Immunogenic and Functional Organization of Hepatitis C Virus (HCV) Glycoprotein E2 on Infectious HCV Virions

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1 JOURNAL OF VIROLOGY, Jan. 2007, p Vol. 81, No X/07/$ doi: /jvi Copyright 2007, American Society for Microbiology. All Rights Reserved. Immunogenic and Functional Organization of Hepatitis C Virus (HCV) Glycoprotein E2 on Infectious HCV Virions Zhen-Yong Keck, 1 Jinming Xia, 1 Zhaohui Cai, 2 Ta-Kai Li, 1 Ania M. Owsianka, 3 Arvind H. Patel, 3 Guangxiang Luo, 2 and Steven K. H. Foung 1 * Department of Pathology, Stanford University School of Medicine, Stanford, California ; Department of Microbiology, Immunology, and Molecular Genetics, University of Kentucky College of Medicine, Lexington, Kentucky ; and MRC Virology Unit, Institute of Virology, University of Glasgow, Church Street, Glasgow G11 5JR, United Kingdom 3 Received 8 August 2006/Accepted 23 October 2006 Development of full-length hepatitis C virus (HCV) RNAs replicating efficiently and producing infectious cell-cultured virions, HCVcc, in hepatoma cells provides an opportunity to characterize immunogenic domains on viral envelope proteins involved in entry into target cells. A panel of immunoglobulin G1 human monoclonal antibodies (HMAbs) to three immunogenic conformational domains (designated A, B, and C) on HCV E2 glycoprotein showed that epitopes within two domains, B and C, mediated HCVcc neutralization, whereas HMAbs to domain A were all nonneutralizing. For the neutralizing antibodies to domain B (with some to conserved epitopes among different HCV genotypes), the inhibitory antibody concentration reducing HCVcc infection by 90%, IC 90, ranged from 0.1 to 4 g/ml. For some neutralizing HMAbs, HCVcc neutralization displayed a linear correlation with an antibody concentration between the IC 50 and the IC 90 while others showed a nonlinear correlation. The differences between IC 50 /IC 90 ratios and earlier findings that neutralizing HMAbs block E2 interaction with CD81 suggest that these antibodies block different facets of virus-receptor interaction. Collectively, these findings support an immunogenic model of HCV E2 having three immunogenic domains with distinct structures and functions and provide added support for the idea that CD81 is required for virus entry. Hepatitis C virus (HCV) is a positive-stranded RNA virus containing at least three structural proteins core and two envelope glycoproteins, E1 and E2 and six nonstructural proteins (1). Studies with infectious retroviral particles pseudotyped with HCV E1E2 (HCVpp) showed that virus entry requires both E1 and E2 glycoproteins associated as a heterodimer, involves interactions with CD81, is low ph dependent, and is blocked by antibodies to HCV E2 and sera from HCV-infected individuals (3, 8, 13). Detailed information on the immunogenic and functional organization of HCV envelope glycoproteins and in particular E2 is needed to facilitate immunotherapeutics and vaccine development. Cross-competition studies with a panel of HCV E2 human monoclonal antibodies (HMAbs) showed that the HCVpp E2 glycoprotein contains three immunogenic conformational domains, designated A, B, and C, that are accessible on the surface of HCVpp (10). Each domain carries epitopes that are either highly conserved among diverse HCV genotypes or more restrictedly conserved. Epitopes within two domains, B and C, are targets of HCVpp-neutralizing antibodies, and the other domain, A, contains nonneutralizing epitopes that participate in structural changes as part of a ph-dependent virus envelope fusion process (9). Recently, three groups developed full-length HCV RNA genomes replicating efficiently when transfected into human hepatoma cells (Huh7) and producing infectious virions (11, 15, 17). The availability of these and other cell-cultured * Corresponding author. Mailing address: Stanford Medical School Blood Center, 800 Welch Rd., Palo Alto, CA Phone: (650) Fax: (650) sfoung@stanford.edu. Published ahead of print on 1 November infectious HCV virions, HCVcc, should greatly accelerate studies of HCV biology (4, 11, 14 17). This report focuses on the immunogenic and functional organization of HCV E2 on HCVcc virions. Three immunogenic conformational domains on HCV virion. It has been reported elsewhere that stable human hepatoma cell lines containing a chromosomally integrated cdna of HCV genotype 2a (JFH1) RNA constitutively secrete infectious virions into the medium (4). This provides a robust source of virus to study each aspect of the entire HCV life cycle. HCVcc virions from stable cell lines could reinfect naïve Huh7.5 cells, and viral replication could be suppressed by alpha interferon. We examined whether HCVcc infectivity can be neutralized by a panel of immunoglobulin G1 (IgG1) HMAbs to three distinct immunogenic domains on HCV E2 glycoprotein. Four antibodies (CBH-2, -5, -8C, and -11) to domain B, three antibodies (CBH-4B, -4D, and -4G) to domain A, one antibody to domain C (CBH-7), and a negativecontrol isotype-matched HMAb to cytomegalovirus (R04) were tested. Production of HCVcc, titration of infectious units, and HCV infection assays were performed as described previously (4). To determine HCV-neutralizing activities of HMAbs, HCVcc-containing culture medium (HCV titer, IU/ml) was used to dilute HMAbs to different concentrations and then added to Huh7.5 cells in a 12-well cell culture plate. After 3hof incubation, the HCV and antibody mixture was removed, and the cells were washed twice with phosphate-buffered saline (PBS) and incubated with 1 ml Dulbecco modified Eagle medium containing 10% fetal bovine serum. Infectivity was determined by measuring the levels of positive-stranded HCV RNA using an RNase protection assay (RPA) (4). Some of the antibodies 1043

2 1044 NOTES J. VIROL. FIG. 1. Neutralization of genotype 2a HCVcc infectivity as determined by RPA using [ - 32 P]UTP-labeled HCV-specific RNA probes. HCVcc were incubated with anti-e2 HMAbs at increasing concentrations prior to infection of Huh7.5 cells preseeded in a six-well plate. Antibody concentrations used were 0, 0.016, 0.08, 0.4, and 2.0 g/ml for CBH-2, -5, and -8C; 0.08, 0.4, 2, and 10 g/ml for CBH-11; and 0, 0.4, 2, 10, and 50 g/ml for CBH-7, -4G, -4B, -4D, and R04 (an isotype-matched HMAb to a cytomegalovirus epitope), respectively. At 3 h p.i., the HCVantibody-containing medium was removed, and the cells were washed with PBS, refed with fresh medium, and incubated at 37 C. At 72 h p.i., total RNA was extracted from the HCV-infected Huh7.5 cells using Trizol reagent (Invitrogen). The presence of positive-strand HCV RNA in the HCV-infected Huh7.5 cells was determined by RPA using an RPA III kit following the manufacturer s procedures (Ambion). As described previously (4), a total of 10 g of total cellular RNA extracted from HCV-infected Huh7.5 cells was hybridized with 10 5 cpm of the [ 32 P]UTPlabeled negative-strand 3 -untranslated-region RNA probe and cpm of human -actin RNA probe. After RNase digestion, the RNA products were analyzed in a 6% polyacrylamide-7.7 M urea gel, autoradiographed, and quantified with a phosphorimager. Probes for HCV and -actin (P hcv and P actin ) are indicated on the right. Numbers at left are molecular sizes in base pairs. (CBH-7, -4G, -4B, and -4D and control antibody R04) were assessed at concentrations up to 50 g/ml. The total cellular RNA was extracted from the HCV-infected Huh7.5 cells in six-well cell culture plates at 3 days postinfection (p.i.) and quantified by an RPA using an [ - 32 P]UTP-labeled RNA probe containing the negative-stranded HCV 3 -untranslatedregion RNA (Fig. 1). The HMAbs to domain B, CBH-2, -5, -8C, and -11, neutralized HCVcc infectivity with high potency, while antibodies to domain A, CBH-4G, -4B, and -4D, had no neutralizing activities. The HMAb to domain C, CBH-7, showed a modest HCVcc-neutralizing activity. HCVcc neutralization was confirmed with NS3 