乙肝病毒核心抗原作为载体用于病毒样颗粒疫苗研究的主要进展

乙肝病毒核心抗原(Hepatitis B virus core antigen,HBcAg)是乙肝病毒的核壳结构蛋白,由183～185个氨基酸组成,大小约21～23kD。HBcAg由于其能自我组装成病毒样颗粒(Virus-like particle,VLP)、高表达、易纯化以及强免疫原性等特点,使其成为一个高效安全且应用广泛的VLP载体,可用于各种病原的疫苗研发。发展至今已有数十种病毒、细菌以及寄生虫的相关基因的抗原表位成功表达在HBcAg VLP颗粒上,成为新型疫苗研发的重要平台。     

Hepatitis B virus screening in contacts of blood donors with antibodies against core protein (anti-HBC), but without surface antigen (HBsAg)

To increase blood safety Brazil introduced screening for anti-HBc among blood donors in 1993. There was a decrease in the hepatitis B virus (HB V) transmission, but this measure identified a great number ofHBsAg-negative, anti-HBc-positive donors. Surveillance policy determines that contacts of HBV carriers should be screened to HBV markers, but there is no recommendation about how to guide contacts of HBsAg-negative, anti-HBc-positive donors. Aiming to evaluate whether the contacts of this group are at greater risk for HB V infection, a cross-sectional study was performed to compare prevalence of HBV infection between contacts of HBsAg-positive blood donors (group I) and contacts of HBsAg-negative, anti-HBc-positive donors (group II). Contacts were submitted to a questionnaire and blood tests for HBV markers. In group I (n = 143), 53 (37.1%) were anti-HBc-positive and 11 (7.7%) were HBsAg-positive. In group II (n = 111), there were 9 and 0.9%, respectively. HB V exposure was associated with group I, sexual activity, blood transfusion, being one of the donor's parents, and living for more than ten years with the donor. Regarding the families as sample units, it was more common to find at least one member with HBV markers (p < 0.05) among the families of group I compared to group II. Contacts of HBsAg-negative, anti-HBc-positive individuals presented a much lower risk of having already been exposed to HBV and there is no need to screen them for HBV in low to moderate prevalence populations.     

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.     

Suitability of yeast- and Escherichia coli-expressed hepatitis B virus core antigen derivatives for detection of anti-HBc antibodies in human sera

Antibody to hepatitis B virus core antigen (anti-HBc) is one of the most important serological markers during hepatitis B virus (HBV) infection. The quality of the hepatitis B virus core antigen (HBcAg; diagnostic antigen) is crucial to the accuracy of anti-HBc detection. In an attempt to explore the suitability of recombinant HBcAg (rHBcAg) for diagnostic purposes, HBcAg was expressed in Escherichia coli (E. coli) and Pichia pastoris (P. pastoris) and evaluated for the detection of anti-HBc. The expression level of the recombinant protein satisfied the criteria for large-scale biologic production. P. pastoris- and E. coli-derived rHBcAg were purified with gel filtration followed by sucrose gradient (reagents A and C) or with a monoclonal anti-HBc antibody binding (reagents B and D) and were utilized to detect anti-HBc in competitive inhibition enzyme-linked immunosorbent assay (ELISA) format. The ELISA using P. pastoris-derived rHBcAg had a higher specificity and sensitivity than that using E.coli-derived rHBcAg to detect the anti-HBc standard panel. Serum specimens were collected from HBV-infected patients and healthy individuals (voluntary blood donors). Anti-HBc was detected in those specimens using P. pastoris- and E. coli-derived rHBcAg. The positive rate of anti-HBc detection in HBV-infected patients' sera was 100% with reagents A and B, 96.4% with reagent C, and 93.6% with reagent D. The negative rate in healthy control sera was 100% with reagents A and B, 97.0% with reagent C, and 99.7% with reagent D. These data indicate that P. pastoris-derived rHBcAg is superior to E.coli-derived rHBcAg for the detection of anti-HBc using the diagnostic ELISA.     

Hepatitis B virus core antigen has two nuclear localization sequences in the arginine-rich carboxyl terminus.

Expression of the hepatitis B virus core antigen (HBcAg) in mouse NIH 3T3 fibroblasts has been shown previously (A. McLachlan et al., J. Virol. 61:683-692, 1987) to result in the nuclear localization of this polypeptide. Since the carboxyl terminus of HBcAg contains four clusters of arginine residues which resemble nuclear localization sequences identified in other nuclear proteins, a series of carboxyl-terminus-truncated HBcAg polypeptides were expressed in mouse fibroblasts to examine the role of these sequences in the cellular localization of HBcAg. By immunofluorescence and cell fractionation analysis, it was demonstrated that regions of the HBcAg polypeptide including the most carboxyl-terminal (cluster 1) and amino-terminal (cluster 4) clusters of arginine residues represent distinct and independent nuclear localization sequences for this polypeptide. Substitution of a threonine residue for the second arginine residue in cluster 4 inactivates the nuclear localization signal in this region of the HBcAg polypeptide, demonstrating the importance of this residue to this signal sequence. However, HBcAg fails to accumulate in the nucleus only when both nuclear localization signal sequences are simultaneously deleted or disrupted by mutation. The possible significance of the nuclear localization sequences identified in the HBcAg polypeptide is discussed in the context of the role of the nucleocapsid in the hepatitis B virus life cycle.     

Hepatitis B core antigen. Detection of antibody by radioimmunoprecipitation.

Radioactive cores were prepared from concentrated Dane particles by DNA polymerase reaction followed by CsCl density gradient centrifugation. Two radioactive peaks were obtained: one peak with an average density of 1.36 g/cm-3 in CsCl contained cores that possessed full serologic reactivity; the other peak, with a density of 1.28 to 1.32 g/cm-3, contained cores associated with globulin. A double antibody immunoprecipitation test was developed, using the radioactive heavy cores as a source of antigen. The test was at least 300 times as sensitive as complement fixation for detecting antibody to core.     

Hepatitis B virus core protein interacts with the C-terminal region of actin-binding protein

Hepatitis B viral core protein is present in the nucleus and cytoplasm of infected hepatocytes. There is a strong correlation between the intrahepatic distribution of core protein and the viral replication state and disease activity in patients with chronic hepatitis. To understand the role of core protein in the pathogenesis of HBV, we used a yeast two-hybrid system to search for cellular proteins interacting with the carboxyl terminus of core protein, as this region is involved in a number of important functions in the viral replication cycle including RNA packaging and DNA synthesis. A cDNA encoding the extreme C-terminal region of human actin-binding protein, ABP-276/278, was identified. This interaction was further confirmed both in vitro and in vivo. In addition, the extreme C-terminal region of ABP-276/278 interacted with the nearly full-length HBV core protein. Since this region is present in both the core and the precore proteins, it is likely that both core and precore proteins of HBV can interact with the C-terminal region of ABP-276/278. The minimal region of ABP-276/278 which interacted with the HBV core protein was the C-terminal 199 amino acid residues which correspond to part of the 23rd repeat, the entire 24th repeat and the intervening hinge II region in ABPs. The potential functional outcome of ABP interaction in HBV replication and its contribution to the pathological changes seen in patients with chronic HBV infection are discussed.     

Hepatitis B virus core gene mutations which block nucleocapsid envelopment

Recently we generated a panel of hepatitis B virus core gene mutants carrying single insertions or deletions which allowed efficient expression of the core protein in bacteria and self-assembly of capsids. Eleven of these mutations were introduced into a eukaryotic core gene expression vector and characterized by trans complementation of a core-negative HBV genome in cotransfected human hepatoma HuH7 cells. Surprisingly, four mutants (two insertions [EFGA downstream of A11 and LDTASALYR downstream of R39] and two deletions [Y38-R39-E40 and L42]) produced no detectable capsids. The other seven mutants supported capsid formation and pregenome packaging/viral minus- and plus-strand-DNA synthesis but to different levels. Four of these seven mutants (two insertions [GA downstream of A11 and EHCSP downstream of P50] and two deletions [S44 and A80]) allowed virion morphogenesis and secretion. The mutant carrying a deletion of A80 at the tip of the spike protruding from the capsid was hepatitis B virus core antigen negative but wild type with respect to virion formation, indicating that this site might not be crucial for capsid-surface protein interactions during morphogenesis. The other three nucleocapsid-forming mutants (one insertion [LS downstream of S141] and two deletions [T12 and P134]) were strongly blocked in virion formation. The corresponding sites are located in the part of the protein forming the body of the capsid and not in the spike. These mutations may alter sites on the particle which contact surface proteins during envelopment, or they may block the appearance of a signal for the transport or the maturation of the capsid which is linked to viral DNA synthesis and required for envelopment.     

Antigenic determinants and functional domains in core antigen and e antigen from hepatitis B virus.

The precore/core gene of hepatitis B virus directs the synthesis of two polypeptides, the 21-kilodalton subunit (p21c) forming the viral nucleocapsid (serologically defined as core antigen [HBcAg]) and a secreted processed protein (p17e, serologically defined as HBe antigen [HBeAg]). Although most of their primary amino acid sequences are identical, HBcAg and HBeAg display different antigenic properties that are widely used in hepatitis B virus diagnosis. To locate and to characterize the corresponding determinants, segments of the core gene were expressed in Escherichia coli and probed with a panel of polyclonal or monoclonal antibodies in radioimmunoassays or enzyme-linked immunosorbent assays, Western blots, and competition assays. Three distinct major determinants were characterized. The single conformational determinant responsible for HBc antigenicity in the assembled core (HBc) and a linear HBe-related determinant (HBe1) were both mapped to an overlapping hydrophilic sequence around amino acid 80; a second HBe determinant (HBe2) was assigned to a location in the vicinity of amino acid 138 but found to require for its antigenicity the intramolecular participation of the extended sequence between amino acids 10 and 140. It is postulated that HBcAg and HBeAg share common basic three-dimensional structure exposing the common linear determinant HBe1 but that they differ in the presentation of two conformational determinants that are either introduced (HBc) or masked (HBe2) in the assembled core. The simultaneous presentation of HBe1 and HBc, two distinctly different antigenic determinants with overlapping amino acid sequences, is interpreted to indicate the presence of slightly differently folded, stable conformational states of p21c in the hepatitis B virus nucleocapsid.     

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     

乙型肝炎病毒核心蛋白作为表位疫苗载体的应用

乙型肝炎病毒核心蛋白(Hepatitis B viruscore protein,HBc)可以形成二十面体对称的颗粒样结构,由于其N端、C端和主要免疫显性区域(Major immunodominant region,MIR)允许一定程度的缺失和外源插入,并且能够将外源序列重复且高密度地暴露在颗粒的表面,诱发强烈的外源序列特异的体液和细胞免疫反应,从上世纪80年代中期就开始被运用于表位疫苗的研究。以下主要从影响HBc作为表位疫苗载体的因素,包括HBc长度、外源插入位点和表位序列的性质等来介绍HBc作为表位疫苗载体的应用。     

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.     

The core antigen of hepatitis B virus as a carrier for immunogenic peptides

The core antigen of hepatitis B virus (HBcAg) made in Escherichia coli yields particles that closely resemble the viral nucleocapsid. Extensive modifications can be made to the primary structure of HBcAg without impairing particle assembly. This enables other peptide sequences, including very long sequences, to be added, substituted, or inserted into the nucleocapsid subunit while retaining the ability to form highly immunogenic particles. These also retain the T cell epitopes of HBcAg and constitute powerful delivery systems for a diverse range of immunogenic epitopes and have significant potential for development of multicomponent vaccines.     

Hepatitis B virus surface antigen binds to apolipoprotein H.

We have previously demonstrated that a plasma membrane-enriched fraction isolated from human liver is capable of binding recombinant hepatitis B surface antigen (rHBsAg) (P. Pontisso, M. A. Petit, M. Bankowski, and M. E. Peeples, J. Virol. 63:1981-1988, 1989). In this study we have separated the plasma membrane proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and used a ligand-blotting technique to identify a 46-kDa rHBsAg-binding protein. This protein could be removed from the membranes with a weakly acidic buffer, implying that it is peripherally bound. Examination of human serum revealed that the 46-kDa binding protein is a serum protein. Isolation of plasma lipoproteins revealed that the binding protein is in part associated with chylomicrons and high-density lipoproteins, both of which are targeted to the hepatocyte during the normal course of lipid metabolism. The binding protein was identified as apolipoprotein H (apo H), also known as beta 2-glycoprotein I, on the basis of copurification of the rHBsAg-binding activity with the apo H protein and the ability of cDNA-expressed apo H to bind rHBsAg. Serum-derived HBsAg also binds to apo H, indicating that binding is not unique to rHBsAg. Binding is saturable, requires only the small S protein of rHBsAg, and is inhibited by excess rHBsAg, antibodies to HBsAg, and antibodies to apo H. The binding activity of apo H is destroyed upon reduction, indicating that 1 or more of its 22 disulfide bonds are required for interaction with rHBsAg. The possibility that an interaction between hepatitis B virus particles and lipoprotein particles may facilitate entry of the virus into hepatocytes is discussed.     

戊型肝炎病毒与HBV重叠感染患者的血清学分析

目的了解戊型肝炎病毒(HEV)感染情况以及乙型肝炎病毒(HBV)重叠感染戊型肝炎病毒的血清学特点及临床意义。方法对HEV、HBV感染以及HBV重叠HEV感染进行血清学检测并进行统计学分析。结果 HBV重叠感染HEV病例多分布青壮年,且女性多于男性;重叠感染患者的年龄高峰都在21~40岁;HEV感染组与HBV-HEV重叠感染组比较,ALP、TB IL存在明显差异,胆汁淤积增多,黄疸程度加深,肝细胞损伤明显。结论 HEV主要侵犯青壮年,HBV重叠HEV感染后肝细胞损害加重,病情趋向重症化。