Maturation of recombinant hepatitis B virus surface antigen particles

The major surface antigen of Hepatitis B virus (HBsAg) is a cysteine-rich, lipid-bound protein with 226 amino acids. Recombinant HBsAg (rHBsAg) with associated lipids can self-assemble into 22-nm immunogenic spherical particles, which are used in licensed Hepatitis B vaccines. Little is known about the structural evolvement or maturation upon assembly beyond an elevated level of disulfide formation. In this paper, we further characterized the maturation of HBsAg particles with respect to their degree of cross-linking, morphological changes, and changes in conformational flexibility. The lipid-containing rHBsAg particles undergo KSCN- and heat-induced maturation by formation of additional intra- and inter-molecular disulfide bonds. Direct measurements with atomic force microscopy (AFM) revealed morphological changes upon maturation through KSCN-induced and heat-/storage-incurred oxidative refolding. Particle uniformity and regularity was greatly improved, and protrusions formed by the protein subunits were more prominent on the surface of the mature particles. Decreased conformational flexibility in the mature rHBsAg particles was demonstrated by millisecond-scale unfolding kinetics in the presence of an environment-sensitive conformation probe. Both the accessible hydrophobic cavities under native conditions and the changeable hydrophobic cavities upon denaturant-induced unfolding showed substantial decrease upon maturation of the rHBsAg particles. These changes in the structural properties may be critical for the antigenicity and immuno-genicity of this widely-used vaccine component.     

Serum hepatitis B surface antigen levels in the natural history of chronic hepatitis B infection

BACKGROUND The production of hepatitis B surface antigen (HBsAg) may evolve during long-lasting virus-host interactions in chronic hepatitis B (CHB). The impact of age on HBsAg production remains unclear. AIM To determine the age-specific distribution patterns of HBsAg and related factors during the natural course of CHB infection. METHODS Seven hundred and sixty-eight untreated HBsAg carriers were enrolled in the study. The parameters and distribution patterns of HBsAg were evaluated in relation to age and immune phases. RESULTS The HBsAg levels were significantly lower in the HBeAg-negative stage, with the lowest levels in inactive carriers. The HBsAg tended to decrease from hepatitis to cirrhosis and to hepatocellular carcinoma, and from Child-Pugh class A to B and to C. Age and HBV DNA were independently associated with HBsAg levels. In HBeAg-positive patients, the HBsAg levels were distributed in a triphasic-like decline pattern by 2 logs across age strata. For HBeAg-negative patients, the titres in inactive carriers exhibited a 2-log reduction, but remained unchanged over age strata in patients with HBeAg-negative hepatitis. The ratios of HBsAg/HBV-DNA were highest, but steadily decreased with age in inactive carriers, whereas the levels remained largely unchanged over the entire age strata in patients with HBeAg-negative hepatitis. CONCLUSIONS Age and HBV DNA levels are independent parameters of HBsAg levels. During the natural course of CHB infection, HBsAg levels decrease with age and disease progression, but the patterns are significantly different between the immune phases of CHB. This information may contribute to our understanding of the immunopathogenesis of chronic hepatitis B and management involving HBsAg quantification.     

Treatment of early stage chronic hepatitis C virus infection

Introduction: Treatment of Hepatitis C Virus (HCV) with direct acting antivirals (DAAs) is able to achieve the cure of infection in almost the totality of patients, independently of the characteristics of the individual and the virus, using short treatment schedules, and without the need of ribavirin. The high cost of DAAs is the main limiting factor for universal treatment of HCV. However, there is a strong evidence that treatment of infection at the early stage of disease may be the most rewarding approach.

Areas covered: This review evaluates the aspects underlying the benefit of treating chronic HCV infection at the early stage of disease. It outlines the considerations that have to be taken into account when planning treatment in patients with HCV and minimal liver disease, assessing the positive reflex of viral eradication on several HCV-associated extra-hepatic conditions such as the risk of lymphoma, insulin-resistance and glycaemic control, and renal function. Lastly, it also covers the improvement of patients’ quality of life and the pharmaco-economic aspects associated with early treatment.

Expert commentary: Treatment of patients with HCV and minimal liver disease is associated with a beneficial, pleiotropic effect of viral eradication that goes beyond the simplistic consideration of the improvement in liver disease-related outcomes.     

Laparoscopical and histological abnormalities of the liver in chronic HBs ag (hepatitis B surface antigen) carriers

Previously, I have reported the laparoscopical and histological findings of 16 chronic HBs Ag carriers in whom the time of HBV infection could be assumed, HBs Ag in the sera persisted for at least 6 months, and laparoscopy with liver biopsy was performed. In these carriers, no abnormal results of liver function tests were found and early history of hepatitis was unknown. Besides these 16 HBs Ag carriers, I have encountered 15 chronic HBs Ag carriers in whom the time of HBV infection could be assumed, HBs Ag in the sera persisted for at least six months, and laparoscopy with liver biopsy was performed. In these carriers, no early history of hepatitis could be found and normal results of liver function tests were observed at least at the time of the first laparoscopy with liver biopsy, but blood chemical analyses resulted in slightly abnormal values at rare intervals during the clinical observations after the first laparoscopy with liver biopsy. In six cases of 15 chronic HBs Ag carriers, laparoscopy revealed a white liver; in four cases, a multicolored liver and in the remaining five cases, a nodulated liver. Histology of the white liver in six cases showed chronic persistent hepatitis (CPH) in three cases, chronic aggressive hepatitis (CAH) in one case and chronic persistent hepatitis with fatty metamorphisis in two cass. Histology of the nodulated liver showed liver cirrhosis in five cases. Histology of the multicolored livers showed CAH in all four cases. The question remains unanswered as to how such morphological alterations of the liver developed in these chronic HBs Ag carriers with normal liver chemistries.     