protein expression measurements by Western blot analysis. Huh7.5 cells were infected in the presence of each of the antibodies as described above; cells were lysed at 3 days p.i. and analyzed. The abilities of each domain A and representative domain B (CBH-5) and domain C (CBH-7) HMAbs to neutralize HCV infectivity to Huh7.5 cells are shown in Fig. 2A and B. All domain A HMAbs showed no effect on NS3 protein levels (Fig. 2A). Strikingly, HCVcc was completely neutralized at low antibody concentrations by all domain B HMAbs as represented by CBH-5 in Fig. 2B and more fully discussed below. However, the domain C HMAb, CBH-7, showed neutralizing activity only at an IgG concentration of 5 g/ml (Fig. 2B). The glyceraldehyde-3- phosphate dehydrogenase (GAPDH) protein used as an internal control was detected by using an anti-gapdh monoclonal antibody in Fig. 2A and B (Abcam, Cambridge, MA). To determine whether a reduction in HCV RNA and NS3 expression by neutralizing antibodies is correlated with a reduction in infectious virion production, CBH-4G and CBH-5 as respective representatives of domains A and B HMAbs were assessed in an infectivity reduction assay (Table 1). HCVcc were incubated with each HCV HMAb and R04 (control) at 0.1 to 100 g/ml for 1 hour prior to Huh7.5 cell infection. Huh7.5 cells were preseeded the previous day at a density of 32,000 cells/well in a 24-well plate. At 3 h p.i., HCV-containing medium was replaced with 1 ml of Dulbecco modified Eagle medium containing 10% fetal calf serum. At day 3 p.i., 1 ml of supernatant from each well was centrifuged at 1,000 g for 5 min to remove cell debris and 350 l was then added to a second corresponding well containing uninfected Huh7.5 cells. Following another 3 days, cell culture medium was removed. The cells were harvested and fixed in triplicate sets onto a 24-spot Teflon-coated microscope slide as described previously (7). Infection was assessed for NS3, NS5A, and E2 expression by an indirect immunofluorescence assay as described previously (7), employing a mouse monoclonal antibody to NS3 (4), a polyclonal sheep anti-ns5a serum (12), and antibody CBH-2 to E2. Detection was with fluorescein isothiocyanate-conjugated anti-species IgG (Jackson ImmunoResearch Laboratories), and counterstaining was done with Evans blue. Bound antibody was revealed by fluorescence microscopy. At all tested antibody concentrations for CBH-4G and R04, the HCVcc yield in the first set of infected cells was not reduced, as sufficient amounts of virus were passed to the second set of Huh7.5 cells, leading to the majority of the cells being infected. For CBH-5, reduced HCVcc infection was observed starting at 0.5 to 1 g/ml, with complete infectivity reduction at concentrations above 1 g/ml. Collectively, these findings support an immunogenic model of HCVcc E2 containing three distinct functional domains as previously shown with HCVpp studies. Virus neutralization potency with HCV virions. A more quantitative measure of neutralization potency was determined by calculating IC 90, the inhibitory antibody concentration reducing HCV infection by 90% (Fig. 2B and C). HCVcc virions were incubated for 2 h with each HMAb at to 100 g/ml prior to Huh7.5 cell infection. Figure 2B shows representative antibodies, with CBH-5 blocking NS3 expression at 0.02 g/ml but CBH-7 requiring 10 g/ml. NS3 reduction as quantified by phosphorimager analysis for each HMAb is summarized in Fig. 2C. The four antibodies to domain B, CBH-2, -5, -8C, and -11, completely neutralized HCVcc with respective IC 90 s at 0.4, 0.1, 1, and 4 g/ml. The average Ig concentration of domain B HMAbs for the IC 90 was 1.37 g/ml or 9.14 nmol/liter. For domain C HMAb CBH-7, the IC 90 was at 50 g/ml or 332 nmol/liter. The neutralization potency is in the order CBH-5 CBH-2 CBH-8C CBH-11 CBH-7, which is in agreement with HCVpp pseudotyped with JFH1 2a E1E2, with domain B HMAbs having greater activities than domain C-specific CBH-7 (Fig. 3A and B).

