Hepatitis C Virus E2 Has Three Immunogenic Domains Containing Conformational Epitopes with Distinct Properties and Biological Functions

Mechanisms of virion attachment, interaction with its receptor,and cell entry are poorly understood for hepatitis C virus (HCV)because of a lack of an efficient and reliable in vitro systemfor virus propagation. Infectious HCV retroviral pseudotypeparticles (HCVpp) were recently shown to express native E1E2glycoproteins, as defined in part by HCV human monoclonal antibodies(HMAbs) to conformational epitopes on E2, and some of theseantibodies block HCVpp infection (A. Op De Beeck, C. Voisset,B. Bartosch, Y. Ciczora, L. Cocquerel, Z. Y. Keck, S. Foung,F. L. Cosset, and J. Dubuisson, J. Virol. 78:2994-3002, 2004).Why some HMAbs are neutralizing and others are nonneutralizingis looked at in this report by a series of studies to determinethe expression of their epitopes on E2 associated with HCVppand the role of antibody binding affinity. Antibody cross-competitiondefined three E2 immunogenic domains with neutralizing HMAbsrestricted to two domains that were also able to block E2 interactionwith CD81, a putative receptor for HCV. HCVpp immunoprecipitationshowed that neutralizing and nonneutralizing domains are expressedon E2 associated with HCVpp, and affinity studies found moderate-to-high-affinityantibodies in all domains. These findings support the perspectivethat HCV-specific epitopes are responsible for functional stepsin virus infection, with specific antibodies blocking distinctsteps of virus attachment and entry, rather than the perspectivethat virus neutralization correlates with increased antibodybinding to any virion surface site, independent of the epitoperecognized by the antibody. Segregation of virus neutralizationand sensitivity to low pH to specific regions supports a modelof HCV E2 immunogenic domains similar to the antigenic structuraland functional domains of other flavivirus envelope E glycoproteins.

INTRODUCTION

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Introduction

Hepatitis C virus (HCV) infects over 170 million individualsworldwide. Although acute infection is usually silent, mostHCV infections progress to chronicity that is not cleared byan apparently robust immune response (3, 24). The virus is amember of the family Flaviviridae (37), with a 9.5-kb positive-strandRNA genome that encodes three structural proteins, the capsidand viral envelope proteins E1 and E2, and at least six nonstructuralproteins, NS2 to NS5b (29). The envelope proteins are thoughtto be the primary mediators of virion attachment and cell entry(13). HCV E2 is a $~$ 70-kDa glycoprotein that shows large variationsamong HCV genotypes and contains a 27-amino-acid (aa) sequenceat its amino terminus that is highly variable and is designatedthe hypervariable region 1, or HVR1 (reviewed in references3 and 6). This linear region on E2 is likely to be involvedin virus infection, since neutralizing antisera to HVR1 havebeen reported in in vitro and in vivo models, although otherstudies showed that HCV with HVR1 deleted remains infectious(15, 19, 34, 41, 47). Unfortunately, a leading contributor todisease progression is the emergence of new viral mutants or"quasispecies" in HVR1 induced by immune selection. Increaseddiversity or mutations in HVR1 correlate with progressive disease,and decreased diversity correlates with resolving disease (14).HCV E2 is thought to mediate attachment to target cells andbinds to human CD81, a member of the tetraspannin family ofproteins (28). Interaction of E2 with CD81 on B or T cells hasbeen reported to result in B-cell aggregation and a loweringof the threshold for T- and B-cell activation (17, 43). Otheralternative receptors that have been proposed include the low-densitylipoprotein receptor (1, 44), two receptors on HepG2 cells,the scavenger receptor type B class I (5, 40), and two closelyrelated membrane-associated C-type mannose-binding lectins,DC-SIGN and L-SIGN (20, 30, 33).

Mechanisms of virion attachment, entry, and virus replicationhave been difficult to study because of difficulties in havingan efficient and reliable in vitro system for virus propagation.The development of infectious HCV retroviral pseudotype particlesexpressing E1E2 (HCVpp) has permitted a more detailed characterizationof functional envelope glycoproteins involved in virion attachmentand entry (4, 25). HCVpp preferentially infect human hepatocytesand hepatocellular cell lines and express noncovalent E1E2 heterodimersas defined in part by HCV human monoclonal antibodies (HMAbs)to conformational epitopes on E2 (32). Production of HMAbs providesinformation on the immune response to native E1 and E2 proteins,as they are recognized during natural infection and should beuseful in determining the function and structure of specificimmunogenic domains of E1 or E2. HMAbs and recombinant antibodiesto E2 have been isolated to conformational epitopes that areconserved between subtypes 1a and 1b (2, 7, 9, 22) and genotypes1 and 2 (23). These HMAbs include antibodies that are effectiveand ineffective in inhibiting the binding of E2 to CD81 andHCVpp entry into target cells (2, 7, 22, 23, 32). Our investigatorsdeveloped a panel of HMAbs to HCV E2, of which the majoritywere to conformational epitopes (23). Each of the HCV HMAbswas secreted from a human hybridoma expressing a unique immunoglobulinG1 (IgG1) gene that had undergone affinity maturation in vivo(10). Some of the epitopes recognized by the HMAbs were broadlyconserved across different HCV genotypes and were able to inhibitthe binding of E2 to human CD81, but only three blocked HCVppentry into Huh-7 cells (32). Why only a subset of the HCV HMAbsto E2 was able to block HCVpp entry remains unclear. One explanationis the availability of the different antibody binding epitopeson the surface of HCVpp. A second possibility is the bindingaffinity of the antibodies, and a third possibility is thatvirus entry is mediated by only specific epitopes on the virussurface. For some viruses, the prevailing view is that inhibitionof virus entry or virus neutralization correlates with increasedantibody binding to any virion surface site, independent ofthe epitope recognized by the antibody. Neutralization is thenthe result of a critical number of binding sites being occupiedand virus entry being prevented through steric hindrance (8).Higher-affinity antibodies will have higher neutralizing activities.Nonneutralizing antibodies either do not bind to the virionsurface or are poor binders with low affinity. In contrast,the role of specific epitopes responsible for functional stepsin virus entry has been documented for other viruses, with specificantibodies blocking distinct steps of virus attachment, interactionwith receptor and coreceptor, and initiation of viral envelopefusion with the cellular membrane (26).

