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

<b>OBJECTIVEb>To access the antibody persistence 24-month after revaccination with 3-dose of hepatitis B vaccine (HepB) among non-response adults.

<b>METHODSb>A total of 24 237 healthy adults who had no histories of hepatitis B infection and hepatitis B vaccination, resided in the local area for more than six months and were aged 18-49 years were selected from 79 villages of Zhangqiu county, Shandong province, China in 2009. Blood samples were obtained and hepatitis B surface antigen (HBsAg), antibody against hepatitis B surface antigen (anti-HBs) and antibody against hepatitis B core antigen (anti-HBc) were detected using ELISA method. A total of 11 590 persons who were negative for all of these indicators were divided into four groups by cluster sampling methods. Each group was vaccinated with one of the following four types of HepB at 0-, 1-, 6-months schedule: 20 µg HepB derived in Saccharomyces Cerevisiae (HepB-SC), 20 µg HepB derived in Chinese hamster ovary cell (HepB-CHO), 10 µg HepB-SC and 10 µg HepB derived in Hansenula Polymorpha (HepB-HP). Blood samples were collected one month after the third dose of primary immunization and tested for anti-HBs using chemiluminescence microparticle immunoassay (CMIA). The non-responders were revaccinated with three doses of HepB at 0-, 1-, 6-months schedule and the type of HepB was the same as which was used for primary immunization. Blood samples were collected one month (T1) and two years (T24) after revaccination and anti-HBs, antibody against hepatitis B core antigen (anti-HBc) and hepatitis B surface angtigen (HBsAg) (if anti-HBs < 10 mU/ml) were detected by CMIA. χ(2) test was used to compared age, gender and body mass index (BMI) between different groups and the anti-HBs positive rate at T1 and T24; analysis of variance (ANOVA) was used to compare the geometric mean concentration (GMC) of anti-HBs between difference groups. The risk factors associated with positive rate of anti-HBs and GMC of anti-HBs were identified by multiple logistic regression analysis and multifactor linear regression model analysis respectively.

<b>RESULTSb>A total of 900 non-responders were identified and 71.7% (645/900) of them completed three-dose revaccination and blood collection after revaccination. 467 (72.4%) non-responsive adults were followed up at T24. The anti-HBs positive rate decreased from 85.65% (95% CI: 82.14%-88.71%) at T1 to 60.60% (95% CI: 56.01%-65.06%) at T24 and the corresponding GMC decreased from 175.62 (95% CI: 139.03-221.84) mU/ml to 21.43 (95% CI: 17.62-26.06) mU/ml. Multivariate analysis showed that positive rate of anti-HBs at T24 was associated with gender, HepB type for revaccination and anti-HBs level at T1, but only anti-HBs level at T1 was associated with the anti-HBs titer at T24. No subject showed HBsAg seroconversion and anti-HBc conversion rate was 3.64% (17/467) at T24.

<b>CONCLUSIONb>Anti-HBs titer decreases rapidly two years after HepB revaccination among non-responsive adults, but more than half non-responderd still kept anti-HBs above protective level. The immunity durability after revaccination was associated with gender, HepB type for revaccination and anti-HBs titer one month after revaccination.

Adolescent ; Adult ; Animals ; Body Mass Index ; CHO Cells ; China ; Cricetinae ; Cricetulus ; Enzyme-Linked Immunosorbent Assay ; Female ; Follow-Up Studies ; Hepatitis B ; prevention & control ; Hepatitis B Antibodies ; blood ; Hepatitis B Core Antigens ; immunology ; Hepatitis B Surface Antigens ; immunology ; Hepatitis B Vaccines ; administration & dosage ; classification ; Humans ; Immunization, Secondary ; Male ; Middle Aged ; Multivariate Analysis ; Pichia ; Risk Factors ; Saccharomyces cerevisiae ; Seroconversion ; Vaccination ; Young Adult

<b>OBJECTIVEb>To investigate the epidemic status of Hepatitis B in children aged 1-14 in 3 counties of Guangdong province in 2013, and to evaluate the effect of hepatitis control in children aged 1-14 after hepatitis B vaccine was integrated into the national immunization program in 2002 and catch-up vaccination was conducted from 2009 to 2011.

