The infection of Hepatitis B virus (HBV) can result in severe consequences, including chronic hepatitis, liver fibrosis, cirrhosis, and even liver cancer. Effective antiviral treatment has the potential to slow down the progression of the disease. HBV serum biomarkers play a crucial role in the dynamic management of chronic hepatitis B (CHB) patients. However, the conventional hepatitis B virus markers, such as hepatitis B serologic testing and HBV DNA, are insufficient to meet the clinical requirements. This review provided a comprehensive overview of the current research on the quantification of HBsAg and anti-HBc, HBV RNA and HBV core-associated antigen, which summarized the crucial role these markers play in the administration of antiviral medications, predicting the efficacy of treatment and anticipating the likelihood of virologic rebound following drug cessation, as well as assessing disease progression in CHB patients.
Humans ; Hepatitis B virus/genetics* ; Clinical Relevance ; Hepatitis B, Chronic/drug therapy* ; Hepatitis B Core Antigens/therapeutic use* ; Biomarkers ; Liver Cirrhosis/drug therapy* ; Antiviral Agents/therapeutic use* ; Hepatitis B Surface Antigens/therapeutic use* ; DNA, Viral/therapeutic use* ; Hepatitis B e Antigens/therapeutic use* ; Hepatitis B/drug therapy*

BACKGROUND: Hepatitis B core-related antigen (HBcrAg) is a promising disease-monitoring marker for chronic hepatitis B (CHB). We investigated correlations between HBcrAg with antiviral efficacy and virological and histological variables. METHODS: One hundred and forty-five CHB patients from the mainland of China between August 2013 and September 2016 who underwent liver biopsy received entecavir therapy and had paired liver biopsy at 78 weeks. We analyzed correlations between HBcrAg and virological and histological variables in hepatitis B e antigen (HBeAg)-positive and HBeAg-negative patients. We also explored the predictors of HBeAg loss after 78 weeks of antiviral therapy. Pearson correlation analysis and logistic forward stepwise regression were the main statistic methods. RESULTS: HBeAg-positive patients (n = 93) had higher baseline HBcrAg (median 7.4 vs. 5.3 log10 U/mL P < 0.001) and greater HBcrAg declines (median 1.6 vs. 0.9 log10 U/mL P = 0.007) than HBeAg-negative patients after 78 weeks of therapy. At baseline, HBcrAg correlated with hepatitis B virus (HBV) DNA in both HBeAg-positive (r = 0.641, P < 0.001) and -negative patients (r = 0.616, P < 0.001), with hepatitis B surface antigen (HBsAg) in HBeAg-positive patients (r = 0.495, P < 0.001), but not with anti-hepatitis B virus core antibody (anti-HBc). Weak correlations existed between HBcrAg, histology activity index (HAI; r = 0.232, P = 0.025), and Ishak fibrosis score (r = -0.292, P = 0.005) in HBeAg-positive patients. At 78 weeks, significant correlations existed only between HBcrAg and anti-HBc in HBeAg-positive (r = -0.263, P = 0.014) and HBeAg-negative patients (r = -0.291, P = 0.045). Decreased HBcrAg significantly correlated with reduced HBV DNA (r = 0.366, P = 0.001; r = 0.626, P < 0.001) and HBsAg (r = 0.526, P = 0.001; r = 0.289, P = 0.044) in HBeAg-positive and -negative patients, respectively, and with reduced HAI in HBeAg-positive patients (r = 0.329, P = 0.001). Patients with HBeAg loss (n = 29) showed a larger reduction in HBcrAg than those without (median 2.3 vs. 1.3 log10 U/mL, P = 0.001). In multivariate analysis, decreased HBcrAg was an independent predictor of HBeAg loss (P = 0.005). CONCLUSIONS: HBcrAg reflects viral replication and protein production. Decreased HBcrAg could predict HBeAg loss after antiviral therapy. TRIAL REGISTRATION Clinical Trials.gov: NCT01962155; https://www.clinicaltrials.gov/ct2/show/NCT01962155?term=NCT01962155&draw=2&rank=1.
Antiviral Agents/therapeutic use* ; Biomarkers ; China ; DNA, Viral ; Hepatitis B Core Antigens/therapeutic use* ; Hepatitis B Surface Antigens ; Hepatitis B e Antigens ; Hepatitis B virus/genetics* ; Hepatitis B, Chronic/drug therapy* ; Humans ; Virus Replication