In this report, antibody competition studies showed three immunogenicdomains on HCV E2 that contained conserved conformational epitopes.The lack of HVR1 involvement with these domains was determinedby binding studies to HVR1 deletion mutants. Expression of theseepitopes on native proteins was analyzed by the ability of HCVHMAbs to immunoprecipitate HCVpp. Affinity studies of HCV HMAbswere performed to correlate antibody binding affinity and blockingof HCVpp entry to target cells. Also, a collective analysisof previous and present findings suggests that the three immunogenicdomains are associated with distinct properties similar to theantigenic structure and function of other flavivirus envelopeE glycoproteins.

MATERIALS AND METHODS

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Materials and Methods

Cell culture and viruses. HeLa and HEK293T cells were from the American Type Culture Collection(Manassas, Va.). Cells were grown in minimal essential medium(MEM; Invitrogen, Carlsbad, Calif.) and Dulbecco's MEM (Invitrogen),respectively, supplemented with 10% fetal calf serum (GeminiBioproducts Inc., Calabasas, Calif.). A cell line constitutivelyexpressing sf1b-E2 on the cell surface was derived by transfectingChinese hamster ovary (CHO) cells with plasmid expressing sf1b-E2.After selection with Geneticin (Invitrogen), CHO cells expressingE2 were identified via fixed-cell immunofluorescence using CBH-5and hemagglutinin (HA) MAbs, essentially as described elsewhere(23). Cells expressing high levels of E2 were subjected to single-cellcloning. Recombinant vaccinia virus expressing HCV envelopeproteins was constructed and grown as described previously (23).Vaccinia virus 1488 expressing the structural proteins of HCV1a strain H (21) was generously provided by Charles Rice (RockefellerUniversity).

MAbs. The production, purification, and biotinylation of the HCV HMAbswere performed as described previously (23). Rat MAb 3/11 toHCV E2 was cultured as described elsewhere (17) and generouslyprovided by Jane McKeating (Rockefeller University). Rat MAbto the influenza virus HA epitope was from Roche Applied Sciences(Indianapolis, Ind.). Murine MAb to the c-myc epitope was fromSanta Cruz Biotechnology (Santa Cruz, Calif.).

Competition assays. Monolayers of HeLa cells were grown to 80% confluence and infectedat 5 PFU/cell with both wild-type virus and recombinant vacciniavirus or wild-type virus only. Cells were harvested after 1day of infection. Extracts were prepared by washing the cellswith phosphate-buffered saline (PBS) and then resuspending $~$ 25x 10⁶ cells in 1 ml of lysis buffer. Extracts prepared in thismanner contained approximately 25 µg of E2 protein/ml.Nuclei were pelleted by centrifugation at 18,000 x g at 4°Cfor 10 min, and resulting cytoplasmic extracts were stored at4°C and used for enzyme-linked immunosorbent assays within24 h of preparation. Microtiter plates were prepared by coatingwells with 500 ng of purified Galanthus nivalis lectin (GNA;Sigma, St. Louis, Mo.) in 100 µl of PBS for 1 h at 37°C.Wells were washed with Tris-buffered saline (TBS; 150 mM NaCl,20 mM Tris-HCl; pH 7.5) and then blocked with BLOTTO (TBS, 0.1%Tween 20, 2.5% normal goat serum, and 2.5% nonfat dry milk)by incubation for 1 h at room temperature (RT). Plates werewashed twice with TBS, followed by the addition to each wellof 15 µl of cytoplasmic extract containing E2 dilutedwith 85 µl of BLOTTO. After 1.5 h at RT, plates were washedthree times with TBS followed by the addition to each well of50 µl of BLOTTO containing competing antibodies at variousconcentrations. After 30 min, 50 µl of a 2-µg/mlsolution of the biotinylated test antibody was added. Afterincubation for 1.5 h at RT, the plates were washed three timeswith TBS, and 100 µl of 1/1,000-diluted alkaline phosphatase-conjugatedstreptavidin (Amersham-Pharmacia Biotech, Piscataway, N.J.)was added. After 1 h at RT, the plates were washed four timeswith TBS followed by a 30-min incubation with a 1-mg/ml solutionof p-nitrophenyl phosphate. Absorbance was measured at 405 nmwith a multiwell plate reader (BioTek Instruments, Winooski,Vt.). Each test HMAb at 2 µg/ml with competing HMAb rangingfrom 0.2 to 50 µg/ml was tested in duplicate in at leasttwo different experiments. To develop a cross-competition matrixfor percentage of test antibody bound to E2, the mean signalwith biotinylated test antibody to E2 with competing antibodyat 20 µg/ml was divided by the signal in the absence ofthe competing HMAb, followed by multiplying by 100.