Physicochemical properties of recombinant hepatitis B surface antigen expressed in mammalian cell (C127)

The physicochemical properties of recombinant hepatitis B surface antigen (r-HBsAg), which was expressed in C127 mammalian cell were studied. Using roller bottle culture in DMEM supplemented with fetal bovine serum, 10–15 mg/L of r-HBsAg was produced with about 31% of purification yield. The purity of r-HBsAG by HPLC was 99.8% and electron microscopic examination showed homogeneous spherical particle with 22 nm in diameter, a morphological characteristic of HBsAg. The density of r-HBsAg by CsCl density gradient method was 1.19 g/ml and the isoelectric point by Mono P^TM HR 5/20 column was 4.6. The analysis of subunit protein pattern using SDS-PAGE followed by scanning densitometry gave 81.3% of S protein and 18.7% of pre-S protein. Fluorophore-assisted-carbohydrate-electrophoresis analysis showed the relative amount of carbohydrate to protein was 1.7% and its major component was N-acetyl glucosamine, which was about 39% of total carbohydrate. The relative amount of lipid to protein determined by vanillin phosphoric acid method was 32.5% and its major component was phospholipid, which was about 70% of total lipid. The physicochemical properties of C127 mammalian cell-derived r-HBsAg are similar to those of p-HBsAg, suggesting that the r-HBsAg can be used in developing a new preventive vaccine against hepatitis B.     

Immunopathogenesis of hepatitis B e antigen negative chronic hepatitis B infection.

Hepatitis B e antigen (HBeAg) negative chronic hepatitis B represents currently the predominant form of chronic hepatitis due to the hepatitis B virus (HBV) in several parts of the world. In this review, recent data regarding the process of HBeAg negative HBV strain selection during the course of chronic HBV infection are presented and the potential virus and/or host-mediated mechanisms that lead to chronic liver necroinflammation, i.e. chronic hepatitis are outlined.     

Immunological properties of recombinant hepatitis B surface antigen expressed in mammalian cell (C127)

The physicochemical properties of recombinant hepatitis B surface antigen (r-HBsAg), which was expressed in C127 mammalian cell were studied. Using roller bottle culture in DMEM supplemented with fetal bovine serum, 10–15 mg/L of r-HBsAg was produced with about 31% of purification yield. The purity of r-HBsAG by HPLC was 99.8% and electron microscopic examination showed homogeneous spherical particle with 22 nm in diameter, a morphological characteristic of HBsAg. The density of r-HBsAg by CsCl density gradient method was 1.19 g/ml and the isoelectric point by Mono P^TM HR 5/20 column was 4.6. The analysis of subunit protein pattern using SDS-PAGE followed by scanning densitometry gave 81.3% of S protein and 18.7% of pre-S protein. Fluorophore-assisted-carbohydrate-electrophoresis analysis showed the relative amount of carbohydrate to protein was 1.7% and its major component was N-acetyl glucosamine, which was about 39% of total carbohydrate. The relative amount of lipid to protein determined by vanillin phosphoric acid method was 32.5% and its major component was phospholipid, which was about 70% of total lipid. The physicochemical properties of C127 mammalian cell-derived r-HBsAg are similar to those of p-HBsAg, suggesting that the r-HBsAg can be used in developing a new preventive vaccine against hepatitis B.     

Influence of surface charge of PLGA particles of recombinant hepatitis B surface antigen in enhancing systemic and mucosal immune responses

This study investigates the efficacy of surface-modified microspheres of hepatitis B surface antigen (HBsAg) in eliciting systemic and mucosal immune responses. Positively charged poly(d,l-lactic-co-glycolic acid) microspheres were prepared by a double-emulsion solvent-evaporation method with cationic agents—stearylamine and polyethylenimine—in the external aqueous phase. Formulations were characterized for morphology, size, density, aerodynamic diameter, entrapment efficiency and in vitro drug-release profile. Immunization was performed after pulmonary administration of the formulations to female Sprague–Dawley rats and the immune response was monitored by measuring IgG levels in serum and secretory (sIgA) levels in salivary, vaginal and bronchoalveolar lavage fluids. The cell-mediated immune response was studied by measuring cytokine levels in spleen homogenates, and a cytotoxicity study was performed with Calu-3 cell line. The aerodynamic diameter of the particles was within the respirable range, with the exception of stearylamine-modified particles. Zeta potential values moved from negative (−6.76 mV) for unmodified formulations to positive (+0.515 mV) for polyethylenimine-modified particles. Compared to unmodified formulations, polyethylenimine-based formulations showed continuous release of antigen over a period of 28–42 days and increased levels of IgG in serum and sIgA in salivary, vaginal and bronchoalveolar lavage. Further, cytokine levels—interferon γ and interleukin-2—were increased in spleen homogenates. The viability of Calu-3 cells was not adversely affected by the microparticles. In summation, this study establishes that positive surface charges on poly(d,l-lactic-co-glycolic acid) particles containing HBsAg enhances both the systemic and mucosal immune response upon immunization via the respiratory route.     

