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Abstract

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Scavenger receptor class B type I (SR-BI) is a high-density lipoprotein (HDL) receptor highly expressed in the liver and modulating HDL metabolism. Hepatitis C virus (HCV) is able to directly interact with SR-BI and requires this receptor to efficiently enter into hepatocytes to establish productive infection. A complex interplay between lipoproteins, SR-BI and HCV envelope glycoproteins has been reported to take place during this process. SR-BI has been demonstrated to act during binding and postbinding steps of HCV entry. Although the SR-BI determinants involved in HCV binding have been partially characterized, the postbinding function of SR-BI remains largely unknown. To uncover the mechanistic role of SR-BI in viral initiation and dissemination, we generated a novel class of anti–SR-BI monoclonal antibodies that interfere with postbinding steps during the HCV entry process without interfering with HCV particle binding to the target cell surface. Using the novel class of antibodies and cell lines expressing murine and human SR-BI, we demonstrate that the postbinding function of SR-BI is of key impact for both initiation of HCV infection and viral dissemination. Interestingly, this postbinding function of SR-BI appears to be unrelated to HDL interaction but to be directly linked to its lipid transfer function. Conclusion: Taken together, our results uncover a crucial role of the SR-BI postbinding function for initiation and maintenance of viral HCV infection that does not require receptor-E2/HDL interactions. The dissection of the molecular mechanisms of SR-BI–mediated HCV entry opens a novel perspective for the design of entry inhibitors interfering specifically with the proviral function of SR-BI. (HEPATOLOGY 2013)

Hepatitis C virus (HCV) is a major cause of liver cirrhosis and hepatocellular carcinoma. Preventive modalities are nonexistent, and the current antiviral treatment is limited by resistance, toxicity, and high cost.1 Viral entry is required for initiation, spread, and maintenance of infection, and thus is a promising target for antiviral therapy. HCV binding and entry into hepatocytes is a complex process involving the viral envelope glycoproteins E1 and E2, as well as several host factors, including highly sulfated heparan sulfate, CD81, the low-density lipoprotein receptor, scavenger receptor class B type I (SR-BI), claudin-1, occludin, and receptor tyrosine kinases.2, 3

Human SR-BI is a glycoprotein that is highly expressed in tissues with a high cholesterol need for steroidogenesis and the liver.4 SR-BI is a multifunctional molecule well known to modulate high-density lipoprotein (HDL) metabolism. SR-BI binds a variety of lipoproteins and mediates selective uptake of HDL cholesterol ester (CE) as well as bidirectional free cholesterol transport at the cell membrane. Genetic SR-BI variants have been associated with HDL levels in humans, and a recent study uncovered a functional mutation in SR-BI impairing SR-BI function and affecting cholesterol homeostasis.5 SR-BI also interacts with different pathogens, including HCV,6-8 and mediates their entry and uptake into host cells. SR-BI is relevant for HCV infection in vivo, and its potential as an antiviral target has been reported.9

SR-BI directly binds HCV E2,6, 8 but virus-associated lipoproteins also contribute to host cell binding and uptake.10, 11 Moreover, physiological SR-BI ligands modulate HCV infection.12-14 This suggests the existence of a complex interplay between lipoproteins, SR-BI, and HCV envelope glycoproteins for HCV entry. SR-BI has also been demonstrated to mediate postbinding events during HCV entry.15-17 HCV–SR-BI interaction during postbinding steps occurs at similar time points as the HCV utilization of CD81 and claudin-1, suggesting that HCV entry may be mediated through the formation of coreceptor complexes.15, 18, 19 These data suggest that SR-BI plays a multifunctional role during HCV entry at both binding and postbinding steps.15, 20 This is corroborated by the fact that murine SR-BI does not bind E2,20, 21 although it is capable of promoting HCV entry.20, 22

To elucidate the mechanistic function of SR-BI in the HCV entry process and to explore its potential as an antiviral target, we generated a novel class of monoclonal antibodies directed against human SR-BI that inhibit HCV entry during postbinding steps without preventing E2 binding to target cells.

Material and Methods

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Additional details are provided in the Supporting Information.

Cells.

HEK293T, Chinese hamster ovary, Buffalo rat liver, Huh7, Huh7.5-GFP, and Huh7.5.1 cells were cultured as described.18, 23-25 Primary human hepatocytes were isolated as described.18 Chinese hamster ovary and Buffalo rat liver cells expressing SR-BI were produced as described.11, 15, 23

Antibodies.