3 VOL. 81, 2007 NOTES 1045 FIG. 2. Effect of HMAbs on genotype 2a HCVcc infectivity as determined by NS3 expression. HCVcc were incubated with each HMAb at increasing concentrations prior to infection of Huh7.5 cells preseeded in a 24-well plate. At 3 h p.i., the HCVcc-antibody-containing medium was removed, and the cells were washed twice with PBS followed by incubation at 37 C in fresh medium. Cells were harvested for Western blotting analysis at 72 h p.i. (A) Domain A HMAbs showed no reduction of NS3 expression. (B) Quantitative HCVcc virus neutralization with CBH-5 and -7 shown as respective representative domain B and C antibodies. HCV NS3 protein expression was determined by a monoclonal antibody specific to NS3 (4). The GAPDH protein used as an internal control was detected using an anti-gapdh monoclonal antibody (Abcam). The expression level for NS3 protein was quantified by phosphorimager analysis, and the percent neutralization relative to control cells without antibody treatment (100%) was calculated. (C) Table of CBH-2, -5, -8C, -11, and -7 antibody concentrations to reach 50% and 90% HCVcc neutralization, IC 50 and IC 90, respectively, as calculated using Prism software (GraphPad). Although these HCV HMAbs were derived from a genotype 1b-infected donor, the IC 50 s of domain B HMAbs for 2a strain JFH1 were lower than with genotype 1b E1E2-pseudotyped HCVpp (Fig. 3B). Also, these antibodies were not able to TABLE 1. HCV infectivity reduction Infection level at antibody concn ( g/ml) a : Antibody CBH-4G CBH-5 / R04 a, 60% infected cells;, 50% infected cells; /, 20% infected cells;, 0.5% infected cells. completely neutralize 1b HCVpp (9, 13) in contrast to complete 2a HCVpp neutralization. Maximum neutralization of genotype 1b HCVpp ranged between 50 and 85% at a saturating IgG concentration of 50 g/ml or higher as shown in Fig. 3A. The IC 50 s for 1b HCVpp neutralization for CBH-2, -5, -8C, -11, and -7 were 1.33, 1.77, 34.58, 16.74, and g/ml, respectively. The respective IC 50 s for JFH1 2a HCVpp neutralization for CBH-2, -5, -8C, -11, and -7 were 0.35, 0.10, 1.50, 1.50, and 60 g/ml. This genotypic difference cannot be explained by antibody affinity, as the antibody disassociation constant, K D, is similar for each antibody with both genotypes except for CBH-8C (Fig. 3C). Studies are under way to assess the relative density of each epitope as identified by these HMAbs on the surface of both genotype 1b and 2a HCVpp, as

4 1046 NOTES J. VIROL. FIG. 3. Virus neutralization of genotype 2a and 1b HCVpp. HCVpp production, purification, and neutralization were performed as previously described (2, 9). (A) HCVpp were incubated with each E2 HMAb at increasing concentrations (0, 0.01, 0.1, 1, 5, 10, 20, 50, and 100 g/ml) for 60 min at 37 C prior to infection of Huh7 cells preseeded in a 96-well plate. At 3 h p.i., the HCVcc-antibody-containing medium was removed, and the cells were washed twice with PBS. Samples were harvested for luciferase activities assay at 72 h p.i. Neutralization activity was determined by percent reduction of luciferase activity relative to no-antibody control. (B) IC 50 s of CBH-2, -5, -8C, -11, and -7 HMAbs for genotype 2a or 1b HCVpp as calculated using Prism software (GraphPad). (C) Antibody K D measurements were performed with sucrose density equilibrium gradient-purified genotype 1b and JFH1 2a HCVpp as previously described (9). the critical number of binding sites being occupied is another contributing factor for virus neutralization. The IC 50 of these antibodies for either 1b or JFH1 2a HCVpp (Fig. 3B) was higher than that