Characterization of the Hepatitis C Virus E2 Epitope Defined by the Broadly Neutralizing Monoclonal...

Characterization of the Hepatitis C Virus E2 EpitopeDefined by the Broadly Neutralizing Monoclonal

Antibody AP33Alexander W. Tarr,1 Ania M. Owsianka,2 Judith M. Timms,1 C. Patrick McClure,1 Richard J. P. Brown,1

Timothy P. Hickling,1 Thomas Pietschmann,3 Ralf Bartenschlager,3 Arvind H. Patel,2 and Jonathan K. Ball1

The mouse monoclonal antibody (MAb) AP33, recognizing a 12 amino acid linear epitope inthe hepatitis C virus (HCV) E2 glycoprotein, potently neutralizes retroviral pseudoparticles(HCVpp) carrying genetically diverse HCV envelope glycoproteins. Consequently, this an-tibody and its epitope are highly relevant to vaccine design and immunotherapeutic devel-opment. The rational design of immunogens capable of inducing antibodies that target theAP33 epitope will benefit from a better understanding of this region. We have used comple-mentary approaches, which include random peptide phage display mapping and alaninescanning mutagenesis, to identify residues in the HCV E2 protein critical for MAb AP33binding. Four residues crucial for MAb binding were identified, which are highly conservedin HCV E2 sequences. Three residues within E2 were shown to be critical for binding to therat MAb 3/11, which previously was shown to recognize the same 12 amino acid E2 epitopeas MAb AP33 antibody, although only two of these were shared with MAb AP33. MAb AP33bound to a panel of functional E2 proteins representative of genotypes 1-6 with higheraffinity than MAb 3/11. Similarly, MAb AP33 was consistently more efficient at neutralizinginfectivity by diverse HCVpp than MAb 3/11. Importantly, MAb AP33 was also able toneutralize the cell culture infectious HCV clone JFH-1. In conclusion, these data identifyimportant protective determinants and will greatly assist the development of vaccine candi-dates based on the AP33 epitope. (HEPATOLOGY 2006;43:592-601.)

Hepatitis C virus (HCV) infection is a majorcause of severe chronic liver disease, includingcirrhosis and hepatocellular carcinoma.1 With

an estimated 3 to 4 million new infections each year,1 an

urgent need exists for the development of an effectiveprophylactic vaccine.

HCV, a member of the Flaviviridae family, has a 9-kbgenome encoding a polyprotein precursor that is cleavedto yield three structural proteins, core, E1, and E2, to-gether with at least six non-structural proteins. E1 and E2are highly glycosylated membrane-anchored proteins thatmediate viral entry.2-6 E2 has been shown to bind to anumber of cell surface molecules.7-10 Whereas the exactmechanism of viral entry is unknown, mounting evidenceindicates that CD81 and SR-BI are key mole-cules.2,4-6,11-14

In an infected individual, HCV exists as a viral quasi-species.15 HCV can be classified into at least six majorgenotypes that exhibit extensive genetic variability, par-ticularly in E1 and E2.16 E1 and E2 are the principletargets for neutralizing antibodies, and identification ofprotective epitopes conserved across different strains ofHCV is a major challenge for vaccine design.17 A numberof antibodies that are capable of blocking E2 binding tocells or cell receptors have been described,18-23 some ofwhich neutralize HCV entry in animal or in vitro mod-els.6,24-26 The first hypervariable region of E2 containspotent neutralizing epitopes, and antibodies raised against

Abbreviations: HCV, hepatitis C virus; MAb, monoclonal antibody; HCVpp,retroviral pseudoparticles; p-NPP, p-nitrophenol phosphate; MLV, murine leuke-mia virus; HCVcc, cell culture infectious HCV; CDR, complementarity determin-ing; GNA, Galanthus nivalis.

From 1The Institute of Infection, Immunity and Inflammation, School of MolecularMedical Sciences, The University of Nottingham, Queen’s Medical Centre, Notting-ham, NG7 2UH, UK; 2MRC Virology Unit, Institute of Virology, University of Glas-gow, Church Street, Glasgow G11 5JR, UK; and the 3Department for MolecularVirology, University Heidelberg, Im Neuenheimer Feld, Heidelberg, Germany.

