Inhibition of the HCV Core Protein on the Immune Response to HBV Surface Antigen and on HBV Gene Expression and Replication In Vivo

The hepatitis C virus (HCV) core protein is a multifunctional protein that can interfere with the induction of an immune response. It has been reported that the HCV core protein inhibits HBV replication in vitro. In this study, we test the effect of the HCV core gene on the priming of the immune response to hepatitis B surface antigen (HBsAg) and on the replication of HBV in vivo. Our results showed that the full-length HCV core gene inhibits the induction of an immune response to the heterogeneous antigen, HBsAg, at the site of inoculation when HCV core (pC191) and HBsAg (pHBsAg) expression plasmids are co-administered as DNA vaccines into BALB/c mice. The observed interference effect of the HCV core occurs in the priming stage and is limited to the DNA form of the HBsAg antigen, but not to the protein form. The HCV core reduces the protective effect of the HBsAg when the HBsAg and the HCV core are co-administered as vaccines in an HBV hydrodynamic mouse model because the HCV core induces immune tolerance to the heterogeneous HBsAg DNA antigen. These results suggest that HCV core may play an important role in viral persistence by the attenuation of host immune responses to different antigens. We further tested whether the HCV core interfered with the priming of the immune response in hepatocytes via the hydrodynamic co-injection of an HBV replication-competent plasmid and an HCV core plasmid. The HCV core inhibited HBV replication and antigen expression in both BALB/c (H-2d) and C57BL/6 (H-2b) mice, the mouse models of acute and chronic hepatitis B virus infections. Thus, the HCV core inhibits the induction of a specific immune response to an HBsAg DNA vaccine. However, HCV C also interferes with HBV gene expression and replication in vivo, as observed in patients with coinfection.     

DNA Prime-Adenovirus Boost Immunization Induces a Vigorous and Multifunctional T-Cell Response against Hepadnaviral Proteins in the Mouse and Woodchuck Model

Induction of hepatitis B virus (HBV)-specific cytotoxic T cells by therapeutic immunization may be a strategy to treat chronic hepatitis B. In the HBV animal model, woodchucks, the application of DNA vaccine expressing woodchuck hepatitis virus (WHV) core antigen (WHcAg) in combination with antivirals led to the prolonged control of viral replication. However, it became clear that the use of more potent vaccines is required to overcome WHV persistence. Therefore, we asked whether stronger and more functional T-cell responses could be achieved using the modified vaccines and an optimized prime-boost vaccination regimen. We developed a new DNA plasmid (pCGWHc) and recombinant adenoviruses (AdVs) showing high expression levels of WHcAg. Mice vaccinated with the improved plasmid pCGWHc elicited a stronger WHcAg-specific CD8⁺ T-cell response than with the previously used vaccines. Using multicolor flow cytometry and an in vivo cytotoxicity assay, we showed that immunization in a DNA prime-AdV boost regimen resulted in an even more vigorous and functional T-cell response than immunization with the new plasmid alone. Immunization of naïve woodchucks with pCGWHc plasmid or AdVs induced a significant WHcAg-specific degranulation response prior to the challenge, this response had not been previously detected. Consistently, this response led to a rapid control of infection after the challenge. Our results demonstrate that high antigen expression levels and the DNA prime-AdV boost immunization improved the T-cell response in mice and induced significant T-cell responses in woodchucks. Therefore, this new vaccination strategy may be a candidate for a therapeutic vaccine against chronic HBV infection.     

