Title: High Density Lipoprotein Inhibits Hepatitis C Virus-neutralizing Antibodies by Stimulating Cell Entry via Activation of the Scavenger Receptor BI
Abstract: Hepatitis C virus (HCV) exploits serum-dependent mechanisms that inhibit neutralizing antibodies. Here we demonstrate that high density lipoprotein (HDL) is a key serum factor that attenuates neutralization by monoclonal and HCV patient-derived polyclonal antibodies of infectious pseudo-particles (HCVpp) harboring authentic E1E2 glycoproteins and cell culture-grown genuine HCV (HCVcc). Over 10-fold higher antibody concentrations are required to neutralize either HCV-enveloped particles in the presence of HDL or human serum, and less than 3–5-fold reduction of infectious titers are obtained at saturating antibody concentrations, in contrast to complete inhibition in serum-free conditions. We show that HDL interaction with the scavenger receptor BI (SR-BI), a proposed cell entry co-factor of HCV and a receptor mediating lipid transfer with HDL, strongly reduces neutralization of HCVpp and HCVcc. We found that HDL activation of target cells strongly stimulates cell entry of viral particles by accelerating their endocytosis, thereby suppressing a 1-h time lag during which cell-bound virions are not internalized and can be targeted by antibodies. Compounds that inhibit lipid transfer functions of SR-BI fully restore neutralization by antibodies in human serum. We demonstrate that this functional HDL/SR-BI interaction only interferes with antibodies blocking HCV-E2 binding to CD81, a major HCV receptor, reflecting its prominent role during the cell entry process. Moreover, we identify monoclonal antibodies targeted to epitopes in the E1E2 complex that are not inhibited by HDL. Consistently, we show that antibodies targeted to HCV-E1 efficiently neutralize HCVpp and HCVcc in the presence of human serum. Hepatitis C virus (HCV) exploits serum-dependent mechanisms that inhibit neutralizing antibodies. Here we demonstrate that high density lipoprotein (HDL) is a key serum factor that attenuates neutralization by monoclonal and HCV patient-derived polyclonal antibodies of infectious pseudo-particles (HCVpp) harboring authentic E1E2 glycoproteins and cell culture-grown genuine HCV (HCVcc). Over 10-fold higher antibody concentrations are required to neutralize either HCV-enveloped particles in the presence of HDL or human serum, and less than 3–5-fold reduction of infectious titers are obtained at saturating antibody concentrations, in contrast to complete inhibition in serum-free conditions. We show that HDL interaction with the scavenger receptor BI (SR-BI), a proposed cell entry co-factor of HCV and a receptor mediating lipid transfer with HDL, strongly reduces neutralization of HCVpp and HCVcc. We found that HDL activation of target cells strongly stimulates cell entry of viral particles by accelerating their endocytosis, thereby suppressing a 1-h time lag during which cell-bound virions are not internalized and can be targeted by antibodies. Compounds that inhibit lipid transfer functions of SR-BI fully restore neutralization by antibodies in human serum. We demonstrate that this functional HDL/SR-BI interaction only interferes with antibodies blocking HCV-E2 binding to CD81, a major HCV receptor, reflecting its prominent role during the cell entry process. Moreover, we identify monoclonal antibodies targeted to epitopes in the E1E2 complex that are not inhibited by HDL. Consistently, we show that antibodies targeted to HCV-E1 efficiently neutralize HCVpp and HCVcc in the presence of human serum. Hepatitis C virus (HCV), 7The abbreviations used are: HCV, hepatitis C virus; HDL, high density lipoprotein; HCVpp, HCV