Received August 31, 2005; accepted December 14, 2005.Supported by the Medical Research Council, European Union contracts QLK2-

CT-2001-01120 (ENHCV) and LSHM-CT-2004-503359 (“VIRGIL EuropeanNetwork of Excellence on Antiviral Drug Resistance”) and the University of Not-tingham Biomedical Research Committee. JT was supported by a Clinical TrainingFellowship from the Wellcome Trust.

Address reprint requests to: Jonathan K. Ball, The University of Nottingham —The Institute of Infection, Immunity and Inflammation, Microbiology and Infec-tious Diseases, Queen’s Medical Center, Nottingham NG7 2UH, UK. E-mail:[email protected]; fax: (44) 115-970 92 or to: Arvind H. Patel,MRC Virology Unit, Institute of Virology, University of Glasgow, Church Street,Glasgow G11 5JR, UK. E-mail: [email protected]; fax: (44) 141-330-4026.

Copyright © 2006 by the American Association for the Study of Liver Diseases.Published online in Wiley InterScience (www.interscience.wiley.com).DOI 10.1002/hep.21088Potential conflict of interest: Nothing to report.

592

this region are capable of preventing infection. However,antibodies to HVR1 tend to be strain specific and havelimited cross-neutralizing potency. Most antibodies thatdemonstrate broad neutralization of cell binding and/orinfection are directed against conformational epitopeswithin E2.27-31 Induction of antibodies recognizing con-formational epitopes is a challenging task, as these aredifficult to mimic, and protein subunit vaccines are morelikely to generate strain-restricted responses due to theimmuno-dominance of the variable regions.32 However,we have recently shown that the mouse monoclonal anti-body (MAb) AP33 can potently neutralize retroviral pseu-do-particles (HCVpp) bearing genetically diverse E1E2glycoproteins.33 The epitope recognized by MAb AP33has been mapped to a 12 amino acid region correspond-ing to residues 412 to 423 located immediately down-stream of HVR1.21,22,34-37 This region is also recognizedby rat MAb 3/11.21 Like AP33, MAb 3/11 is capable ofneutralizing HCVpp carrying strain H77 E1E2, althoughwhether its neutralization capacity extends to geneticallydiverse E1E2 molecules is unknown.3 Identification of ahighly conserved linear neutralizing epitope is a signifi-cant milestone in the development of a cross-reactive vac-cine. Rational development of an AP33-epitope–basedvaccine will require a thorough understanding of the in-teraction between MAb AP33 and its epitope. In the cur-rent report, we have finely mapped the amino acidresidues recognized by MAb AP33 and demonstrated thatMAb 3/11 recognizes a distinct yet overlapping epitope.Importantly, we also show that MAb AP33 has a higherneutralizing ability than MAb 3/11, highlighting variabil-ity in the specificity and quality of the antibody responseagainst this region of the E2 glycoprotein.

Materials and Methods

Antibodies. The anti-E2 monoclonal antibodiesAP33, ALP98, 3/11, and H53 have been previously de-scribed.21,22,38 The dengue type 2–specific MAb 46D (hy-bridoma 3H5-1. ATCC No: HB-46) and the CD81-specific MAb JS-81 (Pharmingen, Oxford, UK) were usedas control antibodies in the HCV JFH-1 cell culture neu-tralization assay. MAbs from non-commercial sourceswere purified from hybridoma supernatants using a pro-tein G column (GE Healthcare, UK).

Peptides. The branched peptides used in immunoas-says corresponded to the AP33 epitope QLINTNG-SWHVN22 encompassing amino acids 412-423 of thegenotype 1a Glasgow strain,36 the control peptide VNL-HDFRSDEIE, and a peptide HLANHQGKWRLH,which represented the most frequent peptide selected by

MAb AP33 from a random peptide phage display library(below).

