Human Interleukin-6 Facilitates Hepatitis B Virus Infection in Vitro and in Vivo

Virology 270, 299–309 (2000) doi:10.1006/viro.2000.0210, available online at http://www.idealibrary.com on

Human Interleukin-6 Facilitates Hepatitis B Virus Infection in Vitro and in Vivo Eithan Galun,* ,† ,1 Orit Nahor,* Ahmed Eid,‡ Oded Jurim,‡ Stefan Rose-John,§ Hubert E. Blum, ¶ Ofer Nussbaum,㛳 Ehud Ilan,㛳 Nili Daudi,* Daniel Shouval,* Yair Reisner,** and Shlomo Dagan㛳 *Liver Unit and †Goldyne Savad Institute of Gene Therapy, Hadassah University Hospital, Ein-Kerem, Jerusalem 91120, Israel; ‡Department of Surgery, Hadassah University Hospital, Jerusalem, Israel; §Medical Clinic-Section Pathophysiology, Mainz University, Mainz, Germany; ¶ Medizinische Universitatsklinik Hugstetter, Freiburg i.Br., Germany; 㛳XTL Biopharmaceuticals Ltd, Kiryat Weizmann, Rehovot, Israel; and **Department of Immunonology, The Weizmann Institute of Science, Rehovot, Israel Received October 28, 1999; returned to author for revision December 17, 1999; accepted January 20, 2000 Background and aim. Research on hepatitis B virus (HBV) infection in vivo has been limited due to the absence of a suitable animal model. We have developed a human–mouse radiation chimera in which normal mice, preconditioned by lethal total body irradiation and radioprotected with SCID mouse bone marrow cells, are permissive for engraftment of human hematopoietic cells and solid tissues. This resulting human–mouse model, which comprises three genetically disparate sources of tissue, is therefore termed Trimera. This study was aimed at assessing the effect of human IL-6 on HBV infection in vivo in Trimera mice. Methods. Trimera mice were transplanted with human liver tissue fragments or with HepG2-derived cell lines, which had been previously infected ex vivo with HBV in the presence or absence of human interleukin-6 (hIL-6) and in the presence of anti-IL-6-neutralizing antibodies. Results. HBV sequences appeared in the sera of animals in which the liver tissue was incubated with both HBV and hIL-6 prior to transplantation. A similar result was obtained when a human hepatoblastoma cell line (HepG2), expressing the hIL-6 receptor, was infected ex vivo with HBV in the presence of hIL-6 prior to their injection into spleens of Trimera mice. However, when liver fragments were infected ex vivo and simultaneously treated with neutralizing antibodies against hIL-6 or were incubated with HBV prior to transplantation without hIL-6, the rate of mice positive for HBV DNA in their sera was lower. Human mononuclear cells are also permissive for HBV infection in vitro: in the presence of hIL-6 the infection of these cells is enhanced; and this infection is suppressed by the chimeric protein named Hyper-IL-6, generated by the fusion of hIL-6 to the soluble hIL-6 receptor (sIL-6R␣, gp80). Conclusion. hIL-6 facilitates HBV infection in vitro and in vivo. © 2000 Academic Press Key Words: viral receptor; viral infection; chimeric mice.

showing that human interleukin-6 (hIL-6) binds to the pre-S1 (aa 21–47) segment of the HBV envelope. Putative candidates for HBV receptors, including annexin V (Hertogs et al., 1993), apolipoprotein H (Mehdi et al., 1994), and asialoglycoprotein receptor (Treichel et al., 1994), have recently been reported. Binding experiments demonstrated that the pre-S1 region of the viral envelope protein contains a recognition site for the host cell (Neurath et al., 1986; Petit et al., 1990, 1992). Although previous studies have suggested that HepG2 cells (Bchini et al., 1991) and human hepatocytes (Galle et al., 1994; Gripon et al., 1988, 1993; Ochiya et al., 1989) could support HBV infection in vitro, no cellular receptor has yet been defined in either system. Previous investigations conducted in an attempt to establish HBV infection in small animal models (Takahashi et al., 1995; Walter et al., 1996) resulted in a very short viremia of a few days, followed by early seroconversion with the development of anti-HBs antibodies. Alternative models for chronic HBV viremia using transgenic mice, expressing the complete HBV genome, as a transgene, were constructed (Guidotti et al., 1995). Al-

INTRODUCTION Hepatitis B virus (HBV) infection in humans can cause chronic liver disease, which may in some cases progress to hepatocellular carcinoma. The initial steps of HBV attachment to cells and the targeting of the viral genome to the host cell nucleus have yet to be elucidated. Thus, a specific receptor for HBV has not been identified, even though various serum proteins and cellular membrane glycoproteins have been suggested to mediate cell penetration or viral receptor, respectively. HBV envelope proteins were reported to contain residues capable of interacting with polymerized albumin (Pontisso et al., 1981) enabling virus penetration into cells via their respective receptors, probably in a nonspecific manner. HBV-related proteins interact with human serum proteins, as reported by Neurath et al. (1992),

1 To whom correspondence and reprint requests should be addressed at Goldyne Savad Institute of Gene Therapy, Hadassah University Hospital, Ein-Kerem, POB 12000, Jerusalem 91120, Israel. Fax: ⫹972-2-6430982. E-mail: [email protected].

