INAUGURAL-DISSERTATION Zur Erlangung des Medizinischen Doktorgrades der Medizinichen Fakultät der Albert-Ludwigs-Universität Freiburg i. Br.

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1 Aus der Medizinischen Universitätsklinik und Poliklinik Abteilung für Gastroenterologie/Hepatologie/Endokrinologie der Albert-Ludwigs-Universität Freiburg i. Br. Hepatitis B Virion and cccdna Formation in Primary Tupaia Hepatocytes and Human Hepatoma Cell Lines upon HBV Genome Transduction with Replication-defective Adenovirus Vectors and in vivo Infection of Tupaias INAUGURAL-DISSERTATION Zur Erlangung des Medizinischen Doktorgrades der Medizinichen Fakultät der Albert-Ludwigs-Universität Freiburg i. Br. Vorgelegt 2000 Von Shaotang Ren geboren in Liaoning, China

2 Dekan: Prof. Dr. med. Dr. h.c. H. E. Blum 1. Gutachter: Prof. Dr. M. Nassal 2. Gutachter: Prof. Dr. med. D. Neumann-Haefelin Jahr der Promotion: 2001

3 1 1 INTRODUCTION Hepatitis B and HBV biology Animal models of hepadnavirus infection Adenovirus vector system STUDY OBJECTIVE MATERIALS AND METHODS Cells and cell lines Bacteria and plasmids Animals Generation and purification of recombinant adenoviruses Generation of recombinant adenoviruses CsCl Gradient Purification of Recombinant Adenovirus Infection of cultured cells Assay of secreted HBV antigens Extracellular HBV DNA detection Dose dependence Time course analysis Intracellular replication-competent core particles analysis HBV DNA detection by Southern blotting HBcAg immunoblotting assay Virions in the culture medium HBsAg ELISA analysis Western blot analysis of HBsAg, HBVpreS1, HBVpreS2 and HBcAg Detection of HBV DNA by Southern blot Nuclear cccdna detection Dose-dependence... 25

4 Time-course analysis In vivo infection of tupaias with recombinant adenoviruses ELISA analysis of HBsAg and HBeAg Assay of Anti-HBs in sera from infected tupaias Detection of HBV DNA in the sera Biosafty analysis of recombinant adenovirus Nose-washing samples Sera samples RESULTS Construction of recombinant Adenovirus vectors carrying complete HBV genomes Amplification and purification of recombinant adenoviruses Infection and Ad-HBV mediated production of HBV antigens in the culture medium Generation of intracellular replication-competent nucleocapsids Formation of extracellular enveloped virions in the culture medium HBV cccdna formation in primary tupaia hepatocytes and hepatoma cell lines Long term observation of HBV within the sera of recombinant adenovirus infected tupaias Dynamic changing of recombinant adenovirus within infected tupaia DISCUSSION The Ad-HBV vector system Competence of primary tupaia hepatocytes for HB virion formation HBV cccdna formation in primary tupaia hepatocytes In vivo infection of tupaias by recombinant adenoviruses carrying HBV complete genomes SUMMARY... 64

5 3 7 REFERENCES ABBREVIATIONS ACKNOWLEDGMENTS CURRICULUM VITAE... 76

6 4 1 INTRODUCTION 1.1 Hepatitis B and HBV biology Hepatitis B virus (HBV) is the pathogenic agent of B-type hepatitis in humans. It causes transient and chronic infections of liver. Transient infections may produce serious illness, and approximately 0.5% terminate with fatal, fulminant hepatitis. Chronic infections are wide-spread and associated with a greatly increased risk for the development of liver cirrhosis and, eventually, primary liver carcinoma (Blumberg, 1997; Seeger and Mason, 2000). More than 350 million people are infected worldwide (Blumberg, 1997; Buendia, 1998). Alone in China, there are 120 million of HBsAg carriers and 28 million patients (Luo, 1996). Worldwide deaths from liver cancer caused by HBV infection probably exceed one million per year (Parkin, Pisani, and Ferlay, 1999; Seeger and Mason, 2000). Even though an effective vaccine has been available for about 20 years, HBV infection still is one of the most important infectious diseases in the world. HBV is an enveloped 3.2kb double-stranded DNA virus that replicates through an RNA intermediate (Summers and Mason, 1982). It is the prototype member of the hepadnaviridae. A characteristic of the HBV genome is its small size and compact organization (Fig. 1). During hepadnavirus infection, early events of the viral life cycle, including entry, uncoating, and delivery of the viral genome into the cell nucleus, are not well understood. This is, in part, due to the absence of cell lines that are susceptible to hepadnavirus infection. The relaxed circular partially double stranded DNA genome is transported to the nucleus of the hepatocyte, where it is converted into a covalently closed circular molecule, named cccdna, which serves as template for the transcription of all viral RNAs (Ganem, 1996)). The synthesis of the new relaxed circular viral DNA genome is mediated by the viral P-protein, which has reverse transcriptase and DNA-dependent DNA polymerase activity. Synthesis

7 5 A. ENVELOPE L S M Lipid Hsc70 CORE Core protein P protein TP domain DNA-genome B. genomic RNA (-)-DNA (+)-DNA poly A ε C 1 1 pres1 prec X 2 pres2 P HBV Enh II 3182 bp PRE β S Enh I PRE α subgenomicrna's Fig. 1. Structural and genetic organization of the hepadnaviruses. A. HBV virion. The envelope contains the surface proteins L, M, and S. The pres domain of L occurs in an inward and an outward topology. The irregularly shaped objects symbolize the chaperone Hsc70 that co-purifies with virions and S particles. B. HBV genome. The HBV genome inside the capsid is present as partially doublestranded circular DNA. The 5' end of the complete (-)DNA strand is covalently linked to the TP domain of P protein. In the lower panel, the heavy black lines represent the DNA genome with the four major open reading frames (ORFs) indicated in the centre. Boxes marked `1 and `2 symblize the direct repeats DR1 and DR2, Enh I and Enh II indicate the two enhancer elements. The outer lines depict the genomic and subgenomic RNAs. Arrowheads indicate transcriptional start sites, the symbolic hairpin the encapsidation signal e, and PREa and b the location of the post-transcriptional regulatory element (PRE) and on the pres/s mrnas. (This figure was kindly provided by Prof. M. Nassal (Nassal, 2000)).

