Protective Efficacy of DNA Vaccines against Duck Hepatitis B Virus Infection

The efficacy of DNA vaccines encoding the duck hepatitis B virus (DHBV) pre-S/S and S proteins were tested in Pekin ducks. Plasmid pcDNA I/Amp DNA containing the DHBV pre-S/S or S genes was injected intramuscularly three times, at 3-week intervals. All pre-S/S and S-vaccinated ducks developed total anti-DHBs and specific anti-S antibodies with similar titers reaching 1/10,000 to 1/50,000 and 1/2,500 to 1/4,000, respectively, after the third vaccination. However, following virus challenge, significant differences in the rate of virus removal from the bloodstream and the presence of virus replication in the liver were found between the groups. In three of four S-vaccinated ducks, 90% of the inoculum was removed between <5 and 15 min postchallenge (p.c.) and no virus replication was detected in the liver at 4 days p.c. In contrast, in all four pre-S/S-vaccinated ducks, 90% of the inoculum was removed between 60 and 90 min p.c. and DHBsAg was detected in 10 to 40% of hepatocytes. Anti-S serum abolished virus infectivity when preincubated with DHBV before inoculation into 1-day-old ducklings and primary duck hepatocyte cultures, while anti-pre-S/S serum showed very limited capacity to neutralize virus infectivity in these two systems. Thus, although both DNA vaccines induced high titers of anti-DHBs antibodies, anti-S antibodies induced by the S-DNA construct were highly effective in neutralizing virus infectivity while similar levels of anti-S induced by the pre-S/S-DNA construct conferred only very limited protection. This phenomenon requires further clarification, particularly in light of the development of newer HBV vaccines containing pre-S proteins and a possible discrepancy between anti-HBs titers and protective efficacy.     

鸭乙型肝炎病毒基因组结构及其编码蛋白的研究进展

鸭乙型肝炎病毒(DHBV)属于禽嗜肝病毒属、嗜肝DNA病毒科病毒,与人乙型肝炎病毒(HBV)具有相同的复制方式及相似的基因结构、抗原结构。DHBV基因组是闭合环状不完全双链DNA,3个主要ORF-PreS/S、ORF-PreC/C、ORF-P位于负链,编码L蛋白、S蛋白、C蛋白和P蛋白。本文通过对DHBV基因组结构特征及其编码病毒重要蛋白的基本特性、结构特点与功能进行全面阐释,旨在对DHBV的深入研究提供重要参考和以DHBV人工感染建立的动物模型研究HBV提供重要理论依据。     

安徽庐江鸭乙型肝炎病毒LJ-76转染细胞系的建立

为建立鸭乙型肝炎病毒ＬＪ－７６的转染细胞系,将ＬＪ－７６病毒ＤＮＡ插入到ｐＵＣ１９的ＥｃｏＲⅠ位点上,分离得到含有双拷贝ＬＪ－７６ＤＮＡ的重组质粒．通过磷酸钙沉淀方法,将经ＣｓＣｌ等密度离心纯化的ＬＪ－７６ＤＮＡ双体导入到人肝癌细胞ＢＥＬ７４０２中．收集转染细胞的培养液进行蔗糖密度梯度离心,所得沉淀经检测发现含有ＬＪ－７６ＤＮＡ并具有特异性ＤＨＢＶ内源性ＤＮＡ多聚酶活性;对上述样品通过ＤｏｔＥＩＡ检测ＤＨＢＶ核心抗原及表面抗原结果为阳性．Ｓｏｕｔｈｅｒｎｂｌｏｔ分析表明转染细胞内存在病毒ＤＮＡ复制中间体ｃｃｃＤＮＡ、ｓｓＤＮＡ和ｒｃＤＮＡ,而ｃｃｃＤＮＡ被认为是复制活动较为活跃的标志．电镜观察转染细胞的上清发现有病毒颗粒的存在．     

Tumor Necrosis Factor-Alpha Induced by Hepatitis B Virus Core Mediating the Immune Response for Hepatitis B Viral Clearance in Mice Model