Polyclonal15 and monoclonal antibodies (mAbs) directed against the extracellular loop of SR-BI were raised by genetic immunization of Wistar rats and Balb/c mice as described15 according to proprietary technology (Aldevron GmbH, Freiburg, Germany). Anti–SR-BI mAbs were purified using protein G affinity columns and selected via flow cytometry for their ability to bind to human SR-BI.15 To determine the affinity of the anti–SR-BI mAbs for human SR-BI, Huh7.5.1 cells were incubated with increasing concentrations of mAbs, and binding was assessed using flow cytometry. Kd values were determined as half-saturating concentrations of the mAbs.26, 27 Antibodies will be provided on request using a material transfer agreement (MTA). Anti-CD81 (JS-81), anti–SR-BI (CLA-1), and phycoerythrin-conjugated anti-mouse antibodies were from BD Biosciences. Anti-His and fluorescein isothiocyanate–conjugated anti-His antibodies were obtained from Qiagen, rabbit anti-actin (AA20-30) antibodies were obtained from Sigma-Aldrich, and mouse anti-NS5A antibodies were obtained from Virostat. Anti-E1 (IGH520, IGH526, Innogenetics), anti-E2 (IGH461, Innogenetics; AP33, Genentech; CBH23, a kind gift from S.K.H. Foung), and patient-derived anti-HCV immunoglobulin G (IgG) have been described.16, 25, 27

Cell Culture–Derived HCV and Pseudoparticle Production and Infection.

Luciferase reporter cell culture-derived HCV (HCVcc), HCV pseudoparticles (HCVpp), murine leukemia virus pseudoparticles, and vesicular stomatitis virus glycoprotein pseudoparticles (VSV-Gpp), infection and kinetic experiments have been described.15, 18, 25, 27, 28 Unless otherwise stated, HCVcc experiments were performed using Luc-Jc1, and infection was analyzed in cell lysates via quantification of luciferase activity.29 For combination experiments, each antibody was tested individually or in combination with a second antibody. Huh7.5.1 cells were preincubated with anti–SR-BI or control mAb for 1 hour and then incubated for 4 hours at 37°C with HCVcc (Luc-Jc1) or HCVpp (P02VJ) (preincubated for 1 hour with or without anti-envelope antibodies). Synergy was assessed using the combination index and the method of Prichard and Shipman.30-32 Cell viability was assessed using a MTT test.2

Cellular Binding of Envelope Glycoprotein E2.

Recombinant His-tagged soluble E2 (sE2) was produced as described.23 Huh7.5.1 cells were preincubated with control or anti–SR-BI serum (1:50), anti–SR-BI or control mAbs (20 μg/mL) for 1 hour at room temperature, and then incubated with sE2 for 1 hour at room temperature. Binding of sE2 was revealed using flow cytometry as described.18, 23

HCVcc Binding.

Huh7.5.1 cells were preincubated with heparin (100 μg/mL), control or anti–SR-BI serum (1:50), or anti–SR-BI or control mAbs (20 μg/mL) for 1 hour prior to incubation with HCVcc at 4°C as described.18 Nonbound HCVcc were removed by washing of cells with phosphate-buffered saline, and cell bound HCV RNA was then quantified by reverse-transcription polymerase chain reaction.18

HCV Cell-to-Cell Transmission.

HCV cell-to-cell transmission was assessed as described.2, 24 Producer Huh7.5.1 cells were electroporated with Jc1 RNA33 and cultured with naïve target Huh7.5-GFP cells in the presence or absence of anti–SR-BI or control mAbs. An HCV E2-neutralizing antibody (AP33, 25 μg/mL) was added to block cell-free transmission.24 After 24 hours of coculture, cells were fixed with paraformaldehyde, stained with an NS5A-specific antibody (Virostat), and analyzed via flow cytometry.2, 24

Immunofluorescence of Viral Dissemination.

Cell spread was assessed by visualizing Jc1-infected Huh7.5.1 cells by immunoflorescence using anti-NS5A (Virostat) and anti-E2 (CBH23) antibodies as described.2

HDL Binding.

HDL was labeled using Amersham Cy5 Mono-Reactive Dye Pack (GE Healthcare). Unbound Cy5 was removed by applying labeled HDL on illustra MicroSpin G-25 Columns (GE Healthcare). Blocking of Cy5-HDL binding with indicated reagents was performed for 1 hour at room temperature prior to Cy5-HDL binding for 1 hour at 4°C on 106 target cells.

Lipid Transfer Assays.

Selective HDL-CE uptake and lipid efflux assays were performed as described.23, 34 HDL-CE uptake was assessed in the presence or absence of anti–SR-BI mAbs (20 μg/mL) and 3H-CE-labeled HDL (60 μg protein) for 5 hours at 37°C. Selective uptake was calculated from the known specific radioactivity of radiolabeled HDL-CE and is denoted in μg HDL-CE/μg cell protein. For lipid efflux assay, Huh7 cells were labeled with 3H-cholesterol (1 μCi/mL) and incubated at 37°C for 48 hours as described.23, 35 Cells were incubated with anti–SR-BI mAbs (20 μg/mL) for 1 hours prior to incubation with unlabeled HDL for 4 hours. Fractional cholesterol efflux was calculated as the amount of label obtained in the medium divided by the total in each well (radioactivity in the medium + radioactivity in the cells) regained after lipid extraction from cells.

Statistical Analysis.

Unless otherwise stated, data are presented as the means ± SD of three independent experiments. Statistical analyses were performed using a Student t test and/or Mann-Whitney test; P < 0.01 was considered statistically significant.

Results

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Production of SR-BI–Specific Monoclonal Antibodies Interfering with the Postbinding Steps of Viral Entry.