8 6 of the viral DNA takes place in the cytoplasm of the hepatocytes within immature nucleocapsids (Summers and Mason, 1982). To establish a sufficient pool of nuclear cccdna, some of the newly synthesized viral DNAs need to be retransported to the nucleus of the infected hepatocyte (Tuttleman, Pourcel, and Summers, 1986). Therefore, the replicative forms of viral DNA are also precursors to cccdna (Fig. 2). Since cccdna is the template for the transcription of viral mrnas, its formation indicates a successful initiation of infection. The mechanism of DNA repair involved in the conversion of rcdna (relaxed circular DNA) to cccdna is not known. Whatever the mechanism for the nuclear transport of virion DNA may be, formation of cccdna, which accumulates only in the nucleus, completes the initiation of infection. The most intriguing aspect of the hepadnavirus replication cycle is the regulation of cccdna synthesis. It is the basis for the establishment and persistence of hepadnaviruses in infected hepatocytes and consequently can be expected to play a major role in recovery from an infection. The nuclei of duck hepatitis B virus (DHBV) -infected hepatocytes contain on average 10 to 50 copies of cccdna per cell while 10 times as much of the rcdna precursor to cccdna may reside in the cytoplasm. Amplification of cccdna levels takes place in the first few days following infection of primary duck hepatocyte cultures with DHBV (Tuttleman, Pourcel, andsummers, 1986). Viral cccdna is thought to be stable in infected and nonproliferating hepatocytes. Studies employing primary woodchuck hepatocyte cultures indicated that the cccdna was rather stable in nonproliferating cells when new woodchuck hepatitis B virus (WHV) DNA synthesis was inhibited by lamivudine or dideoxycytidine treatment (Moraleda et al., 1997). One of the unknown factors is whether cccdna is passed to daughter cells when hepatocytes divide. A recent study (Dandri et al., 2000) not only confirmed that a new reverse transcriptase inhibitor, adefovir, had a very strong inhibitory effect on WHV-DNA synthesis in cultured chronically infected primary woodchuck hepatocytes, but also showed, indirectly, that cccdna was a very stable molecule

9 7 Infectious virion Receptor(s) S particles GOLGI ER/IC ccc-dna Reverse transcription RNA precore, L,M,S + X proteins ε cap Capsid protein RNA pregenome P protein A n Fig. 2. The HBV infectious cycle. Virions infecting primary hepatocytes release nucleocapsids into the cytoplasm. Thses deliver the DNA genome to nucleus where it is converted into cccdna to serve as transcriptional templete. After nuclear export, all transcripts are translated; the RNA pregenome, in addition, interacts with its two gene products to form immature, RNA-containing nucleocapsids. Inside the capsids, the RNA is reverse transcribed into DNA by P protein. The DNA genome can either be re-delivered to the nucleus, or the matured nucleocapsids, via interaction with the surface proteins, bud into a pre-golgi compartment of the secretory pathway and are exported as enveloped virions. Budding of the surface proteins without capsids yields empty spherical and filamentous S particles. (This figure was kindly provided by prof. M. Nassal (Nassal, 2000)).

10 8 (the half-life was longer than 24 days) that appeared to be efficiently transmitted to the dividing hepatocytes. In contrast, cccdna in nondividing duck hepatocytes infected with DHBV appeared to have a half-life of only 3 to 5 days (Civitico and Locarnini, 1994). However, a moer recent report (Addison et al., 2000) showed that the half-life of the DHBV cccdna in infected liver cells is days in vivo. HBV transgenic mice, expressing all kinds of HBV antigens and secreting HBV virions in several organs at different level, do not accumulate cccdna, suggesting that the pathway responsible for the conversion of rcdna to cccdna is defective in these animals (Guidotti et al., 1995). 1.2 Animal models of hepadnavirus infection Within the past three decades, great progress has been made in hepatitis B research, especially in HBV biology. Several fundamental aspects of the HBV replication cycle have been elucidated, mainly by genetic techniques using transfection of cloned HBV DNA into suitable human hepatoma cell lines such as HuH7 and HepG2 (Nassal, 1999; Nassal, 2000). These cells support virus production but neither they, nor other cell lines can be infected. A major focus of HBV research is to understand the virus-host interactions that determine whether an infection will persist or terminate. A related and similarly central problem is the lack of a feasible small animal model of human HBV infection. Historically, major obstacles in the study of HBV have been the inability of the virus to infect cells in vitro, and the lack of animal model systems due to a strict virus-host range. All hepadnaviruses (liver-tropic DNA viruses) have a very narrow host range. Efficient infection by HBV is well documented for only humans and chimpanzees and, in cell culture, for primary hepatocytes from these hosts. Thus, many aspects of HBV biology have been unraveled by studying related hepadnaviruses, such as the duck hepatitis virus (DHBV,an Avihepadnavirus) which is capable of in vitro infection (Tuttleman, Pugh, and Summers, 1986), and the woodchuck hepatitis virus (WHV, an

11 9 Orthohepadnavirus) which allows for the in vivo study in an animal model system (Tyler, Snyder, and Summers, 1986). These current generic animal models (Nassal, 2000) are useful in many respects but suffer from two differing limitations: woodchucks are difficult to keep and their use is restricted to few appropriately equipped facilities; even then, most studies used out-bred, wild-caught animals (Roggendorf and Tolle, 1995). Ducks, by contrast, are convenient experimental animals (Jilbert and Kotlarski, 2000) but very distantly related to humans. The DHBV and WHV systems were instrumental in developing an understanding of the hepadnavirus lifecycle and remain valuable models for HBV infection. However, many significant differences exist between animal hepadnaviruses and HBV. For example, the avian hepatitis viruses do not encode the X gene (Sprengel et al., 1985) and encode only two envelope proteins, analogous to S and L. Major transcriptional differences between woodchuck hepatitis virus and HBV have been reported (Di et al., 1997). An ideal experimental system would consist of HBV, or a very closely related virus, plus a suitable host that is as easily kept and as well characterized as the mouse. Some mouse-based surrogate systems are available; however, although HBVtransgenic mice (Akbar and Onji, 1998; Chisari, 1996) produce substantial amounts of virus (Guidotti et al., 1995) they are not infectable; mice carrying heterologous liver cells (Ilan et al., 1999; Ohashi et al., 2000; Petersen et al., 1998) require immune suppression to prevent rejection of the xenotransplant. Because hepadnaviral host-range is controlled on several levels many barriers would have to be surmounted in order to establish a productive infection in a nonauthentic host. Certainly important is the interaction with cell surface molecules that mediate virus entry (Fig. 2). The large viral envelope protein is essential for these early steps (Bruss et al., 1996) and, for DHBV, a cellular membrane-associated carboxypeptidase seems crucially involved in, but not sufficient for, infection (Breiner,

