Regulation of hepatitis B virus mRNA expression in a hepatitis B surface antigen transgenic mouse model by IFN-gamma-secreting T cells after DNA-based immunization

The immunotherapeutic effect of DNA-mediated immunization against chronic hepatitis B virus (HBV) infection has been evaluated in transgenic mice expressing the sequences that code for the envelope proteins of HBV in the liver. In this model of HBV chronic carriers, a single i.m. injection of plasmid DNA encoding HBV envelope proteins is sufficient to generate specific immune responses leading to the clearance of the transgene expression product and the control of HBV mRNA. The relative contributions of the T cell subpopulations induced by DNA immunization were examined using adoptive transfer experiments. It was shown that either CD8+ or CD4+ T lymphocytes from immunocompetent DNA-immunized animals were sufficient to control viral gene expression in the livers of the recipient transgenic mice. This effect was mediated by a cytokine-dependent mechanism common to both T cell subpopulations; this mechanism did not require cell lysis, but did involve the production of IFN-gamma by the activated T cells.     

Evaluation of immunochromatography assay technique for detection of antibody to hepatitis B surface antigen (HBsAg)

A new immunochromatography assay (Dainascreen Ausab Dainabot) has been recently introduced for the detection of the presence of antibody to HBsAg. To evaluate the feasibility of using the Dainascreen Ausab, we carried out comparison tests with this method and PHA. In the test of 439 sera from HB vaccinees, inhabitants in Iki Island, Nagasaki Pref., patients with autoimmune diseases and with acute hepatitis B, 154 (31.2%) were positive by Dainascreen Ausab, 145 (29.4%) were positive by PHA and 145 (29.4%) were positive by both Dainascreen Ausab and PHA. Nine (1.8%) were positive by only Dainascreen and there were none positive by only PHA. A good correlation was observed between the titer of the antibody by this method and IMx. The anti-HBs assay by this method was able to be completed within 15 minutes and the procedure was very simple. The results indicate that the sensitivity of Dainascreen is superior to PHA and that it is easy to use.     

The prospects for using the genetically engineered surface antigen of the hepatitis B virus (HBsAg) expressed by recombinant poxvirus strains as the basis for vaccines against hepatitis B

The use of a recombinant poxvirus (RPV) strain, expressing HBsAg in the process of reproduction in different bioreactor systems under stationary and bioreactor conditions of cultivation, made it possible to obtain highly purified HBsAg. The identity and purity of HBsAg was confirmed by the analysis of its amino acid composition, SDS electrophoresis in polyacrylamide gel, electron microscopy and high-performance liquid chromatography. Good prospects of the use of RPV-expressed gene engineering HBsAg as the basis vaccines against hepatitis B was demonstrated in 10 experimental batches of vaccine. All batches of the preparation had pronounced immunogenicity and were safe and nontoxic in animal experiments. The ID50 of experimental batches did not exceed 211 ng/ml, which, according to the data of comparative experiments, was lower than, or equal to, corresponding values of analogous foreign commercial preparations, based on plasma or yeast HBsAg.     

Serial changes in titers of antibody to hepatitis B surface antigen after immunization of infants born to mothers with hepatitis B e antigen

The purpose of this study is to identify the existence of hepatovenous intrahepatic anastomosis in normal men. A total of thirteen livers were investigated during the early autopsies of normal men who died in accidents. Perfusion venography of branches of hepatic veins using meglucamine diatrizoate was done in six cases; this method we used had not been reported in the literature. In one case, portal venography was performed. And in the other six cases, liver substance staining was done by injecting the ink through the middle hepatic vein, and such staining of the liver was observed by light microscope. The results show, (1) there are intrahepatic anastomoses between the hepatic veins within the liver; (2) there are anastomoses between the middle hepatic vein and the accessory hepatic veins; and (3) shunts exist between portal veins and hepatic veins. The above findings provide an anatomical basis for the performance of irregular hepatectomy and the rationale for one or two hepatic veins ligation should such veins were traumatized or invaded by liver cancer.     

Proteins of hepatitis B surface antigen

Purified 22-nm forms of hepatitis B surface antigen (Hbsag) representing the three major antigenic subtypes (adw, ayw, and adr) were analyzed for their constituent polypeptides by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. No consistent difference in either the number or relative distributions of the polypeptides was observed for the various subtypes. Seven polypeptides were designated as P-1 through P-7 in order of their decreasing mobilities. By comparison with protein standards, their molecular weights were estimated as 23, 29.5, 36, 41.5, 53.5, 72, and 97 thousand. The P-1 and P-2 components represented the major polypeptides; P-2 and P-5 might by glycoproteins, based on their reaction with periodic acid-Shiff reagent. Each polypeptide contains cysteine residues. HBSAg was radiolabeled with 3H or 14C by reductive methylation or iodinated with 125I by the chloramine-T or lactoperoxidase procedures. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of labeled HBSAg yielded patterns identical to those obtained with protein stain. Comparison of HBSAg labeled by the chloramine-T and lactoperoxide procedures indicated that there was no distinction between internal or external components within the 22-nm structure.     

CpG DNA can induce strong Th1 humoral and cell-mediated immune responses against hepatitis B surface antigen in young mice

Successful neonatal immunization of humans has proven difficult. We have evaluated CpG-containing oligonucleotides as an adjuvant for immunization of young mice (1-14 days old) against hepatitis B virus surface antigen. The protein-alum-CpG formulation, like the DNA vaccine, produced seroconversion of the majority of mice immunized at 3 or 7 days of age, compared with 0-10% with the protein-alum or protein-CpG formulations. All animals, from neonates to adults, immunized with the protein-alum vaccine exhibited strong T helper (Th)2-like responses [predominantly IgG1, weak or absent cytotoxic T lymphocytes (CTL)]. Th2-type responses also were induced in young mice with protein-CpG (in 1-, 3-, and 7-day-old mice) and protein-alum-CpG (in 1- and 3-day-old mice) but immunization carried out at older ages gave mixed Th1/Th2 (Th0) responses. DNA vaccines gave Th0-like responses when administered at 1 and 7 days of age and Th1-like (predominantly IgG2a and CTL) responses with 14-day-old or adult mice. Surprisingly, the protein-alum-CpG formulation was better than the DNA vaccine for percentage of seroconversion, speed of appearance, and peak titer of the antibody response, as well as prevalence and strength of CTL. These findings may have important implications for immunization of human infants.     

孝感市2009年城区学生乙肝表面抗原检测结果分析

乙型肝炎是由乙肝病毒(HBV)引起的以肝脏炎性病变为主并可引起多器官损害的一种传染病.其传染性强,传播途径广,发病率高且目前无治愈乙肝的特效药(1).我国是乙型肝炎高度流行区,HBV感染已成为普遍关注的公共卫生问题(2).     

Immune responses to the hepatitis B surface antigen and liver-specific lipoprotein in acute type B hepatitis

A serial prospective study of cellular immunity to HBsAg and liver-specific membrane lipoprotein was undertaken in 21 adults with acute hepatitis type B. Cellular immunity to HBsAg as determined by leucocyte migration inhibition with partially purified HBsAg as antigen was detected in all the patients during the recovery phase of the illness and was already detectable at the time of admission in 13 (62%) of the cases. In five of the remaining eight the titre of HBsAg in the serum at this time was high and in the whole series there was an inverse correlation between the degree of migration inhibition on admission and the peak HBsAg titre suggesting that antigen or possibly antigen/antibody complexes might be interfering with the demonstration of cellular immunity in vitro. Using a combination of minimum migration index recorded during the recovery period peak HBsAg titre, it was possible to compute the peak aspartate aminotransferase level with reasonable accuracy, a finding consistent with the hypothesis that the severity of the illness is related to both the number of infected hepatocytes and the vigour of the immune response to HBsAg. Evidence of an immune response to the liver-specific hepatocyte membrane lipoprotein was present in 50% of the patients tested at the time of admission, but was transient, having disappeared in every case by four weeks. The minimum migration index recorded with HBsAg as antigen was significantly lower in those with detectable sensitisation to the lipoprotein and it is possible that this autoimmune reaction is also generated by the interaction of T cells with viral antigenic determinants on the liver cell surface.     

Electron microscopy study of hepatitis B antigen (Australia antigen) in serum

Experiments were performed on loosely restrained, conscious dogs in the femoral artery of which a chronic catheter was implanted. All substances were administered through the catheter and vocalization responses to them were used as a principal measure of nociception. In 10 experiments done on 7 dogs, after a single injection of 3 or 10 mumol of 5-hydroxytryptamine, threshold doses (about 0.5 mumol) of acetylcholine for vocalization produced vocalization responses comparable with those to about 3 times threshold doses. In 5 experiments on 5 dogs, after 10 mumol of 5-hydroxytryptamine, threshold doses (about 0.3 nmol) of bradykinin for vocalization produced vocalization responses comparable with those to about twice threshold doses. These doses of 5-hydroxytryptamine produced small vocalization responses in only 3 of the 12 dogs. These results indicate that 5-hydroxytryptamine, although almost devoid of the activity producing noceceptive responses, sensitizes somatic nociceptors for pain-producing substances.     

CpG DNA is a potent enhancer of systemic and mucosal immune responses against hepatitis B surface antigen with intranasal administration to mice

Mucosal immunity is difficult to induce with subunit vaccines unless such vaccines are administered with a mucosal adjuvant such as cholera toxin (CT); however, CT is toxic in humans. Synthetic oligodeoxynucleotides containing immunostimulatory CpG motifs (CpG) are potent adjuvants for the induction of Th1-like systemic immune responses against parenterally delivered proteins. Here, we show in mice that intranasal delivery of hepatitis B surface Ag, which alone has no effect, elicits good immune responses when given with CpG oligodeoxynucleotides and/or CT. Overall, CpG is superior to CT for the induction of humoral and cell-mediated systemic immunity as well as mucosal immune responses (IgA) at local (lung) and distant (feces) sites. Furthermore, CpG and CT act synergistically, giving stronger responses than those observed with 10 times more of either adjuvant alone. Ab isotypes were predominantly IgG1 (Th2-like) with CT, mixed IgG1/IgG2a (Th0) with CpG, and predominantly IgG2a (Th1-like) with CpG and CT together.     

Relation between laboratory test results and histological hepatitis activity in individuals positive for hepatitis B surface antigen and antibodies to hepatitis B e antigen

BACKGROUND: Hepatitis B surface antigen (HBsAg) and antibodies to hepatitis B e antigen (anti-HBe) commonly coexist, and laboratory tests are often requested to assess histological hepatitis activity. An optimum panel of tests has not been found and the usefulness of hepatitis B virus (HBV) DNA assays in this context has not been established. We assessed various blood tests to find which best predicted hepatitis activity. METHODS: Routine plasma biochemical liver tests and serum HBV DNA (hybridisation and PCR assays) were assessed prospectively in 123 patients positive for HBsAg and anti-HBe. We scored histological hepatitis activity (hepatitis activity index) and determined whether chronic active hepatitis (chronic hepatitis with portal and periportal lesions) was present. We analysed the relation between laboratory data and the hepatitis activity index or risk of chronic active hepatitis by multiple regression and multiple logistic regression, respectively. FINDINGS: The analyses provided models for predicting either the hepatitis activity index or the risk of chronic active hepatitis. Aspartate aminotransferase was the most important test in the two models. The contribution of HBV DNA and other assays, especially alanine-aminotransferase activity, were of no practical importance. INTERPRETATION: Because screening by aspartate-aminotransferase activity could not be improved by the addition of other assays or HBV DNA, patients positive for HBsAg and anti-HBe could be screened for chronic active hepatitis with a single assay and counselling of patients can be improved if proper reference values are used.     