pseudo-particles; HCVcc, cell culture-grown genuine HCV; SR-BI, scavenger receptor BI; LDL, low density lipoprotein; mAb, monoclonal antibody; HS, human serum; FCS, fetal calf serum; MLV, murine leukemia virus; GFP, green fluorescent protein; FACS, fluorescence-activated cell sorter; PBS, phosphate-buffered saline; NOB, neutralization of binding; HA, hemagglutinin. a member of the Flaviviridae family, is transmitted during parenteral exposures to infected material, such as contaminated blood or needles. Its genome encodes a precursor polyprotein of ∼3,000 amino acids (1Lindenbach B.D. Rice C.M. Knipe D.M. Howley P.M. Fields Virology. 2001: 991-1042Google Scholar). Cleavage of this polyprotein generates 10 polypeptides, including a core protein, two surface glycoproteins, E1 and E2, and the nonstructural proteins. HCV is an enveloped virus, implying that specific cell surface molecules mediate the capture of viral particles and their penetration inside the infected cells. Several molecules have been proposed as cell entry receptors of HCV, and most of them have been isolated based on binding studies with soluble recombinant E2 protein or HCV-like particles. Potential receptors include the CD81 tetraspanin (2Pileri P. Uematsu Y. Campagnoli S. Galli G. Falugi F. Petracca R. Weiner A.J. Houghton M. Rosa D. Grandi G. Abrignani S. Science. 1998; 282: 938-941Crossref PubMed Scopus (1806) Google Scholar), the low density lipoproteins (LDL) receptor (3Agnello V. Abel G. Elfahal M. Knight G.B. Zhang Q.X. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 12766-12771Crossref PubMed Scopus (810) Google Scholar), the scavenger receptor BI (SR-BI) (4Scarselli E. Ansuini H. Cerino R. Roccasecca R. Acali S. Filocamo G. Traboni C. Nicosia A. Cortese R. Vitelli A. EMBO J. 2002; 21: 5017-5025Crossref PubMed Scopus (961) Google Scholar) that binds HDL, native, or modified LDL and very low density LDL (vLDL) (5Calvo D. Gomez-Coronado D. Lasuncion M.A. Vega M.A. Arterioscler. Thromb. Vasc. Biol. 1997; 17: 2341-2349Crossref PubMed Scopus (214) Google Scholar), and several "capture" molecules that induce concentration of viral particles at the cell surface, hence allowing virions to find the cell entry receptors (6Bartosch B. Cosset F.L. Virology. 2006; 348: 1-12Crossref PubMed Scopus (135) Google Scholar). Experimental data using infectious HCV pseudo-particles (HCVpp) harboring authentic E1E2 glycoproteins (7Bartosch B. Dubuisson J. Cosset F.L. J. Exp. Med. 2003; 197: 633-642Crossref PubMed Scopus (957) Google Scholar, 8Hsu M. Zhang J. Flint M. Logvinoff C. Cheng-Mayer C. Rice C.M. McKeating J.A. Proc. Natl. Acad. Sci. U. S. 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Among infected individuals, only 20% recover from infection spontaneously, whereas most patients progress to chronic infection. The viral and host factors that determine HCV persistence or clearance at the acute stage of infection need to be understood in detail to improve antiviral therapy and to develop efficient vaccines. Studies focusing on innate and cellular immune responses have shown that HCV is able to evade or subvert the host defenses. Spontaneous HCV clearance is associated with a strong, early cellular immune response to multiple HCV epitopes, and both CD4+ and CD8+ responses are maintained for several years after viral clearance (12Bowen D.G. Walker C.M. Nature. 2005; 436: 946-952Crossref PubMed Scopus (647) Google Scholar). The recent development of infection assays based on HCVpp and HCVcc provides new opportunities to study the humoral response. There is evidence that neutralizing antibodies could play a role in disease control. 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Hepat. 