Hepatitis B virus core protein can self-assemble into icosahedral symmetrical viral-like particles (VLPs) in vitro, and display exogenous sequences repeatedly and densely on the surface. VLPs also have strong immunogenicity and biological activity. When the nanoparticles enter the body, they quickly induce specific humoral and cellular immune responses to exogenous antigens. In this study, we designed an HBc-VLPs that can be coupled with antigens at specific sites, and developed a set of efficient methods to prepare HBc-VLPs. Through site-specific mutation technology, the 80th amino acid of peptide was changed from Ala to Cys, a specific cross-linking site was inserted into the main immunodominant region of HBc-VLPs, and the prokaryotic expression vector pET28a(+)-hbc was constructed. After expression and purification, high purity HBc(A80C) monomer protein was assembled into HBc-VLPs nanoparticles in Phosphate Buffer. The results of particle size analysis show that the average particle size of nanoparticles was 29.8 nm. Transmission electron microscopy (TEM) showed that HBc-VLPs formed spherical particles with a particle size of about 30 nm, and its morphology was similar to that of natural HBV particles. The influenza virus antigen M2e peptide as model antigen was connected to Cys residue of HBc-VLPs by Sulfo-SMCC, an amino sulfhydryl bifunctional cross-linking agent, and M2e-HBc-VLPs model vaccine was prepared. The integrity of HBc-VLPs structure and the correct cross-linking of M2e were verified by cell fluorescence tracing. Animal immune experiments showed that the vaccine can effectively stimulate the production of antigen-specific IgG antibody in mice, which verified the effectiveness of the vaccine carrier HBc-VLPs. This study lays a foundation for the research of HBc-VLPs as vaccine vector, and help to promote the development of HBc-VLPs vaccine and the application of HBc-VLPs in other fields.
Animals ; Hepatitis B Core Antigens ; genetics ; immunology ; Immunity, Cellular ; immunology ; Immunoglobulin G ; blood ; Mice ; Mice, Inbred BALB C ; Vaccines, Virus-Like Particle ; genetics ; immunology

In recent years, Hepatitis B virus (HBV) persistent infection mouse model with recombinant adeno-associated virus 8 carrying 1.3 copies of HBV genome (rAAV8-1.3HBV) is concerned. We studied and compared the efficacy among HBV persistent infection mice models by other serotypes except AAV8. First, we prepared and purified five viruses: rAAV1-1.3HBV, rAAV2-1.3HBV, rAAV5-1.3HBV, rAAV8-1.3HBV and rAAV9-1.3HBV. Then we injected each virus into 3 C57BL/6J mice with the dose of lx 1011 vg (Viral genome, vg) per mouse. We detected HBsAg and HBeAg in sera by enzyme-linked immunosorbent assay (ELISA) at different time points post injection. We killed mice 8 weeks post injection and took blood and livers for assay. We detected copies of HBV DNA by real-time quantitative PCR in sera and livers. Meantime, we detected HBcAg in the livers of mice by immunohistochemistry and further performed pathology analysis of these livers. The five groups of mice, HBeAg and HBsAg expression sustained 8 weeks in serological detection and HBV DNA was both detected in sera and livers at the time of 8 weeks post injection. HBeAg, HBsAg, HBV DNA copies expression levels in descending order were AAV8>AAV9>AAV1>AAV5>AAV2. HBcAg expression was detected in livers as well. Varied degrees of liver damage were shown in five groups of mice. This study provides more alternative AAV vector species to establish a persistent infection with hepatitis B model.
Animals ; Dependovirus ; classification ; Disease Models, Animal ; Enzyme-Linked Immunosorbent Assay ; Genetic Vectors ; Genome, Viral ; Hepatitis B ; virology ; Hepatitis B Core Antigens ; metabolism ; Hepatitis B Surface Antigens ; blood ; Hepatitis B e Antigens ; blood ; Hepatitis B virus ; genetics ; Mice ; Mice, Inbred C57BL ; Serogroup ; Virus Replication

<b>OBJECTIVEb>To investigate the effect of protein transduction domain-hepatitis B virus core antigen (CTP-HBcAg18-27)-Tapasin fusion protein-induced specific cytotoxic T lymphocyte (CTL) response on hepatitis B virus (HBV) replication in HBV transgenic mice.