<b>METHODSb>A multi-stage stratified random sampling was designed to survey 1 621 children aged 1-14 in rural area of Nanxiong county, Haifeng county and Xinxing county by questionnaires including general information, medical history and risk factors. The samples were tested with chemiluminescence method to detect hepatitis B virus (HBV) surface antigen (HBsAg), antibody to HbsAg (anti-HBs) and antibody to HBV core antigen (anti-HBc). Chi-square test was used to compare the positive rate of HBV serum markers in different age groups, vaccine histories, birth weight and HBV infection status of mother.

<b>RESULTSb>Among the children aged 1-14 in 3 counties rural regions of Guangdong province, the positive rate of HBsAg, anti-HBs, and anti-HBc was 1.11% (18/1 621), 60.69% (982/1 618) and 1.92% (31/1 617), respectively. The HBsAg positive rate of vaccinated children (0.84%, 13/1 547) was lower than that of unvaccinated children (1/13) or children with unknown vaccination status (6.56%, 4/61) (χ² = 22.64, P < 0.001). The HBsAg positive rate (0.45%, 5/1 118) of the children with birth-dose given within 24 hours was lower than those that of children given beyond 24 hours (2.63%, 61/190) (χ² = 10.21, P < 0.001). The HBsAg positive rate (5/18) of children with birth weight under 2 kilogram was higher than that of children with birth weight above 2 kilogram (0.78%, 12/1 548) (χ² = 120.8, P < 0.001). The HBsAg positive rate of children born to HBsAg-positive mothers (2.80%, 3/107) was higher than that of children born to HBsAg-negative mothers (0.21%, 1/470) (χ² = 8.50, P = 0.004). With the age increasing, the coverage and timely birth-dose coverage of Hepatitis B vaccine (HepB) decreased, and the positive rate of anti-HBs gradually decreased.

<b>CONCLUSIONb>After the catch-up vaccination was conducted in unvaccinated children aged 1-14 years from 2009 to 2011, the HBsAg and anti-HBc positive rate decreased, while the anti-HBs positive rate increased significantly.

Adolescent ; Birth Weight ; Child ; Child, Preschool ; China ; epidemiology ; Hepatitis B ; epidemiology ; Hepatitis B Antibodies ; blood ; Hepatitis B Core Antigens ; immunology ; Hepatitis B Surface Antigens ; immunology ; Hepatitis B Vaccines ; Humans ; Immunization Programs ; Infant ; Risk Factors ; Rural Population ; Seroepidemiologic Studies ; Surveys and Questionnaires

<b>OBJECTIVEb>To analyze the information of the supplementary card for hepatitis B and the laboratory confirmed result of immunoglobulin M antibody to hepatitis B virus (HBV) Core Antigen (anti-HBc IgM) for the suspected acute hepatitis B to evaluate the hepatitis B report data on pilot surveillance.

<b>METHODSb>200 counties were established in China for hepatitis B pilot surveillance and 63 641 cases were reported. We added a supplementary card in National Notificable Disease Reporting System (NNDRS) and all the reported hepatitis B cases in NNDRS were required to fill the supplementary card. Venous blood 5 ml was collected and a confirmed test of anti-HBc IgM was made for suspected acute hepatitis B. We made confirmed diagnosis for the suspected acute hepatitis B according to the supplementary card information of the reporting card and the confirmed test result of anti-HBc IgM.