for JFH1 2a HCVcc (Fig. 2C). The basis for the global differences between HCVcc and HCVpp is not known but could reflect differences in how these particles are assembled, leading to differences in the relative copy number of the incorporated viral glycoproteins and in amino acid alignment and altered accessibility of these epitopes. This could include differences in E2 glycosylation with HCVpp and HCVcc affecting the surface exposure of their epitopes. Lending support to differences in how these particles are packaged are earlier studies with CD81, a putative HCV receptor (6). All domain B (CBH-2, -5, -8C, and -11) and C (CBH-7) antibodies blocked recombinant E2 protein binding to CD81-expressing cells or to CD81-coated plates and were designated as neutralization-of-binding (NOB) positives. All antibodies to domain A, CBH-4B, -4D, and -4G, were NOB negative as they failed to block E2-CD81 interaction. While an antibody designated NOB positive or negative was generally predictive of HCVpp and HCVcc neutralization, there are subtle differences. CBH-7 blocks recombinant 1a E2 binding to CD81 and neutralizes 1a HCVpp but did not block 1a plasma-derived HCV virion binding to CD81 (6). While multiple factors can account for a lack of binding to plasmaderived virions, if HCVcc is more structurally similar to plasmaderived virions than HCVpp is, the findings with CBH-7 are consistent with the view that the structural arrangement of envelope glycoproteins is different with HCVpp and HCVcc and certainly with the CBH-7 epitope. Another observation was the HCVcc neutralization profiles for CBH-5 and CBH-7 displaying a linear relationship with antibody concentration from IC 50 to IC 90 (Fig. 2B and C). However, the profiles for CBH-2, -8C, and -11 displayed a nonlinear relationship between IC 50 and IC 90, in which 5 to 14 times more antibodies were required to achieve IC 90 than IC 50. The possible contributions to differences with neutralization profiles include affinity of antibody binding to different viral epitopes, kinetics of antibody association and dissociation with targeted antigen, IgG subclass, and mechanisms of neutralization whereby these antibodies inhibit different facets of HCVcc interaction with its putative receptor(s) (5). However, IgG subclass or antibody affinity is an unlikely factor in this case as these antibodies are all IgG1 and their relative K D values are similar apart from CBH-5 (Fig. 3C). More studies will be required to find out whether these antibodies block at distinct steps in virus-receptor interactions. In summary, the immunogenic organization of HCVcc E2 glycoprotein consists of three distinct domains with epitopes mediating virus neutralization restricted to two domains as described for HCVpp. The fact

5 VOL. 81, 2007 NOTES 1047 that the ability to block E2 binding to CD81 is predictive of neutralization provides additional support for the idea that CD81 is a required molecule for HCVpp and HCVcc entry. We are grateful to Charles Rice, Rockefeller University, for providing the Huh7.5 cells; Mark Harris, University of Leeds, for the sheep anti-ns5a serum; and Neil Greenspan, Case Western Reserve University, for helpful discussion, and we appreciate the technical assistance of H. Y. Chen and Dana Bangs. This work was supported in part by National Institutes of Health grants HL and AI47355 to S.K.H.F. and CA93712 to G.L. and by the Medical Research Council, United Kingdom. REFERENCES 1. Bartenschlager, R Hepatitis C virus molecular clones: from cdna to infectious virus particles in cell culture. Curr. Opin. Microbiol. 