用rAAV8-1.3HBV制备两种品系小鼠乙型肝炎病毒感染模型的比较研究

我们先前用rAAV8-1.3HBV静脉注射C57BL/6小鼠成功地制备了慢性乙型肝炎病毒(Hepatitis B virus,HBV)感染模型。为了探讨不同品系的小鼠对rAAV8-1.3HBV静脉注射是否具有不同反应,本研究比较了C57BL/6和BALB/c小鼠静脉注射重组病毒后外周血中HBV抗原和抗体水平、病毒载量和肝脏组织HBcAg表达情况,以及不同剂量重组病毒注射与这些指标的关系。将低(4×109 Viral genome,vg)、中(4×1010vg)和高(4×1011vg)三种剂量的rAAV8-1.3HBV通过尾静脉注射至C57BL/6和BALB/c小鼠,分别利用ELISA和荧光定量PCR方法检测血清中的HBV抗原、抗体水平以及HBV DNA,利用免疫组化检测肝脏组织HBcAg的表达。结果发现,对于C57BL/6小鼠,三种不同剂量rAAV8-1.3HBV注射均可造成100%小鼠出现HBV持续感染;血清HBsAg、HBeAg和HBV DNA以及肝组织HBcAg稳定表达超过8个月,其表达水平随重组病毒注射剂量的增加而升高,高剂量注射时可造成超过40%的肝细胞感染HBV,血清中HBV DNA可达105 IU/mL以上;未检测到针对HBV的抗体。对于BALB/c小鼠,三种不同剂量rAAV8-1.3HBV注射也可造成100%小鼠出现HBV持续感染;血清HBeAg和HBV DNA以及肝组织HBcAg稳定表达超过8个月,但是血清HBsAg在重组病毒注射2周之后显著下降甚至消失;在中剂量注射组的BALB/c小鼠血清中检测到低水平的Anti-HBs;血清HBeAg和肝组织HBcAg的表达水平随重组病毒注射剂量的增加而增高,并且各剂量组表达水平均高于C57BL/6小鼠,高剂量注射时可造成超过50%的肝细胞感染HBV。本研究表明,低至4×109 vg剂量的rAAV8-1.3HBV注射即可造成C57BL/6和BALB/c两种品系小鼠出现HBV持续感染,并且HBV复制水平随重组病毒注射剂量增加而增高;BALB/c小鼠对HBV的免疫反应强于C57BL/6小鼠,可以产生针对HBsAg的体液免疫反应而使血清HBsAg转阴,但无法清除携带HBV的肝细胞。     

比较不同血清型AAV携带HBV基因组建立乙肝小鼠模型效果

近年来,用8型腺相关病毒携带1.3拷贝HBV(Hepatitis B virus)基因组建立的HBV持续感染小鼠模型受到越来越多的关注。本研究比较了除AAV8之外的其他4种血清型重组腺相关病毒(Recombinant adeno-associated virus,rAAV)建立乙肝小鼠模型效果。首先,将携带1.3拷贝ayw亚型HBV基因组的1型、2型、5型、8型、9型腺相关病毒分别以1×10~(11) vg/只(Viral genome,vg)的剂量尾静脉注射C57BL/6J小鼠;利用ELISA方法监测小鼠血清中HBeAg和HBsAg表达水平;用定量PCR方法检测小鼠血清和肝脏中HBV DNA拷贝数;用免疫组化方法检测小鼠肝脏中HBc Ag的表达;用HE染色检测小鼠肝脏病理变化。结果显示,在持续8周中,5组小鼠血清中都检测到HBeAg和HBsAg的表达,血清和肝脏中均检测到HBV DNA的存在。HBeAg、HBsAg、HBV DNA表达水平高低依次为AAV8AAV9AAV1AAV5AAV2。5组小鼠用免疫组化方法都检测到肝脏中HBcAg表达,HE染色病理检测均观察到不同程度的肝损伤。本研究扩大了能用于建立乙肝小鼠持续感染模型可选择的AAV载体种类,发现虽然AAV1、2、5、9的建模效果不如AAV8,但它们都可以介导建立持续感染的乙肝小鼠模型,建模效果依次为AAV8AAV9AAV1AAV5AAV2。其中AAV9介导的建模效果与AAV8载体最为接近,可以替代AAV8载体用于有效地建立HBV持续感染的小鼠模型。     