Persistent hepatitis B viral (HBV) infection results in chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma (HCC). An efficient control of virus infections requires the coordinated actions of both innate and adaptive immune responses. In order to define the role of innate immunity effectors against HBV, viral clearance was studied in a panel of immunodeficient mouse strains by the hydrodynamic injection approach. Our results demonstrate that HBV viral clearance is not changed in IFN-α/β receptor (IFNAR), RIG-I, MDA5, MYD88, NLRP3, ASC, and IL-1R knock-out mice, indicating that these innate immunity effectors are not required for HBV clearance. In contrast, HBV persists in the absence of tumor necrosis factor-alpha (TNF-α) or in mice treated with the soluble TNF receptor blocker, Etanercept. In these mice, there was an increase in PD-1-expressing CD8⁺ T-cells and an increase of serum HBV DNA, HBV core, and surface antigen expression as well as viral replication within the liver. Furthermore, the induction of TNF-α in clearing HBV is dependent on the HBV core, and TNF blockage eliminated HBV core-induced viral clearance effects. Finally, the intra-hepatic leukocytes (IHLs), but not the hepatocytes, are the cell source responsible for TNF-α production induced by HBcAg. These results provide evidences for TNF-α mediated innate immune mechanisms in HBV clearance and explain the mechanism of HBV reactivation during therapy with TNF blockage agents.     

Inhibition of Duck Hepatitis B Virus Infection by a Myristoylated Pre-S Peptide of the Large Viral Surface Protein

We have used the duck hepatitis B virus (DHBV) model to study the interference with infection by a myristoylated peptide representing an N-terminal pre-S subdomain of the large viral envelope protein. Although lacking the essential part of the carboxypeptidase D (formerly called gp180) receptor binding site, the peptide binds hepatocytes and subsequently blocks DHBV infection. Since its activity requires an amino acid sequence involved in host discrimination between DHBV and the related heron HBV (T. Ishikawa and D. Ganem, Proc. Natl. Acad. Sci. USA 92:6259-6263, 1995), we suggest that it is related to the postulated host-discriminating cofactor of infection.     

An Engineered Non-Toxic Superantigen Increases Cross Presentation of Hepatitis B Virus Nucleocapsids by Human Dendritic Cells

Virus like particles (VLPs) are potent immunogens capable of priming strong protective antibody responses due to their repetitive structural arrangement and affinity for specific B cell receptors. By contrast, T cell responses to VLPs can be weak due to inefficient uptake and processing by antigen presenting cells. We report here a novel strategy for increasing the T cell reactivity of a VLP, the nucleocapsid of hepatitis B virus, through covalent coupling of M1, an engineered form of the Streptococcal superantigen SMEZ2, that binds MHC II with high affinity but lacks its T cell mitogenic capability. M1:HBcAg conjugates bound to dendritic cells and were efficiently endocytosed into late endosomes. Human dendritic cells pulsed with M1:HBcAgs stimulated HBV-specific CD8⁺ T cells more effectively than cells pulsed with native capsids indicating that the modified VLP was more effectively cross presented by APCs. Coupling of M1 was also able to induce significantly greater reactivity of human CD4⁺ T cells specific for a common T-helper epitope. These studies indicate the potential of recombinant superantigens to act as flexible molecular adjuvants that can be incorporated into various subunit vaccine platforms leading to enhanced T cell reactivity in humans.     

乙型肝炎病毒S基因与截短C基因融合的穿梭表达载体的构建

研究目的是构建HBVS基因和截短C基因融合的胞壁型和分泌型大肠杆菌和分枝杆菌(E．coli-BCG)穿梭质粒。采用PCR方法从结核分枝杆菌MTB毒株H37Rv的基因中扩增出相对分子质量为19000的抗原胞壁区及其上游调控元件基因,克隆入穿梭载体pOLYG中。以含HBV基因组的质粒pCP10序列为模板,PCR扩增获得5基因片段Sw和C基因编码氨基端的部分基因片段Ct,克隆入胞壁型穿梭表达载体pCW和分泌型穿梭表达载体pDE22。经酶切和序列测定证实,胞壁型和分泌型载体pCW-Sw-Ct和pDE22-Sw-Ct构建成功。为进一步研究含S基因和截短C基因融合的重组BCG活疫苗奠定了基础。     