Phylogenetic grouping of HCV HMAbs. Without knowing the exact conformational epitopes targeted bythese antibodies, we attempted to determine their spatial relationshipbased on competition study results. When two antibodies cross-competed,the extent of bidirectional inhibition was interpreted as theextent of epitope overlap by the competing antibodies. For unidirectionalinhibition or enhancement, effects were interpreted as proximal,but not overlapping epitopes (31). Using the principles of UPGMA(unweighted pair-group method using arithmetic averages) toperform a sequential cluster analysis, spatial relationshipswere developed as a phylogenetic tree to correlate the relatednessof the epitopes as identified by this panel of antibodies (16,42). In this analysis, antibodies with the highest bidirectionalinhibition were placed next to each other (see Fig. 1C). Thepaired antibodies were averaged and used to compare other antibodiesaccording to the degree of their cross-competition with thepaired antibodies. The (third) identified antibody with thestrongest bidirectional inhibition was placed next to the firstpair of antibodies, and a new average was obtained between thethird antibody and the average of the first pair. The new averagewas then used for another cycle of comparison with the otherantibodies in this panel until the matrix was completely reduced.

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FIG. 1. Competition analysis of HCV HMAbs. (A) Binding of biotinylated test antibody (as indicated on top of each panel) to HCV Q1b E2 protein captured onto GNA lectin-coated microtiter plates was competed by various concentrations of competing antibodies, as described in Materials and Methods. The bound biotinylated test antibody was detected by using alkaline phosphatase-conjugated streptavidin and p-nitrophenyl phosphate as the substrate. The y axis value (mean ± standard deviation; n = 2) is the absorbance reading obtained in the presence of competing antibody as a percentage of the reading in the absence of competing antibody. Competing antibodies were CBH-2 ( ${blacktriangleup}$ ), CBH-5 ( ${blacksquare}$ ), CBH-8C ( ${diamondsuit}$ ), CBH-8E ( ${blacktriangledown}$ ), CBH-11 (•), CBH-7 ( ${square}$ ), CBH-4G ( ${triangledown}$ ), CBH-4B ( ${triangleup}$ ), and R04 (*). R04 is an isotype-matched control antibody to a cytomegalovirus protein. (B) Cross-competition matrix. (C) Phylogenetic grouping of HCV HMAbs based on the competitive binding assay. Solid lines with numbers indicate the relatedness of the two adjoining antibodies. Circles are clusters of antibodies in a specific domain. Competition results used for calculation are the mean values obtained from two to five separate experiments.

Isolation and cloning of HCV E2 HVR1 deletion constructs. The vaccinia virus recombinant Q1b (GenBank accession no. AF348705)(23), sf1B-E2, and the HCV 1b deletion constructs were derivedfrom the same HCV genotype 1b-positive serum. DNA encoding theE2 protein was prepared by reverse transcription-PCR using PfuTaq polymerase (Stratagene, La Jolla, Calif.) with HCV-specificoligonucleotide primers (forward_1b, 5'-AGATCTACCACCTACACGACGGGGGGGGC-3';forward_1b411, 5'-AGATCTATCCAGCTCATAAACACCAACGGC-3'; reverse_1b,5'-CTGCAGCTCTGATCTGTCCCTATCCTCCAAG-3'). HCV 1a constructs wereamplified by PCR from viral stocks of vaccinia virus constructvv1488 (21) with the HCV-specific oligonucleotide primers forward_1a(5'-AGATCTGAAACCCACGTCACCGGGGG-3'), forward_1a411 (5'-AGATCTATCCAACTGATCAACACCAAC-3'),and reverse_1a (5'-CTGCAGCTCGGACCTGTCCCTGTCTTC-3'). FlankingBglII or PstI restriction sites in the primer sequences areunderlined. Amplified DNA fragments were ligated into the pDisplayvector (Invitrogen) in frame with HA and c-myc as tags, whichwere used for purification and detection of expressed proteins.In-frame HCV inserts and deletion sites were confirmed by DNAsequencing (PE-Applied Biosystems, Foster City, Calif.).