应用微环DNA技术体外诱导和制备重组的乙型肝炎病毒共价闭合环状DNA

乙型肝炎病毒(hepatitis B virus,HBV)共价闭合环状DNA(covalently closed circular DNA,cccDNA)是病毒慢性感染的分子基础。本课题组前期研究通过Cre/loxP介导的位点特异性DNA重组策略,在细胞核内由前体质粒诱导重组cccDNA(rcccDNA^loxP)产生,首次建立了HBV cccDNA的体外培养细胞和小鼠实验模型。本研究基于大肠埃希菌ZYCY10P3S2T PhiC31重组酶诱导表达系统,建立了一种体外诱导HBV rcccDNA(rcccDNA^attR)微环产生和纯化的策略。纯化的rcccDNA^attR微环具有超螺旋结构,细胞培养实验证实其能支持功能性的HBV复制和抗原表达。与普通的线性HBV复制子编码质粒相比,rcccDNA^attR尾静脉高压注射小鼠模型能诱导显著延长的病毒抗原血症。因此,本研究在原核表达系统和实验小鼠水平提供了一种更为简化的HBV cccDNA实验模型系统,并再次显示rcccDNA具有显著的稳定性,能作为一种基本策略在小鼠模型中诱导病毒持续感染。     

Nucleotide sequence of an infectious molecularly cloned genome of ground squirrel hepatitis virus.

We have determined the complete nucleotide sequence of an infectious cloned genome of ground squirrel hepatitis virus (GSHV), a nonpathogenic member of the hepadnavirus group. The genome is 3,311 base pairs long and contains the major open reading frames described for the related human and woodchuck hepatitis B viruses (HBV and WHV, respectively). These reading frames include genes for the major structural proteins (the surface and core antigens), unassigned open reading frames (A and B), the longer of which is presumed to encode the viral DNA polymerase, and an open reading frame preceding and continuous with the surface antigen gene. The arrangement of these open reading frames is similar to that encountered in the genomes of HBV and WHV: all of the reading frames are encoded on the same strand, they are positioned in the same fashion with respect to each other, and a large portion (at least 51%) of the genome can be translated in two reading frames. Comparisons of the predicted translational products of the three mammalian hepadnaviruses reveal 78% amino acid homology between the proteins of GSHV and WHV and 43% homology between those of GSHV and HBV. In addition, a perfect direct repeat of 10 to 11 base pairs, separated by ca. 46 to 223 base pairs, is present in the three mammalian viruses and in duck hepatitis B virus; the position of the repeats near the 5' termini of the two strands of virion DNA suggests a role in viral replication.     

pRNA介导的RNA干扰抑制HBV表达和复制的研究

为了研究由pRNA携带的siRNA(HBVsi18-42)所介导的RNAi过程能有效地抑制HBV的基因表达和病毒复制,我们利用细胞模型和高压注射小鼠模型评价HBVsi18-42对HBV复制和基因表达的抑制作用。通过Western印迹检测细胞内的HBsAg含量,用ELISA检测细胞培养上清和小鼠血清中的HBsAg水平,采用Southern印迹检测HBV的复制中间体,通过免疫组织化学检测肝组织切片中HBcAg的表达情况。试验结果显示,HBVsi18-42能以剂量依赖的方式在293T细胞中抑制HBsAg的表达以及在HepG2细胞中下调病毒HBsAg和HBeAg的表达和病毒复制中间体的水平。在小鼠模型中,注射后的3d内HBVsi18-42使小鼠血清中HBsAg的水平分别下降了98.98%、77.07%和60.73%,免疫组织化学检测显示,在注射后的第3天小鼠肝组织内HBcAg阳性细胞数减少了79.1%。初步结果显示HBVsi18-42无论是在细胞或是在小鼠模型中都能下调HBV的复制和基因的表达。本研究为我们下一步实现由pRNA介导的靶向RNAi及基因治疗提供了理论和技术支持。     