12 10 Urban, and Schaller, 1998; Kuroki et al., 1995; Lucito and Schneider, 1992). Virtually nothing is known about the receptors for the mammalian hepadnaviruses. Intracellularly, hepadnaviral replication depends on host factors that can interact with diverse cis elements on the viral genome. These are not only tissue- but also species-specific, as shown, e.g., by the inactivity of WHV enhancer I in human hepatoma cells (Di et al., 1997). Further virus-host interactions may also contribute to species specificity. An example is the apparent lack of cccdna formation in HBVtransgenic mice. The cccdna is a central intracellular intermediate in the HBV replication cycle (Fig. 2). It is initially formed after nuclear deposition, by the nucleocapsid (Kann et al., 1999), of the partially double-stranded DNA genome present in extracellular virions. During natural infection, new capsids deliver progeny genomes to the nucleus such that a pool of some 10 to 100 copies of cccdna is established (Tuttleman, Pourcel, andsummers, 1986; Wu et al., 1990). Hence mice apparently lack factors required for both HBV uptake and intracellular amplification. Chances for trans-species transmission are therefore likely to be higher for a more closely related virus-host pair. One potential nonauthentic host for human HBV is the Asian tree shrew (Tupaia, Fig. 3). Tupaias, classified in an own order (Scandentia), are the closest relatives of the primate lineage (Novacek, 1992) (Fig. 3). Breeding colonies of the rat-sized animals have been established at numerous places. Some evidence for their susceptibility to infection with human HBV has been reported (Walter et al., 1996; Yan et al., 1996). Though the in vivo data were not really conclusive, results obtained with primary tupaia hepatocytes (PTHs) suggest that infection is principally possible (J. Köck, F. v. Weizsäcker, H.E. Blum, personal communication). However, the very low efficiency made it difficult to directly prove the net formation of complete progeny virions. It is therefore reasoned that replacing the authentic entry process with a more effective HBV genome delivery procedure should allow us to investigate the principal ability of

13 11 Tupaia Fig. 3. phylogenetic tree showing relationships among the major mammalian clades. The solid horizontal bars indicate the age range of the clade on the basis of dated first appearance in the fossil record. Solid lines indicate the branching sequence, although the date of the actual splitting event can only be inferred from the relationships of the clades and their known ages. Dashed lines indicate relatively more ambiguous relationships. Arrow indicates tupaia location within the tree. This relationships tree-figure was copied from the reference (Novacek, 1992).

14 12 PTHs to support all intracellular steps of the hepadnaviral replication cycle. If so, this would provide a rational basis for in vivo experiments aimed at artificially initiating an HBV infection in this species. 1.3 Adenovirus vector system None of the established methods for delivery of cloned HBV DNA is ideal for in vivo applications. Naked DNA (Will et al., 1985) and liposomes (Takahashi et al., 1995) reach only a limited number of hepatocytes. Recombinant baculoviruses are effective in cultured cells (Delaney and Isom, 1998; Delaney, Miller, and Isom, 1999) but in vivo the current vectors are subject to complement inactivation. By contrast, replication-defective adenovirus vectors (Ad-vectors) are a versatile tool for gene delivery and expression. They are widely used in gene transfer in vitro, vaccination in vivo, and gene therapy studies (Benihoud, Yeh, and Perricaudet, 1999). As a vector, they have several advantages, such as a large insert capacity for foreign genes (5-10 kb (He et al., 1998)), relatively easy manipulation, high viral titers (more than pfu/ml) and high-level gene expression, a broad spectrum of cell types for infection independent of active cell division, several admitted-pathways, little chromosomal integration, safety to its host, and the possibility for in vivo infection. But cytotoxicity at high dose infection and host-immunity (especially in the adult) to Adenoviruses are important hurdles to overcome for successful in vivo applications. Ad-vectors were selected in this research mainly due to their high efficiency of hepatocyte infection in vitro and in vivo. The wild type adenovirus genome is a linear duplex DNA of 36 kb organized into six major transcription units. There are 51 serotypes of human Adenoviruses. The serotypes 5 (Ad5) and Ad2 belonging to subgroup C, are commonly studied. The most commonly used adenoviral vector, based on human adenovirus serotype 5, is rendered replication defective by the deletion of the E1 and E3 genes. The early E1 gene is essential for the expression of later transcription units, and is complemented

15 13 in vivo by an adenovirus packaging cell, such as HEK293. HEK293 cells are human embryonic kidney cells that have been transformed by sheared Ad5 DNA. The cells contain and express the transforming genes of Ad5, including E1, which complements the deleted E1 gene from the Ad-vectors for assembly of replicationdefective adenovirus. The E3 gene encodes proteins involved in evading host immunity and is dispensable. Not only do these deletions prevent the virus from replicating itself, they also create space for up to 10 kb of foreign genes. Two methods have traditionally been used to generate recombinant adenoviruses. The first involves direct ligation of the gene of interest into the adenoviral genome. The scarcity of unique restriction sites and the size of the genome make this method technically challenging. The second and more widely used method involves cloning the gene of interest into a shuttle vector and transferring the gene into the adenovirus genome by means of homologous recombination in vivo (Berkner, 1988). To isolate and identify recombinant adenovirus by this method, multiple plaque isolations must be performed, which is an extremely laborious and time-consuming process. A new method, developed by C. Chartier (Chartier et al., 1996) and T.C. He (He et al., 1998), is that homologous recombination between an adenoviral backbone plasmid vector and a shuttle vector carrying the gene of interest is performed in E. coli, usually in BJ5183. The E. coli strain BJ5183 was selected for its recombination capabilities and higher transformation efficiency. This method generates recombinant adenoviral plasmid vectors that obviate the need for plaque purification and significantly decrease the time required to generate virus. The interactions between adenovirus and its host are very complicated. In natural infection, Ad5 causes mild acute illness of upper respiratory tract in young children and usually is asymptomatic. Adult people can produce Ad-antibodies after infection by Adenovirus. Meanwhile, the infected cells and host respond to Adenovirus (vector) infection. Initially, cells respond to Ad-infection by producing IFN-α and IFN β to limit