Cell Culture. Human hepatoma cells Huh-7,39 Huh-Lunet cells, and human epithelial kidney (HEK) 293FTcells (Invitrogen, Paisley, UK) were grown in Dulbecco’smodified Eagle’s medium, GIBCO BRL, Paisley, UK)supplemented with 10% fetal calf serum, 5% non-essen-tial amino acids, and 200 mmol/L L-glutamine (Sigma,Dorset, UK).

Enrichment of Random Peptide Phage Display Li-braries Using AP33 and 3/11. A commercially avail-able 12-mer, M13 gene 3–based random peptide phagedisplay library (New England Biolabs, Hitchin, Hertford-shire, UK) was enriched for MAb AP33- and MAb 3/11-specific peptides by three to four rounds of affinity selec-tion according to the manufacturer’s instructions. Afterthe final round of enrichment, individual phage cloneswere isolated, and the sequence of the peptide insert de-duced following automated DNA sequencing using the�96 sequencing primer supplied by the manufacturer.Deduced peptide insert amino acid sequences, togetherwith the sequence corresponding to amino acids 412-423of the H77c strain, were aligned using ClustalX,40 fol-lowed by manual adjustment.

Phage Immunoassay. To determine the reactivity ofselected phage with the selecting antibody, a phage cap-ture enzyme immunoassay was performed. Approxi-mately 1 � 1011 phage particles were added to microtiterplate wells coated with 10 �g/mL of MAb AP33, 3/11, orALP98. Bound phage were detected by anti-fd antibodyfollowed by an alkaline phosphatase conjugated anti-mouse antibody and Sigmafast p-nitrophenol phosphate(p-NPP) substrate (all from Sigma). Absorbance valueswere determined at 405 nm.

Peptide Enzyme Immunoassay. Branched peptide(500 ng/mL) was coated onto wells of a microtiter plate asdescribed above and then used to capture test MAb.Bound MAb was detected using an alkaline-phosphatase–conjugated anti-human IgG antibody diluted 1/1,000(Sigma), followed by p-NPP substrate. Absorbance wasmeasured at 405 nm.

Alanine-Scanning Mutagenesis. Plasmid pCR3.1(Invitrogen) containing E1E2 from the infectious H77cstrain of HCV was mutated using the QuickchangeII mu-tagenesis kit (Stratagene, Amsterdam, Netherlands), ac-cording to the manufacturer’s protocol. Primers weredesigned using the Stratagene primer design program(www.bioinformatics.org/primerx).

GNA Capture ELISA. To detect MAb binding to E2glycoprotein, an ELISA was performed essentially as pre-viously described.36 Briefly, E1E2 glycoproteins from theclarified lysates of transfected HEK 293FT cells were cap-

HEPATOLOGY, Vol. 43, No. 3, 2006 TARR ET AL. 593

tured onto GNA (Galanthus nivalis) lectin (Sigma)-coated microtiter plates then detected by MAb AP33 or3/11, followed by an anti-species IgG-alkaline phospha-tase conjugate (Sigma) and p-NPP substrate. Absorbancevalues were determined at 405 nm.

Reactivity of MAbs to alanine replacement mutantE1E2 was detected in the same way, except that E1E2normalized for the amount of E2 protein according toreactivity to H53 MAb was used.

To assess the effect of E1E2 denaturation on MAbbinding, E1E2 was denatured as previously described,41

then reactivity to MAbs determined using the GNA cap-ture enzyme immunoassay.

Competition Assays. Competition assays were carriedout using the peptide and sE2/E1E2 ELISAs described,except that immobilized target antigens were used to cap-ture a mixture of biotinylated target MAb and un-biotin-ylated competing MAb or negative control MAb.

HCVpp Infection and Neutralization Assays. Full-length E1E2 (representing amino acid residues 170 to746 of the HCV open reading frame referenced to strainH77c42) clones were generated and their nucleotide se-quence determined as previously described.13 HCVppwere produced essentially as previously described2 by co-transfection of plasmids expressing the full-length E1E2,murine leukemia virus (MLV) Gag-Pol and the MLVtransfer vector carrying the luciferase gene under the con-trol of human cytomegalovirus promoter. Antibody-me-diated neutralization of HCVpp was carried out aspreviously described.33 The neutralizing activity was ex-pressed as % infectivity compared with “no antibody”control.