克隆鸭乙型肝炎病毒DNA双体体内转染的研究

用一种含头尾相连DHBVDNA双体的质粒体内转染2日龄芙蓉鸭,大多数鸭（86％）产生了短暂病毒血症。血清DHBs／preSAg和DHBVDNA于转染后第9天出现,第12～14天达峰值,第28天时多数转阴;少数鸭的病毒血症可持续50天以上。转染鸭肝组织中也检测到复制中间型DHBVDNA的存在。用转染鸭病毒血症期的血清作磷钨酸负染电镜观察,找到了完整的DHBV病毒颗粒,并且用此血清腹腔注射1日龄鸭,60％的鸭被感染成功,证明体内转染后有生物活性的DHBV病毒颗粒的产生。该研究方法的建立．对于研究DHBV变异株．DHBV基因结构与功能的关系等,均有一定理论意义及应用价值。     

DNA Polymerase in the Core of the Human Hepatitis B Virus Candidate

Experiments were done to show that the human hepatitis B antigen (HBAg)-associated DNA polymerase is a component of Dane particles and their antigenically distinct cores prepared by Nonidet P-40 detergent treatment of Dane particles. Before detergent treatment, the DNA polymerase was precipitated by serum containing anti-HB surface antigen (anti-HB(s)) but not with serum containing anti-HB core antigen (anti-HB(c)). After detergent treatment, the enzyme was precipitated by anti-HB(c)- and not by anti-HB(s)-containing serum. Highly purified 16- to 25-nm HBAg particles blocked only the precipitation of DNA polymerase in untreated HBAg preparations. The 110S structure with which the DNA reaction product remains associated in Nonidet P-40-treated preparations was identified as Dane particle core by immunoprecipitation with serum containing anti-HB(c). The DNA polymerase and the radioactive DNA reaction product were used as markers for core in immunoprecipitation tests for anticore. In such assays, 8 of 11 human sera with anti-HB(s) activity and all of 10 sera from chronic HBAg carriers were found to contain anti-HB(c) activity.     

三链DNA的形成抑制DNA结合蛋白与启动子的结合

电泳迁移分析方法及DNaseⅠ足迹实验表明21nt脱氧寡核苷酸G3TG2T GT2G5TG2TGT(CP1)与乙肝病毒(HBV)核心启动子(Cp)片段之间三链DNA的形成有较高的特异性及稳定性.凝胶滞留实验显示, 在大鼠肝细胞核提取物体外转录系统中, CP1可特异地抑制DNA结合蛋白与Cp片段的结合, 而不能与Cp结合形成三链DNA的脱氧寡核苷酸CP3(TGTG2TG5T2GTG2TG3)对蛋白与Cp的结合并无抑制作用.这些结果表明, 三链DNA的形成有可能抑制HBV DNA的转录.     

Duck Hepatitis B Virus cccDNA Amplification Efficiency in Natural Infection Is Regulated by Virus Secretion Efficiency

Previous mutation based studies showed that ablating synthesis of viral envelope proteins led to elevated hepadnaviral covalently closed circular DNA (cccDNA) amplification, but it remains unknown how cccDNA amplification is regulated in natural hepadnaviral infection because of a lack of research system. In this study we report a simple procedure to prepare two identical duck hepatitis B virus inocula, but they possess 10-100-fold difference in cccDNA amplification in infected cell culture. We demonstrate that the infected cells with higher cccDNA amplification significantly reduce the virus secretion efficiency that results in higher accumulation of relaxed circular DNA (rcDNA) and DHBsAg in the cells. The infected cells with lower cccDNA amplification significantly increase the virus secretion efficiency that leads to lower intracellular rcDNA and DHBsAg accumulation. In contrast with the findings generated in the mutation based experimental system, the regulation of cccDNA amplification in natural hepadnaviral infection bypasses direct regulation of the cellular envelope proteins concentration, instead it modulates virus secretion efficiency that ultimately impacts the intracellular rcDNA concentration, an important factor determining the destination of the synthesized rcDNA in infected cells.     

