79.83±2.12%, CD80 at 48.33±7.34% and CD86 at 44.21±5.35%. The treatment of MD-DCs with HBsAg resulted in a CD80 and CD86 enhanced expression, enhanced lymphocyte stimulatory capacity, upregulated expression of Tim-3 and nuclear factor-κB (NF-κB), as well as enhanced cytokine secretion of IL-6, IL-10 and interferon (IFN)-γ. However, a reduced immune response of MD-DCs in response to HBsAg stimulation was observed when the Tim-3 signaling pathway was inhibited prior to stimulation. The expression of NF-κB was decreased and the cytokine secretion level of IL-6, IL-10 and IFN-γ were downregulated. The treatment with HBsAg in vitro resulted in an enhanced immune response of MD-DCs, which may be positively regulated by the Tim-3 signaling molecule. [ABSTRACT FROM AUTHOR]

The aim of this study was to prepare poly(lactic-co-glycolic acid) (PLGA) microspheres containing hepatitis B virus surface antigen (HBsAg) using human serum albumin (HSA) as a stabilizer. Lyophilization and emulsification of HBsAg solution with dichloromethane caused a considerable loss of HBsAg antigenicity. Thus, the effects of HSA and trehalose on HBsAg recovery during lyophilization and emulsification were investigated. Adding HSA to HBsAg solutions significantly improved antigen recovery to >90% during lyophilization and emulsification. The effects of co-encapsulated HSA on the characteristics of the PLGA microspheres and stability of HBsAg released from the microspheres were also investigated. The in vitro release test showed that HBsAg was released from the PLGA microspheres continuously over seventy days. A large amount of released HBsAg was inactive without co-encapsulation of HSA. On the contrary, with HSA co-encapsulation, the released HBsAg retained approximately 90% of its antigenicity. The single injection of the HBsAg-HSA-loaded PLGA microspheres in rats resulted in higher anti-HBsAg IgG and Th1 cytokine levels than the single injection of the HBsAg-loaded microspheres or two injections of the conventional aluminum-adjuvanted HBsAg vaccine. Based on these findings, the HBsAg-HSA-loaded PLGA microspheres could be an effective carrier for HBsAg and form a promising depot system. [ABSTRACT FROM AUTHOR]

The Malaria Vaccine Technology Roadmap calls for a 2015 landmark goal of a first-generation malaria vaccine that has protective efficacy against severe disease and death, lasting longer than one year. This review focuses on product development efforts over the last five years of RTS,S, a pre-erythrocytic, recombinant subunit, adjuvanted, candidate malaria vaccine designed with this goal of a first-generation malaria vaccine in mind. RTS,S recently completed a successful pivotal Phase III safety, efficacy and immunogenicity study. Although vaccine efficacy was found to be modest, a substantial number of cases of clinical malaria were averted over a 3–4 years period, particularly in settings of significant disease burden. European regulators have subsequently adopted a positive opinion under the Article 58 procedure for an indication of active immunization of children aged 6 weeks up to 17 months against malaria caused by Plasmodium falciparum and against hepatitis B. Further evaluations of the benefit, risk, feasibility and cost-effectiveness of RTS,S are now anticipated through policy and financing reviews at the global and national levels. [ABSTRACT FROM AUTHOR]

Bio-nanocapsules (BNCs) are a hollow nanoparticle consisting of about 100-nm liposome (LP) embedding about 110 molecules of hepatitis B virus (HBV) surface antigen (HBsAg) L protein as a transmembrane protein. Owing to the human hepatocyte-recognizing domains on the N-terminal region (pre-S1 region), BNCs have recently been shown to attach and enter into human hepatic cells using the early infection mechanism of HBV. Since BNCs could form a complex with an LP containing various drugs and genes, BNC–LP complexes have been used as a human hepatic cell-specific drug and gene-delivery system in vitro and in vivo . However, the role of BNCs in cell entry and intracellular trafficking of payloads in BNC–LP complexes has not been fully elucidated. In this study, we demonstrate that low pH-dependent fusogenic activity resides in the N-terminal part of pre-S1 region (NPLGFFPDHQLDPAFG), of which the first FF residues are essential for the activity, and which facilitates membrane fusion between LPs in vitro . Moreover, BNC–LP complexes can bind human hepatic cells specifically, enter into the cells via clathrin-mediated endocytosis, and release their payloads mostly into the cytoplasm. Taken together, the BNC portion of BNC–LP complexes can induce membrane fusion between LPs and endosomal membranes under low pH conditions, and thereby facilitate the endosomal escape of payloads. Furthermore, the fusogenic domain of the pre-S1 region of HBsAg L protein may play a pivotal role in the intracellular trafficking of not only BNC–LP complexes but also of HBV. [ABSTRACT FROM AUTHOR]