To further explore the role of HCV–SR-BI interaction during HCV infection, we generated five rat and three mouse monoclonal antibodies (mAbs) directed against the human SR-BI (hSR-BI) ectodomain (Table 1). These antibodies bound to endogenous SR-BI on human hepatoma Huh7.5.1 cells and primary human hepatocytes but did not bind to mouse SR-BI (mSR-BI) expressed on rat BRL cells (Fig. 1A,B and Supporting Fig. 1). Three rat mAbs (QQ-4A3-A1, QQ-2A10-A5, and QQ-4G9-A6) and one mouse mAb (NK-8H5-E3) significantly (P < 0.01) inhibited HCVcc infection in a dose-dependent manner with 50% inhibitory concentrations (IC50) between 0.2 and 8 μg/mL (Fig. 1C,D and Table 1). The apparent Kd (Kdapp) corresponding to the half-saturating concentrations for binding to Huh7.5.1 cells ranged from 0.5 to 7.4 nM, demonstrating that these antibodies recognize SR-BI with high affinity (Table 1). It is noteworthy that there seems to be a correlation between the antibody affinity and inhibitory capacity, with the low affinity antibodies unable to block HCV infection. We next aimed to characterize the viral entry steps targeted by these anti–SR-BI mAbs. We first assessed their ability to interfere with viral binding. To reflect the complex interaction between HCV and hSR-BI during viral binding, we studied the effect of anti–SR-BI mAbs on HCVcc binding to Huh7.5.1 cells at 4°C. Incubation of Huh7.5.1 cells with anti–SR-BI mAbs before and during HCVcc binding did not inhibit virus particle binding (Fig. 2A). Similar results were obtained using sE2 as a surrogate model for HCV (Supporting Results and Supporting Fig. 1). These data suggest that, in contrast to described anti–SR-BI mAbs,20 these novel anti–SR-BI mAbs do not inhibit HCV binding but interfere with HCV entry during postbinding steps. Next, to characterize potential postbinding steps targeted by these anti–SR-BI mAbs, we assessed HCVcc entry kinetics into Huh7.5.1 cells in the presence of anti–SR-BI mAbs inhibiting HCV infection (QQ-4A3-A1, QQ-2A10-A5, QQ-4G9-A6, and NK-8H5-E3) added at different time points during or after viral binding (Fig. 2B). This assay was performed side-by-side with an anti-CD81 mAb inhibiting HCV postbinding15, 18, 29 and proteinase K36 to remove HCV from the cell surface. HCVcc binding to Huh7.5.1 cells was performed for 1 hour at 4°C in the presence or absence of compounds. Subsequently, unbound virus was washed away, cells were shifted to 37°C to allow HCVcc entry, and compounds were added every 20 minutes for up to 120 minutes after viral binding. These kinetic experiments indicate that anti–SR-BI mAbs inhibited HCVcc infection when added immediately after viral binding as well as 20-30 minutes after initiation of viral entry (Fig. 2C), demonstrating that QQ-4A3-A1, QQ-2A10-A5, QQ-4G9-A6, and NK-8H5-E3 indeed target postbinding steps of the HCV entry process. This time frame is comparable to the kinetics of resistance of internalized virus to proteinase K (Fig. 2C), indicating that these postbinding steps precede completion of virus internalization. Taken together, these data indicate that a postbinding function of SR-BI is essential for initiation of HCV infection. In contrast to previous anti–SR-BI mAbs inhibiting HCV binding20 as well as polyclonal anti–SR-BI antibodies and small molecules interfering with both viral binding and postbinding,15, 17, 23 these antibodies are the first molecules exclusively targeting the postbinding function of SR-BI and thus represent a unique tool to more thoroughly assess the relevance of this function for HCV infection.

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Figure 1. Binding of monoclonal anti–SR-BI antibodies to human hepatocytes and inhibition of HCV infection. (A) Huh7.5.1 cells and (B) primary human hepatocytes were incubated with anti–SR-BI mAbs, and antibody binding was assessed using flow cytometry. Results are expressed as the net mean fluorescence intensity (ΔMFI) of a representative experiment. (C) Inhibition of HCVcc infection by anti–SR-BI mAbs. Huh7.5.1 cells were preincubated for 1 hour at 37°C with anti–SR-BI or control mAbs (100 μg/mL) before infection with HCVcc (Luc-Jc1) for 4 hours at 37°C. HCV infection was assessed by luciferase activity in lysates of infected Huh7.5.1 cells 72 hours postinfection. Results are expressed as the mean ± SD % HCVcc infectivity in the absence of antibody of three independent experiments. (D) Dose-dependent inhibition of HCVcc infection by anti–SR-BI mAbs. Huh7.5.1 cells were preincubated for 1 hour at 37°C with anti–SR-BI or control mAbs at the indicated concentrations before infection with HCVcc (Luc-Jc1) for 4 hours at 37°C. HCV infection was assessed by luciferase activity in lysates of infected Huh7.5.1 cells 72 hours postinfection. Results are expressed as the mean ± SD % HCVcc infectivity in the absence of antibody of three independent experiments performed in triplicate. *P < 0.01.