Production of Cell Culture Infectious HCV andNeutralization Assays. Cell culture infectious HCV(HCVcc) were generated as previously described usingthe plasmid pFK-Luc-JFH1. pFK-Luc-JFH1�E1-E2 wasalso used in the analyses to control for non-specific trans-duction of reporter activity.5 Neutralization assays wereperformed by infecting Huh7-Lunet cells43,44 with virusstocks that were pre-incubated with or without antibodiesat room temperature for 1 hour before application. Fourhours post-infection, the inoculum was removed and re-placed with culture medium. Seventy-two hours later, in-fectivity conferred by the virus inoculum was determinedusing a luciferase assay, as previously described.5

Sequence Analysis of the VH and VL Regions ofMAbs AP33 and 3/11. cDNA was generated frommRNA obtained from approximately 1 � 106 hybridomacells using Thermoscript (Invitrogen) with the poly-dToligonucleotide primer included in the kit. Two microli-ters of resulting cDNA was used as template in polymer-ase chain reaction designed to amplify the variable regions

of the light and heavy chains. Heavy chain amplificationwas achieved as previously described,45 using the senseprimer VH1BACK (5�-AGG TSM ARC TGC AGSAGT CWG G-3�) with antisense primer VH1FoR-2 (5�-GGG GCC AAG GGA CCA CGG TCA CCG TCTCCT CA-3 �). Light chain amplification was achieved aspreviously described,46 using the primers Mk (5 �-GGGAGC TCG AYA TTG TGM TSA CMC ARW CTAMCA-3 �) with reverse primer Kc (5 �-GGT GCA TGCGGA TAC AGT TGG TGC AGC ATC-3 �). Polymer-ase chain reaction products were cloned into thepGEM-T vector (Promega, Madison, WI) and two clonesfor each heavy and light chain were sequenced using theT7 forward and M13 reverse primer (Promega) and theABI PRISM BigDye Terminator Cycle SequencingReady Reaction Kit (Perkin Elmer Applied Biosystems,Boston, MA), according to the manufacturer’s protocol.Framework (FWR), and complementarity determining(CDR) regions were identified using the IgBLAST(http://www.ncbi.nlm.nih.gov/igblast/) and Web Anti-body Modelling tool (http://www.bioinf.org.uk/abs/)with subsequent manual adjustment.

Results

Enrichment of Random Peptide Phage Display Li-braries Identifies E2 Residues Associated With MAbsAP33 and 3/11 Binding. Both MAb AP33 and 3/11have previously been shown to bind a 12mer peptide cor-responding to amino acids 412-423, immediately down-stream of HVR1, and were raised after immunizationwith soluble E2 from the Gla and H strains of HCV,respectively.21,22 To determine whether these antibodiesmight recognize overlapping, yet distinct epitopes en-compassed within this region, each antibody was used toenrich random peptide phage display libraries to identifycritical residues involved in mediating MAb binding. Af-ter three to four rounds of affinity selection against eachantibody, individual phage clones were isolated and theirreactivity to the selecting antibody assessed by phage cap-ture ELISA (Fig. 1). Nineteen and 40 clones selected byAP33 and 3/11, respectively, were analyzed, and of these17 and 35 were reactive to the selecting antibody. None ofthe AP33-selected clones reacted with 3/11 and vice versa,suggesting that these antibodies recognized distinctamino acid residues within this region. None of the en-riched clones reacted with the control antibody ALP98(not shown).

DNA sequence analysis of random peptide insertspresent in selected antibody-reactive phage clones wasperformed. Amino acid sequences aligned to the region ofH77 E2 corresponding to the putative MAb AP33 and

594 TARR ET AL. HEPATOLOGY, March 2006

3/11 epitopes are presented in Fig. 2. Phage selected byMAb AP33 could be divided into three groups based onthe amino acid sequence of the random peptide insert.Multiple alignment of the phage sequences showed abso-lute conservation of four amino acids, namely L413(Leucine corresponding to position 413 of the H77 E1E2sequence), N415, G418, and W420.