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0042-6822/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.

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though such transgenic animals are an important tool for understanding the biology of the virus and the effect of various components of the immune systems in the inflammatory reaction in the liver related to HBV infection (Guidotti et al., 1996), the early steps of infection could not be assessed in these animals. Infection models have been established for avian and mammalian members of the hepadnavirus family other than HBV, in vitro (Tuttleman et al., 1986) as well as in vivo (Mason et al., 1983). However, the viral attachment mechanism of each member of this viral family is probably different, as suggested from genomic comparison (Sprengel et al., 1984) and reports on the duck hepatitis B virus receptor (Breiner et al., 1998; Ishikawa et al., 1994; Tong et al., 1999; Urban et al., 1998) and coreceptor (Li et al., 1996, 1999). Recently we have shown that normal human liver tissue that is incubated ex vivo with HBV and then transplanted under the kidney capsule of Trimera mice can support the generation of viremia for a number of weeks (Ilan et al., 1999). The experimental strategy for the development of a small animal model for HBV infection was based on the evidence that although the primary infection site for HBV are hepatocytes, lymphocytes (Korba et al., 1986; Laskus et al., 1997, 1999; Romet Lemonne et al., 1983), and endothelial cells (Blum et al., 1983; Mason et al., 1993) have both been shown to harbor HBV transcripts and viral related proteins or pre-S1 binding protein (Pontisso et al., 1991), suggesting a common specific cell membrane receptor mechanism that supports viral penetration on all three cell types. The tissue specificity is probably related to the early steps of infection, as suggested from experiments suggesting that receptor-negative cells are permissive to HBV replication following transfection (Galun et al., 1992). All three cell types that are naturally susceptible to HBV, i.e., hepatocytes, lymphocytes, and endothelial cells, also respond to IL-6 through the human IL-6 receptor (hIL-6R) (Bauer et al., 1989; Kishimoto et al., 1992; Mackiewicz et al., 1992). In addition, hIL-6 binds to HBV through pre-S1, as previously shown (Neurath et al., 1992). Human IL-6 is a member of the family of four-helical cytokines with pleiotropic biological effects. In the liver, IL-6 modulates the transcription of acute-phase response genes and stimulates liver regeneration. IL-6 binds to a specific IL-6R␣ (gp80) and subsequently induces homodimerization of gp130 (IL-6R␤). We hypothesized that hIL-6 might have a role in HBV infection, directly or indirectly. In this study we have employed our HBV Trimera system (Ilan et al., 1999) in order to investigated the possible role of hIL-6 in HBV infection. RESULTS Human IL-6 supports ex vivo. HBV infection of human liver in Trimera mice. Human liver tissue fragments were incubated with HBV DNA-positive sera in the presence of hIL-6. After infection these fragments were transplanted

FIG. 1. hIL-6 mediates HBV viremia in SCID ⬎ BNX chimeric mice transplanted with human tissue. PCR amplification products of HBV precore/core region following DNA extraction from sera of mice, 16 and 31 days after subcapsular kidney transplantation of normal human liver fragments. The human liver fragments were incubated ex vivo with human HBV-positive serum prior to transplantation (A) or with HBV DNA-positive serum and hIL-6 simultaneously (B) or liver fragments were initially preincubated with hIL-6 and later with HBV (C). In each section (A to C), the top panel is an EtBr staining, and the bottom panel shows the results of Southern hybridization analysis of the same gel using a 32P-labeled HBV DNA probe. The molecular marker size (m) is indicated by an arrow; numbers at the head of each panel indicate mice ID No.; ⫹, positive serum control; and ⫺, negative serum control.

in Trimera mice. HBV DNA sequences were detected from day 16 to day 31 posttransplantation in the sera of about 50% (9/19, animals 14 to 19 were negative and are not shown in the figure) of the transplanted animals (Fig. 1B). Incubation of liver tissue with hIL-6 prior to ex vivo HBV infection increased the efficiency of infection to about 80% (9/11) of transplanted animals (Fig. 1C). In contrast, animals transplanted with liver tissue fragments incubated ex vivo with HBV-positive sera, in the absence of exogenous hIL-6, remained free of detectable levels of HBV DNA sequences for a period of at least 31 days posttransplantation (Fig. 1A). The number of ani-

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FIG. 2. HBsAg staining of an ex vivo HBV incubation of a normal liver fragment with hIL-6, 1 month following implantation under the kidney subcapsule of SCID ⬎ BNX chimeric mice.

mals in group A for both day 16 and day 31 in a number of experiments is over 4; additional experiments are described in the following sections. Immunohistochemical analysis revealed that animals, which contained HBV sequences in their serum at day 31, also expressed HBsAg in the implanted livers (Fig. 2). It is hard to determine the exact nature of the cells in which HBsAg is expressed. According to our previous publications and unpublished data on hepatitis C virus (HCV) and HBV infection in Trimera mice it is suggested that the HBsAg-positive cells are probably hepatocytes. The absence of detectable HBsAg staining in liver fragments incubated ex vivo with HBV under the above conditions and fixed for immunohistochemical analysis prior to transplantation, i.e., negative controls, indicates that the HBsAg observed in 31 days posttransplantation in HBVpositive mice is most likely due to de novo HBsAg synthesis. Subsequent experiments utilizing over 350 Trimera mice transplanted with liver fragments, following ex vivo incubation with HBV-positive serum without addition of exogenous hIL-6, resulted in an overall infection efficiency of 30% (hIL-6 levels in the sera of patients that

was our source of HBV per infection was in the range of 5 to 120 pg/ml). To further assess the facilitating role of hIL-6 in HBV infection, mice sera were analyzed for HBV DNA sequences, following infection. Human liver fragments were infected ex vivo with HBV following short incubation with hIL-6 and then transplanted under the kidney capsule of the chimeric mice. HBV DNA was measured in sera of these mice on days 11, 18, and 25 posttransplantation. The addition of hIL-6 increased the rate of infection by more than 50% compared to the control group infected without the cytokine, as shown in Fig. 3. This increase is detectable at days 11 and 18 after transplantation. In long-term experiments, circulating HBV DNA sequences were detected up to 2 months following transplantation (data not shown). The effect of anti-hIL-6 on HBV infection. Figure 3 shows that HBV sequences were detected also in the group of animals in which the liver fragment was not incubated with hIL-6 prior to transplantation. One possible explanation could be the presence of hIL-6 in sera of HBV-infected patients, which is our viral source for these experiments. In order to reduce the possible effect of the

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FIG. 5. Detection of HBV ccc DNA in human liver fragments preincubated with hIL-6 and HBV and implanted in Trimera mice. Two weeks posttransplantation DNA was extracted from fragments, pooled, and analyzed for the presence of HBV ccc DNA and HBV RC DNA by dot blot hybridization as described under Material and Methods. Lane 1, pooled DNA from five grafts harvested from infected Trimera mice; lane 2, pooled DNA from five grafts harvested from noninfected Trimera mice; lane 3, DNA from intrinsically HBV-infected human liver tissue; lane 4, DNA from human liver tissue; lane 5, DNA from mouse kidney tissue. FIG. 3. Detection of HBV DNA in mice sera at days 12, 18, and 25, following the transplantation of normal human liver tissue which was ex vivo incubated with HBV infectious serum in the presence (black bars) or the absence (gray bars) of hIL-6.

intrinsic hIL-6, which could have originated from the HBV infectious serum and/or the human liver tissue, mice were transplanted with liver fragments infected with HBV serum that had been pretreated with a monoclonal antibodies against hIL-6 prior to the incubation with the liver tissue. In this experimental group of animals, the infection step was also without the addition of hIL-6. Control mice were transplanted with HBV-infected liver fragments using the previously described protocol in the presence of exogenous hIL-6. The results in Fig. 4 show

FIG. 4. The effect of anti-hIL-6 on HBV infection in chimeric mice. The experimental protocol is described in the text. Detection of HBV DNA in mice sera at days 10, 17, and 24, following the transplantation of normal human liver tissue which was ex vivo incubated with HBV infectious serum in the presence of hIL-6 (black bars) or in which the HBV infectious serum was initially treated with anti-hIL-6-neutralizing monoclonal antibodies and then incubated with the liver tissue in the absence of hIL-6 (gray bars).

that in the presence of antibodies to hIL-6, the infection rate is reduced. Thus, for this experiment, added hIL-6 increased the efficiency of infection by ⬃200% for day 10, ⬃140% for day 17, and ⬃400% for day 24 posttransplantation, compared to mice transplanted with liver fragments infected with anti-hIL-6-treated virus. In experiments described in Figs. 3 and 4 as well as in all other experiments described with the “Trimera” system all animals were infected with the same serum and transplanted with the same liver tissue. Detection of HBV ccc DNA in implanted livers preinfected with hIL-6 and HBV. The small size of the implanted liver fragment restricted our methods for HBV replication to those techniques combining amplification steps to detect HBV replication intermediates. Animals positive for HBV DNA in sera were also analyzed for viral replication in implanted liver fragments applying specific primers for HBV ccc DNA. As shown in Fig. 5 HBV ccc DNA was detected in implants preincubated with HBV and human IL-6 and only in mice positive for HBV DNA in serum. The HBV ccc DNA species was not detected in implants from animals in which HBV DNA was not detected in serum. Induction of HBV DNA viremia in Trimera mice by transplantation of HBV-infected HepG2-derived cell lines. Mice were implanted with HepG2-hIL-6R␣ (stably transfected HepG2 cells with hIL-6 receptor ␣) subsequent to incubation with HBV, in the presence of hIL-6. The cells were then injected into the spleen of mice and migrated through the portal system to the liver (Fig. 6). HBV DNA sequences were analyzed in serum 13 days after transplantation by PCR. The results of these experiments are summarized in Table 1. HBV sequences were not detected in sera of mice that were treated similarly but without hIL-6. Viremia could not be detected also in mice transplanted with HepG2-PDI cells (which do not express the gp80-binding protein (Ehlers et al., 1994; RoseJohn et al., 1993), but do express the gp130 subunit of the hIL-6 receptor (Dittrich et al., 1994). However, in experi-

HBV INFECTION IN VIVO TABLE 1 HBV DNA Sequences in Sera of Chimeric Mice Following Intrasplenic Injection of HepG2 and HepG2-Derived Cell Lines Incubated ex Vivo with HBV with or without hIL-6 Cell lines HepG2 With (⫹) or without (⫺) hIL-6 HBV DNA Positive

⫹ 2/2

⫺ 2/2

HepG2-IL6-R

HepG2-PDI

⫹ 4/4

⫹ 2/2

⫺ 0/2

⫺ 0/2

Note. Mice were implanted with HepG2 and HepG2-derived cell lines subsequent to incubation with HBV in the presence or absence of hIL-6. Following the ex vivo incubation of the cells, they were injected into the spleen of mice and migrated through the portal system to the liver (Fig. 6). HBV DNA sequences were detected in serum 13 days after transplantation. All parts of the experiment were conducted with the same HBV infectious serum.

ments in which HepG2 cells were incubated with HBV in the presence or absence of hIL-6, HBV DNA sequences could be detected in mice sera. The effect of hIL-6 and hyper-IL-6 (HIL-6) on HBV infection of human mononuclear cells. To further determine the effect of hIL-6 on HBV infection an additional human cell lineage was assessed for its permissiveness for HBV infection in the presence of this cytokine. From previous studies we had learned that lymphocytes support HBV infection and also harbor HBV-integrated sequences (Korba et al., 1986; Laskus et al., 1997, 1999). Lymphocytes were also shown to express the IL-6␣ and ␤ receptors (Peters et al., 1998). The fact that human lymphocytes express the human IL-6 receptor and are permissive for HBV infection suggested to us that these cells could be applied to evaluate the role of hIL-6 in viral entrance to cells. Incubation of HBV with human mononuclear cells resulted in the generation of RC HBV DNA from day 4 postinfection. The highest level of HBV DNA was detected at day 7 and decreased late after, as shown for both RC and ccc DNA species. In other experiments we were able to detect HBV DNA, RC, and ccc up to 21 days following infection (data not shown). HBV RC DNA was detected earlier and the level of HBV ccc DNA was higher when the virus was incubated with hIL-6 (Fig. 7). However, when human mononuclear cells were incubated with HBV in the presence of hyper-hIL-6 (IL6/ sIL6R␣ fusion protein, Fischer et al., 1997), RC and ccc HBV DNA viral species were reduced to undetectable levels. The morphology of the HBV-infected and noninfected human mononuclear cells as well as their number at each time point was identical for all three groups. DISCUSSION In our previous reports, we have shown that Trimera mice are permissive for the growth of normal human