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Figure 2. Monoclonal anti–SR-BI antibodies do not interfere with HCV binding to SR-BI but inhibit HCV entry at postbinding steps. (A) To assess the effect of anti–SR-BI mAbs on viral binding, Huh7.5.1 cells were preincubated with heparin (100 μg/mL), anti–SR-BI or control (CTRL) serum (1:50), or anti–SR-BI or control (CTRL IgG) mAbs (20 μg/mL) for 1 hour prior to incubation with HCVcc (Jc1) at 4°C in the presence of compounds. Nonbound HCVcc were removed by washing of cells with phosphate-buffered saline, and HCVcc binding was then quantified by reverse-transcription polymerase chain reaction of cell-bound HCV RNA. Results are expressed as the mean ± SD of one representative experiment performed in quintuplicate. (B) Schematic drawing of the experimental setup. To discriminate between virus binding and postbinding events, HCVcc (Luc-Jc1) binding to Huh7.5.1 cells was performed in the presence or absence of anti-CD81 (5 μg/mL), anti–SR-BI (20 μg/mL) or control mAbs (20 μg/mL), or proteinase K (50 μg/mL) for 1 hour at 4°C, before cells were washed and incubated for 4 hours at 37°C with compounds added at different time points during infection. Compounds were then removed and cells were cultured for an additional 48 hours. Dashed lines indicate the time intervals where compounds were present. (C) HCV entry kinetics. Time course of HCVcc infection of Huh7.5.1 cells following addition of the indicated antibodies at different time points during infection is shown. HCV infection was assessed by luciferase activity in lysates of infected Huh7.5.1 cells 48 hours postinfection. Results are expressed as the mean % HCVcc infectivity in the absence of antibody of three independent experiments performed in triplicate. *P < 0.01.

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Table 1. mAbs Directed Against hSR-BI
mAbIsotypeKdapp Huh7.5.1 (nM)IC50 HCVcc (μg/mL)Inhibition of HDL-CE Uptake (Mean % ± SD)Inhibition of Cholesterol Efflux (Mean % ± SD)
  1. Isotype, binding affinity to Huh7.5.1 cells (Kdapp), and inhibition of HCVcc infection (IC50) and lipid transfer of anti–SR-BI mAbs are shown. Huh7.5.1 cells were incubated with increasing concentrations of mAbs, and Kd values were determined as half-saturating concentrations of the mAbs. IC50 was determined after incubation of Huh7.5.1 cells with serial dilutions of anti–SR-BI mAbs for 1 hour at room temperature before infection with HCVcc. The results represent the mean of three independent experiments performed in triplicate. Lipid uptake and efflux were assessed in Huh7 cells as described in Materials and Methods in the presence of anti–SR-BI mAbs (20 μg/mL). The results are expressed as % inhibition of lipid transfer relative to cells incubated in the absence of antibody and represent the mean ± SD of three independent experiments.

  2. Abbreviation: NA, not applicable.

QQ-4A3-A1Rat IgG2b1.00.744.18 ± 1.4240.97 ± 0.92
QQ-2A10-A5Rat IgG2b0.50.247.64 ± 1.240 ± 1.01
QQ-4G9-A6Rat IgG2b0.51.044.64 ± 1.5739.02 ± 1.14
PS-6A7-C4Rat IgG2bNANA10.24 ± 1.52−2.52 ± 1.25
PS-7B11-E3Rat IgG2bNANA11.73 ± 2.15.04 ± 0.83
NK-8H5-E3Mouse IgG2b7.48.056.28 ± 0.844.74 ± 0.55
NK-6B10-E6Mouse IgG1NANA1.28 ± 1.6918.41 ± 0.81
NK-6G8-B5Mouse IgG1NANA5.64 ± 1.0413.08 ± 0.77

A Postbinding Function of SR-BI Is Essential for Cell-to-Cell Transmission and Viral Spread.

