Hepatitis B virus core antigen: enhancement of its production in Escherichia coli,and interaction of the core particles with the viral surface antigen

The core antigen (HBcAg) of hepatitis B Virus (HBV) can be expressed in Escherichia coil where it assembles into icosahedral particles containing 240 or 180 subunits. Analysis of the two kinds of particles by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) showed that a substantial proportion of their subunits were smaller than the full-length HBcAg monomer and of variable size, but all had the same N-terminal sequence showing that the smaller species were heterogeneous in their arginine-rich C-terminal regions. Around 50% of these arginine residues are encoded by the triplet AGA which is rare in E. coli. Supplementation of the level of AGA tRNA in the cell by transformation with plasmids expressing the T4 AGA tRNA gene significantly enhanced the yield of HBcAg. Fusion phage carrying a ligand specific for HBcAg showed no significant difference in the affinity for the two sizes of HBcAg particles, but in similar reactions in solution HBV surface antigen exhibited differential affinities for the same two HBcAg preparations.     

Expression and characterization of hepatitis C virus core protein fused to hepatitis B virus core antigen

Recombinant plasmids were constructed by fusing the gene fragments encoding the full-length (1-191aa) and the truncated (1-40aa and 1-69aa) HCV core proteins (HCc) respectively to the core gene of HBV at the position of amino acid 144 and expressed in E. coli. The products were analyzed by ELISA, Western blotting as well as the immunization of the mice. The results showed that those fusion proteins (B144C191, B144C69, B144C40) possessed the dual antigenicity and immunogenicity of both hepatitis B virus core antigen (HBcAg) and hepatitis C virus core protein (HCc). Analysis by electron microscopy and CsCl density gradient ultra-centrifugation revealed that similar to the HBcAg itself, all fusion proteins were able to form particles. Comparison of the antigenicity and immunogenicity of those fusion proteins showed that the length of HCc gene fused to HBeAg had no much effect on the antigenicity and immunogenicity of HBcAg, however, B144C69 and B144C40 induced higher titres antibodies against HCc than B14d     

Expression and characterization of hepatitis C virus core protein fused to hepatitis B virus core antigen

Recombinant plasmids were constructed by fusing the gene fragments encoding the full-length (1-191aa) and the truncated (1-40aa and l-69aa) HCV core proteins (HCc) respectively to the core gene of HBV at the position of amino acid 144 and expressed inE. coli. The products were analyzed by ELISA, Western blotting as well as the immunization of the mice. The results showed that those fusion proteins (B144C191, B144C69, B144C40) possessed the dual antigenicity and immunogenicity of both hepatitis B virus core antigen (HBcAg) and hepatitis C virus core protein (HCc). Analysis by electron microscopy and CsCI density gradient ultra-centrifugation revealed that similar to the HBcAg itself, all fusion proteins were able to form particles. Comparison of the antigenicity and immunogenicity of those fusion proteins showed that the length of HCc gene fused to HBcAg had no much effect on the antigenicity and immunogenicity of HBcAg, however, B144C69 and B144C40 induced higher titres antibodies against HCc than B144C191. Using those fusion proteins, ELISA for screening of antibodies against both HBV and HCV in human sera was also established.     

乙肝病毒核心蛋白病毒样颗粒表面抗原密度对抗体应答水平的影响

【目的】为了探究乙肝病毒核心蛋白(HepatitisBviruscoreprotein,HBc)病毒样颗粒(Virus-like particles,VLPs)表面抗原密度对免疫后抗体应答水平的影响,制备了不同抗原密度的HBc VLPs疫苗,并检测了其在小鼠体内的抗体应答水平。【方法】首先制备了N端带有3个甘氨酸的人巨细胞病毒重组抗原域AD-4作为模式抗原,接着通过Sortase A的介导将AD-4连接到HBc VLPs表面上。将系列浓度梯度AD-4抗原在SortaseA介导下分别与相同浓度的HBcVLPs发生反应,制备不同抗原密度的HBc-AD-4 VLPs。将其分别免疫6–8周龄BALB/c小鼠3次,每次免疫间隔2 w,间接ELISA法检测被免疫小鼠血清的抗体应答水平。【结果】结果表明,当HBc VLPs表面抗原密度为44.4%时,即HBc反应浓度∶AD-4反应浓度为1:0.5时,不足以引起高滴度的抗体产生;当HBc VLPs表面抗原密度为64.2%时,即HBc反应浓度∶AD-4反应浓度为1:1时,HBc-AD-4 VLPs诱导的AD-4特异性抗体滴度与100%抗原密度的HBc-AD-4VLPs所引起的抗体滴度相当;当HBcVLPs表面抗原密度大于64.2%时,引起的抗体应答水平不因抗原密度增加而进一步增强。【结论】发现了HBcVLPs表面抗原密度与免疫后抗体滴度呈正相关,然而免疫64.2%抗原密度的HBc VLPs所产生的抗体滴度可达峰值,抗原密度进一步增加,抗体应答水平不会进一步加强。     