Analyses of HCV E2 deletion constructs. HEK293T cells were seeded to obtain 60 to 70% confluence bythe following day. For transfection of a T-75 flask, a mixtureof 40 µg of the plasmid DNA and 240 µg of PerFectLipid Pfx-2 (Invitrogen) were combined in 1 ml of serum-freeDulbecco's MEM. After 4 h of incubation at 37°C, the transfectionsolution was replaced with 20 ml of complete medium and cellswere grown for 24 h. Cell extracts were prepared by washingcells with PBS and resuspending them in 1 ml of lysis buffer.Nuclei were pelleted by centrifugation at 18,000 x g at 4°Cfor 10 min. For microtiter plate assays, the plates were preparedby coating wells with 500 ng of purified GNA lectin in 100 µlof PBS for 1 h at 37°C. Wells were washed with TBS and thenblocked with 150 µl of BLOTTO by incubation for 1 h atRT. Wells were washed twice with TBS, followed by the additionof 25 µl of extract from HEK293 cells transfected withE2 deletion constructs diluted in 75 µl of BLOTTO. After1.5 h at RT, plates were washed three times with TBS followedby the addition of 100 µl of BLOTTO containing variousMAbs at 10 µg/ml. Plates were incubated for 1.5 h andwashed three times with TBS, and then 100 µl of alkalinephosphatase-conjugated secondary antibody, diluted in BLOTTOas recommended by the manufacturer, was added (for anti-humanand anti-mouse antibodies [Promega, Madison, Wis.] and for anti-ratantibody [Kirkegard & Perry, South San Francisco, Calif.]).Bound secondary antibody was detected and quantified as describedabove.

For Western blot analysis, 293T cells were transfected witheither full-length E2 or constructs with HVR1 deleted overnightusing a calcium phosphate transfection kit (Clontech, Palo Alto,Calif.). After washing once with PBS, the cells were lysed inlysis buffer. Ten micrograms of the proteins was denatured inLaemmli sodium dodecyl sulfate (SDS) sample buffer and loadedonto SDS-polyacrylamide gels. Proteins were separated by electrophoresisand transferred to nitrocellulose membranes. Blots were blockedfor 1 h in 5% (wt/vol) nonfat dry milk dissolved in TBS-0.1%Tween 20 (TBST). Blots were then probed with rat anti-HA antibodyovernight at 4°C in blocking buffer. After washing withTBST three times, the blots were probed with a secondary antibody(horseradish peroxidase-conjugated anti-mouse IgG from SantaCruz Biotech) for 1 h at RT. Blots were washed three times inTBST and then developed with enhanced chemiluminescence. Westernblot images were captured using ChemiDoc imager system (Bio-Rad,Richmond, Calif.).

Immunoprecipitation. Production of HCVpp was carried out as described previously(4). Briefly, 293T cells were transfected with expression vectorsencoding the viral components, i.e., E1E2 glycoproteins, retroviralcore proteins, and packaging-competent green fluorescent protein-containingretroviral transfer vectors, by using a calcium-phosphate transfectionprotocol. 293T cells were metabolically labeled from 16 to 40h posttransfection with 50 µCi of ³⁵S-labeled proteinlabeling mix (Amersham Biosciences)/ml. Metabolically labeled293T cells and supernatant containing HCVpp were lysed with0.5% Igepal CA-630 in TBS (50 mM Tris-HCl [pH 7.5], 150 mM NaCl).Approximately 10⁵ infectious pseudotype particles were usedper immunoprecipitation reaction mixture. Immunoprecipitationswere carried out as described elsewhere (12, 13). Briefly, 7µg of MAb was incubated with protein A-Sepharose (Sigma)for 1 h at 4°C in TBS containing 0.2% Igepal CA-630. Beadswere then incubated with the antigen for 1 h at 4°C. Betweeneach step, beads were washed once with TBS-Igepal. After thelast step, they were washed three times with this buffer andonce with distilled water. The precipitates were then heatedat 70°C for 5 min in SDS-polyacrylamide gel electrophoresissample buffer and run on a polyacrylamide gel. After electrophoresis,gels were treated with sodium salicylate, dried, and exposedat –80°C to an autoradiograph (Amersham).

Antibody affinity measurements. A range of 0.001 to 100 µg of each HCV HMAb/ml was incubatedwith either genotype 1b E2 constitutively expressed in CHO cellsor transiently expressed 1a E2 in 293T cells for 45 min andwashed twice, followed by incubation with fluorescein isothiocyanate-labeledgoat anti-human IgG (4 µg/ml; Jackson Immunoresearch,West Grove, Pa.) for 45 min on ice. Cells were then washed inPBS containing 1% fetal calf serum at 4°C and resuspendedin fixative solution. Fluorescence of HMAb-bound cells was analyzedby flow cytometry using a FACSCalibur (Becton-Dickinson, SanJose, Calif.), and the mean fluorescence intensity (MFI) valuesof cell populations were obtained. The MFI value of nonspecificfluorescence was measured by using an isotype-matched controlHMAb (RO4), the fluorescence of which was subtracted from theMFI values of the specific HMAbs. The MFI values of the cellpopulations incubated with different amounts of antibodies wereanalyzed using Prism software, and the saturation binding curveswere fit by nonlinear regression.