16 14 Ad infection (Wold and Chinnadurai, 2000). The IFN-α, β and γ can also be produced later by immunized cells to interfere with Ad-replication. At the early innate inflammatory phase (first few days), macrophages become activated and secrete cytokines, such as TNF-α, IL-1b, IL-6, and IL-8. These cytokines stimulate other macrophage, neutrophils and NK cell to move into infected tissues and through phagocytosis and apoptosis to kill infected cells. At the late immune-specific phase, CD4-helper cells and CD8-CTL attack the infected cells, via the perforin-granzyme pathway, or Fas-Fasl pathway, or minor TNF-TNFR1 pathway. Ad-proteins regulate apoptosis of infected cells (Wold and Chinnadurai, 2000). Some Ad-expressed proteins inhibit apoptosis of infected cells to maintain Ad replication. For example, among the various Ad proteins, the E1B-19k protein plays an important role in suppression of apoptosis during viral infection. The Bcl-2 protein can functionally substitute for the E1B-19k protein during viral replication. The E1B-55k protein inhibits p53 gene's transcriptional activation function to inhibit apoptosis. E3- coded proteins inhibit immune-mediated apoptosis. E3-gp19k protein blocks MHC class I antigen presentation. The RID protein removes and degrades Fas. E3-14.7k protein inhibits TNF-induced apoptosis. On the other hand, some Ads-expressed proteins induce apotosis to propagate and release Ads to infect other cells. The E4 proteins provide functions essential for efficient viral DNA replication, shut-off of host protein synthesis and cause viral cytopathicity. E4 proteins may be responsible for induction of apoptosis. E1A prevents transcription of IFN-inducible genes, and VA- RNAI prevents the IFN/PKR-mediated shut-off of protein synthesis. The adenovirus death protein (ADP) induces of cell lysis and virus release. The ADP is located in E3 unit. The peak period of ADP synthesis occurs at very late stage when much of the virus assembly is complete. Within the E3 deleted adenovirus vector, which lacks ADP, cell lysis is postponed from 24 hours (Shenk, 1996a) to 3 days. This is of benefit for E3-deleted-Ad-vector preparation.

17 15 2 STUDY OBJECTIVE The purpose of this study was to confirm the possibility of establishing HBV replication within transduced tupaia hepatocytes from a HBV1.3 X genome, and to set up a new HBV-infection animal model. Therefore, replication-defective adenovirus vectors that carry complete HBV genomes under control of the authentic regulatory elements were generated. These Ad-HBV vectors were used to transduce primary tupaia hepatocytes and, for comparison, human hepatoma cell lines which are known to support the HBV replication circle except that they cannot be infected. Generation of HBV gene products would first demonstrate that the Advectors can efficiently infect primary hepatocytes from tupaias, and second that the HBV regulatory elements are functional in this host. The next question was whether these Primary Tupaia Hepatocytes Ad-HBV subgenomic RNAs + pgrna TP core protein P cccdna? RT mature capsid ER Golgi? HBV + HBsAg + HBeAg + naked cores???? Fig. 4. Schematic outline of Ad-vector mediated HBV genome transduction system. The replication-defective Ad-HBV vector infects PTH or Huh7 cell and deposits its recombinant genome in the nucleus. The authentic HBV regulatory elements on the transduced HBV genome control transcription of subgenomic and genomic HBV RNAs which serve as mrnas. pgrna is used to translate core and P protein, and then is packaged into newly forming immature nucleocapsids. Using its terminal protein (TP) domain as primer, P protein reverse transcribes the pgrna, yielding mature DNA containing nucleocapsids. These can either bud into the secretory pathway and be exported as enveloped virions, or redirect the DNA genomes to the nucleus for conversion into cccdna. In addition to virions, HBsAg and HBeAg and naked cores are released from the transduced cells. The aim of the following workes will confirme whether this system suports all the authentic HBV replication circles.

18 16 gene-products assembled into replication-competent nucleocapsids, and whether these are released as complete enveloped virions. The most important issue was whether the primary tupaia hepatocytes are able to support hepadnaviral cccdna formation. If so, this would demonstrate that tupaias are a more appropriate host for HBV than mice (Fig. 4). HBV transgenic mice fail to produce cccdna. The final aim was to determine whether the recombinant adenoviruses could be used to transduce tupaias in vivo with concomitant induction of HBV antigenmia and virionmia. This would form a basis for developing tupaias into a new animal model of HBV infection.