<b>RESULTSb>63 641 hepatitis B cases were reported in 200 hepatitis B pilot surveillance counties in 2013. Among 1 723 cases which were filled with the HBsAg positive within six months in supplementary card, 735 cases were reported as chronic hepatitis B, the proportion was 42.66%. Among 4 582 cases which were filled with anti-HBc IgM positive in supplementary card, 2 436 cases were reported as acute hepatitis B, the proportion was 53.16%. 1 829 cases were reported as chronic hepatitis B, the proportion was 39.92%. The validity cases of the information for liver puncture and the HBV surface antigen (HBsAg) transform during the recovery period in supplementary cards for all the reporting cases were 579 and 4 961, and the rate were 0.91% and 7.80%, respectively. 4 302 suspected acute cases were made confirmed diagnosis, and 1 197 cases (27.82%) were confirmed acute and 2 590 cases (60.20%) were confirmed chronic.

<b>CONCLUSIONb>Clinical doctors failed to make full use of the information of supplementary cards to make classification diagnose for hepatitis B. Suspected acute hepatitis B with anti-HBc IgM positive should be pay attention to follow up and further distinguish acute or chronic hepatitis B according to the HBsAg transform.

China ; epidemiology ; Hepatitis B ; epidemiology ; Hepatitis B Antibodies ; blood ; Hepatitis B Core Antigens ; immunology ; Hepatitis B Surface Antigens ; immunology ; Humans ; Immunoglobulin M ; blood ; Sentinel Surveillance

<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

<b>OBJECTIVEb>To compare the antibody response between adults with hepatitis B virus (HBV) core antibody (anti-HBc) single positivity and healthy adults after primary immunization and revaccination of hepatitis B vaccine(HepB).

<b>METHODSb>Adults aged from 18 to 49 who were both negative for HBV surface antigen (HBsAg) and antibody to HBsAg (anti-HBs), but positive for anti-HBc and narrated no history of HepB immunization by themselves, were selected as single anti-HBc positive group ('anti-HBc alone'). Adults who were negative for HBsAg, anti-HBs and anti-HBc, with age differences within 2 years, and same gender under the 1 : 1 matching program, were selected to form the control group. Both groups were vaccinated on 0-1-6 schedule with the same HepB. Those who were non-response to HepB at primary immunization were revaccination on 0-1-6 schedule. Response rates and geometric mean concentrations (GMC) between the two groups were compared.

<b>RESULTSb>In total, the number of anticipants were 228 pairs. Rates on non-response, low-response, normal-response and high-response after the primary immunization were 8.77% , 11.84%, 31.14% and 48.25% in the control group respectively. The corresponding rates were 8.33%, 30.70%, 35.96% and 25.00% in the 'anti-HBc alone'. The rate of low-response in the control group was lower than that in the 'anti-HBc alone' (χ(2) = 22.28, P < 0.01), while the rate of high-response was higher than that in the control group (χ(2) = 24.43, P < 0.01). GMC of anti-HBs in the control group (534.07 mIU/ml) was higher than that in the 'anti-HBc alone' (183.99 mIU/ml) (u = 4.42, P < 0.01). The anti-HBs conversion rates were 82.35% and 41.18% in the control group and in the 'anti-HBc alone' respectively after the first-dose revaccination, but increased to 90.00% and 82.35% after the third-dose revaccination. The anti-HBs conversion rates in the control group were higher than that in the 'anti-HBc alone' after the first-dose revaccination (P < 0.05), while there was no difference seen between the two groups after the third-dose revaccination (P > 0.05).

<b>CONCLUSIONb>Immune response in the anti-HBc positive adults after primary immunization was weaker than that in common adults. However, immune response induced by HepB was enough to prevent them from infecting HBV. The rates of response showed an obvious increase after revaccination, hence the same HepB immunization strategy could be used.