RESULTS

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Results

Competition analyses of HCV HMAbs. Competition studies were performed to determine the spatialproximity of each conformational epitope to other epitopes onHCV E2 as defined by a panel of HCV HMAbs (Table 1) (23). Theseantibodies were derived from peripheral B cells of an individualwho had an asymptomatic HCV genotype 1b infection. Sequenceanalysis of the IgG1 genes of the HMAbs confirmed that theywere derived from independent B cells (10, 23). As describedpreviously, the antibodies varied in the breadth of their reactivitieswith different genotypes of HCV E2, with some broadly reactivewith genotypes 1a, 1b, 2a, and 2b and in their ability to inhibitthe interaction of HCV E2 with human CD81 (Table 1). Genotype-specificplasmids used to produce HCV E2 in the vaccinia virus expressionsystem in this analysis were derived from sera of other patientsinfected with different HCV genotypes. Complete inhibition ofantibody reactivity to denatured E2 protein confirmed that allHMAbs were to conformational epitopes except for CBH-17, whichretained reactivity. Competition studies were performed withgenotype 1b E2, since the original B cells were from an individualinfected with HCV 1b. Each HMAb was purified and biotinylated,and the binding of the antibodies in increasing concentrationsof competing antibody was determined. Representative bindingcurves are presented in Fig. 1A. The cross-competition matrix(Fig. 1B) shows the mean signals of biotinylated test HMAb withcompeting HMAb divided by signals without competing HMAb frommultiple experiments. The antibodies are approximately orderedon both axes to reflect bidirectional inhibition along the diagonalaxis of the matrix. Strong inhibition is indicated by valuesof $≤$ 50%, and enhancement is indicated by values of $≥$ 125%. HMAbsCBH-2, -5, -8C, -8E, and -11 (as shown for CBH-2 and -5 in Fig.1A) form one cluster, where each antibody showed strong bidirectionalinhibition with all other antibodies in the cluster (Fig. 1B).No significant inhibition was observed with a control HMAb,R04, or CBH-4B -4D, -4G, and -7. HMAbs CBH-4B, -4D, and -4Gform another cluster with strong bidirectional inhibition toeach other (as shown for CBH-4B in Fig. 1A). CBH-7 is to itselfand is not significantly inhibited by CBH-2, -5, -8C, -8E, -11,or the control antibody. The relationship between CBH-7 andthe CBH-4B, -4D, and -4G cluster is unusual, with either stronglyinhibitory (CBH-4B) or enhancing (CBH-4G) effects on binding.While uneven bidirectional inhibition was observed within eachcluster, a predominately unidirectional effect was observedbetween CBH-7 and the CBH-4B, -4D, and -4G cluster (Fig. 1B).

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TABLE 1. Selected characteristics of HCV HMAbs

Many techniques have been developed for determining phylogeneticrelationships in comparative biology. The UPGMA algorithm isa straightforward approach for cluster analysis, weighting eachdata point in the cluster equally (16, 42). The competitiondata were analyzed and spatially placed by pairing antibodieswith the highest bidirectional inhibition as most related (shownnext to each other in Fig. 1C). For example, the binding ofCBH-5 to E2 was reduced to 25% by CBH-11, and the binding ofCBH-11 was reduced to 9% by CBH-5. The average of these twovalues is 17%, or 0.17 (as shown above the line in the figure).A relationship tree was generated with the closest two antibodies(e.g., CBH-5 and -11) paired together. Their inhibition percentagesagainst each of the other antibodies were averaged and addedto the matrix in proximity to the original pair. This cyclewas repeated until all HMAbs were assigned in this tree. Thisapproach identified three distinct immunogenic domains. HMAbsCBH-4G, -4B and -4D constitute domain A. A second domain, B,includes CBH-2, -5, -8C, -8E, and -11; CBH-7 is in a separatedomain, C. Within each domain, strong bidirectional inhibitionwas observed, with values generally less than 0.5. Virtuallyno cross-competition was observed between domains A and B, witha relational value of 0.84. While domain C had a theoreticalrelational value with the other two domains of 0.97, domainC has a more complex relationship with domain A, as CBH-7 cross-competeswith CBH-4B but enhances CBH-4G binding. Because CBH-7 affectsthese antibodies in opposite directions, it is possible thatCBH-7 is either in proximity to domain A or induces structuralchanges that affect domain A. CBH-17 was the only antibody toa linear epitope and did not influence the binding of the otherantibodies (data not shown). These studies suggest that eightHCV HMAbs define three immunogenic domains containing conformationalepitopes on the HCV E2 glycoprotein.