Considerably more sequence diversity was evident inthe phage clones selected by MAb 3/11 (Fig. 2B). Onegroup of clones was identical to the corresponding regionof H77c at positions 420 (W), 421 (H), and 423 (N) ofthe H77c polyprotein. The remaining phage clone pep-tide inserts had varying degrees of similarity to the H77E2 region, although commonly shared residues were thetryptophan or histidine at positions 420 and 421. Onesequence present in clones isolated during both rounds ofselection (e.g., 3/11.27), had extensive homology to the412-423 region, including positions 415 and 418-421.This phage peptide was unreactive to MAb AP33 inELISA, even though it contained three of the four puta-tive contact residues recognized by MAb AP33 and wasreactive to MAb 3/11.

To verify that the phage-displayed peptides were spe-cific for each antibody, a branched peptide representingthe most prevalent random peptide sequence (cloneAP33.12) was synthesized and used in ELISA. MAbAP33, but not MAb 3/11 reacted with this peptide(Fig. 3).

Reactivity of MAb AP33 and 3/11 to Alanine Re-placement Mutant E1E2 Proteins. To further delineatethe MAb reactive region, a panel of H77 E1E2 mutantclones was generated, in which each residue of the puta-

Fig. 1. Selection of MAb-reactive phage clones by enrichment ofrandom peptide phage display libraries. A 12mer random peptide phagedisplay library was enriched using the monoclonal antibodies AP33 (A)and 3/11 (B), and resulting phage clones tested for reactivity in anenzyme immunoassay to monoclonal antibody AP33 (solid square) and3/11 (open square). WT, wild-type M13 phage.

Fig. 2. Amino acid sequences of random peptide inserts of MAb-reactive phage selected by MAb AP33 (A) and 3/11 (B). Amino acidsequences are aligned to the region containing the putative MAb AP33and 3/11 epitope encompassing amino acids residues 412-423. Res-idues conserved between the H77c sequence and random peptide insertare shown in bold type. Phage clones were analyzed after three roundsof selection by MAb AP33 and after three and four rounds of selection byMAb 3/11.


tive MAb AP33 and 3/11 epitope was substituted in turnby alanine. Mutants were expressed in HEK 293FT cellsand reactivity of the resulting proteins to MAb AP33 and3/11 assessed using a GNA capture ELISA (Fig. 4). Bind-ing of AP33 was reduced by more than 75% comparedwith wild-type for mutants L413A, N415A, G418A, andW420A, indicating that these residues were critical forbinding. Mutation T416A and N417A also reducedAP33 binding, although this effect was not as marked asthe other four mutations. Substitution of glutamine byalanine at position 412 (Q412A) consistently enhancedbinding of AP33 to E1E2 by approximately 50% com-pared with the wild-type H77 protein. By contrast, thissubstitution had no effect on MAb 3/11 binding. Alaninereplacement of the remaining five residues had negligibleeffect on AP33 recognition.

Consistent and significant reduction of binding byMAb 3/11 compared with wild-type H77 protein wasobserved for mutant N415A, W420A, and H421A, high-lighting the importance of these residues in binding byMAb 3/11. Substitution of the isoleucine at position 422resulted in moderate enhancement of 3/11 binding. Ala-nine replacement of the remaining residues either had noeffect or resulted in moderate reductions in binding com-pared with wild-type.

Comparison of Binding Affinities and HCVpp Neu-tralization Efficiencies of MAb AP33 and MAb 3/11.The fine epitope mapping experiments indicated thatMAbs AP33 and 3/11 were recognizing different contactresidues within the E2 protein, so we went on to assesswhether these differences might translate into differencesin binding affinity or neutralizing potency. Bindingcurves of reactivity of each MAb against E1E2 and 412-423 branched peptide, together with a MAb competitionassay, are presented in Fig. 5. The concentration of MAb

AP33 required to obtain 50% binding to the 412-423branched peptide was more than 10-fold lower than thatrequired for MAb 3/11 (Fig. 5A). Similarly, in biotinyl-ated MAb binding assays, AP33 was more efficient atcompeting for binding than 3/11 (Fig. 5B). Comparedwith MAb 3/11, competition with MAb AP33 resulted ingreater reduction in binding by both biotinylated MAbAP33 and 3/11. The MAbs also exhibited marked differ-ences in affinity to E1E2 representative of diverse HCVgenotypes (Fig. 5C). Concentrations of MAb AP33needed to obtain 50% binding to the E1E2 proteinsranged from approximately 1 � 101 to 1 � 103 ng/mL,whereas 50% binding was achievable using 3/11 at con-centrations ranging from approximately 1 � 102 to 1 �104 ng/mL. Together, these data indicate that AP33 has ahigher affinity for E2 than MAb 3/11.