RNA干扰抑制人乙型肝炎病毒复制的研究

采用表达shRNA的载体构建了表达针对病毒HBsAgmRNA保守区的shRNA的质粒psiHBs,利用细胞模型和高压注射小鼠模型评价RNA干扰对HBV复制和基因表达的抑制作用。通过Western印迹检测细胞内的HBsAg,用ELISA检测细胞培养上清和血清中的HBsAg,采用Southern印迹检测HBV的复制中间体,最后通过免疫组化的方法检测肝组织切片中HBcAg的表达情况。结果显示pHBV1.3和psiHBs共转染HepG2后,与对照组相比病毒HBsAg和HBeAg的表达和病毒复制中间体的水平下降了90%以上,并且shRNA的作用效率存在序列特异性和剂量依赖性。在高压注射小鼠模型中,psiHBs表达的shRNA使小鼠血清中HBsAg的水平下降了80%以上,免疫组化检测显示,小鼠肝组织内HBcAg阳性细胞数减少了75.1%,而且shRNA的抑制作用至少能持续4d。研究显示载体表达的shRNA无论是在细胞或是在小鼠模型中都能对HBV的复制和基因的表达发挥序列特异性的抑制作用。本研究为我们下一步实现由RNAi介导的基因治疗提供了理论和技术支持。     

Inhibition of cellular proteasome activities enhances hepadnavirus replication in an HBX-dependent manner

The X protein (HBX) of the hepatitis B virus (HBV) is not essential for the HBV life cycle in vitro but is important for productive infection in vivo. Our previous study suggests that interaction of HBX with the proteasome complex may underlie the pleiotropic functions of HBX. With the woodchuck model, we demonstrated that the X-deficient mutants of woodchuck hepatitis virus (WHV) are not completely replication defective, possibly behaving like attenuated viruses. In the present study, we analyzed the effects of the proteasome inhibitors on the replication of wild-type and X-negative HBV and WHV. Recombinant adenoviruses or baculoviruses expressing replicating HBV or WHV genomes have been developed as a robust and convenient system to study viral replication in tissue culture. In cells infected with either the recombinant adenovirus-HBV or baculovirus-WHV, the replication level of the X-negative construct was about 10% of that of the wild-type virus. In the presence of proteasome inhibitors, the replication of the wild-type virus was not affected, while the replication of the X-negative virus of either HBV or WHV was enhanced and restored to the wild-type level. Our data suggest that HBX affects hepadnavirus replication through a proteasome-dependent pathway.     

Novel Woodchuck Hepatitis Virus (WHV) Transgene Mouse Models Show Sex-Dependent WHV Replicative Activity and Development of Spontaneous Immune Responses to WHV Proteins

The woodchuck model is an informative model for studies on hepadnaviral infection. In this study, woodchuck hepatitis virus (WHV) transgenic (Tg) mouse models based on C57BL/6 mice were established to study the pathogenesis associated with hepadnaviral infection. Two lineages of WHV Tg mice, harboring the WHV wild-type genome (lineage 1217) and a mutated WHV genome lacking surface antigen (lineage 1281), were generated. WHV replication intermediates were detected by Southern blotting. DNA vaccines against WHV proteins were applied by intramuscular injection. WHV-specific immune responses were analyzed by flow cytometry and enzyme-linked immunosorbent assays (ELISAs). The presence of WHV transgenes resulted in liver-specific but sex- and age-dependent WHV replication in Tg mice. Pathological changes in the liver, including hepatocellular dysplasia, were observed in aged Tg mice, suggesting that the presence of WHV transgenes may lead to liver diseases. Interestingly, Tg mice of lineage 1281 spontaneously developed T- and B-cell responses to WHV core protein (WHcAg). DNA vaccination induced specific immune responses to WHV proteins in WHV Tg mice, indicating a tolerance break. The magnitude of the induced WHcAg-specific immune responses was dependent on the effectiveness of different DNA vaccines and was associated with a decrease in WHV loads in mice. In conclusion, sex- and age-dependent viral replication, development of autoimmune responses to viral antigens, pathological changes in the liver in WHV Tg mice, and the possibility of breaking immune tolerance to WHV transgenes will allow future studies on pathogenesis related to hepadnaviral infection and therapeutic vaccines.     