HCV disseminates via direct cell-to-cell transmission.24, 37 To assess the role of SR-BI postbinding function in viral dissemination, we first investigated the ability of anti–SR-BI mAbs to interfere with neutralizing antibody-resistant viral spread by studying direct HCV cell-to-cell transmission in the presence of QQ-2A10-A5 and QQ-4G9-A6. Viral “producer” cells containing replicating HCV Jc1 (Pi) are cocultured with green fluorescent protein (GFP)-expressing “target” cells (T) in the presence of E2-neutralizing mAb (AP33, 25 μg/mL) to prevent cell-free HCV transmission.24 AP33 reduces cell-free transmission by >90%, and infectivity of producer cell supernatants is minimal at the time of coculture; viral transmission thus occurs predominantly via cell-to-cell transmission in this assay.2, 24 HCV cell-to-cell transmission is assessed by quantifying HCV-infected, GFP-positive target cells (Ti) by flow cytometry.2, 24 Both anti–SR-BI mAbs (10 μg/mL) efficiently blocked HCV cell-to-cell transmission (Fig. 3A and Supporting Fig. 2A,B), indicating that these antibodies may prevent viral spread in vitro. Because these anti–SR-BI mAbs do not block HCV–SR-BI binding (Fig. 2A) but inhibit HCV entry during postbinding steps (Fig. 2C), these data suggest that an SR-BI postbinding function plays an important role during HCV cell-to-cell transmission. To ascertain the importance of the SR-BI postbinding function in this process, we performed additional cell-to-cell transmission assays using mSR-BI, which in contrast to hSR-BI is unable to bind E2. Cells lacking SR-BI and robustly replicating HCV, which would be an ideal model cell to study cell-to-cell transmission by mSR-BI in the absence of hSR-BI, have not been described. However, hSR-BI has been reported to be a limiting factor for HCV spread in Huh7-derived cells, as overexpression of hSR-BI increases cell-to-cell transmission.37 We thus used Huh7.5 cells or Huh7.5 cells overexpressing either mSR-BI or hSR-BI as target cells. Cell-to-cell transmission was enhanced in Huh7.5 cells overexpressing either hSR-BI (2.04 ± 0.03 fold) or mSR-BI (1.92 ± 0.19 fold) compared with parental cells (Fig. 3B). These data indicate that E2–SR-BI binding is not essential for viral dissemination and confirm the crucial role of SR-BI postbinding function in this process. Furthermore, to assess whether anti–SR-BI mAbs prevent viral dissemination in already HCV-infected cell cultures when added postinfection, we performed a long-term analysis of HCVcc infection by culturing Luc-Jc1–infected Huh7.5.1 cells in the presence or absence of control or anti-SR-BI mAbs QQ-4G9-A6 and NK-8H5-E3 as previously described.2 When added 48 hours after infection and maintained in cell culture medium throughout the experiment, these anti–SR-BI mAbs efficiently inhibited HCV spread over 2 weeks in a dose-dependent manner without affecting cell viability (Fig. 3C,D and Supporting Fig. 2C,D). We also assessed Jc1 spread in Huh7.5.1 cells via immunostaining of infected cells as described.2 While 74.5 ± 2.3% and 70.0 ± 3.2% of cells incubated with control rat or mouse mAbs stained positive for NS5A and E2, respectively, incubation with QQ-4G9-A6 and NK-8H5-E3 markedly reduced the number of NS5A-positive (14.2 ± 3.4%) and E2-positive (16.7 ± 2.6%) cells (Fig. 3E,F). Taken together, these data indicate that a postbinding function of SR-BI is required for HCV cell-to-cell transmission and spread.

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Figure 3. The SR-BI postbinding function is relevant for HCV cell-to-cell transmission and viral spread. (A) Quantification of HCV-infected target cells (Ti) after cocultivation with HCV (Jc1) producer cells (Pi) during incubation with control or anti–SR-BI mAbs (10 μg/mL) in the presence of E2-neutralizing antibody AP33 (25 μg/mL) via flow cytometry. Data are expressed as % infected target cells and represent the mean ± SD of three independent experiments. (B) Quantification of HCV cell-to-cell transmission in parental target cells compared with target cells overexpressing mSR-BI or hSR-BI. Data are expressed as the mean ± SD from three different experiments. (C,D) Long-term analysis of HCVcc (Luc-Jc1) infection in the presence or absence of control (10 μg/mL) or anti–SR-BI mAbs (C) QQ-4G9-A6 or (D) NK-8H5-E3 at the indicated concentrations. Antibodies were added 48 hours after HCVcc infection and control medium or medium containing antibodies were replenished every 4 days. Luciferase activity was determined in cell lysates every 2 days. Data are expressed as log10 RLU and represent the mean ± SD of one representative out of three different experiments performed in duplicate. (E,F) Cell spread in the presence or absence of anti-SR-BI mAbs. Antibodies were added 48 hours after HCVcc (Jc1) infection and control medium or medium containing antibodies were replenished every 4 days. HCV-infected cells were visualized 7 days postinfection via immunofluorescence using (E) anti-NS5A or (F) anti-E2 (CBH23) antibodies. The percentage of infected cells was calculated as the number of infected cells relative to the total number of cells as assessed by 4′,6-diamidino-2-phenylindole staining of the nuclei. *P < 0.01.

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SR-BI Determinants Relevant for HCV Postbinding Steps May Be Linked to the Lipid Transfer Function of the Entry Factor.