Objectives In the case of coinfection with HIV and hepatitis B virus ( HBV) and/or hepatitis C virus ( HCV), hepatic disease progression is often accelerated, with higher rates of liver cirrhosis and liver-related mortality. We aimed to evaluate the performance of the rapid tests used routinely to detect HBV surface antigen ( HBsAg) and anti- HCV among HIV-infected patients in Guinea- Bissau. Methods Blood samples from HIV-infected patients in Guinea- Bissau were stored after testing for HBsAg and anti- HCV with rapid tests. Samples were subsequently re-tested for HBsAg and anti- HCV in Denmark. Results Two rapid tests were used in Guinea- Bissau: HBsAg Strip Ref 2034 ( VEDA. LAB, Alençon, France; sensitivity 62.3%; specificity 99.2%) and HEPA-SCAN ( Bhat Bio- Tech, Bangalore, India; sensitivity 57.1%; specificity 99.7%). In the two tests the ability to obtain the correct outcome depended on the antigen and antibody concentrations, respectively. Sex, age, CD4 cell count and antiretroviral therapy status did not differ between false negative and true positive samples in either of the tests. The study is limited by a low number of anti- HCV positive samples. Conclusions New diagnostic rapid tests should always be evaluated in the setting in which they will be used before implementation. [ABSTRACT FROM AUTHOR]

Occult hepatitis B virus infection (OBI) is highly prevalent worldwide. In some cases, it is a consequence of infection with variant viruses mutated in the S gene and producing a surface antigen not recognized by diagnostic kits. In most cases, OBI is due to a strong inhibition of hepatitis B virus (HBV) activities exerted by host defense mechanisms. OBI may reactivate in patients undergoing immunosuppressive therapy and/or chemotherapy with the possibility of a consequent development of acute hepatitis that may lead to hepatic failure. Hematological malignancies and therapeutic schedules including rituximab are the conditions most frequently associated with OBI reactivation. However, this event may occur in a large number of additional clinical and therapeutic settings. Identification of patients prone to undergo reactivation is of great importance for promptly starting a proper antiviral therapy that may stop the HBV reactivation and prevent its clinical sequelae. © 2014 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Highlights: [•] Flow cytometry was used for simultaneous detection of multiple analytes using E. coli cells. [•] E. coli cells with and without fluorescence in cytosol were used for multiple analyte detection. [•] E. coli cell with autodisplayed Z-domains was used for hyper sensitive immunoassay. [•] Flow cytometry based multiple detection was demonstrated with HBsAg and CRP as model analytes. [Copyright &y& Elsevier]

Abstract: The S domain of hepatitis B virus surface antigen (sHBsAg) is the primary component for vaccine development against virus infection. For stable expression of sHBsAg in recombinant Saccharomyces cerevisiae, new accessory genes necessary for foreign protein expression were screened by DNA microarray. Among 600 genes of interest, genes coding for an activating protein of ATPase in Hsp90 (Aha1p), S. cerevisiae DnaJ (Scj1p), thioredoxin 2 (Trx2p) and a GTPase-activator specific for Sar1 (Sec23p) as well as Pdi1p were selected in transcriptome analysis, which are known to facilitate disulfide bond formation or induce protein transport in the secretion pathway. Individual and combinatorial expression of SEC23, TRX2 and PDI1 increased total sHBsAg concentration by 1.9–6.5-fold, relative to the control strain expressing sHBsAg only. Additionally, moderate expression of Kex2p protease able to cut off the signal peptide enhanced the portion of the authentic sHBsAg to total sHBsAg. Fed-batch fermentation of the S. cerevisiae 2805 strain coexpressing the sHBsAg, SEC23, PDI1 and KEX2 genes resulted in 70.6mg/L final sHBsAg concentration which was 5.6 times higher than that of the control. Transmission electron microscopic analysis of the yeast cells elucidated the effects of the accessory gene coexpression on the intracellular localization of sHBsAg. Like PDI1, coexpression of both SEC23 and/or TRX2 newly isolated in this study is expected to improve the target protein expression in S. cerevisiae. [Copyright &y& Elsevier]

Abstract: A bio-nanocapsule (BNC) is a hollow nanoparticle consisting of an approximately 100-nm-diameter liposome with about 110 molecules of hepatitis B virus (HBV) surface antigen L protein embedded as a transmembrane protein. BNC can encapsulate various drugs and genes and deliver them specifically to human hepatic cells based on the ability of HBV to recognize human hepatocyte, which is integrated in the N-terminal region of L protein. However, it is elusive whether the cellular attachment and entry into hepatic cells of BNC utilize the early infection mechanism of HBV. In this study, we have found that while all human hepatic cells show distinct affinities for BNC compared to non-hepatic cells, primary hepatocytes shows the highest efficiency for cellular binding and incorporation of BNC. Amounts of BNCs bound weakly and strongly to cell membranes and those entered into the cells varied significantly depending on the types of human hepatic cells. The weak and strong binding modes of BNC are likely mediated through binding to two distinct HBV receptors (heparin-mediated low-affinity and unidentified high-affinity receptors), which play major roles in the early infection mechanism of HBV. The rates of cellular uptake of BNC are similar to those reported for HBV. The BNCs incorporated into the cells are swiftly sorted to either early endosomes or macropinosomes and then to late endosomes and/or lysosomes. These findings strongly suggest that BNC is bound to and incorporated into human hepatic cells according to the early infection mechanism of HBV. [Copyright &y& Elsevier]

Abstract: Bio-nanocapsules (BNCs) are hollow nanoparticles composed of the L protein of hepatitis B virus (HBV) surface antigen (HBsAg), which can specifically introduce genes and drugs into various kinds of target cells. Although the classic electroporation method has typically been used to introduce highly charged molecules such as DNA, it is rarely adopted for proteins due to its very low efficiency. In this study, a novel approach to the preparation of BNC was established whereby a target protein was pre-encapsulated during the course of nanoparticle formation. Briefly, because of the process of BNC formation in a budding manner on the endoplasmic reticulum (ER) membrane, the association of target proteins to the ER membrane with lipidation sequences (ER membrane localization sequences) could directly generate protein-encapsulating BNC in collaboration with co-expression of the L proteins. Since the membrane-localized proteins are automatically enveloped into BNCs during the budding event, this method can be protect the proteins and BNCs from damage caused by electroporation and obviate the need for laborious consideration to study the optimal conditions for protein encapsulation. This approach would be a useful method for encapsulating therapeutic candidate proteins into BNCs. [Copyright &y& Elsevier]

Abstract: Bio-nanocapsules (BNCs) are hollow particles (approx. 50nm diameter) consisting of hepatitis B virus surface antigen (HBsAg) large (L, pre-S1+pre-S2+S) proteins embedded in a unilamellar liposome, sharing the same transmembrane S region with an immunogen of hepatitis B vaccine (i.e., HBsAg small (S) protein particle). BNCs can incorporate drugs and genes into the hollow space and systemic administration of the BNCs can deliver the products to human liver via the human hepatocyte-specific receptor within the pre-S (pre-S1+pre-S2) region displayed on BNC’s surface. Thus, BNCs are expected to offer efficient and safe non-viral nanocarriers to deliver human liver-specific genes and drugs. To date, BNCs have been purified from the crude extract of BNC-overexpressing yeast cells by fractionation with polyethylene glycol followed by one CsCl equilibrium and two sucrose density gradient ultracentrifugation steps. However, the process was inefficient in terms of yield and time, and was not suitable for mass production because of the ultracentrifugation step. Furthermore, trace contamination with yeast-derived proteinases degraded the pre-S region, which is indispensable for liver-targeting, during long-term storage. In this study, we developed a new purification method involving heat treatment and sulfated cellulofine column chromatography to facilitate rapid purification, completely remove proteinases, and enable mass production. In addition, the BNCs were functional for at least 14months after lyophilization with 5% (w/v) sucrose as an excipient. This new process will significantly contribute to the development of forthcoming BNC-based nanomedicines as well as hepatitis B vaccines. [Copyright &y& Elsevier]