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tissue. Human tissues transplanted into these animals continue their differentiated state as in the host animal (Lubin et al., 1991). This advantage was employed for the development of a HCV infection model in a small animal, with continuous viral replication for over 2 months (Galun et al., 1995), and the development of a new HIV infection model system (Shapira-Nahor et al., 1997). The possible role of hIL-6 in HBV infection and the potential application of the chimeric animal were both suggested in our recently reported in vivo HBV infection model (Ilan et al., 1999). The present study was aimed to further assess the potential role of hIL-6 in HBV infection in our HBV small animal model. The effect of hIL-6 on HBV infection is shown in the experiments in which HBV was incubated ex vivo with human liver fragments and resulted in generation of HBV sequences in the sera of transplanted chimeric animals. In these animals we were also able to detect a viral surface protein in the transplanted tissue (Fig. 2). In addition, when a hIL-6R␣-expressing cell line were incubated ex vivo with HBV and injected into the spleen of chimeric animals, HBV sequences were detected in mouse sera when hIL-6 was present. In all three cell lines the addition of hIL-6 to cells in the presence of HBV supported HBV replication (Table 1). Furthermore, antihIL-6-neutralizing monoclonal antibodies reduced the efficiency of HBV infection. However, we were also able to detect HBV sequences in some chimeric animals that were transplanted with liver tissue or injected with HepG2-derived cells in the absence of exogenous supplementation of hIL-6. Viremia observed in hIL-6-untreated animals may result from endogenous hIL-6, which is naturally present in human sera. The ability of mAb’s against hIL-6 to reduce the efficiency of infection supports this conclusion. These results are in agreement with those previously reported by Bchini et al. (1991), who showed that sera from HBV DNA-positive patients supported HBV infection of HepG2 cells in vitro. In addition to the possible effect of hIL-6 on the infection of hepatocyte lineage cells with HBV, we had investigated the role of hIL-6 on HBV infection of mononuclear hematopoietic cells. These cells were applied for the experiments knowing that they respond to hIL-6 and were shown to harbor on their cell membrane the hIL-6 receptor ␣ (gp80) and ␤ (gp130) (Peters et al., 1998). As a first step to this end we had developed an in vitro infection assay by incubating human lymphocytes in vitro with HBV-positive sera (Fig. 7). The amount of HBV increased until day 7 and decreased later on. In this infection system we were able to detect HBV ccc DNA. We were not able to detect HBsAg in these cells, a similar result to previous reported studies assessing HBV replication in mononuclear hematopoietic cells from HBV DNA-positive patients (Korba et al., 1986; Laskus et al., 1997, 1999). The supplementation of hIL-6 to the infection process facilitated the infection of lymphocytes

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FIG. 6. Liver histology of a HepG2-hIL-6R tumor that developed 1 month following intrasplenic injection into a SCID ⬎ BNX chimeric mice (H & E staining).

in vitro, enabling us to detect HBV DNA earlier and possibly to a higher level. Although the exact role of hIL-6 in HBV infection could not be determined from the results of this study, it could be speculated that hIL-6 might serve as a bridge for viral internalization (Hibi et al., 1990; Rose-John et al., 1991), by interacting with HBV (Neurath et al., 1992), and with the

hIL-6R␤ subunit gp130, through a membrane-anchored hIL-6R␣ subunit (gp80) (Kalai et al., 1997) or the secretory hIL-6R (shIL-6R) that later interacts with gp130 (Cichy et al., 1996; Mackiewicz et al., 1992; May et al., 1992; Somers et al., 1997). On the other hand, direct interaction between hIL-6 and the gp130 membrane glycoprotein has also been reported (D’Alessandro et al., 1993) and

FIG. 7. Infection of human lymphocytes with HBV. Human lymphocytes were incubated in the presence of hIL-6 or without the addition of the cytokine. Cells were kept in culture in the presence of PHA and IL-2. HBV incubation was also conducted in the presence of the superagonist HIL-6. RC and ccc HBV DNA species were analyzed in the cells.

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FIG. 8. Illustration of the possible interactions between HBV and hIL-6. This hypothesis argues that hIL-6 is a potential mediator for HBV infection in a direct or indirect mode. (Option I) HBV first interacts with hIL-6; this complex later finds the receptor for hIL-6 (gp80), the cell membrane-anchored type (hIL-6R␣), or the secretory type (shIL-6R␣). In both cases the viral–receptor complex attaches to the gp130 protein on the cell membrane. (Option II) hIL-6 through a signal transduction mechanism up-regulates an unknown factor to be expressed at the cell surface to enable viral attachment. A through D are potential site for antiviral agents to interfere with viral entry to cells.

could serve as another mode of attachment of HBV to the host cell. Alternatively, hIL-6 could mediate the activation of Jak-Tyk and STAT family protein kinases (Cressman et al., 1996; Lutticken et al., 1994; Narazaki et al., 1994; Stahl et al., 1994; Zhong et al., 1994) or the expression of various cellular proteins (Caldenhoven et al., 1994; Suto et al., 1993), that further increase the expression of an unidentified membrane receptor or ligand, respectively, thus facilitating HBV infection. Hyper-IL-6 is a superagonist to hIL-6 and as such increases the effect of this cytokine on hepatocytes in vitro (⬎100 fold) as well as in vivo (Maione et al., 1998). The result that the hyper-IL-6 (HIL-6) superagonist molecule inhibited HBV infection in vitro supports the possibility that the hIL-6R␣ is essential in the physical interaction between that virus and the cell membrane, i.e., a direct effect. In case that HBV infection was enhanced by HIL-6, we were not able to suggest if it was a direct or an indirect effect. Our working hypothesis on the possible relationship between the hIL-6 signal transduction system and HBV infection is illustrated in Fig. 8. Based on this hypothesis various modes of antiviral strategies could be employed. The interaction of hIL-6 with gp80 or gp130 could be blocked by peptides derived from gp80 or gp130 (Fig. 8, A1 and A2) and hIL-6 amino acid sequence (Fig. 8, C1, C2, and C3) or from those competing with hIL-6 interaction with pre-S1 (Fig. 8, B). Conjugating hIL-6 with various