<b>METHODSb>Twenty HBV-transgenic mice were randomly divided into two groups for a 3-week course of once weekly subcutaneous immunizations with either CTP-HBcAg18-27-Tapasin fusion protein or CTP-HBcAg18-27. Mice administered isotonic saline served as blank controls. Expressions of cytokines in splenocytes were analyzed by flow cytometry. Serum levels of hepatitis B surface antigen (HBsAg) and HBV DNA were determined by microparticle enzyme immunoassay and real-time fluorescent PCR assay, respectively. Expression of HBsAg in hepatic tissues was detected by immunohistochemistry.

<b>RESULTSb>Immunization with 100 mug of CTP-HBcAg18-27-Tapasin fusion protein led to a significant increase in proportions of CTLs in spleen (2.70%+/-0.20% vs. 50 mug of CTP-HBcAg18-27-Tapasin: 1.66%+/-0.53%, 50 mug of CTP-HBcAg18-27: 1.26%+/-0.56%, and blank controls: 0.75%+/-0.71%; F = 741.45, P = 0.000) and up-regulation of inflammatory cells in hepatic tissue. In addition, both immunizations of CTP-HBcAg18-27-Tapasin led to significant decreases in serum HBsAg and HBV DNA levels compared to those in the CTP-HBcAg18-27 group.

<b>CONCLUSIONb>HBV-related modification of the expression of the molecular chaperone Tapasin may affect its interaction with intracellular antigen peptides, thereby leading to increases the number of specific CTLs in the spleen, decreases in serum HBsAg and HBV DNA levels, and down-regulation of HBsAg expression in hepatic tissue. These results obtained in HBV-transgenic mice suggest that the CTP-HBcAg18-27-Tapasin fusion protein has anti-HBV activity.

Animals ; DNA, Viral ; blood ; Female ; Hepatitis B ; immunology ; Hepatitis B Core Antigens ; genetics ; Hepatitis B Surface Antigens ; blood ; Hepatitis B virus ; physiology ; Male ; Membrane Transport Proteins ; genetics ; Mice ; Mice, Inbred C57BL ; Mice, Transgenic ; Recombinant Fusion Proteins ; genetics ; immunology ; T-Lymphocytes, Cytotoxic ; immunology ; Transfection ; Virus Replication

Cytotoxic T cells (CTLs) play a key role in the control of Hepatitis B virus (HBV) infection and viral clearance. However, most of identified CTL epitopes are derived from HBV of genotypes A and D, and few have been defined in virus of genotypes B and C which are more prevalent in Asia. As HBV core protein (HBc) is the most conservative and immunogenic component, in this study we used an overlapping 9-mer peptide pool covering HBc to screen and identify specific CTL epitopes. An unconventional HLA-A2-restricted epitope HBc141-149 was discovered and structurally characterized by crystallization analysis. The immunogenicity and anti-HBV activity were further determined in HBV and HLA-A2 transgenic mice. Finally, we show that mutations in HBc141-149 epitope are associated with viral parameters and disease progression in HBV infected patients. Our data therefore provide insights into the structure characteristics of this unconventional epitope binding to MHC-I molecules, as well as epitope specific CTL activity that orchestrate T cell response and immune evasion in HBV infected patients.
Adult ; Amino Acid Sequence ; Animals ; Binding Sites ; Epitopes ; chemistry ; immunology ; metabolism ; Female ; Genotype ; HEK293 Cells ; HLA-A2 Antigen ; metabolism ; Hepatitis B Core Antigens ; chemistry ; immunology ; metabolism ; Hepatitis B virus ; genetics ; metabolism ; Humans ; Hydrogen Bonding ; Male ; Mice ; Mice, Inbred BALB C ; Mice, Transgenic ; Middle Aged ; Molecular Dynamics Simulation ; Mutation ; Protein Binding ; Protein Structure, Tertiary ; T-Lymphocytes, Cytotoxic ; immunology ; metabolism

BACKGROUND/AIMS: The prevalence of occult HBV infection depends on the prevalence of HBV infection in the general population. Hemodialysis patients are at increased risk for HBV infection. The aim of this study was to determine the prevalence of occult HBV infection in hemodialysis patients. METHODS: Total of 98 patients undergoing hemodialysis in CHA Bundang Medical Center (Seongnam, Korea) were included. Liver function tests and analysis of HBsAg, anti-HBs, anti-HBc and anti-HCV were performed. HBV DNA testing was conducted by using two specific quantitative methods. RESULTS: HBsAg was detected in 4 of 98 patients (4.1%), and they were excluded. Among 94 patients with HBsAg negative and anti-HCV negative, one (1.1%) patient with the TaqMan PCR test and 3 (3.2%) patients with the COBAS Amplicor HBV test were positive for HBV DNA. One patient was positive in both methods. Two patients were positive for both anti-HBs and anti-HBc and one patient was negative for both anti-HBs and anti-HBc. CONCLUSIONS: The present study showed the prevalence of occult HBV infection in HBsAg negative and anti-HCV negative patients on hemodialysis at our center was 3.2%. Because there is possibility of HBV transmission in HBsAg negative patients on hemodialysis, more attention should be given to prevent HBV transmission.
Adult ; Aged ; Aged, 80 and over ; Antibodies/blood ; DNA, Viral/analysis ; Feces/*virology ; Female ; Hepatitis B/complications/*epidemiology/transmission ; Hepatitis B Core Antigens/immunology ; Hepatitis B virus/genetics/immunology ; Hepatitis C Antibodies/blood ; Humans ; Kidney Failure, Chronic/*complications/diagnosis ; Male ; Middle Aged ; Polymerase Chain Reaction ; Prevalence ; Renal Dialysis ; Risk Factors

This paper is aimed to investigate the inhibitory effects of hepatitis B virus (HBV) preC and C genes-specific antisense locked nucleic acid (LNA) on HBV replication and expression in transgenic mice. The antisense LNA, which was complementary to the preC and C gene region of HBV, was designed, synthesized, and injected into transgenic mice via the tail vein. Serum HBV DNA was tested with real-time PCR, and Serum HBsAg was tested with time-resolved fluorescence immune assay (TRFIA). Then the expression of HBcAg in the liver was detected with immuneohistochemistry. Serum ALB, ALT, BUN and CRea were measured with an antomatic biochemicall analyzer. It was found that 5 days after LNA injection, serum HBV DNA levels in the dual-target group were reduced by 53.72%, and serum HBsAg levels were decreased by 71.57%. These values were significantly higher than those in the control groups (P<0.05) and the expression levels of HBcAg in the liver were significantly lower than those in the control groups (P<0.05). The result also showed that there were no significant differences discovered in serum ALB, ALT, BUN and CR between the experiment groups and the control groups. The present study provides that antisense LNA targeting to both preC and C genes has shown strong inhibition on HBV replication and expression in transgenic mice, and stronger than target at single gene site.
Animals ; Antiviral Agents ; pharmacology ; DNA, Viral ; blood ; Female ; Gene Targeting ; Hepatitis B Core Antigens ; metabolism ; Hepatitis B Surface Antigens ; blood ; Hepatitis B virus ; drug effects ; genetics ; physiology ; Liposomes ; Male ; Mice ; Mice, Transgenic ; Oligonucleotides ; pharmacology ; Oligonucleotides, Antisense ; pharmacology ; Virus Replication ; drug effects

<b>OBJECTIVEb>To investigate the differential expression of programmed death-1 (PD-1) in the hepatitis B core antigen (HBcAg)17-28-specific CD8+ T cell subsets of adolescent patients with chronic hepatitis B virus (HBV) infection during the immune tolerant phase and the immune clearance phase.