HVR1 is not involved in conformational epitopes on E2. To determine whether HVR1 is involved in the conformationalepitopes as defined by domain A to C antibodies, constructsof 1b (pDN-411) and 1a strain H (pDNH-411) E2 without theirHVR (from aa 384 to 410) (Fig. 2A) were produced with HA andc-myc tags at the N and C termini, respectively. To test whethertag proteins have an effect on antibody binding, intracellularlyexpressed 1a and 1b E2s without tags were tested and shown tobe without detectable differences by immunofluorescence assay(data not shown). DNA sequencing confirmed the junction of deletion,and the expected sequences resulted in no frameshifts or prematureterminations. The expression of the E2 deletion constructs wasverified by Western blot analysis of cytoplasmic extracts oftransiently transfected HEK293 cells by using a MAb to the tag-HAepitope (Fig. 2B). Wild-type and HCV E2 deletion constructswere then transfected into HEK293T cells, and intracellularforms of E2 and E2 deletions were captured onto GNA lectin-coatedmicrotiter plates. The reactivities of the HCV HMAbs with E2and the E2 deletion were then determined. Anti-tag antibodies(HA and c-myc) were used as positive controls for protein expressionand for GNA capture (Fig. 2C and D). All HCV HMAbs reacted withwild-type genotype 1b sf1b E2 protein, and none reacted withproteins captured from extracts of mock-transfected HEK293 cells(Fig. 2C). All HCV HMAbs retained reactivity with E2 producedby the pDN-411 deletion construct, indicating that the epitopesrecognized by the antibodies did not include HVR1, althoughCBH-2 and -8E were reduced. No reactivity was observed witha control antibody (R04) to either wild-type E2 or the proteinwith E2 deleted. Antibody 3/11 was used as a positive controlin these studies, since the epitope of this antibody has beendefined as being outside of HVR1 (aa 412 to 423) (17). Next,the same panel of antibodies was tested against analogous genotype1a E2 derived from strain H with (sfH1a-E2) or without (pDNH-411)HVR1 (Fig. 2D). HMAbs CBH-8C and CBH-11 did not recognize eithersfH1a-E2 or pDNH-411; CBH-2 had a significant reduction. ForCBH-11 this was expected, since this antibody does not reactto 1a E2. However, the reduced or lack of reactivity for CBH-2and -8C was more isolate specific, as previous studies showedthese antibodies binding equally well to other 1a E2 proteins(Table 1) (23). The other HCV HMAbs and control antibodies hadequivalent reactivities with the strain H-derived E2 proteinsand the genotype 1b E2 proteins. All HMAbs reactive with sfH1a-E2retained reactivity with the HVR1-deficient construct pDNH-411,confirming that the epitopes recognized by these HMAbs wereoutside HVR1. The reduction in binding of CBH-2 and -8E to genotype1b and not to 1a HVR1 proteins with E2 deleted may reflect structuraldifferences between genotypes and the involvement of HVR1 inthese two epitopes in 1b E2 but not with the other E2 HMAbs.

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FIG. 2. Epitopes recognized by HCV HMAbs are located outside of HVR1. (A) Construct designation (on the left) showing the first amino acid sequence included in the two E2 deletion constructs employed in this study. All constructs were expressed in the pDisplay vector, which includes a heterologous signal sequence and TM domain (solid black lines) as well as epitopes recognized by MAbs to influenza virus HA (vertical bars) and c-myc (horizontal bars). HCV 1b E2 sequences are indicated in white, and strain H E2 sequences are shaded. (B) Western blot analysis showing the expression of HCV E2 proteins. Wild-type 1b and 1a (strain H) E2 proteins are shown as sf1b and sfH1a, respectively. The HVR1 1b and 1a deletions (aa 384 to 410 were deleted) are shown as pDN-411 and pDNH-411, respectively. A protein size marker is indicated in kilodaltons. (C) Reactivity of HCV HMAbs with E2 deletion constructs. HEK293 cells were mock transfected (white bars) or transfected with the HCV E2 constructs sf1b (stippled bars) or pDN-411 (black bars). Twenty-four hours posttransfection, cytoplasmic extracts were prepared and equivalent aliquots were captured onto GNA lectin-coated microtiter plates as described in Materials and Methods. The captured E2 proteins were then incubated with 10 µg of the indicated HCV HMAb/ml (x axis), and the amount of bound antibody was determined. Bars represent the mean absorbance values obtained from duplicate wells. Error bars indicate one standard deviation from the mean. (D) Same experiment as in panel C, except that HEK293 cells were transfected with sfH1a E2 (stippled bars) or pDNH-411 (black bars).

Conformational epitopes on E2 associated with HCVpp. Only eight of nine HCV HMAbs to conformational epitopes weretested with HCVpp, because of an inadequate amount of CBH-8Eantibodies. The hybridoma producing this HMAb was unstable andwill require alternative production for further studies. Ofthe remaining eight antibodies, only CBH-5 and -7 had strongactivity, and CBH-2 had weak activity, to block HCVpp entryto target cells as shown previously (32). The other antibodieshad no neutralizing activity. To assess whether virus neutralizationis caused by the expression or lack of expression of their respectiveepitopes on E2 associated with HCVpp, immunoprecipitation studieswere performed with HCVpp and cell lysate-associated E1E2 glycoproteins(Fig. 3). It is worth noting that HCVpp-associated E2 had aslower and more diffuse migration pattern than the cell-associatedform. This is due to modifications of the glycans by Golgi enzymes,as previously shown (32). With HCVpp (Fig. 3A), CBH-4B, -4D,and -7 showed strong binding, CBH-5 showed moderate binding,CBH-2 and -4G showed weak binding, and no detectable bindingwas seen with CBH-8C or -11. The lack of reactivity with CBH-8Cand -11 was expected, since the HCVpp were derived from the1a H strain, which is not recognized by these antibodies (Fig.2D). For CBH-4G, a lower antibody affinity could be a contributingfactor, as discussed below. Another explanation is the maskingof this epitope on the surface of HCVpp. From these studies,it is reasonable to conclude that conformational epitopes asidentified by antibodies to domains A, B, and C, CBH-2, -4B,-4D, -5, and -7, are present to some degree on the virion surface,although virus neutralization is restricted to domains B andC, CBH-2, -5, and -7. The strong binding of domain A antibodiesCBH-4D and -4B, while being nonneutralizing, supports the viewthat virus neutralization for HCV is mediated in part by restrictedvirion surface E2 epitopes in specific domains.