9: Bartosch, B., J. Dubuisson, and F. L. Cosset Infectious hepatitis C virus pseudo-particles containing functional E1-E2 envelope protein complexes. J. Exp. Med. 197: Bartosch, B., A. Vitelli, C. Granier, C. Goujon, J. Dubuisson, S. Pascale, E. Scarselli, R. Cortese, A. Nicosia, and F. L. Cosset Cell entry of hepatitis C virus requires a set of co-receptors that include the CD81 tetraspanin and the SR-B1 scavenger receptor. J. Biol. Chem. 278: Cai, Z., C. Zhang, K. S. Chang, J. Jiang, B. C. Ahn, T. Wakita, T. J. Liang, and G. Luo Robust production of infectious hepatitis C virus (HCV) from stably HCV cdna-transfected human hepatoma cells. J. Virol. 79: Greenspan, N. S Affinity, complementarity, cooperativity, and specificity in antibody recognition. Curr. Top. Microbiol. Immunol. 260: Hadlock, K. G., R. E. Lanford, S. Perkins, J. Rowe, Q. Yang, S. Levy, P. Pileri, S. Abrignani, and S. K. Foung Human monoclonal antibodies that inhibit binding of hepatitis C virus E2 protein to CD81 and recognize conserved conformational epitopes. J. Virol. 74: Hadlock, K. G., J. Rowe, S. Perkins, P. Bradshaw, G. Y. Song, C. Cheng, J. Yang, R. Gascon, J. Halmos, S. M. Rehman, M. S. McGrath, and S. K. Foung Neutralizing human monoclonal antibodies to conformational epitopes of human T-cell lymphotropic virus type 1 and 2 gp46. J. Virol. 71: Hsu, M., J. Zhang, M. Flint, C. Logvinoff, C. Cheng-Mayer, C. M. Rice, and J. A. McKeating Hepatitis C virus glycoproteins mediate ph-dependent cell entry of pseudotyped retroviral particles. Proc. Natl. Acad. Sci. USA 100: Keck, Z. Y., T. K. Li, J. Xia, B. Bartosch, F. L. Cosset, J. Dubuisson, and S. K. Foung Analysis of a highly flexible conformational immunogenic domain a in hepatitis C virus E2. J. Virol. 79: Keck, Z. Y., A. Op De Beeck, K. G. Hadlock, J. Xia, T. K. Li, J. Dubuisson, and S. K. Foung Hepatitis C virus E2 has three immunogenic domains containing conformational epitopes with distinct properties and biological functions. J. Virol. 78: Lindenbach, B. D., M. J. Evans, A. J. Syder, B. Wolk, T. L. Tellinghuisen, C. C. Liu, T. Maruyama, R. O. Hynes, D. R. Burton, J. A. McKeating, and C. M. Rice Complete replication of hepatitis C virus in cell culture. Science 309: Macdonald, A., K. Crowder, A. Street, C. McCormick, K. Saksela, and M. Harris The hepatitis C virus non-structural NS5A protein inhibits activating protein-1 function by perturbing ras-erk pathway signaling. J. Biol. Chem. 278: Op De Beeck, A., C. Voisset, B. Bartosch, Y. Ciczora, L. Cocquerel, Z. Keck, S. Foung, F. L. Cosset, and J. Dubuisson Characterization of functional hepatitis C virus envelope glycoproteins. J. Virol. 78: Pietschmann, T., A. Kaul, G. Koutsoudakis, A. Shavinskaya, S. Kallis, E. Steinmann, K. Abid, F. Negro, M. Dreux, F. L. Cosset, and R. Bartenschlager Construction and characterization of infectious intragenotypic and intergenotypic hepatitis C virus chimeras. Proc. Natl. Acad. Sci. USA 103: Wakita, T., T. Pietschmann, T. Kato, T. Date, M. Miyamoto, Z. J. Zhao, K. Murthy, A. Habermann, H. G. Krausslich, M. Mizokami, R. Bartenschlager, and T. J. Liang Production of infectious hepatitis C virus in tissue culture from a cloned viral genome. Nat. Med. 11: Yi, M., R. A. Villanueva, D. L. Thomas, T. Wakita, and S. M. Lemon Production of infectious genotype 1a hepatitis C virus (Hutchinson strain) in cultured human hepatoma cells. Proc. Natl. Acad. Sci. USA 103: Zhong, J., P. Gastaminza, G. Cheng, S. Kapadia, T. Kato, D. R. Burton, S. F. Wieland, S. L. Uprichard, T. Wakita, and F. V. Chisari Robust hepatitis C virus infection in vitro. Proc. Natl. Acad. Sci. USA 102: