Potential conflict of interest: Nothing to report.
Endstage liver disease caused by chronic hepatitis C virus (HCV) infection is the leading indication for liver transplantation in the Western world. However, immediate reinfection of the grafted donor liver by circulating virus is inevitable and liver disease progresses much faster than the original disease. Standard antiviral therapy is not well tolerated and usually ineffective in liver transplant patients, whereas anti-HCV immunotherapy is hampered by the extreme genetic diversity of the virus and its ability to spread by way of cell-cell contacts. We generated a human monoclonal antibody against scavenger receptor class B type I (SR-BI), monoclonal antibody (mAb)16-71, which can efficiently prevent infection of Huh-7.5 hepatoma cells and primary hepatocytes by cell-culture-derived HCV (HCVcc). Using an Huh7.5 coculture system we demonstrated that mAb16-71 interferes with direct cell-to-cell transmission of HCV. Finally we evaluated the in vivo efficacy of mAb16-71 in “human liver urokinase-type plasminogen activator, severe combined immune deficiency (uPA-SCID) mice” (chimeric mice). A 2-week anti-SR-BI therapy that was initiated 1 day before viral inoculation completely protected all chimeric mice from infection with serum-derived HCV of different genotypes. Moreover, a 9-day postexposure therapy that was initiated 3 days after viral inoculation (when viremia was already observed in the animals) suppressed the rapid viral spread observed in untreated control animals. After cessation of anti-SR-BI-specific antibody therapy, a rise of the viral load was observed. Conclusion: Using in vitro cell culture and human liver-chimeric mouse models, we show that a human mAb targeting the HCV coreceptor SR-BI completely prevents infection and intrahepatic spread of multiple HCV genotypes. This strategy may be an efficacious way to prevent infection of allografts following liver transplantation in chronic HCV patients, and may even hold promise for the prevention of virus rebound during or following antiviral therapy. (HEPATOLOGY 2012)
With approximately 3% of the world's population infected with the hepatitis C virus (HCV), endstage liver disease caused by this infection is currently the most common indication for liver transplantation.1 However, the donor liver almost inevitably becomes infected by circulating virus and disease progression is accelerated in immune-suppressed transplant patients.2 Less than 30% of liver transplant patients treated with pegylated interferon therapy with or without ribavirin will achieve a sustained virological response and this combination therapy is often not well tolerated.3-5 Therefore, new strategies to prevent graft reinfection are urgently needed. In the coming years, new direct antiviral compounds will considerably improve therapy outcome in patients without severe liver disease,6-8 but the side effects and potential drug-drug interactions associated with triple therapy may severely complicate their use in liver transplant patients with endstage liver disease.9-12
Because of the extreme genetic diversity of HCV and its ability to spread by way of cell-cell contacts, successful immunotherapy with polyclonal or monoclonal HCV-specific antibodies may be difficult to achieve.13-17 In contrast, viral (co-)receptors are genetically conserved and may represent better therapeutic targets. HCV entry is a multistep process in which different putative attachment factors and viral receptors are involved (reviewed18-20). Although heparan sulfate proteoglycans and the LDL-receptor are considered primary attachment factors, scavenger receptor class B type I (SR-BI)/Cla1,21-27 CD81,28 claudin-1,29 and occludin30 seem to be actively involved in the entry process. After initial attachment, the viral particle directly and/or indirectly interacts with SR-BI, which together with CD81 triggers downstream events involving both claudin-1 and occludin.
We have previously shown that blockade of the tetraspanin CD81 can prevent in vivo infection by different HCV strains. However, the beneficial effect of this approach was virtually abolished when CD81 antibody was administered 6 hours after the virus injection,31 a likely consequence of the ability of HCV to efficiently disseminate by way of cell-cell contacts in a CD81-independent manner.32, 33 Although the role of CD81 in direct cell-to-cell transmission is still a matter of debate, claudin-1, occludin, and especially SR-BI seem to play a prominent role in this process.34 We have generated a human immunoglobulin G4 (IgG4) monoclonal antibody (mAb16-71) that targets SR-BI. Using the HCV cell culture system (HCVcc),35-37 primary human hepatocyte cultures that faithfully recapitulate the polarized nature of hepatocytes in vivo,38, 39 and a human liver-chimeric mouse model,40-42 we show here that mAb16-71 prevents infection and viral spread of multiple HCV genotypes. Thus, this antibody is an attractive candidate molecule for preventing infection of allografts and recurrent chronic hepatitis following liver transplantation in chronic HCV patients, and for preventing the emergence of escape mutants and virus rebound during or following antiviral therapy.
ALT, alanine aminotransferase; AST, aspartate aminotransferase; CETP, cholesteryl ester transfer protein; HCVcc, cell culture produced HCV; HDL, high-density lipoprotein; IgG, immunoglobulin G; mAb, monoclonal antibody; MID100, 100% mouse infectious dose; SCID, severe combined immune deficiency; SR-BI, scavenger receptor class B type I; TCID50, 50% tissue culture infectious dose; uPA, urokinase-type plasminogen activator.
Materials and Methods
A detailed description of the methods used can be found in the online Supporting Materials.
Cells and Antibodies.
Huh-7.5 cells were maintained at 37°C, 5% CO2 in Dulbecco's Modified Eagle Medium (DMEM, Invitrogen) containing 10% fetal bovine serum (FBS) and 0.1 mM nonessential amino acids (NEAA). EGFP-IPS/CD81neg cells have been described43 and were grown in complete media containing 6 μg/mL blasticidin.
Primary adult and fetal cell cultures were established as described.38, 39 Jc1 and J6/JFH-1 Clone 244 HCVcc stocks were produced by electroporation of in vitro transcribed RNA into Huh-7.5 cells as described.35
Chimeric mice were produced as described.40 All animals used in this study received hepatocytes from a single donor and the study protocol was approved by the Animal Ethics Committee of the Faculty of Medicine and Health Sciences of the Ghent University.
The effectiveness of the different antibodies was evaluated in a prophylactic and postexposure setting. For the prophylactic treatment, chimeric mice received a 2-week antibody therapy consisting of five intraperitoneal injections (days −1, 1, 5, 8, and 12), each containing 400 μg mAb16-71. One day after the first antibody injection (day 0), all mice were inoculated with a viral dose that was previously shown to infect all challenged animals (MID100) of the following HCV strains: mH77C (genotype 1a; 104 IU/mouse), mED43 (genotype 4a; 104 IU/Mouse), or mHK6a (genotype 6a; 105 IU/mouse).16, 17 The challenge viruses mH77C, mED43, and mHK6a were produced by infecting different naïve chimeric mice (hence the prefix “m”) with a pool of acute phase plasma derived from chimpanzees infected with H77C, ED43, and HK6a, respectively.45 For the postexposure treatment, chimeric mice were first infected with mH77C virus, whereas treatment with mAb16-71 or CD81 antibody (clone JS81, BD Biosciences) was initiated 3 days later. Treated animals received five intraperitoneal antibody injections at days 3, 5, 7, 10, and 12; each containing 400 μg antibody.
To analyze whether the difference between treatment groups was statistically significant, the data obtained were analyzed using the unpaired nonparametric two-tailed Mann-Whitney test. Data were analyzed using GraphPad InStat v. 3.0b (GraphPad Software, La Jolla, CA).
Anti-SR-BI Antibody Therapy Prevents HCV Infection In Vitro.
To investigate whether SR-BI blockade could prevent HCV infection we developed a human IgG4 monoclonal antibody that targets SR-BI, designated mAb16-71. The amino acid sequence of mAb16-71 is identical to that of antibody C167 that we produced earlier,24 but the gene sequence was codon-optimized to achieve higher and more efficient production. The antiviral efficacy of mAb16-71 was first evaluated in the HCVcc system. One day after seeding in a 96-well plate, Huh-7.5 cells were incubated with different concentrations of mAb16-71. After 1 hour the antibody was washed away and the Huh-7.5 cells were exposed to H77/JFH1 HCVcc and infection was allowed to proceed for 48 hours. As shown in Fig. 1A, a clear dose-dependent reduction of the amount of HCV-infected cells was observed.
To evaluate the protective efficacy of mAb16-71 in a clinically more relevant in vitro model, we assessed antibody blockade in primary adult hepatocytes which, unlike hepatoma cells, are polarized and in which HCV entry factors localize to similar cellular compartments as in hepatocytes in vivo.38 Micropatterned cocultures were pretreated with mAb16-71, anti-CD81, or isotype control antibody and subsequently infected with Jc1 HCVcc expressing Gaussia luciferase. As shown in Fig. 1B, we observed a 5-fold reduction of HCV infection in the mAb16-71-treated wells compared with the isotype control, and a 7-fold reduction of infection upon anti-CD81 treatment. Although antibody C167, which has the same amino acid sequence as mAb16-71, has previously been demonstrated to be inefficient at blocking HCV entry in adult MPCC cultures, the robustness of HCV infection of these cultures has since been improved. Additionally, donor variability could also help explain this difference.38
We confirmed our data in cultures of primary human fetal hepatocytes, which are a more amenable in vitro culture system, given the more robust infection levels achieved compared with adult hepatocytes.39 Primary human fetal hepatocytes, transduced with the RFPnls-IPS HCV reporter system,43 were preincubated with different concentrations of mAb16-71. Parallel cultures were treated with an isotype-matched antibody (negative control) or JS81, an antibody that targets CD81 and prevents attachment and infection of HCV (positive control). As shown in Fig. 1C, infection of primary fetal hepatocytes by J6/JFH-1 HCVcc was also reduced in a dose-dependent manner.
mAb16-71 Interferes with Direct Cell-to-Cell Transmission In Vitro.
HCV can spread directly from an infected Huh-7.5 cell to uninfected neighboring cells, with possibly all four HCV entry factors CD81, SR-BI, claudin, and occludin, being involved in this process.32-34 However, by using Huh-7.5 target cells in which CD81 was selectively knocked down (EGFP-IPS/CD81neg), we have recently shown that HCV cell-to-cell spread can occur independently of CD81.43 We therefore used this cell line to investigate whether mAb16-71 is capable of inhibiting this alternative transmission route. To this end, HCVcc-infected (Jc1) Huh-7.5 cells were cocultured with uninfected EGFP-IPS/CD81neg cells. EGFP-IPS/CD81neg cells have been previously shown to be essentially nonpermissive to cell-free HCV infection.43 Nevertheless, mixing uninfected EGFP-IPS/CD81neg target cells with infected Huh7.5 cells resulted in an infection of 8%-10% of the target cells. However, in the presence of increasing concentrations of mAb16-71 a dose-dependent reduction in EGFP-IPS/CD81neg target cell infection was observed, whereas no significant changes in HCV transmission were observed in the presence of an isotype-matched control antibody (Fig. 1D). This clearly proves that mAb16-71 not only prevents cell-free HCV infection, but also interferes with the direct cell-to-cell transmission route.
Prophylactic Administration of mAb16-71 Protects Chimeric Mice from Infection by HCV of Different Genotypes.
Given the encouraging results in cell culture, we investigated whether administration of mAb16-71 to “human liver urokinase-type plasminogen activator, severe combined immune deficiency (uPA-SCID)” mice (chimeric mice) could protect these animals from a subsequent challenge with serum-derived virus. These mice have a humanized liver (up to 90% chimerism) and are in addition to the chimpanzee the preferred animal model for reproducible infection with natural HCV isolates.40, 42, 45, 46 Two chimeric mice underwent a 2-week therapy consisting of five intraperitoneal injections, each containing 400 μg of mAb16-71. One day after the first injection, both chimeric mice were challenged with a 100% mouse infectious dose (MID100) of serum-derived genotype 1a HCV (mH77C). In contrast to nontreated control animals, which experienced a rapid increase of viral RNA in their plasma, HCV RNA remained undetectable (<375 IU/mL) in both treated mice in the 12-week observation period (Fig. 2A). mAb16-71 was equally effective in protecting five chimeric mice after challenge with serum-derived genotype 4 (mED43; n = 3) or genotype 6 (mHK-6a; n = 2) HCV, indicating that SR-BI-based immunotherapy is successful most likely irrespective of HCV genotype (Fig. 2B,C).
The 2-week treatment protocol was very well tolerated by the chimeric mice, which showed no signs of overt toxicity. No significant changes in human albumin, transaminases (aspartate aminotransferase [AST] and alanine aminotransferase [ALT]), triglyceride, cholesterol, and high-density lipoprotein (HDL) levels were measured in mice that received a 2-week mAb16-71 therapy when compared with untreated control mice (Table 1).
Table 1. Plasma analysis of control and mAb16-71 treated chimeric mice
Postexposure mAb16-71 Therapy Blocks Viral Dissemination In Vivo.
To substantiate the role of SR-BI in cell-to-cell spread in vivo, we performed a postexposure treatment experiment in chimeric mice. Fifteen chimeric mice were injected with an MID100 dose of mH77C HCV. Three days later, plasma HCV RNA levels were determined and HCV RNA could be detected in all but two animals, which were included in the untreated group (n = 7). Four of the remaining mice received five injections of mAb16-71 at days 3, 5, 7, 9, and 12 and the last four animals were treated with anti-CD81 antibody (clone JS81) using the same dosing protocol. In the untreated group the viral load rapidly increased during the first 2 weeks after virus inoculation, reaching values ranging between 104 and 107 IU/mL (Fig. 3A). Treatment with anti-CD81 mAb caused a minor, statistically nonsignificant, delay in the rise of viral load, possibly due to inhibition of infection by cell-free virus, but all animals experienced an increase in viral load, confirming our previous data that HCV can spread in a CD81-independent manner.31, 33 In contrast, in three out of four mice treated with mAb16-71, HCV RNA levels did not increase but remained positive at unquantifiable levels (<375 IU/mL), whereas in the fourth mouse HCV RNA was undetectable. In this mouse the viral load started to rise 9 days after cessation of anti-SR-BI therapy and reached a level of almost 106 IU/mL 4 weeks after infection (Fig. 3A). In the two other mAb16-71-treated mice the viremia started to rise 16 to 23 days after cessation of therapy, whereas in the fourth mAb16-71-treated mouse HCV RNA remained detectable at unquantifiable levels throughout the 8-week observation period. Statistical analysis using the two-tailed nonparametric Mann-Whitney test showed that the median HCV RNA level of mAb16-71-treated animals differed significantly from that in the control group (P = 0.023, P = 0.0061, and P = 0.016 at days 7, 14, and 21, respectively). No differences were observed between the HCV RNA levels of CD81-treated mice and control mice (P > 0.99, P = 0.164, and P = 0.41 at days 7, 14, and 21, respectively). At the start of therapy (day 3) no statistically significant differences were observed between the different groups (control versus mAb16-71: P = 0.25; control versus anti-CD81: P = 0.45).
In the four mAb16-71-treated mice HCV RNA became detectable at different timepoints after cessation of therapy. Therefore, we quantified the plasma mAb16-71 levels 2 days after the last antibody injection and observed a correlation between these mAb16-71 plasma levels and the duration of protection (Fig. 3B). High levels of circulating antibody indirectly indicate complete saturation of the SR-BI molecules present on the human hepatocytes in the chimeric mouse liver. In addition, sequence analysis of virus recovered from the mAb-16-71-treated mice that became HCV positive at weeks 3 and 5 showed that the deduced amino acid sequence of the envelope region was identical to the sequence of the viral inoculum and that of the viruses found in the control animals (data not shown). The absence of adaptive mutations and the correlation between plasma mAb16-71 levels and the duration of protection argue against virus escape.
A 2-week mAb16-71 therapy of chronically infected chimeric mice had no effect on viral load (data not shown).
Prevention of reinfection of the liver allograft in chronic HCV patients who undergo liver transplantation for endstage liver disease (cirrhosis and/or hepatocellular carcinoma) will be one of the main therapeutic challenges of the next decade. New antiviral therapies consisting of pegylated interferon, ribavirin, and protease inhibitors seem to be very effective in eradicating HCV infection in chronically infected patients without severe liver disease.6-8 However, these new antiviral cocktails elicit considerable side effects and the currently approved protease inhibitors are both inhibitors of cytochrome P450 3A, which is responsible for the metabolism of cyclosporine and tacrolimus.9, 10, 12 This will certainly severely complicate the use of telaprevir and boceprevir in a liver transplant setting. Because of the extreme variability of the viral envelope proteins and probably also because of the association of the viral particles with lipoproteins,47 anti-HCV antibodies with neutralizing capacity hardly induce sterilizing immunity.13-17 Therefore, the genetically highly conserved cellular receptors utilized by the virus to infect the host cell may seem better alternatives to prevent infection of the allograft. Recently, Mensa et al.48 showed a correlation between the viral load decay during the first 24 hours after graft reperfusion and the SR-BI expression levels in the donor liver, suggesting that SR-BI plays a major role in the initial uptake of the virus and making it an attractive therapeutic target.
We developed a human monoclonal antibody that efficiently prevents HCV infection of both Huh-7.5 hepatoma cells and primary hepatocytes. Moreover, this antibody is capable of interfering with direct cell-to-cell transmission of HCV in vitro. Importantly, using chimeric mice, we have clearly shown that anti-SR-BI therapy not only prevents infection by HCV of multiple genotypes, but it also inhibits the spread of the virus in an already established infection in vivo.
Antibody targeting of SR-BI turns out to be superior to anti-CD81 therapy for several reasons. The expression pattern of SR-BI is more restricted than the ubiquitously expressed CD81,49 which may allow for a reduction of the treatment dose and potential side effects. In fact, a 2-week treatment of chimeric mice with mAb16-71 induced no significant changes in hepatic serum markers as compared with untreated mice. Although our limited data suggest that mAb16-71 therapy might be safe in humans, more extensive preclinical toxicity studies must be performed in different animal species, as well as safety and pharmacokinetic studies in healthy volunteers and, ultimately, in liver transplant patients. It needs to be emphasized that mice represent a very stringent model for safety testing of anti-SR-BI mAb therapy because these animals do not express cholesteryl ester transfer protein (CETP), which facilitates cholesterol transport and triglyceride exchange in humans, thus potentially providing an alternative route of lipid metabolism in case of reduced SR-BI function upon mAb16-71 treatment.50
In addition, mAb16-71 remains effective in blocking HCV dissemination, even if administered several days after viral inoculation. This suggests that SR-BI may be a molecule involved in direct cell-to-cell transmission of HCV in vivo and represents an important advantage over anti-CD81 blockade which did not prevent virus spread even when administered therapeutically soon after viral challenge.31 In fact, our antibody seems more effective in vivo than what could be anticipated from cell culture experiments. This implies still unknown discrepancies between the currently used cell culture systems and the in vivo reality, thereby further emphasizing the value of experiments in animal models.
Viruses with mutations or deletions in their envelope proteins have been described to become independent for SR-BI.51-53 However, it remains to be determined whether these mutated viruses are also not reliant on SR-BI in vivo. We could not identify any adaptive mutations in the envelope region of the virus that was recovered from two mAb16-71-treated mice that became HCV-positive 9 days and 29 days after cessation of the 2-week antibody treatment. Furthermore, it is doubtful that such variants would arise and expand in an infected patient, because they are sensitive to neutralizing antibodies that are ubiquitously present in the plasma of all chronically infected patients.13, 51-54 A viral mutant losing its SR-BI dependence would most likely be immediately neutralized by the host's preexisting adaptive immune response.
Besides SR-BI, claudin-1 and occludin may be very attractive targets for antiviral therapy. These tight junction proteins are essential for viral entry and direct cell-to-cell transmission.29, 30, 34, 55 In a recent publication, Lupberger et al.56 identified the epidermal growth factor (EGF)-receptor as another host factor involved in HCV entry and dissemination. However, a 30-day treatment of chimeric mice with erlotinib, a small molecule that specifically inhibits EGF-receptor activity, did not prevent but only delayed the kinetics of infection.
In conclusion, we show here that the human monoclonal antibody mAb16-71 can efficiently block in vitro and in vivo infection by multiple HCV genotypes. In addition, we demonstrate that blockade of SR-BI after infection can prevent rapid virus spread through the liver parenchyma, presumably by interfering with SR-BI-dependent cell-free as well as direct cell-to-cell HCV transmission. Therefore, targeting SR-BI may represent a superior strategy for anti-HCV immunotherapy to prevent the emergence of escape mutants and virus rebound during or following antiviral therapy, and to prevent allograft infection in chronically infected HCV patients undergoing orthotopic liver transplantation.
We thank Dr. Veronique Stove and Yvonne Geybels for the analysis of mouse plasma and Dr. Robert H. Purcell (NIH) and Dr. Jens Bukh (NIH; CO-HEP, Copenhagen) for providing plasma from acutely infected chimpanzees.