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FIG. 3. Epitopes exposed on HCV glycoproteins associated with HCVpp. HEK293T cells transfected to produce HCVpp were labeled for 24 h. Supernatants (HCVpp) (A) and cell lysates (B) were immunoprecipitated with HCV HMAbs as noted.

Immunoprecipitation of intracellular E1E2 showed binding withall conformation-sensitive antibodies except for CBH-8C and-11. Interestingly, differences in reactivity were observedbetween HCVpp-associated envelope proteins and their intracellularforms. Domain B antibodies, CBH-2 and -5, had stronger reactivitiesagainst cell-associated E1E2. Indeed, when compared to CBH-7,these antibodies precipitated 4 to 5 times less HCVpp-associatedE2 protein. Since HCVpp-associated envelope proteins are modifiedby Golgi enzymes (32), the glycans added to these proteins inthis compartment might potentially reduce the accessibilityof the epitopes recognized by CBH-2 and -5. Alternatively, wecannot exclude some local structural changes induced by modifiedglycans in domain B.

Antibody affinity and virus neutralization. To estimate affinity, saturation of HCV HMAb binding to cellsurface-expressed genotype 1a and 1b E2 was measured by flowcytometry, and the data were analyzed using Prism software (GraphPad).Saturation profiles for domain A-, B-, and C-specific HMAbsto 1a E2 are shown in Fig. 4. As summarized in Table 2, theantibodies displayed a wide range of dissociation constants(K_d). In general, affinity tended to be higher to 1b E2 thanto 1a E2, except with CBH-4G and -7. Moderate-to-high-affinityantibodies of <5 x 10^–8 K_d were observed in all threegroups to 1b E2 and in domains A and C to 1a E2. In domain B,CBH-5 had a similar affinity with CBH-2 to 1a E2 but higherneutralizing activity for HCVpp derived from strain H77. Thedifference suggests that the number of CBH-5 epitopes is greaterthan the number of CBH-2 epitopes on the surface of HCVpp. Thisis supported by the observation that CBH-5 has a higher totalbinding than CBH-2 to 1a E2, as shown in Fig. 2D. CBH-4G weaklyprecipitated E1E2 associated with HCVpp and weakly bound tointracellular E1E2 compared to the other two antibodies in domainA. A possible explanation is that this epitope is partly maskedon the surface of HCVpp.

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FIG. 4. Saturation binding of HMAbs to HCV E2. Transiently transfected 293T cells with 1a E2 were incubated with the HCV HMAbs in increasing concentrations as indicated. Staining and flow cytometry analysis were performed as described in Materials and Methods. The data points are means of two determinations and are representative of three independent experiments. Binding affinity data shown in Table 2 were analyzed using Prism software.

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TABLE 2. Comparison of biological activities of HCV HMAbs

Collectively, there are distinct biological activities betweenthe antibody clusters (Table 2). Domain A antibodies, CBH-4B,-4D, and -4G, had no neutralization activity but showed greaterrecognition of HCVpp than intracellular E1E2, except for CBH-4G.One of the antibodies, CBH-4D, in previous studies was low-pHsensitive, with a 40% reduction in binding (32). Domain B antibodies,CBH-5 and CBH-2, have neutralizing activities but show reducedrecognition of HCVpp compared to intracellular E1E2. The domainC antibody, CBH-7, has neutralization activity and equal accessibilityof its epitope on E2 in HCVpp and intracellular E1E2.

DISCUSSION

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Discussion

Precise information on the mechanisms of virion attachment andentry will be critical in the successful development of newertherapeutics and an effective vaccine for HCV. Studies withMAbs on some viruses tend to support two different views onvirus neutralization. One perspective is that virus neutralizationcorrelates with antibody binding affinity to any virion surfacesite and is irrespective of the epitopes recognized by theseantibodies. Antibody binding of a critical number of sites onthe virion surface prevents virus entry through steric hindrance(8). The other perspective is that specific antibodies blockingdistinct steps of virus attachment, interaction with receptorand coreceptor, and initiation of viral envelope fusion leadto virus neutralization (26). The development of HCVpp providesa useful tool to study the functional roles of specific immunogenicdomains on envelope glycoproteins in virion binding and entry.We and other colleagues recently showed that HCVpp-associatedenvelope proteins are noncovalent E1E2 heterodimers, recognizedby a panel of MAbs to conformational epitopes and CD81, andsome epitopes are sensitive to low-pH treatment. Consequently,HCVpp are likely to contain envelope proteins in a similar structureas native virions (32).