Aggregation of recombinant hepatitis B surface antigen in Pichia pastoris

The combination of immunoaffinity and size-exclusion chromatography (SEC) is a powerful tool to analyze multiprotein particle assembly. This approach was used to investigate the source of aggregation of recombinant hepatitis B surface antigen (HBsAg) detected in purified material. As HBsAg aggregation does not originate in the stresses, such as the concentration of HBsAg solutions, temperature and chaotropic agents, it is less probable that the HBsAg aggregate is produced during the process. To test whether aggregation takes place in vivo, crude yeast extract containing the expressed HBsAg was fractioned on a Sephacryl S-400 column just after cell disruption, and each fraction immunopurified individually. As a result, the HBsAg aggregate was isolated from a fraction corresponding to the elution of large particle aggregates only, not native HBsAg particles. It was biologically active, which demonstrates aggregate formation by specific assembly of partially or wholly folded HBsAg intermediates.     

Examination of the polypeptides of hepatitis B surface antigen

When the polypeptides of hepatitis B surface antigen were examined by SDS-polyacrylamide gel electrophoresis under a variety of conditions, anomalous results were found to be due to (i) variable and at times incomplete dissociation of polypeptides after boiling with 1% SDS and reducing agent, (ii) reaggregation of solubilized material under certain electrophoretic conditions and during laboratory manipulations, and (iii) the variable presence of additional components in hepatitis B surface antigen prepared from certain individual donors. When these factors were taken into account, two major components were consistently identified by discontinuous buffer polyacrylamide gel electrophoresis, of apparent mol. wt. 60000 to 70000 and 12000 to 14000. However, in view of the demonstrated limitations of this technique in examining HBsAg polypeptides, alternative methods are necessary to confirm the true mol. wt. of the unique virus-specified amino acid sequence present.     

A humanized antibody with specificity for hepatitis B surface antigen

A murine monoclonal antibody H67 was characterized for the binding specificity, which showed that H67 recognizes a disulfide-bond-dependent conformational epitope of common a antigenic determinant on the hepatitis B surface antigen. The result suggested that this antibody may have the potential of replacing hepatitis B immune globulin in the prevention of hepatitis B virus (HBV) infection. Therefore, we have constructed the humanized antibody HuS10 by grafting the complementarity determining regions and some framework amino acid residues of H67 onto the most homologous human antibody variable regions, 21/28 for heavy chain variable region and B1 and J kappa 2 for light chain variable region, followed by combining with human constant regions C gamma 1 and C kappa. The affinity of the HuS10 was the same as that of the H67, 8 x 10(8) x 10(8)M-1, and the HuS10 neutralized the in vitro infection of adult human hepatocyte primary culture by adr or ayw subtype of HBV. The neutralization assay showed that the HuS10 had approximately 2,000-times higher specific activity than commercially available polyclonal HBIG. These results suggest that the humanized antibody will be useful in the prevention or treatment of HBV infection.     

CpG DNA is a potent enhancer of specific immunity in mice immunized with recombinant hepatitis B surface antigen

Unmethylated CpG dinucleotides in bacterial DNA or synthetic oligodeoxynucleotides (ODN) cause B cell proliferation and Ig secretion, monocyte cytokine secretion, and activation of NK cell lytic activity and IFN-gamma secretion in vivo and in vitro. The potent immune activation by CpG ODN suggests possible utility for enhancing immune responses to vaccines. Mice immunized with recombinant hepatitis B virus surface Ag and a CpG ODN as an immune enhancer have titers of Abs against HBsAg (anti-HBs) that are five times higher than those of mice immunized with HBsAg and the standard adjuvant, aluminum hydroxide (alum). Ab titers in mice immunized with HBsAg and both CpG ODN plus alum were 35 times higher than the titers in mice immunized with alum alone, indicating a strong synergistic interaction between the CpG ODN and alum. ODN without CpG motifs had little or no immune-enhancing activity at the doses used herein. Alum induces a Th2 humoral response (mostly IgG1) and no CTL. In contrast, CpG ODN gives a strong Thl response with predominantly IgG2a Abs and CTL, even when mixed with alum. In vitro studies to determine possible mechanisms of CpG immune-enhancing effects show that CpG ODN induce expression of costimulatory molecules on Ag-presenting cells and drive B cell isotype switching in the appropriate cytokine milieu. These studies demonstrate that CpG ODN are promising new immune enhancers for vaccination applications.