Hepatitis C Virus Core and Envelope Proteins Do Not Suppress the Host's Ability To Clear a Hepatic Viral Infection

Several hepatitis C virus (HCV) proteins have been shown in vitro to interact with host cellular components that are involved in immune regulation. However, there is a paucity of data supporting the relevance of these observations to the in vivo situation. To test the hypothesis that such an interaction suppresses immune responses, we studied a line of transgenic C57BL/6 mice that express the HCV core and envelope proteins in the liver. The potential effects of these proteins on the hepatic immune response were evaluated by challenging these mice with a hepatotropic adenovirus. Both transgenic and nontransgenic mice developed similar courses of infection and cleared the virus from the liver by 28 days postinfection. Both groups of mice mounted similar immunoglobulin G (IgG), IgG2a, interleukin-2, and tumor necrosis factor alpha responses against the virus. Additionally, BALB/c mice were able to clear infection with recombinant adenovirus that does or does not express the HCV core and envelope 1 proteins in the same manner. These data suggest that HCV core and envelope proteins do not inhibit the hepatic antiviral mechanisms in these murine experimental systems and thus favor a model in which HCV circumvents host responses through a mechanism that does not involve general suppression of intrahepatic immune responses.     

Core Particles of Hepatitis B Virus and Ground Squirrel Hepatitis Virus I. Relationship Between Hepatitis B Core Antigen- and Ground Squirrel Hepatitis Core Antigen-Associated Polypeptides by Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis and Tryptic Peptide Mapping

The relationships among the core antigen polypeptides of hepatitis B virus (HBV) and ground squirrel hepatitis virus (GSHV) were studied using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and tryptic peptide mapping. The major core antigen polypeptides of liver-derived HBV (p22) and GSHV (p20.5) shared 56% of the spots in their peptide maps. Comparison of hepatitis B core antigen (HBcAg) p19 or ground squirrel hepatitis core antigen (GSHcAg) p16.5 with their respective major polypeptides indicated that these components probably resulted from cleavage of the major polypeptide of each virus. Other polypeptides smaller than the major component of each virus were often faint on polyacrylamide gels and probably resulted from the cleavage or degradation of components larger than p22 of HBcAg or p20.5 of GSHcAg, since their peptide maps contained spots unique to these high-molecular-weight components. p26 of GSHcAg and p27.5 of HBcAg shared approximately two-thirds of the spots on their peptide maps with those of their respective major core polypeptides. Furthermore, p37.5 of GSHcAg and p40 of HBcAg shared about 60% homology with their respective major polypeptides, and also shared many of the spots that were unique to p26 of GSHcAg or p27.5 of HBcAg but were not found in the peptide map of their respective core antigen polypeptides. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis bands larger than 40,000 daltons were variably present, and peptide mapping indicated that these were aggregates of various smaller core antigen-associated polypeptides. The results suggest that p40 of HBcAg and p37.5 of GSHcAg are the largest unique polypeptides in these core particles, and that they are encoded for by the genome of each virus. That a subset of the spots unique to p40 or p37.5 was also found in p27.5 of HBcAg or p26 of GSHcAg, respectively, as compared to the major core polypeptides, also suggests that p27.5 and p26 are unique proteins encoded by the genome of each virus. It is proposed that the core antigen gene of each virus is larger than that which would encode the major polypeptide of each virus, and that the genetic organizations of the core genes of HBV and GSHV are very similar.     

突变型乙肝病毒核心抗原DNA疫苗的体外表达

目的:构建突变型核心抗原核酸疫苗,观察该核酸疫苗在体外蛋白的表达.方法:采用基因工程定点突变技术,构建5种突变型核酸疫苗,分别去除乙肝病毒核心抗原N端的第1、2位氨基酸,命名为M12,去除3、4位氨基酸命名为M34以及去除5、6位的氨基酸命名为M56,用上述构建的核酸疫苗与野生型HBc核酸疫苗(pJW4303/Hc)及空载体质粒pJW4303分别用脂质体转染293T细胞,应用蛋白印迹法检测核心蛋白的表达.结果:经过pstl和BgI双酶切和测序鉴定结果突变型核心抗原核酸疫苗构建成功.在去除2个氨基酸的核酸疫苗结果中显示:野生型pJW4303/HBe、M12、及M56体外转染293T细胞后,在细胞上清和裂解中能很好的表达,而M34上清未见表达,仅裂解中可见极少量疑似表达条带;在原有基础上分别去除第3位和第4住氨基酸,命名为M3和M4,结果显示M3上清未见表达,裂解液中可见少量表达,而M4在上清和裂解中均可见明显的表达.结论:去除核心抗原N端第3位的氨基酸(M3)可以明显影响核心抗原的表达,HBcAg氨基端第3位氨基酸对蛋白的表达可能起到重要的作用.