Of eight HCV HMAbs to conformational epitopes on E2, only threeantibodies, CBH-2, -5, and -7, were able to inhibit HCVpp attachmentand entry to Huh-7 cells (Table 2) (32). Competition analysesof these HMAbs showed that conformational epitopes on E2 wereclustered into three distinct domains. Domain A consisted ofCBH-4B, -4D, and -4G; domain B contained CBH-2, -5, -8C, -8E,and -11; and domain C contained HMAb CBH-7. In domain B, CBH-2and -5 were neutralizing. The lack of neutralizing activityof the other two tested antibodies, CBH-8C and -11, was explainedby their lack of recognition of the genotype 1a H strain, whichwas used to construct the HCVpp. The neutralizing antibodiesin domains B and C (CBH-2, -5, and -7) are to conformationalepitopes on proteins associated with the virion surface anddo not involve HVR1, as shown by HVR1 deletion studies. Allantibodies in domains B and C inhibited the E2-CD81 interaction(Table 1), supporting the involvement of CD81 in virus entry(46). Domain A antibodies were nonneutralizing and did not blockthe E2-CD81 interaction. Their ability to precipitate E1E2 associatedwith HCVpp suggests that their epitopes are also on the surfaceof virions, supporting the perspective that HCV virion attachmentand entry are restricted to specific virion surface domains.

This perspective is further supported by antibody affinity studies.CBH-4B has a moderate affinity to 1a E2 that is higher thanthat of CBH-2 or -5 (Table 2). But CBH-4B in domain A, whoseepitope is on E2 associated with the virion surface, is nonneutralizing.Nonetheless, higher-affinity antibodies will tend to have higherneutralizing activities, with CBH-7 having higher activity thanCBH-2 or -5 (32). More studies of antibodies in a specific domain,such as domain B, using HCVpp constructed with genotype 1b arerequired to further support this relationship of antibody affinityand virus neutralization activity.

The clustering of these epitopes on HCV E2 into three antigenicdomains is in agreement with topological mapping by similarMAb cross-competition studies with other flavivirus E glycoproteinsand the crystal structure of tick-borne encephalitis virus Eglycoprotein (35, 39). The flavivirus E glycoprotein is thedominant antigen inducing neutralizing antibodies and is theprotein responsible for virus attachment to cell receptors andinitiation of viral envelope fusion leading to cell entry (36,38). Epitopes on flavivirus E protein are clustered into threestructural domains. A central domain I containing nonneutralizingepitopes is felt to be a hinge region involved in low-pH-inducedconformational changes (39). Our HCV E2 domain A has similarproperties for nonneutralizing epitopes, and at least one ofthe epitopes, CBH-4D, was shown to be pH sensitive, with 40%binding reduction under low pH (Table 2). The varied effectsof CBH-7 on CBH-4G (increase) and CBH-4B (decrease) in bindingto E2 suggest that a conformational change is possibly inducedwith CBH-7 binding, consistent with domain A as a possible hingeregion. Flavivirus E protein domain II is involved in dimerizationand membrane fusion and is able to elicit neutralizing and nonneutralizingantibodies (35, 39). For HCV, the identity of the fusion proteinis unclear. At first, a putative fusion peptide in E1 led tothe proposal that this protein is the fusion protein (18). But,other homology studies suggested that E2 is responsible forvirus-induced fusion (27, 45). Flavivirus E protein domain IIIcontaining distal projecting loops from the virion surface elicitsthe strongest neutralizing antibodies, is minimally affectedby low pH, and is felt to be the receptor binding motif (35,38, 39). Currently, it is not possible to correlate our HCVdomain B and C antibodies in a similar manner. Both domainscontain neutralizing antibodies and are able to inhibit theE2-CD81 interaction. One possible clue is that previous studiesshowed that CBH-2 is uniquely able to recognize noncovalentE1E2 heterodimers and not high-molecular-weight E1E2 aggregatesthat are misfolded. Furthermore, CBH-2 will only recognize 1aH strain E2 when complexed with E1 (11). These findings suggestthat the HCV domain B antibodies are potentially correlatedwith the flavivirus E protein domain II involved in envelopeprotein dimerization. Further studies are required to substantiatethis model for HCV. Differences are to be expected in the structuralorganization of HCV E2 and flavivirus E glycoproteins, sincea major determinant is the number of disulfate bonds, whichare different between these glycoproteins. Epitope mapping andmore detailed investigation on structure-function propertiesof this panel of HCV HMAbs will be useful to advance our knowledgeon E2 immunogenic structures, which in turn should facilitateeffective vaccine design.

ACKNOWLEDGMENTS

We thank J. Rowe and S. Rajyaguru for technical assistance.

This work was supported in part by NIH grants HL079381 and AI47355to S.K.H.F. J.D. was supported by EU grant QLRT-2001-01329 andgrants from the Agence Nationale de Recherche sur le Sida andthe Association pour la Recherche sur le Cancer.

FOOTNOTES

* Corresponding author. Mailing address: Stanford Medical School Blood Center, 800 Welch Rd., Palo Alto, CA 94304. Phone: (650) 723-6481. Fax: (650) 725-6610. E-mail: sfoung{at}stanford.edu.

REFERENCES

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