乙型肝炎病毒急性感染小鼠模型的建立

采用高压水注射方法,通过尾静脉将具有复制能力的HBV质粒导入BABL/cJ小鼠体内,应用real-time PCR、ELISA、RIA、Southern Blot、Northern Blot,以及免疫组化等方法,检测小鼠病毒血症、血清和肝组织中HBV 抗原表达动态变化、肝组织中HBV转录和复制情况,以及小鼠免疫应答状况。结果HBV基因可以在小鼠体内表 达和复制,并诱导小鼠产生特异性免疫应答,其应答模式及HBV清除过程与人类的HBV急性感染类似。实验显 示高压注射具有复制能力的HBV质粒可以在小鼠体内建立HBV急性感染模型,这种模型可以用于HBV病毒 学、免疫学以及抗病毒药物筛选等方向的研究。     

Insulin Dominantly Suppresses Hepatitis B Virus Gene Expression in Cultured Human Hepatoma Cells

We have shown previously that insulin suppresses the expression of hepatitis B surface antigen (HBsAg) gene from an endogenous integrated viral genome in cultured human hepatoma Hep3B cells. In this study, we demonstrated that insulin suppresses the viral mRNA transcribed from transiently transfected tandem repeat hepatitis B virus (HBV) dimer DNA or DNA fragment that contains only the major HBsAg gene. Insulin treatment also resulted in a decrease in HBV viral particles produced by the HBV-DNA-transfected cells in a dose-dependent manner. Furthermore, when insulin was simultaneously added with glucocorticoid, which stimulates HBV gene expression, the stimulatory effect of glucocorticoid was completely abolished. Our results suggest that insulin has a dominant negative effect on the HBV gene expression in cultured human liver cells.     

Oncogenesis of mammary glands, skin, and bones by polyomavirus correlates with viral persistence and prolonged genome replication potential.

A correlation between polyomavirus-induced oncogenesis and viral persistence on the one hand and/or prolonged genome replication potential on the other was established with respect to their respective organ distributions. Prolonged replication potential is defined as the capacity of a genome to replicate in a given organ from the time of infection up to the onset of oncogenesis. This conclusion was derived following intraperitoneal infection of BALB/c mice with wild-type strain A2. Viral genomes were used as parameters of persistence and replication and were detected by Southern blotting and PCR analysis. The major tumor target organs (mammary gland, skin, and bone), which have not been previously analyzed for persistence, were compared with other, non-tumor-prone organs (kidney, liver, lung, spleen, and salivary gland). A progressive loss of viral genomes was observed in all tissues as a function of time postinfection; however, genomes were shown to persist through 20 weeks postinfection in the mammary glands, skin, and bones to an extent similar to that in the previously described kidneys (D. J. McCance, J. Virol. 39:958-962, 1981; W. P. Rowe, J. W. Hartley, J. D. Estes, and R. J. Huebner, Natl. Cancer Inst. Monogr. 4:189-209, 1960). Thus, tumors arise among organs that sustain a persistent infection, but not all such organs develop tumors (e.g., the kidney). The capacity of organs to support de novo replication at various ages, including the age reached when the first tumors are detected, was also determined using a 3-day infection period for ages between 0 and 7 weeks. For all organs tested, a higher level of genomes was observed in organs of mice infected as neonates than in those infected after the age of 3 weeks. However, marked organ-specific differences were seen in the degree and timing of loss of replication. In particular, viral genome replication, although reduced, was maintained in the mammary glands, skin, and bones of adult animals, in contrast to the kidneys. We conclude that organ-specific oncogenesis correlates with two organ-specific parameters: persistence of viral genomes and prolonged viral genome replication potential. This may reflect a requirement for continued viral genome replication and/or gene expression for tumorigenesis. In turn, these parameters may be linked to the tissue-specific continued capacity for cellular division.     