<b>METHODSb>A total of 105 patients between the ages of 12-28 years old (mean age 17.20+/-6.35) with chronic HBV infection and 15 healthy age-matched individuals were enrolled in the study. The patients were divided into two groups according to their current status in immune clearance phase (n = 55) or immune tolerant phase (n = 50), as determined by hepatic biopsy pathology. Flow cytometry was used to detect HLA-A2 type and PD-1 expression on peripheral blood mononuclear cells (PBMC) and HBcAg17-28-specific CD8+ T cells. PD-1 mRNA levels in PBMCs were measured by reverse transcription-polymerase chain reaction (RT-PCR). Independent samples t-test was used to compare means between the two groups, and one-way ANOVA was used to compare means among multiple groups. Pearson's correlation coefficient was used to assess the significance of correlation.

<b>RESULTSb>The frequency of HBcAg18-27-specific CD8+ T cells was significantly higher in the immune clearance phase group than in the immune tolerant phase group (t = 18.08, P less than 0.01), but the expression of PD-1 on the HBcAg18-27 specific CD8+ T cells was significantly lower in the immune clearance phase group than in the immune tolerant phase group (t = 4.72, P less than 0.01). A negative correlation existed between the frequency of HBcAg18-27-specific CD8+ T cells and PD-1 expression (r = -0.463, P less than 0.01). A positive correlation existed between HBV viral load and PD-1 expression on the HBcAg18-27-specific CD8+ T cells in chronic HBV infection patients (r = 0.882, P less than 0.01), and there was a negative correlation between PD-1 expression levels on HBcAg18-27-specific CD8+ T cells and hepatic tissue inflammation score (r = -0.76, P less than 0.01). PD-1 mRNA in PBMCs was significantly higher in the immune tolerant phase group than in the immune clearance phase group (t = 30.89, P less than 0.01).

<b>CONCLUSIONb>Up-regulated expression of PD-1 is associated with HBV-specific CD8+ T cells and may play a crucial role in inhibiting their function during the immune tolerance phase of chronic HBV infection in adolescents.

Adolescent ; CD8-Positive T-Lymphocytes ; metabolism ; HLA-A2 Antigen ; Hepatitis B Core Antigens ; Hepatitis B virus ; genetics ; Hepatitis B, Chronic ; blood ; Humans ; Leukocytes, Mononuclear ; metabolism ; T-Lymphocyte Subsets ; metabolism

Occult HBV infection (OBI) is defined as presence of HBV DNA in the liver tissue in patients with serologically undetectable HBsAg. There are differences in virologic and serological profiles of OBI. Majority of OBI are positive for anti-HBs and/or anti-HBc and minor portion are negative for all HBV markers. However, there are no HBV mutations in the surface and its regulatory regions. HBV infection persists by the presence of covalently closed circular DNA (cccDNA) within the infected hepatocytes, which serves as a reservoir for future infection. OBI increases the risk of HBV transmission through transfusion, hemodialysis, and organ transplantation. Therefore effective measures should be employed to screen OBI. Antiviral therapy is needed in HBsAg-negative transplant patients who are anti-HBc positive to prevent the recurrence of HBV infection. Since HBV replication is strongly suppressed by immune surveillance system in OBI patients, immunosuppression results in massive HBV replication. This leads to acute hepatitis and sometimes mortality when immune surveillance is recovered after stopping immunosuppressive drugs/anticancer chemotherapy. Therefore, narrow surveillance is required to recognize the viral reactivation and start antiviral agents during immunosuppressive therapy/anticancer chemotherapy in patients with OBI.
Blood Transfusion ; DNA, Viral/analysis ; Hepatitis B/*diagnosis/transmission ; Hepatitis B Core Antigens/immunology ; Hepatitis B virus/genetics/*physiology ; Humans ; Liver Transplantation ; Renal Dialysis ; Virus Activation