siRNA pool targeting different sites of human hepatitis B surface antigen efficiently inhibits HBV infection

The main objective was to determine whether a pool of small interfering RNAs (siRNAs) targeting different regions of hepatitis B virus surface antigen (HBsAg) efficiently inhibits hepatitis B virus (HBV) infection. siRNAs targeting different regions of HBsAg were transfected into HBV-producing HepG2.2.15 cells and at 72 h post-transfection, the culture medium was collected for ELISA to determine HBsAg, while total RNA was isolated from the cells for real-time PCR. Three siRNA sequences that efficiently inhibited HBV infection were converted into small hairpin RNAs (shRNAs) and then cloned into a single plasmid psiSTRIKE driven by a single U6 promoter. These shRNA expressing plasmids were tested for HBsAg gene silencing in HepG2.2.15 cells. A pool of siRNAs targeting HBsAg efficiently inhibited HBV replication and antigen expression when transfected into HepG2.2.15 cells, compared with the use of single siRNA. Similarly, the plasmid encoding three different shRNAs driven by a single U6 promoter was more effective in silencing HBsAg at DNA, mRNA and protein levels compared with the plasmid encoding single shRNA. No apoptotic change was observed in the cells when the plasmid was transfected at a dose of 0.5-2 microg/1 x 10(6) cells after complex formation with Lipofectamine LTX. Furthermore, transfection with siRNA or shRNA did not increase interferon-gamma (IFNs-gamma) release, suggesting no induction of IFN response. In conclusion, a pool of chemically synthesised siRNAs as well as the shRNA expression plasmid encoding multiple shRNAs targeting different regions of HBsAg showed high gene silencing in HepG2.2.15 cells.     

Hepatitis B surface antigen in notified cases of viral hepatitis.

Eighty-five patients notified to the local medical officer of health as infectious or serum hepatitis during the period 1 September 1974-1 September 1975 were investigated by questionnaire. Serum from 73 cases was tested for the presence of HBsAg. Sixteen cases were notified as serum hepatitis but only 13 were shown to have HBsAg in their serum. Sixty-eight cases were notified as infectious hepatitis and 21 HBsAg positive cases were found amongst the 57 sera tested. Two-thirds of the HBsAg positive cases were not suspected clinically. The epidemiology of hepatitis in Christchurch is discussed and suggestion is made that all notified cases of hepatitis should have blood tested for HBsAg.     

T-cell responses to hepatitis B surface antigen in lung transplant recipients

STUDY OBJECTIVE: To determine whether lung transplant recipients would have a less vigorous T-cell response to hepatitis B surface antigen (HBsAg) than that of patients awaiting lung transplantation and healthy subjects, we sought to measure and compare T-cell responses among these three groups. DESIGN: Prospective study. SETTING: Lung transplant clinic at a university hospital. SUBJECTS: Twelve lung transplant recipients, 12 patients awaiting lung transplantation, and 15 healthy subjects. All participants had received the hepatitis B vaccine series and had a documented antibody response to it. INTERVENTION: Blood samples were obtained from each participant. MEASUREMENTS AND MAIN RESULTS: Participants' sex, age, time since lung transplantation (if applicable), and time since hepatitis B immunization were recorded. Peripheral blood mononuclear cells were isolated from the participants' blood samples for the trans vivo delayed-type hypersensitivity (DTH) assay. These cells were mixed with saline, tetanus toxoid, or HBsAg and injected into the footpads of immunodeficient mice. Resultant swelling of the footpad was used as an index of human T-cell response. The healthy subjects were younger than the patients in both transplant groups. However, we found no significant difference in DTH response elicited by HBsAg among the healthy subjects, patients awaiting lung transplantation, and lung transplant recipients (mean +/- standard error [SE] 34.7 +/- 4.3, 32.1 +/- 3.1, and 33.5 +/- 4.0 x 10(-4) in., respectively, p>0.8) or when tetanus toxoid was used as a positive control (15.7 +/- 2.8, 22.8 +/- 6.5, and 21.7 +/- 3.9 x 10(-4) in., respectively, p>0.3). No correlation between age or time since immunization and DTH response was noted. CONCLUSION: Lung transplant recipients maintained a T-cell response to HBsAg that was similar in vigor to that of both patients awaiting transplantation and healthy subjects even though their antibody concentrations waned rapidly after transplantation. The role of these T cells as a correlate of protection from infection remains to be investigated.