1996; 3: 11-17Crossref PubMed Scopus (136) Google Scholar). On the other hand, a recent study suggested that the interplay of HCVpp with high density lipoprotein (HDL), but not LDL, leads to protection from neutralizing antibodies present in sera of both acute and chronic patients (19Bartosch B. Verney G. Dreux M. Donot P. Morice Y. Penin F. Pawlotsky J.-M. Lavillette D. Cosset F.-L. J. Virol. 2005; 79: 8217-8229Crossref PubMed Scopus (249) Google Scholar). Indeed, HCVpp were more effectively neutralized by purified monoclonal antibodies and immunoglobulins isolated from chronic patients, as compared with the same antibodies in the presence of HDL or human serum (19Bartosch B. Verney G. Dreux M. Donot P. Morice Y. Penin F. Pawlotsky J.-M. Lavillette D. Cosset F.-L. J. Virol. 2005; 79: 8217-8229Crossref PubMed Scopus (249) Google Scholar). Likewise, nonresolving acute phase patients develop low titer neutralizing antibodies that are unable to neutralize HCVpp in infection assays performed in the presence of human serum, as a result of the presence of HDL that completely abrogate their activity (18Lavillette D. Morice Y. Germanidis G. Donot P. Soulier A. Pagkalos E. Sakellariou G. Intrator L. Bartosch B. Pawlotsky J.-M. Cosset F.-L. J. Virol. 2005; 79: 6023-6034Crossref PubMed Scopus (242) Google Scholar, 19Bartosch B. Verney G. Dreux M. Donot P. Morice Y. Penin F. Pawlotsky J.-M. Lavillette D. Cosset F.-L. J. Virol. 2005; 79: 8217-8229Crossref PubMed Scopus (249) Google Scholar). Interestingly, deletion or mutation of HVR1, the hypervariable region-1 of the HCV-E2 glycoprotein that is under strong evolution pressure during disease outcome (29Farci P. Shimoda A. Coiana A. Diaz G. Peddis G. Melpolder J.C. Strazzera A. Chien D.Y. Munoz S.J. Balestrieri A. Purcell R.H. Alter H.J. 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HCVpp, RD114pp, MLVpp, and HApp were produced (7Bartosch B. Dubuisson J. Cosset F.L. J. Exp. Med. 2003; 197: 633-642Crossref PubMed Scopus (957) Google Scholar, 34Sandrin V. Boson B. Salmon P. Gay W. Nègre D. LeGrand R. Trono D. Cosset F.-L. Blood. 2002; 100: 823-832Crossref PubMed Scopus (251) Google Scholar) by transfection in 293T cells of vectors encoding viral glycoproteins, packaging proteins, and GFP transfer vector. Viral particles containing supernatants were used to infect Huh-7 hepatoma cells directly or upon purification by ultracentrifugation through a 20% sucrose cushion. Expression Constructs and Production of HCVcc—The pFK-Luc-Jc1 is a chimeric J6CF/JFH1 HCV genome consisting of codons 1–846, derived from J6CF (AF177036), and codons 847–3033, derived from JFH1 (AB047639). The plasmid pFK-Luc-Jc1 also encodes a bicistronic firefly-luciferase reporter with a design analogous to pFK-Luc-JFH1 as described recently (10Wakita T. Pietschmann T. Kato T. Date T. Miyamoto M. Zhao Z. Murthy K. Habermann A. Krausslich H.G. Mizokami M. Bartenschlager R. Liang T.J. Nat. Med. 2005; 11: 791-796Crossref PubMed Scopus (2424) Google Scholar). HCVcc were produced (10Wakita T. Pietschmann T. Kato T. Date T. Miyamoto M. Zhao Z. Murthy K. Habermann A. Krausslich H.G. Mizokami M. Bartenschlager R. Liang T.J. Nat. Med. 2005; 11: 791-796Crossref PubMed Scopus (2424) Google Scholar) by electroporation of Huh7-Lunet cells. These cells, characterized by high permissiveness for HCV RNA replication (35Lohmann V. Hoffmann S. Herian U. Penin F. Bartenschlager R. J. Virol. 2003; 77: 3007-3019Crossref PubMed Scopus (345) Google Scholar), originally carried a selectable HCV replicon and were cured by treatment with a specific inhibitor. Culture fluid of electroporated cells was harvested 48 h later and used directly in infection assays using Huh7-Lunet target cells or after purification, as described above for HCVpp. Reagents and Antibodies—Preparation of HS was described previously (19Bartosch B. Verney G. Dreux M. Donot P. Morice Y. Penin F. Pawlotsky J.