components that could suppress HBV interaction with any molecule involved in the attachment to the cell membrane is also possible (Fig. 8, D). A different possibility to the role of hIL-6 in HBV infection would be that when the virus buds from the infected cells it incorporates the hIL-6R␣ into the viral envelope and through this integration enables the direct binding of hIL-6 with the virus on one side and the hepatocyte at the other. Additional experiments are needed to further elucidate the exact role of hIL-6, hIL-6R, shIL-6R, and gp130 in HBV infection. Once the early steps of viral infection, including critical factors involved in cell membrane attachment, are unfolded, new additional, antiviral strategies could be developed. MATERIAL AND METHODS Chimeric animals (Trimera mice). We have recently shown that a chimeric mouse, generated by using Beige/ Nude/X linked immunodeficient (BNX) mice preconditioned by total body irradiation (12 Gy) and reconstituted with severe combined immunodeficient (SCID) mice bone marrow (BM) cells, is permissive for normal human T and B cells (Lubin et al., 1991), as well as for normal human liver tissue (Galun et al., 1995). HCV viremia was

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detectable for up to 2 months after implantation under the kidney capsule of the BNX ⬎ SCID chimeric animals of either a human liver fragment with preexisting HCV infection or normal human liver tissue, incubated ex vivo with HCV-positive sera (Galun et al., 1995). Recently we were also able to establish the HBV in vivo infection model in Trimera mice (Ilan et al., 1999). This animal model was applied for the experiments described in this report. HBV infection protocol. We have recently described our HBV infection model using chimeric animals (Ilan et al., 1999). In brief, small fragments (1–3 mm 3) of normal human liver from a patient with no evidence of any HBV-related markers or disease (HBsAg and anti-HBc negative as well as HBV DNA by PCR negative) were selected for the experiments. Liver fragments were incubated ex vivo with 400 ␮l of a high-titer HBV DNApositive serum (⬎10 7 particles/ml) in 1 ml DMEM supplemented with 2 ␮g/ml polybrene, in the absence or presence of hIL-6 (500 ng/ml), for 24 h at 37°C, in a 5% CO 2 incubator. In each experiment described all groups of mice were infected with the same HBV infectious sera. After treatment, the liver fragments were repeatedly washed with DMEM and transplanted under the kidney capsule of chimeric animals. Blood was collected retrobulbarly from each mouse, and 100 ␮l of serum samples was treated with 0.5 mg/ml proteinase K in 10 mM EDTA and 0.25% SDS for 2 h at 55°C or overnight at 37°C and extracted twice with phenol, once with phenol–CHCl 3, and once with CHCl 3. DNA was precipitated with ethanol, using 0.1 vol 5 M NaCl and a DNA microcarrier. DNA was dissolved in 30 ␮l Tris–EDTA, pH 8.0, and was subjected to PCR amplification. In an alternative infection protocol a fragment of normal human liver from a patient with no evidence of any HBV-related markers of disease was infected ex vivo with hepatitis B virus. Small (1–3 mm 3) liver fragments were incubated with 1 ml human serum containing ⬃10 8 HBV particles and 3 ␮g polybrene for 1.5 h at 37°C. To test the effect of IL-6 we added into the infection mixture 500 ng of rhIL-6 (van Dam et al., 1993). In experiments designed to neutralize intrinsic IL-6 effects, the infection mixture was preincubated for 2 h at 25°C with 50 mg of monoclonal anti-human IL-6 antibody (Cat. No. MAB 206, R&D Systems Inc., Minneapolis, MN) in the absence of added IL-6, before the incubation with the liver tissue. Detection of HBV sequences in chimeric mouse serum. HBV sequences were detected by PCR using 20 ␮l of serum in 50 ␮l PCR reaction volume containing 10 pmol of each oligonucleotide primer in reaction buffer containing 10 mM Tris–HCl, pH 8.3; 50 mM KCl; 2.0 mM MgCl 2; 0.01% (w/v) gelatin; 250 ␮M dATP, dGTP, dCTP, and TTP; and 0.5 U of Taq polymerase (Promega, Madison, WI). The reaction mixtures were overlaid with 30 ␮l of mineral oil. PCR cycles included 94°C for 1 min, 55°C for 1 min, and 72°C for 3 min, 35 repeated cycles.

Oligonucleotides used for the precore/core amplification: oligo 1, sense (nt 1778 to 1806) 5⬘-GGAGGCTGTAGGCAAAATTGGTCTGCGC-3⬘; oligo 2, antisense (nt 2446 to 2408) 5⬘-CCCGAGATTGAGATCTTCTGCGACGCGGCGATT-GAG-ACC-3⬘. The sequence originated from an adw subtype; nt numbering starts from EcoRI site. The expected size of the PCR DNA product is 668 bp. The PCR samples were run on a 2% agarose gel, transferred to a nylon membrane (Biodynea), and hybridized with a 32 P-radioactive nick-translated probe. The autoradiogram was exposed, with intensifying screens, at ⫺70°C for 7 h. In order to confirm the PCR results, the mice serum samples were also subjected to PCR amplification with primers spanning the envelope gene region and showed the same results (data not shown). Reproducible results were obtained from similar experiments. There were 10–20 mice in each experimental group. Part of the experiments described were assessed by applying a short HBV DNA sequence detection system which we have recently developed (Klein et al., 1997). In brief, HBV DNA was extracted from 100 ␮l of mouse serum that was incubated in 400 ␮l of extraction solution (0.25% SDS, 5 mM EDTA, 10 mM Tris–HCl, pH 8, 250 mg/ml proteinase K) for 2.5 h at 65°C. A total of 100 ␮l of 5mg/ml of bovine serum albumin (BSA) solution was then added, followed by phenol/chloroform extraction and ethanol precipitation. DNA was collected, dried, and resuspended in 50 ␮l H 2O. PCR was performed using the same core primers in 100 ␮l of a PCR mixture containing 1⫻ Taq polymerase buffer (Promega), 2.5 mM MgCl 2, 0.2 mM each dNTP, 50 pmol of each primer, 1 mg/ml BSA, and 0.5 U of Taq polymerase. The PCR reaction was carried for 32 cycles, 1 min at 94°C, 1.5 min at 55°C, and 2 min at 72°C. The reaction was ended with 10 min at 72°C. Fifty microliters of the PCR mixture was dot-blotted and then hybridized with a 32P-labeled DNA fragment corresponding to nucleotides 1866–2290 of the core sequence, followed by exposure to an X-ray film. To prevent PCR product contaminations, the extraction of DNA and analysis of PCR products were preformed in separate rooms by different personnel. All known enzymatic agents were UV illuminated prior to use for over 10 min. All samplings were conducted using filtered tips. Determination of HBV ccc DNA in engrafted liver fragments. The method for the specific detection HBV ccc DNA was previously reported by Kock and Schlicht (1993) and Mason et al. (1998). We have applied this method with minor modifications. DNA was extracted from liver fragments (3 mm 3 each), using the DNAzol-BD reagent (Molecular Research Center Inc., Cincinnati, OH) (Lubin et al., 1991) and resuspended in 100 ␮l of H 2O. Fifty microliters was subjected to PCR using the 5⬘-end primer 5⬘-CCTCTGCCGATCCATACTGCGGAAC-3⬘ (HBV nucleotides 1257–1282, S2), chosen from within the HBV particle (⫹) strand deleted region, and the 3⬘-end primer 5⬘-GCTTGCCTGAGTGCAGTATGG-3⬘ (HBV nucleotides