Adolescent ; Adult ; Hepatitis B ; immunology ; prevention & control ; Hepatitis B Antibodies ; blood ; Hepatitis B Core Antigens ; immunology ; Hepatitis B Surface Antigens ; blood ; Hepatitis B Vaccines ; immunology ; Humans ; Immunization, Secondary ; Middle Aged ; Vaccination ; Young Adult

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 widespread use of cytotoxic chemotherapy and immunosuppressants has resulted in reactivation of hepatitis B virus (HBV) recently becoming an issue. Although rituximab (an anti-CD20 monoclonal antibody) has revolutionized the treatment of lymphoma, recent reports have suggested that rituximab therapy increases the risk of viral-mediated complications, and particularly HBV reactivation. This study analyzed real clinical practice data for rituximab-related HBV reactivation. METHODS: Between January 2005 and December 2011, 169 patients received treatment with rituximab. Screening status of the HBV infection and frequency of preemptive therapy were determined in these patients, and the clinical features of HBV reactivation were analyzed. RESULTS: Seventy-nine of the 169 patients with chronic or past HBV infection were selected for evaluation of HBV reactivation. Of the 90 patients who were excluded, 22 (13.0%) were not assessed for HBsAg and anti-HBc, and 14 (8.3%) were not assessed for anti-HBc due to seronegativity for HBsAg. The selected patients were divided into those with chronic HBV infection (n=12) and those with past HBV infection (n=67); six patients (7.6%) experienced HBV reactivation. Eight patients received preemptive therapy, but three patients (37.5%) underwent HBV reactivation. Although HBsAg seropositivity was an independent risk factor for HBV reactivation (P=0.038), of the six patients with HBV reactivation, two (33.3%) had past HBV infection and three (50%) died of liver failure. CONCLUSIONS: The findings of this study demonstrate that adherence to guidelines for screening and preemptive therapy for HBV reactivation was negligent among the included cohort. Attention should be paid to HBV reactivation in patients with past as well as chronic HBV infection during and after rituximab therapy.
Adolescent ; Adult ; Aged ; Aged, 80 and over ; Antibodies/blood ; Antibodies, Monoclonal, Murine-Derived/*adverse effects/therapeutic use ; Antineoplastic Agents/adverse effects/*therapeutic use ; Child ; Child, Preschool ; Hepatitis B/etiology/mortality/virology ; Hepatitis B Core Antigens/immunology ; Hepatitis B Surface Antigens/blood ; Hepatitis B virus/*physiology ; Humans ; Lymphoma/*drug therapy ; Middle Aged ; Odds Ratio ; Retrospective Studies ; Risk Factors ; *Virus Activation ; Young Adult

Although DNA vaccination is now a promising strategy against hepatitis B virus (HBV) infection, this approach has relatively modest antiviral effect, indicating that immunosuppressive mechanisms may occur in the long-term established infection. In this study, we studied the immunogenicity and anti-HBV efficiency of a combination of HBV surface (HBsAg) and core (HBcAg) DNA vaccine, enhanced by heat shock protein (HSP) gp96 or HSP70 and mediated by in vivo electroporation. Immunization with gp96 adjuvanted HBsAg/HBcAg DNA formulation induced potent T cell and antibody immunity against HBsAg and HBcAg. Notably, treatment with gp96 or HSP70 as adjuvant resulted in reduction of Treg populations by around 20%. Moreover, compared with nonimmunized control mice, immunization with gp96 or HSP70 adjuvanted DNA vaccine dramatically decreased serum HBsAg and viral DNA levels, and HBcAg expression in liver. These results may therefore provide an effective strategy for designing gp96-based DNA vaccine for immunotherapy of chronic HBV infection.
Adjuvants, Immunologic ; Animals ; Electroporation ; HSP70 Heat-Shock Proteins ; immunology ; Hepatitis B Core Antigens ; immunology ; Hepatitis B Surface Antigens ; immunology ; Hepatitis B Vaccines ; immunology ; Hepatitis B, Chronic ; prevention & control ; Immunization ; Membrane Glycoproteins ; immunology ; Mice ; Mice, Transgenic ; Vaccines, DNA ; immunology

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