-M. Lavillette D. Cosset F.-L. J. Virol. 2005; 79: 8217-8229Crossref PubMed Scopus (249) Google Scholar). The HDL (Calbiochem) preparation (density 1.063–1.2 mg/ml) contained a mixture of HDL2 and HDL3. BLTs (36Nieland T.J. Chroni A. Fitzgerald M.L. Maliga Z. Zannis V.I. Kirchhausen T. Krieger M. J. Lipid Res. 2004; 45: 1256-1265Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar) were obtained from Chembridge. The JS81 CD81-specific mAb was purchased from Pharmingen. The 9/27, 3/11 (8Hsu M. Zhang J. Flint M. Logvinoff C. Cheng-Mayer C. Rice C.M. McKeating J.A. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 7271-7276Crossref PubMed Scopus (693) Google Scholar), AP33 (37Owsianka A. Clayton R.F. Loomis-Price L.D. McKeating J.A. Patel A.H. J. Gen. Virol. 2001; 82: 1877-1883Crossref PubMed Scopus (166) Google Scholar), CBH-2, CBH-5, CBH-7 (30Keck Z.Y. Li T.K. Xia J. Bartosch B. Cosset F.L. Dubuisson J. Foung S.K. J. Virol. 2005; 79: 13199-13208Crossref PubMed Scopus (83) Google Scholar), and H35, H48, H53, H54, H57, and H60 (7Bartosch B. Dubuisson J. Cosset F.L. J. Exp. Med. 2003; 197: 633-642Crossref PubMed Scopus (957) Google Scholar, 38Deleersnyder V. Pillez A. Wychowski C. Blight K. Xu J. Hahn Y.S. Rice C.M. Dubuisson J. J. Virol. 1997; 71: 697-704Crossref PubMed Google Scholar, 39Flint M. Maidens C. Loomis-Price L.D. Shotton C. Dubuisson J. Monk P. Higginbottom A. Levy S. McKeating J.A. J. Virol. 1999; 73: 6235-6244Crossref PubMed Google Scholar, 40Op De Beeck A. Voisset C. Bartosch B. Ciczora Y. Cocquerel L. Keck Z. Foung S. Cosset F.L. Dubuisson J. J. Virol. 2004; 78: 2994-3002Crossref PubMed Scopus (191) Google Scholar), and the E2mAb-1 8C. Granier, G. Verney, and F. L. Cosset, unpublished data. are E2-specific mAbs. H111 (30Keck Z.Y. Li T.K. Xia J. Bartosch B. Cosset F.L. Dubuisson J. Foung S.K. J. Virol. 2005; 79: 13199-13208Crossref PubMed Scopus (83) Google Scholar) and A4 (41Dubuisson J. Hsu H.H. Cheung R.C. Greenberg H.B. Russell D.G. Rice C.M. J. Virol. 1994; 68: 6147-6160Crossref PubMed Google Scholar) are E1-specific mAbs. Polyclonal antibodies against E1 (strain H77) were purified from mouse immune sera (kind gift of G. Verney), obtained by immunization with modified HCVpp harboring E1 glycoproteins only. 8C. Granier, G. Verney, and F. L. Cosset, unpublished data. Antibodies were purified using protein-G-Sepharose (Amersham Biosciences). A pool of HCV immunoglobulins was purified and concentrated from over 30 chronic HCV sera of genotypes 1a, 1b, 2a, 2b, and 3 as described previously (19Bartosch B. Verney G. Dreux M. Donot P. Morice Y. Penin F. Pawlotsky J.-M. Lavillette D. Cosset F.-L. J. Virol. 2005; 79: 8217-8229Crossref PubMed Scopus (249) Google Scholar). The recombinant CD81-LEL fragment (amino-acids 112–202) and a truncated soluble form of E2 glycoprotein (sE2) (amino-acids 384–664) were fused to a His tag, produced in mammalian cells and purified on nickel-nitrilotriacetic acid resin (Qiagen). Binding Assays—Binding of HCVpp was performed as described previously for other types of pseudo-particles (42Lavillette D. Boson B. Russell S. Cosset F.-L. J. Virol. 2001; 75: 3685-3695Crossref PubMed Scopus (70) Google Scholar). Briefly, 50 μl of virus particles purified on a 20% sucrose cushion were incubated to 106 target cells in the presence of 0.1% sodium azide for 1 h. Human serum was added or not at the concentration of 2.5%. Cells were then washed twice with PBFA (PBS, 2% fetal bovine serum, and 0.1% sodium azide) and incubated with the H53 anti-HCV-E2 mAb (40 μg/ml) for 1 h at 4°C. After two washes, cells were incubated with a goat anti-mouse RPE (R0480; Dako A/S Denmark) diluted in PBFA (1:100) for 45 min at 4 °C. Fluorescence of living 10,000 cells was determined by FACS analysis. Binding of soluble E2 glycoprotein to CD81 was performed as described previously (39Flint M. Maidens C. Loomis-Price L.D. Shotton C. Dubuisson J. Monk P. Higginbottom A. Levy S. McKeating J.A. J. Virol. 