Restriction endonuclease mapping of the hepatitis B viral genome isolated from Taiwan

The Eco RI fragment of hepatitis B virus (HBV) DNA isolated from human blood plasma Dane particles were inserted into plasmid pUC8 Eco RI site and transformed into E. coli JM103 host. Two recombinants pTWL1 and pTWL2 were found to carry 3.2 kbp fragment and proved to have HBV genome by Southern hybridization method. The 1.4 kbp Bam HI fragment which carried the hepatitis B viral surface antigen (HBsAg) gene, obtained via Bam HI digestion of Dane particles DNA which was made fully double stranded by endogenous DNA polymerase reaction, was also inserted into plasmid pUC8 Bam HI site. Four recombinant clones, pTWS1, pTWS2, pTWS3, and pTWS4 were found. Only one of the clones pTWS1 carried the HBsAg gene in a correct orientation with respect to the lac promoter sequence. The physical mapping of HBV DNA was performed with several restriction endonucleases. Our results indicated that the HBV DNA insert contains unique XbaI and HpaI cleavage sites and lacks the cleavage sites for the HindIII, SmaI, KpnI, SalI, and SstI endonucleases. The locations of Bam HI, BglII, and HincII endonucleases cleavage sites within the cloned HBV DNA of the pTWL1 plasmid were similar to that HBV DNA of adw and adw2 subtypes.     

特异性表达siRNA的新型乙肝核酸药物的研究

旨在研究靶向于HBsAg的肝脏特异性siRNA,实现核酸药物高靶向性、高特异性基因治疗乙肝。通过重组PCR的方法构建靶向于HBsAg的肝脏特异性表达的siRNA的核酸药物。将目标载体转染HepG2 2.2.15细胞,用ELISA的方法测定转染细胞培养液内HBsAg的含量。PCR鉴定和测序确定重组PAAV-MCS载体PApoE1-ApoE2-hAAT-siHBsAg-U1 3’Box构建成功。重组质粒对HepG2 2.2.15细胞分泌的HBsAg所产生的抑制作用自转染后5 d达到高峰,抑制率达到(30±0.28)%(P<0.01),siRNA特异性表达单元能显著降低HepG2 2.2.15细胞HBV DNA的拷贝数。成功构建的肝脏特异性、siRNA高表达的核酸药物可以有效地抑制HBV基因的表达和复制,为乙肝的进一步治疗研究做了关键性工作。     

Circular Intermediates of Recombinant Adeno-Associated Virus Have Defined Structural Characteristics Responsible for Long-Term Episomal Persistence in Muscle Tissue

Adeno-associated viral (AAV) vectors have demonstrated great utility for long-term gene expression in muscle tissue. However, the mechanisms by which recombinant AAV (rAAV) genomes persist in muscle tissue remain unclear. Using a recombinant shuttle vector, we have demonstrated that circularized rAAV intermediates impart episomal persistence to rAAV genomes in muscle tissue. The majority of circular intermediates had a consistent head-to-tail configuration consisting of monomer genomes which slowly converted to large multimers of >12 kbp by 80 days postinfection. Importantly, long-term transgene expression was associated with prolonged (80-day) episomal persistence of these circular intermediates. Structural features of these circular intermediates responsible for increased persistence included a DNA element encompassing two viral inverted terminal repeats (ITRs) in a head-to-tail orientation, which confers a 10-fold increase in the stability of DNA following incorporation into plasmid-based vectors and transfection into HeLa cells. These studies suggest that certain structural characteristics of AAV circular intermediates may explain long-term episomal persistence with this vector. Such information may also aid in the development of nonviral gene delivery systems with increased efficiency.