The SR-BI ectodomain has been demonstrated to be important for both HDL binding and CE uptake, but the determinants involved in these processes have not yet been defined. To assess whether anti–SR-BI mAbs inhibiting HCV postbinding steps affect HDL binding to SR-BI, we studied Cy5-labeled HDL binding to hSR-BI in the presence or absence of anti–SR-BI mAbs. In contrast to polyclonal anti–SR-BI serum, which inhibited Cy5-labeled HDL binding, none of the anti–SR-BI mAbs markedly interfered with HDL–SR-BI binding at concentrations inhibiting HCV infection by up to 90% (Fig. 4A, statistically not significant). Furthermore, we investigated the effect of these mAbs on CE uptake and cholesterol efflux. Whereas PS-6A7-C4, PS-7B11-E3, NK-6B10-E6, and NK-6G8-B5 had no effect on lipid transfer, QQ-4A3-A1, QQ-2A10-A5, QQ-4G9-A6, and NK-8H5-E3 partially reduced both CE uptake and cholesterol efflux at concentrations inhibiting HCV infection by up to 90% (Fig. 4B,C). These data indicate that the anti–SR-BI mAbs inhibiting HCVcc infection also partially inhibit SR-BI–mediated lipid transfer (Table 1). Taken together, these data suggest that SR-BI determinants involved in HCV postbinding events do not mediate HDL binding but may contribute to lipid transfer, in line with the reported link between the SR-BI lipid transfer function and HCV infection.11, 12, 23

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Figure 4. Anti–SR-BI mAbs do not interfere with HDL binding but partially inhibit lipid transfer. (A) HDL binding to BRL3-hSR-BI cells. BRL3-hSR-BI cells were incubated in the presence or absence of anti–SR-BI mAbs (20 μg/mL) or polyclonal serum (1:50) or respective controls, prior to Cy5-HDL binding for 1 hour at 4°C. Bound Cy5-HDL was quantified using flow cytometry. Results represent the mean ± SD of two different experiments performed in duplicate. (B) Lipid uptake by Huh7 cells. Huh7 cells were incubated with a mixture of anti–SR-BI mAbs (20 μg/mL) and 3H-CE–labeled HDL for 5 hours before incubation with unlabeled HDL for 30 minutes. Selective uptake was calculated from the known specific radioactivity of radiolabeled HDL-CE and is denoted in μg HDL-CE/μg cell protein. Results represent the mean ± SD of three different experiments performed in sextuplicate. (C) Cholesterol efflux from Huh7 cells. Huh7 cells were first incubated with 3H-cholesterol for 48 hours and then with BSA (0.5%) for 24 hours. Subsequently, cells were first incubated with anti–SR-BI mAbs (20 μg/mL) for 1 hour and then with unlabeled HDL for 4 hours. Fractional cholesterol efflux was calculated as the amount of the label obtained in the medium divided by the total label in each well regained after lipid extraction from cells. Results represent the mean ± SD of three different experiments performed in triplicate. *P < 0.01.

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Synergy Between Antibodies Targeting SR-BI Postbinding Function and Neutralizing Antibodies on Inhibition of HCV Infection.

To assess the clinical relevance of blocking SR-BI postbinding function to inhibit HCV infection, we determined the effect of anti–SR-BI mAbs on entry into Huh7.5.1 cells of HCVcc and HCVpp of major genotypes and highly infectious HCV strains selected during liver transplantation (P02VJ). All anti–SR-BI mAbs inhibiting HCVcc genotype 2a (Jc1) infection (QQ-4A3-A1, QQ-2A10-A5, QQ-4G9-A6 and NK-8H5-E3) also inhibited infection of HCVcc and HCVpp of all major genotypes (P < 0.01), whereas VSV-Gpp entry was unaffected (Fig. 5 and Supporting Fig. 3). Moreover, entry of patient-derived HCVpp P02VJ into both Huh7.5.1 cells and primary human hepatocytes was also efficiently inhibited by these anti–SR-BI mAbs (Supporting Fig. 7 and data not shown). Given that combinations of drugs targeting both viral and host factors represents a promising future approach to prevent and treat HCV infection, we next determined whether the combination of anti–SR-BI mAbs NK-8H5-E3 or QQ-2A10-A5 and anti-HCV envelope antibodies results in an additive or synergistic effect on inhibiting HCV infection. Thereto, each antibody was tested individually or in combination with a second antibody in a checkerboard format, and synergy was assessed using the combination index and the method of Prichard and Shipman30-32. Combination of anti–SR-BI and anti-HCV envelope antibodies resulted in a synergistic effect on inhibition of HCVpp P02VJ entry and HCVcc infection as reflected by a combination index of 0.06-0.67 (Supporting Fig. 7), and synergy of low doses was confirmed using the method of Prichard and Shipman (Fig. 6). These combinations reduced the IC50 of anti–SR-BI mAbs by up to 100-fold (Supporting Fig. 7). The marked observed synergy may be explained by the fact that the envelope- and SR-BI–specific antibodies target highly complementary steps during HCV entry. Taken together, these data indicate that interfering with SR-BI postbinding function may hold promise for the design of novel antiviral strategies targeting HCV entry factors.