Antigenicity and immunogenicity of novel chimeric hepatitis B surface antigen particles with exposed hepatitis C virus epitopes

The small envelope protein of hepatitis B virus (HBsAg-S) can self-assemble into highly organized virus like particles (VLPs) and induce an effective immune response. In this study, a restriction enzyme site was engineered into the cDNA of HBsAg-S at a position corresponding to the exposed site within the hydrophilic a determinant region (amino acid [aa] 127-128) to create a novel HBsAg vaccine vector allowing surface orientation of the inserted sequence. We inserted sequences of various lengths from hypervariable region 1 (HVR1) of the hepatitis C virus (HCV) E2 protein containing immunodominant epitopes and demonstrated secretion of the recombinant HBsAg VLPs from transfected mammalian cells. A number of different recombinant proteins were synthesized, and HBsAg VLPs containing inserts up to 36 aa were secreted with an efficiency similar to that of wild-type HBsAg. The HVR1 region exposed on the particles retained an antigenic structure similar to that recognized immunologically during natural infection. VLPs containing epitopes from either HCV-1a or -1b strains were produced that induced strain-specific antibody responses in immunized mice. Injection of a combination of these VLPs induced antibodies against both HVR1 epitopes that resulted in higher titers than were achieved by vaccination with the individual VLPs, suggesting a synergistic effect. This may lead to the development of recombinant particles which are able to induce a broad anti-HCV immune response against the HCV quasispecies or other quasispecies-like infectious agents.     

Inhibition of hepatitis B virus assembly with synthetic peptides derived from the viral surface and core antigens

The long surface antigen (L-HBsAg) of hepatitis B virus (HBV) plays a central role in the production of infectious virions. During HBV morphogenesis, both the PreS and S domains of L-HBsAg form docking sites for the viral nucleocapsids. Thus, a compound that disrupts the interaction between the L-HBsAg and nucleocapsids could serve as a therapeutic agent against the virus based upon inhibition of morphogenesis. Synthetic peptides correspond to the binding sites in L-HBsAg inhibited the association of L-HBsAg with core antigen (HBcAg). A synthetic peptide carrying the epitope for a monoclonal antibody to the PreS1 domain competed weakly with L-HBsAg for HBcAg, but peptides corresponding to a linear sequence at the tip of the nucleocapsid spike did not, showing that the competing peptide does not resemble the tip of the spike.     

Differential formation of disulfide linkages in the core antigen of extracellular and intracellular hepatitis B virus core particles.

Our understanding of the assembly of hepatitis B virus is still very limited. We present evidence to demonstrate that the HBc antigen formed oligomers through disulfide linkages in the extracellular hepatitis B virus core (HBc) particles. However, the intracellular HBV core particles did not contain disulfide-linked HBc antigens. Furthermore, the extracellular particles which had disulfide bonds were more stable than intracellular particles at pH 7.5 and 10 and in 3 M NaCl and 4 M urea. These data suggest that the formation of disulfide bonds in the HBc antigen is important for the integrity of the viral core particles.     

Recombinant core particles of hepatitis B virus exposing foreign antigenic determinants on their surface

Insertion of foreign oligopeptide sequences (40-50 amino acids in length) into the Pro144 position of hepatitis B core antigen (HBcAg) leads to the formation of chimeric capsids in Escherichia coli cells. These capsids are morphologically and immunologically similar to native HBcAg, but expose the inserted oligopeptides on their outer surface and exhibit antigenic and immunogenic characteristics of the latter. As a source of model antigenic determinants, the appropriate DNA copies excised from cloned viral genes such as the pre-S region of hepatitis B virus, the transmembrane protein gp41 of human immunodeficiency virus 1 and the envelope protein gp51 of bovine leukemia virus have been used. The localization of the inserted antigenic determinants on the surface of chimeric capsids does not depend on the presence or absence of the arginine-rich, 39 amino acid-long C terminus of HBcAg.     

