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

OBJECTIVETo investigate the clinical value of serum anti-Ku86 in early detection of hepatocellular carcinoma (HCC).

METHODSExpression levels of Ku86 protein in HCC and adjacent normal liver tissues were detected by Western blotting. Serum anti-Ku86 level in 83 patients with early HCC and 124 patients with liver cirrhosis were detected by enzyme-linked immunosorbent assay (ELISA). Chemiluminescence was used to measure the serum level of α-fetoprotein (AFP).

RESULTSExpression of Ku86 protein in HCC was increased when compared with the adjacent normal liver tissues (0.21 ± 0.05 vs. 0.08 ± 0.02, P < 0.01). Serum anti-Ku86 level was significantly elevated in HCC patients compared with that in liver cirrhosis patients (0.47 ± 0.22 vs. 0.22 ± 0.06 Abs at 450 nm, P < 0.01), but there was no significant difference between HBV infection and HCV infection in HCC patients (0.51 ± 0.19 vs. 0.47 ± 0.24, P = 0.267). Of note, serum anti-Ku86 level was significantly decreased after surgical resection of the tumors in the 30 HCC cases tested (P < 0.01). The results of ROC analysis indicated a better performance of anti-Ku86 (0.857) than AFP (0.739) for early detection of HCC. In 83 HCC patients, the positive rate of anti-Ku86 was 61.4% (51/83), significantly higher than that of the AFP positive rate (27.7%, 23/83). The anti-Ku86 level was positive in 37 of 60 HCC cases with negative AFP. Combination assay of AFP and anti-Ku86 could detect 60 of 83 HCC cases (72.3%, 60/83). There was no significant correlation of anti-Ku86 and AFP (r = 0.156, P = 0.161).

CONCLUSIONSSerum anti-Ku86 level is significantly elevated and is not related to HBV and HCV infection in HCC patients. Serum anti-Ku86 antibody may be a potential biomarker for early detection of HCC, and can be used in combination with AFP in clinics.

Adult ; Aged ; Antigens, Nuclear ; immunology ; Autoantibodies ; blood ; Biomarkers, Tumor ; blood ; Carcinoma, Hepatocellular ; blood ; diagnosis ; virology ; DNA-Binding Proteins ; immunology ; Early Detection of Cancer ; Female ; Hepatitis B ; blood ; Hepatitis C ; blood ; Humans ; Ku Autoantigen ; Liver Cirrhosis ; blood ; Liver Neoplasms ; blood ; diagnosis ; virology ; Male ; Middle Aged ; ROC Curve ; alpha-Fetoproteins ; metabolism

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

The role of hepatic CD69+ natural killer (NK) cells in virus-induced severe liver injury and subsequent hepatic failure is not well defined. In this study, a mouse model of fulminant liver failure (FHF) induced by murine hepatitis virus strain 3 (MHV-3) was used to study the role of hepatic CD69+NK cells in the development of FHF. The CD69 expression in NK cells in the liver, spleen, bone marrow and peripheral blood was detected by using flow cytometry. The correlation between the CD69 level in hepatic NK cells and liver injury was studied. The functional marker (CD107a), and activating and inhibitory receptor (NKG2D and NKG2A) expressed on CD69+NK cells and CD69-NK cells were detected by using flow cytometry. Pro-inflammatory cytokines (IL-9, IFN-γ and TNF-α) were also examined by using intracellular staining. After MHV-3 infection, the number of CD69+NK cells in the liver of BALB/cJ mice was increased markedly and peaked at 72 h post-infection. Similar changes were also observed in the spleen, bone marrow and peripheral blood. Meanwhile, the CD69 expression in hepatic NK cells was highly correlated with the serum level of ALT and AST. The expression of CD107a and NKG2D, as well as the production of TNF-α, IFN-γ and IL-9 in hepatic CD69+NK cells was all significantly up-regulated during 48-72 h post-infection. In contrast, the NKG2A expression was increased in hepatic CD69-NK cells but not in CD69+NK cells. These results suggested that hepatic CD69+NK cells play a pivotal role in the pathogenesis of FHF by enhancing degranulation and cytotoxic ability of NK cells and increasing the production of pro-inflammatory cytokines.
Animals ; Antigens, CD ; immunology ; Antigens, Differentiation, T-Lymphocyte ; immunology ; Coronavirus Infections ; immunology ; Female ; Hepatitis, Viral, Animal ; immunology ; Killer Cells, Natural ; immunology ; Lectins, C-Type ; immunology ; Mice ; Mice, Inbred BALB C ; Murine hepatitis virus ; immunology

We recently developed a mouse model of hepatitis B virus (HBV) chronic infection by intravenous (i.v.) injection with rAAV8-1. 3HBV to C57BL/6 mice. To define the responses of different mouse strains after injection with rAAV8-1. 3HBV, we intravenously injected rAAV8-1. 3HBV at doses of 4 x10(9) (Viral genome,vg), 4 x 10(10) vg and 4 x 10(11) vg to C57BL/6 and BALB/c mice,respectively, and determined the levels of serum HBV antigen and antibody by ELISA,serum viral DNA by real-time PCR,and HBcAg expression in liver by immunohistochemical staining. For C57BL/6 mouse strain with injection of rAAV8-1. 3HBV at three doses, 100% of the mice carried HBV for more than 8 months. The levels of serum HBsAg and HBeAg, serum viral DNA and HBcAg-positive hepatocytes increased in a rAAV8-1. 3HBV dose-dependent manner. For C57BL/6 mice injected with rAAV8-1. 3HBV at the dose of 4 x 10(11) vg,over 40% of hepatocytes expressed HBcAg and serum viral DNA reached over 10(5) IU/mL. No HBV antibody was detected in sera of C57BL/6 mice. For BALB/c mice with injection of rAAV8-1. 3HBV at three doses, serum HBeAg, serum viral DNA and HBcAg-positive hepatocytes persisted for more than 8 months, but serum HBsAg declined remarkably at 2 weeks after injection. The levels of serum HBeAg and HBcAg-positive hepatocytes in BALB/c mice increased in a rAAV8-1. 3HBV dose-dependent manner. Injection with rAAV8-1. 3HBV at the dose of 4 x 10(11) vg resulted in over 50% of BALB/c mice hepatocytes expressing HBcAg. Serum anti-HBsAg were detected in BALB/c mice with rAAV8-1. 3HBV injection at the dose of 4 x10 (10) vg. In conclusion, both C57BL/6 and BALB/c strains can be developed to chronic HBV infection mouse models by i. v. injection with rAAV8-1. 3HBV at doses of 4 x10(9) - 4 x 10(11) vg and the levels of HBV replication increase in a rAAV8-1. 3HBV dose-dependent manner. In contrast to C57BL/6 strain, the BALB/c mice carry out humoral immunity to HBsAg, but fail to mediate HBV clearance.
Animals ; Dependovirus ; genetics ; metabolism ; Disease Models, Animal ; Genetic Vectors ; genetics ; metabolism ; Hepatitis B ; immunology ; virology ; Hepatitis B Antibodies ; immunology ; Hepatitis B Surface Antigens ; immunology ; Hepatitis B e Antigens ; immunology ; Hepatitis B virus ; genetics ; immunology ; physiology ; Hepatocytes ; immunology ; virology ; Humans ; Male ; Mice ; Mice, Inbred BALB C ; Mice, Inbred C57BL ; Transduction, Genetic ; Virus Replication

Natural killer (NK) cells play an important role in innate immunity, especially in the response to viral infections, such as hepatitis C virus (HCV). Killer cell immunoglobulin-like receptors (KIRs) are the primary receptors of NK cells that mediate innate immunity. KIRs are also involved in acquired immunity, because some KIRs are expressed on the surface of certain subsets of T cells. In this study, the frequency of KIR genes, HLA-C allotypes, and combinations of KIR genes with their HLA-C ligands were evaluated in two different groups of the Korean population: controls and patients with chronic HCV infection. The study population consisted of 147 Korean patients with chronic HCV infection. The frequency of KIR2DS2 in patients with chronic HCV infection was 9.5% which was significantly lower than 19.5% of the control (P < 0.01). However, there were no significant differences in the frequency of other KIR genes, HLA-C allotypes or different combinations of KIR genes with their HLA-C ligands. This study can contribute to the further prospective study with a larger scale, suggesting the assumption that KIR2DS2 might aid in HCV clearance by enhancing both the innate and acquired immune responses of people in Korea.
Adult ; Aged ; Female ; Genes, MHC Class I ; Genotype ; HLA-C Antigens/genetics ; Hepacivirus/immunology ; Hepatitis C, Chronic/*genetics/immunology ; Humans ; Killer Cells, Natural/immunology/virology ; Male ; Middle Aged ; Receptors, KIR/*genetics/immunology ; Republic of Korea ; T-Lymphocyte Subsets/immunology

BACKGROUND/AIMS: There have been only few studies on the population-adjusted seroprevalence of HBV and HCV and on the change of them for more than 10 years in Korea. Therefore, this study was performed to evaluate them at a single health center in Daegu and Gyeongbuk province considering the population composition ratio. METHODS: We analyzed the seromarkers of HBV and HCV of 6,237 randomly sampled adults who had received health screening at the health promotion center in Dongsan hospital during the periods from year 1997 to 1999 (Period A) and from 2007 to 2009 (Period B). RESULTS: The seroprevalences of HBsAg, anti-HBs, anti-HBc and anti-HCV were 4.8%, 70.2%, 47.4%, and 0.5%, respectively. There is no difference in the seroprevalence of HBsAg between period A and B. However, downward tendency of prevalence through A to B from 6.8% to 4.5% could be found in 40s, and upward tendency from 4.7% to 6.8% in 50s. As for the seroprevalence of anti-HCV, although there was no difference through A to B, for 50s, it decreasd from 2.0% to 0.3% (p=0.007). However, for 60s, it increased as much as decreasd for 50s. CONCLUSIONS: The seroprevalence of HBsAg in Daegu/Gyeongbuk province was 4.8% and showed no difference through the time period of 10 years. The seroprevalence of anti-HCV was 0.5% and also showed no difference through the time periods.
Adult ; Aged ; Female ; Hepatitis B/*epidemiology ; Hepatitis B Antibodies/*blood ; Hepatitis B Surface Antigens/*blood/immunology ; Hepatitis C/*epidemiology ; Hepatitis C Antibodies/*blood ; Humans ; Male ; Middle Aged ; Republic of Korea ; Rural Population ; Seroepidemiologic Studies ; Urban Population

BACKGROUND/AIMS: We investigated the frequency of occult hepatitis B virus (HBV) infection in anti-hepatitis C virus (HCV)-positive individuals and the effects of occult HBV infection on the severity of liver disease. METHODS: Seventy-one hepatitis B virus surface-antigen (HBsAg)-negative patients were divided according to their HBV serological status into groups A (anti-HBc positive, anti-HBs negative; n=18), B (anti-HBc positive, anti-HBs positive; n=34), and C (anti-HBc negative, anti-HBs positive/negative; n=19), and by anti-HCV positivity (anti-HCV positive; n=32 vs. anti-HCV negative; n=39). Liver biopsy samples were taken, and HBV DNA was quantified by real-time PCR. RESULTS: Intrahepatic HBV DNA was detected in 32.4% (23/71) of the entire cohort, and HBV DNA levels were invariably low in the different groups. Occult HBV infection was detected more frequently in the anti-HBc-positive patients. Intrahepatic HBV DNA was detected in 28.1% (9/32) of the anti-HCV-positive and 35.9% (14/39) of the anti-HCV-negative subjects. The HCV genotype did not affect the detection rate of intrahepatic HBV DNA. In anti-HCV-positive cases, occult HBV infection did not affect liver disease severity. CONCLUSIONS: Low levels of intrahepatic HBV DNA were detected frequently in both HBsAg-negative and anti-HCV-positive cases. However, the frequency of occult HBV infection was not affected by the presence of hepatitis C, and occult HBV infection did not have a significant effect on the disease severity of hepatitis C.
Adult ; Aged ; Cohort Studies ; DNA, Viral/analysis ; Female ; Genotype ; Hepatitis B/*complications/*diagnosis ; Hepatitis B Core Antigens/blood/immunology ; Hepatitis B Surface Antigens/blood/immunology ; Hepatitis B virus/*genetics ; Hepatitis C, Chronic/*complications/genetics/*pathology ; Humans ; Liver/virology ; Male ; Middle Aged ; Polymerase Chain Reaction ; Severity of Illness Index

While currently therapeutic vaccines for chronic hepatitis B virus (HBV) infection are actively being developed to complement standard antiviral treatments, their immune activity, especially T cell activity, remains to be further improved. Here, we investigated the role of heat shock proteins HSP70 and gp96 on cellular and humoral immunity, using the main structure antigens of hepatitis core (HBcAg) and surface (HBsAg) as the DNA vaccine. By ELISPOT (enzyme linked immunospot assay), IFN-gamma intracellular staining, [3H]-thymidine incorporation and ELISA (enzyme linked immunosorbent assay) analyses, we showed that immunization with HBsAg/HBcAg DNA formulation along with HSP70 or gp96 induced significant increase of T-cell (about 1-6-fold) and antibody (about 20%-60%) immunity against HBsAg and HBcAg. These results may provide bases for designing HSP70- and gp96-based vaccines aimed at eliciting T-cell responses for therapeutic applications.
Adjuvants, Immunologic ; pharmacology ; Animals ; Female ; HSP70 Heat-Shock Proteins ; immunology ; Hepatitis B Core Antigens ; immunology ; Hepatitis B Surface Antigens ; immunology ; Hepatitis B Vaccines ; immunology ; Hepatitis B virus ; immunology ; Hepatitis B, Chronic ; immunology ; therapy ; Humans ; Immunoglobulin G ; immunology ; Membrane Glycoproteins ; immunology ; Mice ; Mice, Inbred BALB C ; T-Lymphocytes ; immunology ; Vaccines, DNA ; immunology