1999; 73: 6235-6244Crossref PubMed Google Scholar) and was used to detect the "neutralization of binding" (NOB) activity of antibodies to CD81. Briefly, 5 μg/ml of sE2 harboring a His tag was mixed with 10 μg/ml of tested antibodies and incubated for 1 h at 37 °C. Molt-4 cells (105 cells) were then incubated to the mixture for 1 h at room temperature, and the amount of cell-bound sE2 was determined by FACS analysis using anti-His tag antibody (penta-His; Qiagen). Infection Assays—Huh-7 and Huh7-Lunet cells were seeded 24 h prior to inoculation (7Bartosch B. Dubuisson J. Cosset F.L. J. Exp. Med. 2003; 197: 633-642Crossref PubMed Scopus (957) Google Scholar, 10Wakita T. Pietschmann T. Kato T. Date T. Miyamoto M. Zhao Z. Murthy K. Habermann A. Krausslich H.G. Mizokami M. Bartenschlager R. Liang T.J. Nat. Med. 2005; 11: 791-796Crossref PubMed Scopus (2424) Google Scholar). 2 h before infection, target cells were preincubated in Dulbecco's modified Eagle's medium containing 0.1% FCS. Medium was then removed, and dilutions of viral supernatants and various compounds were added to the cells as indicated. After 4 h, supernatants were removed, and the infected cells were kept in regular medium (Dulbecco's modified Eagle's medium, 10% FCS) for 72 h before analysis. For infection assays with HCVpp, the infectious titers were deduced from the percentage of GFP-positive cells, as determined by FACS analysis (7Bartosch B. Dubuisson J. Cosset F.L. J. Exp. Med. 2003; 197: 633-642Crossref PubMed Scopus (957) Google Scholar). Infections were controlled by pseudo-particles devoid of E1E2 glycoproteins, which resulted in background titers below 5 × 102 infectious units/ml. For analyses of HCVcc infectious titers, cells were lysed for luciferase assays, as described previously (10Wakita T. Pietschmann T. Kato T. Date T. Miyamoto M. Zhao Z. Murthy K. Habermann A. Krausslich H.G. Mizokami M. Bartenschlager R. Liang T.J. Nat. Med. 2005; 11: 791-796Crossref PubMed Scopus (2424) Google Scholar). Surface Plasmon Resonance Binding Assays—Biomolecular interactions were studied using a BIAcore-3000 instrument (BIAcore AB, Uppsala, Sweden), which used surface plasmon resonance as a detection method. The AP33 antibody (100 μg/ml in 10 mm acetate buffer, pH 4.5) was covalently immobilized to the dextran matrix of a CM3 sensor chip (amine coupling kit, BIAcore AB) at a flow rate of 5 μl/min. Activation and blocking steps were performed as described previously (43Ricard-Blum S. Bernocco S. Font B. Moali C. Eichenberger D. Farjanel J. Burchardt E.R. van der Rest M. Kessler E. Hulmes D.J. J. Biol. Chem. 2002; 277: 33864-33869Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). Purified HCVpp were injected over AP33 antibody in PBS containing 0.005% P20 surfactant (BIAcore AB) at a flow rate of 5 μl/min at 25 °C. HCVpp capture levels ranged between 200 and 500 resonance units. A control flow cell was prepared by immobilizing irrelevant antibody (mouse anti-IL2) according to the same procedure. Control sensorgrams representing nonspecific binding to the sensor chip surface were automatically subtracted from the sensorgrams obtained with captured HCVpp. Binding assays of HDL, A4 antibody, and CD81-LEL were performed at 25 °C in PBS with 0.005% P20 surfactant at a flow rate of 5 μl/min. The surface was then regenerated with pulse of 0.025% SDS. We reported previously that an interplay between HDL and HCV-E2 leads to infection enhancement of HCVpp (19Bartosch B. Verney G. Dreux M. Donot P. Morice Y. Penin F. Pawlotsky J.-M. Lavillette D. Cosset F.-L. J. Virol. 2005; 79: 8217-8229Crossref PubMed Scopus (249) Google Scholar, 44Voisset C. Callens N. Blanchard E. Op De Beeck A. Dubuisson J. Vu-Dac N. J. Biol. Chem. 2005; 280: 7793-7799Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar), an event stimulated by HVR1, and to inhibition of neutralizing antibodies (19Bartosch B. Verney G. Dreux M. Donot P. Morice Y. Penin F. Pawlotsky J.-M. Lavillette D. Cosset F.-L. J. Virol. 2005; 79: 8217-8229Crossref PubMed Scopus (249) Google Scholar). An example of neutralization curves deduced from the results of infection assays obtained with a monoclonal antibody (mAb), carried out in the presence or the absence of HDL or HS, is displayed in Fig. 1. These results allowed estimation of IC50 and IC90 values for this mAb (i.e. antibody concentrations that reduce HCVpp infectious titers by 2- and 10-fold, respectively) at 1.3 and 8 μg/ml, respectively, in the absence of HS or HDL. This compared with an IC50 of 6.6 μg/ml for neutralization assays performed in the presence of HS. The attenuation of neutralization was not affected by the mAb concentration, in the range of values tested, i.e. 0.1–50 μg/ml (data not shown). More importantly, no IC90 could be determined from neutralization assays performed in the presence of HS or HDL because the mAb could not neutralize more than 65% HCVpp under these conditions (Fig. 1), even at high antibody concentrations (19Bartosch B. Verney G. Dreux M. Donot P. Morice Y. Penin F. Pawlotsky J.-M. Lavillette D. Cosset F.-L. J. Virol. 2005; 79: 8217-8229Crossref PubMed Scopus (249) Google Scholar). Therefore, such a level of neutralization only reduced the infectious titers by less than 3-fold, which contrasted with the complete inhibition of HCVpp infectivity achieved in serum-free conditions for antibody concentrations of ∼50 μg/ml (19Bartosch B. Verney G. Dreux M. Donot P. Morice Y. Penin F. Pawlotsky J.-M. Lavillette D. Cosset F.-L. J. Virol. 2005; 79: 8217-8229Crossref PubMed Scopus (249) Google Scholar, 31Owsianka A. Tarr A.W. Juttla V.S. Lavillette D. Bartosch B. Cosset F.L. Ball J.K. Patel A.H. J. Virol. 2005; 79: 11095-11104Crossref PubMed Scopus (246) Google Scholar). Finally, similar results were obtained for HCVpp carrying E1E2 glycoproteins of other genotypes and/or when HS was added to purified HCVpp/antibody immune complexes (Table 1).TABLE 1Desensitization of HCVpp neutralization by human serumSequential treatment of viral particles and Huh-7 target cells% neutralizationcResults are expressed as the mean percentages (mean ± S.D.; n = 4) of inhibition of the infectious titers relative to incubation with medium devoid of antibodies.Step A, treatment of HCVppaHCVpp mixed, or not, with 2.5% HS and/or 4 μg/ml of the AP33 mAb, as indicated, were preincubated for 45 min at room temperature. NA indicates not applicable.Step B, infection of Huh-7 cellsbHuh-7 target cells were simultaneously incubated with the indicated reagents for 4 h at 37 °C.NAHCVpp + mAb72.23 ± 10.1NAHCVpp + mAb + HS19.73 ± 10.7Preincubation HCVpp + HSMix step A + mAb21.46 ± 9.4Preincubation HCVpp + mAbMix step A76.48 ± 3.8Preincubation HCVpp + mAbMix step A + HS38.36 ± 7.5Preincubation HCVpp + mAb, then purificationdHCVpp preincubated, or not, with HS and/or the AP33 mAb were purified by ultracentrifugation through a 20% sucrose cushion.Purified mix step A56.92 ± 10,2Preincubation HCVpp + mAb, then purificationdHCVpp preincubated, or not, with HS and/or the AP33 mAb were purified by ultrac