Major polypeptide of duck hepatitis B surface antigen particles

The 40- to 50-nm pleomorphic particles found in the sera of domestic Pekin ducks infected with duck hepatitis B virus were purified by rate zonal and isopycnic centrifugation. Sodium dodecyl sulfate-polyacrylamide gel electrophoretic polypeptide analysis of these particles, called duck hepatitis B surface antigen particles, revealed the major component to be a single 17,500-dalton polypeptide. This result is in contrast to polypeptide analyses of the surface antigens of related mammalian viruses, including hepatitis B, in which a major doublet of polypeptides is seen with molecular weights ranging from 23,000 to 29,000. Tryptic maps of 17,500-dalton polypeptide resembled that of the major non-glycosylated polypeptide of the adw subtype of hepatitis B surface antigen. A serological assay for antibody to the purified duck virus particles is also described.     

Phosphorylation-dependent binding of hepatitis B virus core particles to the nuclear pore complex

Although many viruses replicate in the nucleus, little is known about the processes involved in the nuclear import of viral genomes. We show here that in vitro generated core particles of human hepatitis B virus bind to nuclear pore complexes (NPCs) in digitonin-permeabilized mammalian cells. This only occurred if the cores contained phosphorylated core proteins. Binding was inhibited by wheat germ agglutinin, by antinuclear pore complex antibodies, and by peptides corresponding either to classical nuclear localization signals (NLS) or to COOH-terminal sequences of the core protein. Binding was dependent on the nuclear transport factors importins (karyopherins) alpha and beta. The results suggested that phosphorylation induces exposure of NLS in the COOH-terminal portion of the core protein that allows core binding to the NPCs by the importin- (karyopherin-) mediated pathway. Thus, phosphorylation of the core protein emerged as an important step in the viral replication cycle necessary for transport of the viral genome to the nucleus.     

Neutralization of hepatitis B virus surface antigen with immunoglobulin preparations

The in vitro experiments with immunoglobulin and blood plasma containing hepatitis B virus (HBV) markers, revealed that the control of immunoglobulin preparations for the presence of hepatitis B virus surface antigen (HBsAg), mandatory for Russia, was not sufficiently informative. The neutralization of HBsAg with specific antibodies to the level, not determined by the EIA method, reached not less than 24 ng/ ml in 2 hours of incubation and not less than 49 ng/ml in 24 hours of incubation, which, when evaluated in 1 lU of anti-HBs, was 34.6 +/- 0.9 ng and 70.7 +/- 1.8 ng of HBsAg respectively. The process of the formation of immune complexes depended mainly on the time of incubation of experimental samples and on the antibody--antigen proportion in the system. The neutralization of viruses by antibodies had no influence on the capacity of the polymerase chain reaction to detect HBV DNA.     

The relationship between core promoter mutation of hepatitis B virus, viral load and hepatitis B e antigen status in chronic hepatitis B patients

The aim of this study is to detect the possible association of hepatitis B virus (HBV) core mutation, hepatitis B e antigen (HBeAg) status and the viral load in chronic hepatitis B (CHB) patients. Sixty-six patients with CHB were enrolled. Hepatitis markers and hepatitis C virus antibody (HCV-Ab) were tested using micro particle enzyme immunoassay kits. Viral load was measured by real-time polymerase chain reaction (PCR) and the mutation was analyzed by nested PCR followed by restriction fragment length polymorphism. Most of CHB patients were HBeAg (-ve). The HBeAg status did not have an influence on the presence or absence of T1762/A1764 mutation. HBV-DNA serum level was not significantly different in patients with core mutation and patients without core mutation in HBeAg (-ve) group, while in HBeAg (+ve) group HBV-DNA serum level was significantly higher in patients with core mutation. This study reports the predominance of HBeAg (-ve) and HBV core promoter mutation.     

Circulating hepatitis B surface antigen particles carry hepatocellular microRNAs

Hepatitis B virus (HBV) produces high quantities of subviral surface antigen particles (HBsAg) which circulate in the blood outnumbering virions of about 1\10^3–6 times. In individuals coinfected with the defective hepatitis Delta virus (HDV) the small HDV-RNA-genome and Delta antigen circulate as ribonucleoprotein complexes within HBsAg subviral particles. We addressed the question whether subviral HBsAg particles may carry in the same way cellular microRNAs (miRNAs) which are released into the bloodstream within different subcellular forms such as exosomes and microvescicles. Circulating HBsAg particles were isolated from sera of 11 HBsAg carriers by selective immunoprecipitation with monoclonal anti-HBs-IgG, total RNA was extracted and human miRNAs were screened by TaqMan real-time quantitative PCR Arrays. Thirty-nine human miRNAs were found to be significantly associated with the immunoprecipitated HBsAg, as determined by both comparative DDCT analysis and non-parametric tests (Mann-Whitney, p<0.05) with respect to controls. Moreover immunoprecipitated HBsAg particles contained Ago2 protein that could be revealed in ELISA only after 0.5% NP40. HBsAg associated miRNAs were liver-specific (most frequent = miR-27a, miR-30b, miR-122, miR-126 and miR-145) as well as immune regulatory (most frequent = miR-106b and miR-223). Computationally predicted target genes of HBsAg-associated miRNAs highlighted molecular pathways dealing with host-pathogenThe finding that HBsAg particles carry selective pools of hepatocellular miRNAs opens new avenues of research to disentangle the complex interactions between host and HBV and provides a non invasive tool to study the physiopathology of liver epigenetics.     

Factors affecting the presentation of exogenous hepatitis B virus core antigen

Hepatitis B virus core antigen (HBcAg) plays a critical role in terminating acute Hepatitis B virus infection and may be used as a potential vaccine candidate. The cell surface major histocompatibility complex (MHC) class 1 molecules are thought to be involved in the presentation of HBcAg. Surface MHC class 1 HLA A2 heavy chain (HC) and trimeric molecules were characterized on transfected Hela cells used as antigen presenting cells (APC) for the presentation of HBcAg. The results show that antibodies against HC HLA A2 and trimeric HLA-A2 molecules resulted in increased activation of HBcAg 18-27 minimal peptide specific cytotoxic T lymphocytes (CTLs), while the addition of exogenous beta2-microglobulin decreased the activation of HBcAg specific CTLs. Further, specific CD8+ T cells were activated only when Hela cells as APCs were primed with HBcAg (peptide, soluble or embedded on virosomes) at pH 6.5.     

Lipid composition of hepatitis B virus surface antigen particles and the particle-producing human hepatoma cell lines

More than 90% of lipids of hepatitis B virus surface antigen (HBsAg) particles produced by two human hepatoma cell lines (huGK-14 and PLC/PRF/5) were composed of phospholipids, with phosphatidylcholine being the dominant component, accounting for more than 80% of total membrane lipids. Analysis of subclass compositions of phospholipids of HBsAg particles and the host cell lines revealed that 1,2-diacyl glycerophosphocholine was preferentially incorporated into the membrane of the HBsAg particles, although both host cell lines contained extremely high concentrations (more than 60% of total phospholipids) of ether-linked phospholipids. Phospholipids of other hepatoma cell lines (HuH-7, Hep-G2, and huL-1) which were not associated with hepatitis B virus (HBV) infection, were composed mostly of 1,2-diacylglycerophospholipids. Activities of dihydroxyacetone-phosphate acyltransferase, which is known to be an obligatory enzyme in ether lipid biosynthesis, were found to be elevated by three- to fourfold in both huGK-14 and PLC/PRF/5 cells compared to those of other hepatoma cell lines. The results suggest a possible relationship between HBV-induced hepatocellular carcinogenesis and the drastic change in the metabolism of membrane phospholipids.     

Overcoming T cell tolerance to the hepatitis B virus surface antigen in hepatitis B virus-transgenic mice

The sequence of the hepatitis B virus (HBV) major envelope (Env) protein (ayw subtype) was scanned for the presence of H-2(d,b) motifs. Following binding and immunogenicity testing, two new H-2(d)-restricted epitopes (Env.362 and Env.364) were identified. These epitopes induced CTLs capable of recognizing naturally processed HBV-Env, but were apparently generated with lower efficiency than the previously defined dominant Env.28 epitope. Next, HBV-transgenic mice that express all of the HBV proteins and produce fully infectious particles were immunized with a mixture of lipopeptides encompassing the Env.28, Env.362, and Env.364 epitopes. Significant CTL responses were obtained, but they had no effect on viral replication in the liver, nor did they induce an inflammatory liver disease. However, in adoptive transfer experiments, CTL lines generated from the HBV-transgenic mice following immunization were able to inhibit viral replication in vivo without causing hepatitis. This is in contrast to CTL lines derived from nontransgenic mice that displayed both antiviral and cytopathic effects, presumably because they displayed higher avidity for the viral epitopes than the transgenic CTLs. These results suggest that T cell tolerance to HBV can be broken with appropriate immunization but the magnitude and characteristics of the resultant T cell response are significantly different from the response in HBV-naive individuals since their antiviral potential is stronger than their cytotoxic potential. This has obvious implications for immunotherapy of chronic HBV infection.