19 17 3 MATERIALS AND METHODS 3.1 Cells and cell lines. Human hepatoma cell lines HuH7 and HepG2 were grown under standard conditions as previously described (Nassal, 1992). Cells were maintained in DMEM medium with 10% fatal calf serum and antibiotics. Cell densities at confluence were around 1.2 x 10 7 cells per 10cm-dish. For preparation of primary tupaia hepatocytes, animals from an in-house breeding colony were anaesthesized, the livers were surgically exposed and perfused using a two-step protocol as previously described (Walter et al., 1996). Cells were seeded on Collagen I-coated plates (Becton Dickinson) at a density of 4-5 x10 6 cells/10 cm dish, and maintained in BioCoat hepatocyte defined medium (Becton Dickinson). Viability was usually around %. Cells could routinely be maintained for at least 2 to 3 weeks. After harvesting, cells were stored at 80 C. 3.2 Bacteria and plasmids. Plasmids were amplified in E. coli Top10 cells (Invitrogen) and homologous recombinations were performed in strain BJ5183 (Chartier et al., 1996; He et al., 1998). Shuttle plasmid ptg9530-hbv1.3 was generated by inserting, through several steps, a 1.3x HBV genome (subtype ayw), starting at nt position 2351 (with position 1 corresponding to first nt of the core gene (Pasek et al., 1979)), extending through a unit length genome and ending at position 88 after the poly-adenylation signal, into the unique Sal I cloning site in plasmid ptg9530 (Chartier et al., 1996). Shuttle plasmid padtrack-hbv1.3 was similarly constructed by insertion of the same 1.3x HBV genome between the Sal I and Xho I sites in plasmid padtrack (He et al., 1998). The shuttle plasmids and the corresponding adenovirus backbone plasmids ptg4656 and padeasy1, containing Ad5 genomes with E1A/B plus E3 deletions,

20 18 were kindly provided by M. Lusky (Transgene, Molsheim) and T.C. He (Howard Hughes Medical Institute, Baltimore, MD). 3.3 Animals Tupaias were purchased from China (Guangxi Provice), housed, maintained, and propagated in accordance with guidelines under approved protocols. 3.4 Generation and purification of recombinant adenoviruses Generation of recombinant adenoviruses. Recombination between the shuttle and the adeno-backbone plasmids was performed essentially as described (Chartier et al., 1996; He et al., 1998) with some modifications. Briefly, for the ptg system (Fig. 5), the shuttle plasmid ptg9530- HBV1.3 was digested by Pac I and Apa I and purified by low melting agarose. Adbackbone plasmid ptg4656 was linearized by Cla I. A unique Cla I site, at position 918, located in E1 of Ad5 (nucleotide numbers refer to position on the Ad5 genome (Chroboczek, Bieber, and Jacrot, 1992)) had been deleted during construction of ptg4656 (Chartier et al., 1996). The two fragment homologous recombinations were performed in strain BJ5183 with ampicillin selection. To increase plsmaid yields which are very low in BJ5183, all colonies from a plate were washed off two times by 5 ml LB medium and collected. After centrifuge, plasmids were purified by mini-prep and re-transformed into Top10 (E. coli) competent cell. The target recombinants were preselected by restriction enzyme digestion or colony hybridization using an HBV whole-genome specific probe. Subsequently the desired recombinants were identified by Xhol I and EcoR I restriction digestion analysis and selectively sequencing. Plasmid DNAs from one well characterized clone were used to excise the Ad-vector part using the flanking Pac I restriction sites (Fig. 5). Linear DNAs were transfected into 293 cells using FuGENE 6 Reagent (Roche Diagnostics) according

21 19 ptg9530 (4939 bp) Cla I Cla I Cla I ptg4656 (37524 bp) 1 Pac I Sal I 3328 Cloning 4623 Apa I Apa I 1 Pac I 458 MLP Afl III 458 lacz E1 Bgl II SV40 pa 4623 Apa I 4929 Apa I Digest with Cla I E3 Xba I Xba I Pac I ptg9530hbv1.3 (9058 bp) ptg4656 (Cla I cut) Pac I HBV [Sal I] pa Apa I Apa I Digest with Apa I and Pac I 1 Pac I 458 MLP Cla I Cla I lacz 3328 SV40 pa 4623 Apa I 4929 Apa I Purification E3 Xba I Xba I Pac I Cla I cut large fragment 1 Pac I 458 MLP Cla I Cla I lacz SV40 pa Apa I 4929 Apa I E3 Xba I Xba I Pac I Apa I and Pac I fragment 1 Pac I HBV pa 4623 Apa I Homologous recombination in E. coli BJ5183 Digest with Pac I Ad-HBV Ad5 HBV Ad5 1 E1A/E1B E3 AdGFP-HBV Ad5 egfp CMV HBV 3182 A n pgrna A n sg RNAs Ad5 E Transfect 293 cells Ad-HBV1.3 recombinant ad5 virus replication-defective ( E1, E3) trans-complemented by 293 AdGFP-HBV1.3 Fig. 5 Schematic outline of the construction and generation of Ad-HBVs. The genes of HBV1.3mer was first cloned into the shuttle vector ptg9530. Then it was digested with Apa I and Pac I and subsequently cotransformed into E. Coli BJ5283 with the adenoviral backbone ptg4656, linearized by Cla I. The target recombinants were selected and confirmed. These interesting plasmids were linearized by Pac I and transfected into HEK293 cells. The AdGFP-HBV1.3 was generated similar to this protocol according to reference (He, et al, 1998).

22 20 to the manufacture's recommendations. After 8 to 10 days (sometimes no typical plaques formed, especially without GFP indicator), cells and medium were collected. The vector particles were released from the cells by three freeze-thaw cycles, and the lysates were used to infect 293 cells (Gall, 1998) CsCl Gradient Purification of Recombinant Adenovirus The 293 cell was split into of 150 mm dishes, using DMEM+10%FBS+1%P/S. When the cells reached 100% confluency, ml from 30ml cell lysis /150 mm-dish was added with DMEM (no FBS) to 5 ml /150 mm-dish, and incubated at 37 C-5% CO2 for hour with 15-minutes intervals rocking to redistribute the medium. Later 25 ml medium was added to 30 ml/dish in total and incubated for 3 days. The cells were harvested 3-days post infection at full CPE (cytopathic effect) by centrifugation. The supernatants were collected and autoclaved. The pellets were resuspended with 1ml/dish in HBS buffer (10 mm HEPES, ph7.4, 150 mm NaCl). The samples were stored at 80 C. Before gradient separation, the stored samples were taken through three freeze/thaw/vertex (several seconds) cycles in alcohol-dry ice/37 C water-bath to release the virus from the cells. After centrifugation at 4000 rpm for 5 minutes at 4 C, the supernatants containing viruses were loaded onto the caesium chloride (CsCl) gradient. The samples should be kept at 4 C or on ice from this point on. 10ml light CsCl solution (1.2 g/ml) was pipetted into a 38ml ultracentrifuge tube (BECKMAN 25 x 89mm). Took up 8 ml heavy CsCl (1.46 g/ml) by using 10-ml syringe with 10-cm needle and inserted to the bottom of the 38 ml tube and carefully dispensed the heavy CsCl. After loading the viral stock on the top of the CsCl gradient (maximum ml) and checking the balance, centrifuged at rpm (KONTRON-TST 28.38/17 rotor) for 2 hours at 4 C without brake. Using 18-gauge needle and 5 ml syringe, punctured the tube under the viral band and fetched approximately 2.5 ml 0f virus-containing solution per a tube.

23 21 If the second-round purification was needed, the collected band should be diluted with equal volume of chilled 10 mmtris-buffer. Prepared the gradient in a 12 ml-tubes as above, but 4 ml-light CsCl, 3 ml-heavy CsCl and 5 ml-diluted samples. After centrifugation at rpm (TST rotor) for 1 hour at 4 C without brake, the virus band (0.8 ml /tube, totally 2.5 ml) was collected as above. In order to remove the CsCl in the viral stock, a NAP-25 column (Sephadex G-25 Medium, Pharmacia Biotech) was equilibrated with 5 ml x 5 times of HBS and 2.5ml samples were loaded onto the column. First 0.5 ml HBS was added to the column without collecting the flow-through. The samples were eluted with 3.0 ml of HBS and brought to the final concentration of 40% glycerol, 10 mm Hepes, 150 mmnacl, 0.1% BSA. The viral stock was aliquoted and stored at -20 C. Physical titers were determined via absorbance at 260 nm. Infectious titers were determined by plaque assays on 293 cells (Gall, 1998). When the viral stocks were kept at 20 C more than 24 hours, if the pellet of virus particle formed at the bottom of the tube, the reamplification and purification need repeat. This meant the purified virus had lost their activity, usually due to the exposing to room temperature for a long time during manipulation. 3.5 Infection of cultured cells. Cells were counted after trypsin digestion using a hemocytometer. Virus stock aliquots containing the appropriate number of pfu to obtain the desired multiplicity of infection (moi) were mixed with 2 ml per 10 cm dish Biocoat medium (PTHs), or DMEM (Huh7 and HepG2) and added to the cells. After 1.5 h at 37 C with occasional rocking medium was added to 10 ml. Media were changed every two days. Conditioned media were stored at 4 C. Cells were harvested at the desired times, washed with TBS buffer (25 mm Tris-HCl, ph 7.4, 137 mm NaCl, 2.7 mm KCl), and stored at 80 C. Total protein concentration in cell lysates was determined using the Bradford assay (BioRad).

24 Assay of secreted HBV antigens. HBsAg was determined using the HBsAg monoclonal II ELISA kit (Dade-Behring). Total amounts of HBsAg were calculated by comparison with serial dilutions of a standard HBsAg-positive human serum (gift of W. Gerlich, University of Giessen). HBeAg was assayed using the IMx HBe 2 kit (Abbott) and compared to positive and negative control sera provided by the manufacturer. 3.7 Extracellular HBV DNA detection Dose dependence Adult tuapaia hepatocytes, prepared as above, were seeded in 10 cm-dish. On the second day, cells were counted and infected with 5, 25, 125, and 625 moi of Ad- HBV1.3. The cells were maintained as above. Culture media from day 7 to 8 (48 hours) were collected and 9 ml were used to extract DNA and southern blotting as below Time course analysis Tupaia liver cells were manipulated as above and infected with 100 moi of Ad- HBV1.3. Five milliliters medium was taken for each time course analysis, respectively. For enzymatic enrichment of HBV DNA contained in enveloped particles a procedure similar to a previously reported method was used (Wei, Tavis, and Ganem, 1996). In brief, particles from 5 to 20 ml culture medium were precipitated by 10% polyethylene glycol (PEG 8000) for one hour on ice. After centrifugation, the pellets were resuspended in 800 µl of 50 mm Tris (ph 8.0). Mg 2+ acetate was adjusted to 5 mm and incubated with 750µg/ml pronase at 37 C for 1 hour, and then with 500 µg/ml DNase I for 1 hour. The samples were adjusted to 15 mm EDTA, 0.5% SDS and 500 µg/ml proteinase K, and incubated at 45 C for 1 more hour. After phenol extraction the liberated nucleic acids were precipitated with ethanol and resuspended in TE

25 23 buffer. For Southern blotting, the DIG nonradioactive nucleic acid labeling and detection system was employed (Roche Molecular Biochemicals). The DIG-labeled EcoRI-cut HBV whole genome probe was used to detect HBV DNA samples blotted on positively charged nylon membrane. The protocols for detection were according to the manufacture's recommendation. 3.8 Intracellular replication-competent core particles analysis HBV DNA detection by Southern blotting To release intracellular cores cells were resuspended in lysis buffer (50 mm Tris-HCl, ph 8.0, 10 mm EDTA, 100 µg/ml RNase A, 0.7% NP-40) and incubated at 37 C for 15 minutes. Nuclei and cellular debris were removed by centrifugation. To the supernatants, Mg 2+ acetate (final concentration 10 mm) and DNase I (final concentration 500 µg/ml) were added and incubated at 37 C for 45 minutes to digest nonencapsidated DNA. For native agarose gel electrophoresis, about 1 % of the lysate was loaded on a 1% agarose gel. Gels were blotted on positively charged nylon membrane (Roche Diagnostics) by capillary transfer in TNE buffer (10mM Tris- HCl, ph7.5, 150 mm NaCl, 1mM EDTA). After soaking in 0.2 N NaOH / 150 mm NaCl and neutralization in 0.2 M Tris-HCl, ph M NaCl (5 min each) the membrane was fixed by UV-crosslinking (Stratagene UV1800) and briefly rinsed with water. HBV specific nucleic acids were detected using a DIG-labeled probe as above HBcAg immunoblotting assay The same amounts (1%) of Ad-infected cell lysates as above were separated on an agarose gel and blotted on PVDF membrane (Amersham-Pharmacia) by capillary transfer in TNE buffer. Core protein was detected using the polyclonal rabbit anti- HBcAg antiserum H800 (Birnbaum and Nassal, 1990) and a peroxidase-conjugated secondary antibody with a chemiluminescent substrate (ECL-Plus, Amersham- Pharmacia).

26 3.9 Virions in the culture medium 24 Twenty-five ml of conditioned medium was aliquoted into a 30-ml Falcon tube and centrifuged at 9000 rpm (SS-34 rotor) for 10 minutes at 4 C. Appropriate solid PEG (final concentration of 10% of PEG) was added into another 30 ml-tube and mixed well with the supernatant. After melting completely (incubate at 37 C for several minutes), the mixture was Incubated on ice at least for 1 hour (or overnight). Centrifuged at rpm for 20 min at 4 C and removed the supernatant completely. The pellets were resuspended in 1.5 ml of 50 mm Tris-HCl, ph 8.0, added DAase I to 500 µg/ml, MgAc to 5 mm, and Rnase A to 200 µg/ml, and incubated at 37 C for 1 hour. During the digestion, the CsCl gradient (5 steps each of 2 ml from 1.1 to 1.5g/ml) was prepared as Fig. 10A. The mixture was loaded on CsCl step gradients and centrifuged at rpm for more than 16 hours at 4 C in a Kontron TST rotor. The samples of 0.5 ml/fraction were carefully fetched (total 23 fractions) and stored at 4 C. The densities were measured by using refractometry HBsAg ELISA analysis HBsAg ELISA was detected as above by using 0.02 to 50 µl samples from each fraction per well. The HBsAg-standard was employed to determine the quantities of HBsAg in each fraction Western blot analysis of HBsAg, HBVpreS1, HBVpreS2 and HBcAg. Proteins within 5 µl of CsCl gradient from fractions 7 to 18 were separated by SDS- PAGE (12.5 % polyacrylamide, 0.1 % SDS) and blotted to PVDF membranes (BioRad). Individual envelope proteins were detected using the following antibodies: human monoclonal antibody 4/7B (Paulij et al., 1999), recognizing an epitope around aa of S protein (gift of R.A. Heijtink, Organon Teknika, NL); mouse monoclonal antibody S26 (Meisel et al., 1994), specific for an epitope around the motif QDPR in the pres2 region (provided by V. Bichko, Scriptgen, MA); mouse monoclonal MA18/07 (Heermann et al., 1984; Sominskaya et al., 1992), directed

27 25 against an epitope close the N terminus of the pres1 domain (gift of W. Gerlich), and H800 to HBcAg as above. Detection was performed using peroxidase-conjugated secondary antibodies and the ECL-Plus system Detection of HBV DNA by Southern blot To extract the nucleic acids from the density-gradient 250µl sample from the fraction 7 to 18, respectively, were digested by adding 580 µl of 50 mm Tris-HCl (ph 8.0), 9 µl of 0.5 M EDTA (5 mm EDTA final), 45 µl of 10% SDS (0.5% SDS final), and 23 µl Proteinase K (500 µg/ml final) at 45 C for 1-3 hour. The nucleic acids were extracted by 900 µl of phenol-chloroform and precipitated by 2 µl glycogen, 40 µl of 5 M NaCl (0.2 M final) and 1 ml of isopropanol. After resuspending the pellets in 15 µl TE, the samples were electrophoresed on a 1% agarose gel and assayed by Southern blotting with DIG-labeled HBV probe as above Nuclear cccdna detection Dose-dependence Adult tupaia liver cells were prepared as described above. On the second day post preparation, cells were infected with Ad-HBV1.3 by 5, 25, 125, 625 and 1250 moi, respectively. Cells were harvested at different time points: 7 days post infection of the former four doses, 5 days of the later two doses (because of CPE). Huh7 cells were transduced with 5, 25 and 125 moi of Ad-HBV1.3 and harvested on day 7 post infection Time-course analysis Cells were seeded and infected as above. For the time-course analysis, the tupaia hepatocytes were infected with 100 moi. Cells were maintained for 18 days and harvested in every two days post infection. Episomal DNAs including HBV cccdna were prepared by a modification (J. Summers, personal communication) of a previously described protocol (Yang,

28 26 Mason, and Summers, 1996). In brief, cells were resuspended in lysis buffer containing 0.2% NP-40, mixed with an equal volume of 0.15 N NaOH containing 6% SDS, incubated at 37 C for 20 min, and neutralized by adding 3M K + acetate ph 5.5 to a final concentration of 0.6 M. After 30 min on ice, cellular debris and chromosomal DNA were removed by centrifugation at 20,000 g for 15 min at 4 C. After phenol extraction, soluble nucleic acids in the supernatant were precipitated by adding 0.7 volumes of isopropanol. Pellets were resuspended in 500 µl of restriction buffer 4 (New England Biolabs) containing 40 U/ml of Hpa I, 400 µg/ml RNase A, and 300 U/ml Plasmid-Safe DNase (Epicentre Technologies), and incubated at 37 C for 4-6 hours. Subsequently, a second NaOH treatment was performed by adding 0.2 N NaOH to 0.05 N final concentration. After 10 min at 37 C, the reaction was neutralized by adding 3M K + acetate to 0.6 M final concentration, the samples were extracted with phenol, and nucleic acids were ethanol precipitated. Southern blot analysis was performed as described above In vivo infection of tupaias with recombinant adenoviruses Newborn tupaias, 3 to 5 days old, were injected into the tail vein with µl of purified Ad-HBV1.3 or Ad-GFP-HBV1.3. Total volume was adjusted to µl using PBS. Sera were collected every 7-10 days post injection within the first three months, and every days three months later. Samples were stored at 80 C. For adult tapaia infection 6 months to 1.5 years old tupaias were intravenously (tail or rear leg veins) injected with viral stock diluted to 500 µl with PBS. Sera were collected every days post infection. Liver biopsies were performed 7 days post injection and liver tissues were divided, frozen in liquid nitrogen and stored at 80 C or fixed with 10% formaldehyde.