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Figure 5. Genotype-independent inhibition of HCVpp and HCVcc infection by monoclonal anti–SR-BI antibodies. (A-E) Inhibition of entry into Huh7.5.1 cells of HCVpp bearing envelope glycoproteins from genotypes 1-4. Huh7.5.1 cells were preincubated with control (CTRL IgG) or anti–SR-BI mAbs (50 μg/mL) for 1 hour at 37°C before infection with HCVpp bearing envelope glycoproteins of strains H77 (1a), HCV-J (1b), JFH1 (2a), UKN3A1.28 (3a), or UKN4.21.16 (4) and VSV-Gpp. The results represent the mean ± SD from three experiments performed in triplicate. (F) Inhibition of infection of Huh7.5.1 cells with HCVcc bearing envelope glycoproteins from genotypes 1-4. Huh7.5.1 cells were preincubated with anti–SR-BI mAb (NK-8H5-E3, 50 μg/mL) for 1 hour at 37°C before infection with HCVcc. HCVpp and HCVcc infection was analyzed by luciferase reporter gene expression. Results are expressed as % HCVpp entry or HCVcc infection and represent (A-E) the mean ± SD from three independent experiments performed in triplicate and (F) the mean ± SEM from three independent experiments performed at least in triplicate. *P < 0.01.

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Figure 6. Synergy between anti–SR-BI and neutralizing antibodies in inhibiting HCV infection. HCVcc (Luc-Jc1) were preincubated with increasing concentrations of (A,B) anti-E1 (IGH520) or (C,D) anti-E2 (AP33) mAbs or (E,F) purified heterologous anti-HCV IgG obtained from an unrelated chronically infected subject or isotype control IgGs for 1 hour at 37°C and added to Huh7.5.1 cells preincubated with increasing concentrations of control or anti–SR-BI mAbs (A,C,E) NK-8H5-E3 or (B,D,F) QQ-2A10-A5 in a checkerboard format. HCVcc infection was analyzed by luciferase reporter gene expression. Effects of antibody combinations on HCVcc infection were evaluated using the method of Prichard and Shipman.32 Combination studies for each pair of compounds were performed in triplicate. The theoretical additive effect is calculated from the dose-response curves of individual compounds using the equation z = x + y (1 − x), where x and y represent the inhibition produced by the individual compounds and z represents the effect produced by the combination of compounds. The theoretical additive surface is subtracted from the actual experimental surface, resulting in a horizontal surface that equals the zero plane when the combination is additive. A surface raising more than 20% above the zero plane indicates a synergistic effect of the combination, and a surface dropping lower than 20% below the zero plane indicates antagonism.

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Discussion

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

We generated novel anti–SR-BI mAbs specifically inhibiting HCV entry during postbinding steps that enabled us for the first time, using endogenous SR-BI, to explore and validate the hypothesis that SR-BI has a multifunctional role during HCV entry and to elucidate the functional role of SR-BI postbinding activity for HCV infection. Our data demonstrate that the HCV postbinding function of hSR-BI can indeed be dissociated from its E2-binding function. Moreover, we demonstrate that the postbinding activity of SR-BI is of key relevance for cell-free HCV infection as well as cell-to-cell transmission.

SR-BI mediates uptake of HDL-CE in a two-step process including HDL binding and subsequent transfer of CE into the cell without internalization of HDL. At the same time, SR-BI also participates in HCV binding and entry into target cells. SR-BI is able to directly bind E2 and virus-associated lipoproteins but additional functions of SR-BI have been reported to be at play during HCV infection.11, 12, 15, 23 The results from this study highlight the importance of an SR-BI postbinding function for HCV entry and further extend the relevance of this function for HCV cell-to-cell transmission.

The molecular mechanisms underlying HCV cell-to-cell transmission are only partially understood. A recent study showed that SR-BI contributes to this process37 and that E2–SR-BI interaction and/or SR-BI–mediated lipid transfer likely takes place during HCV dissemination, as antibodies and small molecule inhibitors targeting both SR-BI binding and lipid transfer reduce HCV cell-to-cell transmission.9, 17 However, which SR-BI functions are relevant for this process remain to be determined. Taking advantage of our novel mAbs uniquely inhibiting SR-BI postbinding activity required for HCV entry, we demonstrated that an E2 binding-independent postbinding function is involved in neutralizing antibody-resistant cell-to-cell transmission. E2-independent SR-BI function in HCV dissemination is in line with the observation that cell-to-cell transmission is largely insensitive to E2-specific antiviral mAbs.37 Given that mSR-BI does not bind sE2 but mediates HCV entry and promotes cell-to-cell transmission, the postbinding function of SR-BI seems to be essential for HCV infection and dissemination, while the binding function may be dispensable. Furthermore, since HVR1-deleted HCV is less sensitive to inhibition by anti–SR-BI mAbs (Supporting Results and Supporting Fig. 4), HVR1–SR-BI interaction may play an important role during postbinding steps of HCV entry.

Previous studies using small molecule inhibitors indicated a role for SR-BI lipid transfer function in HCV infection and HDL-mediated entry enhancement.12, 13, 23 Because inhibition of cell-free HCV entry and cell-to-cell transmission by our anti–SR-BI mAbs was associated with interference with lipid transfer, our data suggest that the SR-BI lipid transfer function may be relevant for both initiation of HCV infection and viral dissemination. Of note, our anti–SR-BI mAbs are the first anti–SR-BI mAbs that do not inhibit HDL binding to SR-BI. These data suggest that HCV entry and dissemination can be inhibited without blocking HDL–SR-BI binding. The further characterization of the SR-BI postbinding function will make it possible to determine whether the SR-BI–mediated postbinding steps of HCV entry and dissemination are directly linked to its lipid transfer function.