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2075–2055, AS2). PCR was performed in a 100-␮l reaction mixture containing 1⫻ Taq polymerase buffer, 2.5 mM MgCl 2, 0.2 mM each dNTP, 50 pmol of each primer, 1 mg/ml BSA, and 2.5 U of Taq polymerase (Promega). The PCR reaction was programmed for 2 min at 94°C and then 30 cycles, 1 min at 94°C and 3 min at 72°C. The final elongation reaction was 5 min at 72°C. Analysis on 1.5% agarose gel results in a 820-bp band. The gel was further analyzed by sequencing and by dot blot hybridization, using the 32P-labeled 424-bp DNA fragment corresponding to nucleotides 1866–2290 of the core sequence. Immunohistochemistry. Paraffin-embedded tissues were cut into 4- to 6-␮m-thick sections, deparaffinized in xylene and alcohol, and placed for 15 min in alcohol– H 2O 2 for blocking endogenous peroxidase. Slides were thoroughly washed with tap water and transferred to PBS. Sections were then treated with BSA to prevent background staining and incubated for 1 h with a primary murine anti-HBs antibody (Zymed Lab, Inc., San Francisco, CA) at room temperature, in a humidified chamber. Slides were rinsed with PBS for 3 min and incubated with a biotinylated linked goat anti-mouse antibody for 30 min and with the labeling reagent peroxidase-conjugated streptavidin (Biogenex StrAvigen, from San Ramon, CA), for 30 min. After rinsing, the peroxidase label was demonstrated using 3-amino-9-ethylcarbazol (AEC) for 15 minutes and counterstained with Mayer hematoxylin. AEC produced a red product that is soluble in alcohol and is used with an aqueous mounting medium (Kaiser’s glycerol gelatin). A negative control was run using the same technique, but omitting the primary antibody and adding the streptavidin–biotin complex. Liver biopsy from a HBV-infected patient, with high-titer HBV DNA and HBsAg in serum, was used as a positive control. Incubation of HepG2-derived cells with HBV in the presence or absence of hIL-6. To further assess the role of hIL-6 in supporting HBV infection, a human hepatoblastoma cell line HepG2 (ATCC HB 8065), a HepG2derived, stably transfected hIL-6R␣ (gp80) cell line (HepG2-hIL-6R␣) and a null hIL-6R (a cell line which does not express hIL-6R␣) named HepG2-PDI (RoseJohn et al., 1993), were incubated with HBV DNA-positive sera, with or without hIL-6. It is important to indicate that all cell lines do express the IL-6R␤ (gp130), including the HepG2-PDI cells probably at a similar level; the HepG2 cells express also the IL-6R␣ although at a lower level than the HepG2-hIL-6R␣ cells. Cells were grown as a monolayer culture in DMEM supplemented with 10% fetal bovine serum. For infection experiments, cells were trypsinized, washed with PBS, and incubated with HBVpositive human sera (10 8 particles/ml), in the presence or absence of hIL-6 (500 ng/ml), in a total volume of 1 to 2 ml. After 2 to 4 h, 2 ⫻ 10 6 cells were injected intrasplenicly to chimeric mice to generate hepatoblastoma foci in the liver. Animals were splenectomized following the

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injection. Mice were bled at day 13. DNA was extracted from 100 ␮l sera and subjected to PCR amplification. All groups of mice were infected with the same HBV infectious sera. Long-term human mononuclear culture. Human blood was withdrawn in Li 2⫹–heparin collecting tubes from healthy donors. Serum samples from these donors were assessed for HBV serological and virological markers including HBV DNA by PCR as well as for HCV markers. All samples used in the experiments described were found negative for these viral markers including anti-HBc antibodies. Mononuclear cells were separated from 20–40 ml of blood by Ficoll–Hypaque sedimentation gradient, centrifuged at 800 g for 35 min at 20°C. Cells were collected and washed two times with RPMI 1640 medium containing 10% FCS, glutamine, and antibiotics (penicillin and streptomycin). Cells were used for infection experiment, in part from fresh separation samples and in part from frozen samples stored in liquid nitrogen. Cells were cultured in the same separation medium and additives containing 2 ␮g/ml of phytohemagglutinin (PHA), 20 U/ml of human IL-2, at 37°C with 5% CO 2. After 3 days, mononuclear cells were infected with HBV sera as will be described later. Following infection the culture medium was supplemented with PHA and IL-2 every 3 culture days. Cells were kept in culture for 21 days and over using these conditions. HBV infection protocol human mononuclear cells. Human sera were screened for the presence of HBV DNA sequences. Positive sera were analyzed quantitatively for viral particle concentration as we have described before (Klein et al., 1997). Sera containing over 10 8 particles per milliliter were selected for infection experiments. For each infection experiment between 5 ⫻ 10 6 to 10 7 PMNC were used. The infection experiments included the following groups: (Group 1) Cells were incubated with HBV without the addition of hIL-6 in the presence of 2 mg/ml polybrene, for 90 min at 37°C, in a 5% CO 2 shaking incubator, in a total volume of 1.5 to 2 ml. (Group 2) Cells were incubated with 1 ml of HBV-positive serum, in the presence of hIL-6 (500 ng/ml) and 2 mg/ml polybrene, for 90 min at 37°C, in a 5% CO 2 shaking incubator, in a total volume of 1.5 to 2 ml. (Group 3) Cells were incubated with 1 ml of HBV-positive serum, in the presence of the designer cytokine HIL-6, 1.5 ␮g/ml, which was recently produced by one of the investigators of this study (Fischer et al., 1997; Rakemann et al., 1999; Schirmacher et al., 1998) and 2 mg/ml polybrene, for 90 min at 37°C, in a 5% CO 2 shaking incubator, in a total volume of 1.5 to 2 ml. Following incubation of the viral hIL-6 or viral HIL-6 mixtures, the cells were intensively washed three times with F-12 medium. Cell were continuously incubated in RPMI medium supplemented with 10% FCS, PHA, and hIL-2 as described above. Cells were harvested for HBV DNA extraction every 3 days up to day 21 following infection.

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ACKNOWLEDGMENTS We thank Ofer Ben-Moshe, Yossi Arazi, and Shoshana Berre for their technical support and Professor Amos Panet and Professor Ehud Katz for critical reading of the manuscript.

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