Abstract: Hepatitis B virus, a member of the hepadnavirus family, causes the acute and chronic diseases of the human liver. The S domain of hepatitis B virus surface antigen (sHBsAg) was expressed under the control of the galactose-inducible GAL1 promoter in recombinant Saccharomyces cerevisiae. Batch fermentation of S. cerevisiae 2805/pδMFα-sHBsAg resulted in 4.92gl−1 dry cell mass and 2.21mgl−1 sHBsAg concentration. To improve the expression level of sHBsAg, the pdi1 gene encoding protein disulfide isomerase was coexpressed in recombinant S. cerevisiae. Batch fermentation of S. cerevisiae 2805/pδMFα-sHBsAg+pPDI using 21gl−1 glucose and 33gl−1 galactose resulted in 9.75gl−1 dry cell mass and 13.3mgl−1 sHBsAg concentration, which were 2 and 6 times higher than those for S. cerevisiae 2805/pδMFα-sHBsAg, respectively. Appearance of three sHBsAgs with different molecular sizes of 24kDa, 34kDa and 40kDa in immunoblot assay and their endoglycosidase treatment indicated that sHBsAg might be expressed in three types of the authentic, MFα signal sequence-containing and N-glycosylated MFα signal sequence-containing forms. Fed-batch fermentation of recombinant S. cerevisiae 2805 coexpressing the sHBsAg and pdi1 genes was carried out by feeding 600gl−1 glucose continuously and controlling galactose concentration at around 20gl−1. As a result, 20.2gl−1 dry cell mass and 74.4mgl−1 maximum sHBsAg concentration were obtained, which were 4.1 and 33.7 times higher than those for the batch fermentation of S. cerevisiae 2805/pδMFα-sHBsAg, respectively. [Copyright &y& Elsevier]

The small (S) envelope protein is the major component of hepatitis B virus surface antigen (HBsAg). Some mutations in the S-HBsAg coding region may cause deficiency in the secretion of both viral and empty subviral particles (SVPs) and lead to accumulation of HBsAg inside the cells. In this study, we identified a unique amino acid substitution (L215Q) in the carboxyl-terminal end of S-HBsAg of an HBV genotype F isolate that provoked an inhibitory effect on secretion of SVPs. HBsAg levels were measured daily by enzyme-linked immunosorbent assay in the medium and cell extracts of HuH7 and CHO cells transiently transfected with plasmids containing wild-type or mutated S-HBsAg gene. Wild-type HBsAg was detectable in both medium and cell extracts of transfected cells. In contrast, extracellular levels of mutant HBsAg were not higher than cut-off values. By immunofluorescence with monoclonal antibody, it was shown that wild-type HBsAg was distributed throughout the cytoplasm, whereas mutant HBsAg was concentrated around the nucleus, suggesting retention in the endoplasmic reticulum. Amino acid substitutions that inhibit HBsAg secretion, such as that characterized in this study (L215Q), should have implications in HBV immunological diagnostics. Copyright © 2008 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Hepatitis B virus surface antigen (HBsAg), a specific antigen on the membrane of hepatitis B virus (HBV)-infected cells, provides a perfect target for therapeutic drugs. In order to mediate successful targeted delivery of these therapies, it is essential to have antibodies that recognize HBsAg with high specificity and affinity. In this report, we constructed a natural immune antigen binding fragments (Fab) antibody phage display library against HBsAg and after three rounds of panning, five Fab fragments with significant HBsAg binding ability were selected and analysed. DNA sequencing revealed that all the light chains had the same sequence, while all the Fd genes exhibited different sequences. For further application, all of the Fab antibodies were reconstructed into single chain antibodies (scFvs) and expressed in Escherichia coli BL21 cells. Indirect enzyme-linked immunosorbent assay analysis demonstrated that all five scFvs maintained a high affinity for HBsAg and could bind HBsAg on the membrane of HBV-infected cells. Indirect fluorescent staining analysis revealed that one of the scFvs (scFv15) could be internalized into HBsAg-positive HepG2.2.15 cells through clathrin-mediated endocytosis pathway. The internalizing scFv15 antibody would have great potential for the targeted delivery of therapeutics to HBV-infected cells. [ABSTRACT FROM AUTHOR]

Abstract: This paper evaluates the immunopurification behavior of a plantibody HBsAg specific plantibody coupled to Sepharose CL-4B at different ligand densities. Results show no significant differences in the adsorption and elution capacities, and rHBsAg recovery of immunosorbents at 3.43, 4.45, and 5.31mg/mL of ligand densities compared to its mouse-derived mAb counterpart consistently used in the rHBsAg purification process. Therefore, plantibody ligand densities higher than 3.43mg/mL do not improve the immunopurification behavior of this immunosorbent, but increase the antibody consumption and the Hepatitis B vaccine cost. Immunosorbent of 2.23mg/mL of ligand density demonstrated a poor performance. The IgG leached detectable level never exceeded the approved limit (3ng IgG/μg rHBsAg). Values close to this limit were only observed at the ligand density of 5.31 and 2.27mg/mL. In the case of the ligand density of 2.23mg/mL the IgG leached value was high (2.90ng IgG/μg rHBsAg) due to a low level of eluted antigen. In conclusion, it supports feasibility of using this plantibody at 3.43mg/mL of ligand density for large-scale immunopurification of rHBsAg for human use, avoiding the biosafety and ethical concerns of the massive use of animals for this purpose. [Copyright &y& Elsevier]