Using SR-BI chimeras, we demonstrate that the determinants relevant for HCV postbinding steps lie within the N-terminal half of the human SR-BI ectodomain (Supporting Results and Supporting Figs. 5 and 6). Amino acids 70-87 and residue E210 of SR-BI are required for E2 binding, while distinct protein regions are involved in HDL binding.20, 38 Although the SR-BI determinants involved in HDL binding and CE uptake have not yet been defined, a recent study reported that amino acid C323 is critical for these processes.38 Given that our anti–SR-BI mAbs do not interfere with E2 and HDL binding, amino acids 70-87 and residues E210 and C323 are most likely not part of the targeted epitopes. Interestingly, the amino acid associated with cholesterol homeostasis5 probably also lies outside these epitopes. The further characterization of these epitopes may make it possible to more thoroughly determine the regions of SR-BI relevant for its postbinding function during initiation of HCV infection and spread.

Finally, our data suggest that the SR-BI postbinding function is a highly relevant target for antivirals. Therapeutic options for a large proportion of HCV-infected patients are still limited by drug resistance and adverse effects.1 Furthermore, a strategy for prevention of HCV liver graft infection is absent. Antivirals targeting essential host factors required for the HCV life cycle are attractive because they may increase the genetic barrier to antiviral resistance.2, 3 Indeed, our data demonstrate a marked synergy on the inhibition of HCV entry when combining antibodies directed against the viral envelope and SR-BI. These results suggest that combining molecules directed against viral and host entry factors is a promising strategy for prevention of HCV infection such as liver graft infection. The potent effect on cell-to-cell transmission and viral spread also opens a perspective of SR-BI–based entry inhibitors for treatment of chronic infection.

Small molecules and mAbs targeting SR-BI and interfering with HCV infection have been described.12, 17, 26 A human anti–SR-BI mAb has been reported to inhibit HDL binding, to interfere with cholesterol efflux and to decrease HCVcc entry during attachment steps without having a relevant impact on SR-BI–mediated postbinding steps.20, 26 A codon-optimized version of this mAb has been demonstrated to prevent HCV spread in vivo,9 underscoring the potential of SR-BI as an antiviral target. The mAbs generated in our study are highly novel in their function, as they do not interfere with sE2–SR-BI binding but inhibit HCV entry during postbinding steps of cell-free infection and cell-to-cell transmission. Furthermore, in contrast to described anti–SR-BI mAbs,26 these mAbs do not hinder HDL–SR-BI binding and only partially inhibit lipid transfer at concentrations significantly inhibiting HCV infection. Given their novel mechanism of action and their potential differential toxicity profile, QQ-4A3-A1, QQ-2A10-A5, QQ-4G9-A6, and NK-8H5-E3 define a novel class of anti–SR-BI mAbs for prevention and treatment of HCV infection.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

We thank R. Bartenschlager (University of Heidelberg, Germany) for providing Luc-Jc1 expression vector; T. Wakita (National Institute of Infectious Diseases, Tokyo, Japan) for the JFH1 construct; S. K. H. Foung (Stanford University, Palo Alto, CA) for anti-E2 antibody CBH23; and C. M. Rice (The Rockefeller University, New York, NY) and F. V. Chisari (The Scripps Research Institute, La Jolla, CA) for Huh7.5 and Huh7.5.1 cells, respectively. We also thank A. H. Patel (MRC-University of Glasgow Centre for Virus Research, Glasgow, UK) for Huh7.5-GFP cells and AP33 antibody; J. Ball (University of Nottingham, Nottingham, UK) for providing plasmids for production of different HCVpp genotypes; and D. Trono (Ecole Polytechnique Fédérale de Lausanne, Lausanne, Switzerland) for pWPI plasmid. We also acknowledge E. Schnober (University of Freiburg, Freiburg, Germany) for contributing to sE2 binding assays and S. Durand (INSERM U748, Strasbourg, France), C. Bach (INSERM U748, Strasbourg, France), J. Barths (INSERM, University of Strasbourg, Strasbourg, France), C. Granier (INSERM U758, France), and S. Glauben (Aldevron Freiburg, Freiburg, Germany) for technical assistance.

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  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information
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Supporting Information

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Additional Supporting Information may be found in the online version of this article.

FilenameFormatSizeDescription
HEP_26097_sm_SuppFig1.tif704KSupporting Information Figure 1.
HEP_26097_sm_SuppFig2.tif525KSupporting Information Figure 2.
HEP_26097_sm_SuppFig3.tif240KSupporting Information Figure 3.
HEP_26097_sm_SuppFig4.tif260KSupporting Information Figure 4.
HEP_26097_sm_SuppFig5.tif987KSupporting Information Figure 5.
HEP_26097_sm_SuppFig6.tif249KSupporting Information Figure 6.
HEP_26097_sm_SuppFig7.tif362KSupporting Information Figure 7.
HEP_26097_sm_SuppInfo.doc132KSupporting Information

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