29 ELISA analysis of HBsAg and HBeAg All of the animals infected with recombinant adenoviruses produced HBsAg as detected by HBsAg-ELISA assay as described above. The dynamic changes of HBsAg in sera were analyzed in two newborn tupaias (B201 and A6B6) that were infected with Ad-HBV1.3. HBeAg in serum was also determined in the two baby animals as above Assay of Anti-HBs in sera from infected tupaias The Anti-HBs in sera from tupaias infected with recombinant adenoviruses was assayed by a Anti-HBs kit (Enzygnost Anti-HBs II, Dade-Behring) according to the manufactory recommendation Detection of HBV DNA in the sera The HBV DNAs were purified from sera by proteinase K digestion and phenol extraction. In brief, 50 to 60 µl of sera ten-day post infection were diluted into 500 µl of digestion buffer (50 mm Tris, ph8.0, 10 mm EDTA, 0.9 % SDS, and 1 mg/ml proteinase K) and digested at 45 C for 5 hours. The nucleic acids were extracted with an equal volume of phenol and chloroform. DNA was precipitated with isopropanol. The nucleic acids were analyzed by Southern blotting as described above Biosafty analysis of recombinant adenovirus Nose-washing samples Possible shedding of recombinant adenovirus in the airway epithelium was monitored in two female adult tupaias after injection with Ad-GFP-HBV1.3. One tupaia, number 45C2, was infected with 100 µl (4-5 x pfu/m) viral stock, the other, number B4B4, with 20 µl (1 x of pfu/ml). The nose-washing samples were collected by using a sterile-plastic-stick to take a drop of washing solution (DMEM with 100 IU of penicillin-100 µg of streptomycin/ml) into the tupaia nostril, absorb the liquid with a

30 28 swab, and elute the swab in 1 ml of the washing solution. The samples at time points of 1, 3 and 27 hours, 2, 3, 6 and 7 days post infection were collected and stored at 4 C until used to infect 293 cells. The nose-washing samples were filtered through 0.45 µm filters and left on the 293 cells for 3 three hours. Possible formation of GFP positive cells and/or plaques was monitored by immune fluoresence microscopy at 3, 5, 7 and 10 days post infection Sera samples An adult female tupaia, C115, was infected with 50 µl (2-2.5 x of pfu/ml) viral stock by injection into the left leg vein. One serum sample was collected before injection, then sera samples were collected at 2, 4, 6, and 24 hours, and 4, 7 days post infection, respectively. The sera of 20 µl from each sample were used to infect 293 cell. Seven days later, the animal was killed and several kinds of tissues were collected. One lobe of liver cells was prepared, seeded and cultured as above.

31 29 4 RESULTS 4.1 Construction of recombinant Adenovirus vectors carrying complete HBV genomes In order to test whether all HBV control elements are functional in PTHs a 1.3x overlength HBV genomes (subtype ayw) was used for generating the recombinant vectors. They imitate the circular HBV cccdna as transcriptional template by having duplicated terminal regions (Fig. 5). The borders were essentially the same as on an overlength genome used to generate HBV-transgenic mice producing complete virions (Guidotti et al., 1995). The high liver-specificity of most of the authentic hepadnaviral control elements significantly adds to the biosafety of the Ad-HBV vectors. The previously tedious methods for generating recombinant Ad-vectors have been superseded by procedures in which homologous recombination between a small, manipulatable shuttle plasmid and a large adenovirus backbone plasmid is entirely performed in a suitable E. coli strain (Fig. 5). The resulting plasmid derivative of the recombinant Ad-vector genome is characterized by conventional molecular biology methods, and transfection of the excised vector genome into suitable eukaryotic cells gives rise to a clonal population of Ad-vector particles. The vectors used in this study, human adenovirus type 5 (group C), lack the E1A and E1B regions essential for initiating adenovirus replication and, in addition, the E3 region involved in subverting the host s immune response (Wold and Chinnadurai, 2000). Their replication is therefore restricted to a complementing cell line such as 293. Two different systems were used. The first was that reported by Chartier et al. (Chartier et al., 1996). The 1.3x HBV DNA was cloned into the shuttle plasmid ptg9530 and recombined with the adeno-backbone plasmid ptg4656 (see Fig. 5). The second was the AdEasy system (He et al., 1998), which utilizes a more

32 30 advanced antibiotic selection scheme and, in addition, provides an enhanced green fluorescent protein (egfp) cassette in the adeno part. This allows for direct monitoring of virus production and infection efficiency by fluorescence microscopy. The yields of desired plasmid recombinants were variable with the first system (2-70% positive among 11 different constructs, including WHV or HBV-mutation constructions). Sometimes colony hybridization was required to identify several potential positives within about 100 colonies. An Ad-HBV1.3 was identified within 8 colonies (12.5%) by restriction enzyme analysis. The second system was more robust, yielding between one and four positives among 10 plasmids analyzed. One well-defined clone obtained by each method was used to generate Ad-HBV1.3 and its counterpart Ad-GFP-HBV1.3 (Fig. 5). 4.2 Amplification and purification of recombinant adenoviruses After several rounds of amplification through 293 cell infection, and after concentration on CsCl gradients, both yielded infectious Ad-HBV1.3 and Ad-GFP- HBv1.3 virions. In the 293 cell plaque-assay, 3 x plaque-forming units (pfu) per ml of Ad-GFP-HBV1.3, and 5.5 x pfu/ml of Ad-HBV1.3 were determined. Meanwhile the purification and storing conditions were optimized during preparation of several different recombinant adenoviruses. The second round of CsCl-gradient concentration and removal of CsCl through a Sephadex G-25 Medium column did not seem to reduce the activity of adenovirus. However, temperature was an important factor for getting high titer of viral stocks. After release from the cytoplasm of 293 cells, adenovirus-containing samples should be kept on ice. Each manipulation step should be finished as fast as possible. These optimizations increased the yield of the ratio of viral particle number (based on particles/od m 260 unit) to pfu from 1:50 to 1:5 (usually 1:20 50). Among the 11 different recombinant adenoviruses, the titers ranged from 3 to 70 x pfu/ml. As an example, after removing CsCl through the column, one aliquot of Ad-HBV1.3 viral sample was mixed with storing buffer and