Abstract: An enzyme-linked immunosorbent assay to quantify the mAb CB.Hep-1 during downstream purification process was standardized and validated. This assay is characterized by a short time of incubation at high temperature, allowing the detection of this antibody with high specificity and sensitivity. Detection of antigen–antibody reaction was achieved using a horseradish peroxidase conjugated anti-mouse IgG whose enzyme activity was revealed with o-phenylenediamine substrate. The immunoassay is linear in a range between 3.12 and 50ng/mL, with a recovery of 98.55–107.62%. According to results, it is possible to estimate the mAb CB.Hep-1 concentration with high precision and reproducibility. The intra- and interassay coefficient of variation ranged from 0.25 to 8.64% and 1.84 to 9.43%, respectively. Significant differences were not observed in the plant-derived antibody quantification by HRP-ELISA and PhoA-ELISA (n =18), demonstrating that plant endogenous peroxidases do not produce interferences in the quantification of this molecule. Therefore, both antibodies can be tested with the same immunoassay with high precision, specificity and accuracy during their respective purification processes without interference of the buffers and sample characteristics. [Copyright &y& Elsevier]

Objective: We have reexamined hepatitis B virus subtypes to determine the role of specific HBsAg amino acids in serologic reactivity because of problematic genotype/subtype associations seen in a set of geographically diverse serum specimens. Methods: We obtained DNA sequences for 491 HBsAg-positive specimens from geographically distinct locations, determined their genotypes through phylogenetic analysis, and subtyped the specimens using an algorithm derived from published data on the molecular basis of HBsAg subtype reactivity. Problematic samples were subtyped serologically to resolve conflicts based on the amino acid sequence alone. Results: Three isolates were found to have unusual genotype/subtype associations. Examination of the isolates’ amino acid sequences suggested amino acid positions 122, 127, 140, 159 and 160 can be used to determine subtype reactivity from HBsAg amino acid sequences, while position 134, previously thought to play a role, is no longer important. Conclusions: This re-examination of hepatitis B virus subtypes shows the involvement of amino acid positions 122, 127, 140, 159 and 160 in HBsAg reactivity. While d, y, and r reactivities are controlled by single amino acid changes, w reactivity is determined by positions 122, 127, 140, and 159. Copyright © 2007 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

This paper provides an evaluation of a plant-derived HBsAg-specific antibody in the immunopurification of the recombinant HBsAg for vaccine purposes. This plant-derived antibody was obtained from different batches of 100–200 kg of tobacco leaves and coupled to Sepharose CL-4B with high efficiency. The plant-derived antibody immunoaffinity matrix purification behavior (elution capacity, antigen purity, purification cycles, and ligand leakage) was comparable to that of its mouse-derived monoclonal antibody homolog. This result supports the feasibility of using this plant-derived antibody for the immunopurification of the Hepatitis B surface antigen for human use, opening a new possibility to overcome the constrain of monoclonal antibody production in mice. [Copyright &y& Elsevier]

Background: The aim of this study is to identify the risk factors of survival and recurrence after curative hepatic resection for stage IV-A hepatocellular carcinoma (HCC).Methods: Sixty-five patients with stage IV-A HCC who underwent curative hepatic resection and discharged from hospital were enrolled in this retrospective study. Prognostic factors were evaluated by univariate and multivariate analysis. Clinicopathologic features and survival with stage IV-A HCC were compared with those of 290 patients with stage I to III HCC who underwent curative hepatic resection during the same period.Results: Disease-free and overall survival for patients with stage IV-A HCC was significantly lower than for those with stage I to III HCC. Positive hepatitis B virus (HBV) surface antigen was an independent prognostic factor of poor disease-free and overall survivals in patients with stage IV-A HCC. There were no significant differences in the disease-free and overall survivals between non-HBV-related stage IV-A HCC and stage I to III HCC.Conclusions: Even for patients with highly advanced HCC, curative hepatic resection may be a feasible therapeutic option for those with non-HBV-related HCC. [ABSTRACT FROM AUTHOR]