Similarly, a comparison of the ability of MAbs AP33and 3/11 to neutralize HCVpp carrying E1E2 represen-tative of genotypes 1 to 6 (Fig. 6A) showed that, whereasboth antibodies were capable of broad neutralization,neutralization potency of AP33 was consistently greaterthan MAb 3/11 (P � .001, Wilcoxon’s matched pairs

Fig. 4. Reactivity of MAb AP33 and 3/11 to a panel of E2 mutantscarrying alanine substitution in the region 412-423 identifies residuescritical for binding. Alanine replacement mutants of strain H77c E1E2were transiently expressed, and protein, normalized for reactivity to theMAb H53, was captured on GNA coated microtiter plates followed bydetection with either MAb AP33 (A) or 3/11 (B). Reactivity is expressedas a percentage of the binding observed for wild-type H77 E1E2.

Fig. 3. Monoclonal antibodies AP33 and 3/11 recognize distinctepitopes. Branched synthetic peptides corresponding to the most fre-quent phage mimotope selected by MAb AP33 (peptide A) and to aminoacids 412-423 of the Glasgow strain (peptide B) were coated to thewells of a microtiter plate then detected using MAb AP33 (open) and3/11 (solid).


test). When used at a concentration of 50 �g/mL, MAbAP33 was able to neutralize HCVpp infectivity by be-tween 80% and 99%. By contrast, the same concentra-tion of MAb 3/11 only resulted in 10% to 80%neutralization. As has been previously reported,33 MAbAP33 failed to neutralize HCVpp carrying E1E2 from thegenotype 5 strain UKN.5.14.4; similarly, MAb 3/11 wasalso unable to neutralize HCVpp carrying this E1E2clone. This isolate has a 4–amino acid change(QLIQNGSSWHIN) in the E2 region corresponding toresidues 412 - 423. This mutation alters two of the resi-

dues critical for AP33 (N415 and G418) recognition andone (N415) for 3/11. Consistent with this, our data showthat both MAbs AP33 and 3/11 fail to react withUKN5.14.4 E2 (data not shown).33 Both MAbs also failto neutralize UKN5.14.4 HCVpp.

MAb AP33 Neutralizes HCVcc Infectivity. We alsoassessed the neutralizing capability of MAb AP33 usingthe recently described JFH-1 HCVcc system. MAb AP33was able to reduce HCVcc infectivity by greater than 80%when added to the JFH-1 virus inoculum at a concentra-tion of 50 �g/mL (Fig. 6B).

Fig. 5. Monoclonal antibody AP33 has a higher E2-binding affinity than MAb 3/11. (A) Dilutions of MAb AP33 (circle) and 3/11 (triangle) wereused to detect immobilized 412-423 branched peptide. (B) Binding of biotinylated MAb AP33 (b-AP33) and MAb 3/11 (b-3/11) to a branchedpeptide corresponding to amino acids 412-423 of the Glasgow strain was performed either in the absence (open square) or presence of competingMAb ALP98 (light gray), 3/11 (dark gray), or AP33 (solid). Binding is expressed as the percentage of binding observed in the absence of competingMAb. (C) Dilutions of MAb AP33 (open circle) and 3/11 (open triangle) were used to detect a panel of functional E1E2 clones representative ofgenotypes 1a (UKN1A.20.8), 1b (UKN1B12.16), 2a (UKN2A1.2), 2b (UKN2B2.8), 3 (UKN3.13.6), 4 (UKN4.21.16), 5 (UKN5.15.7), and 6(UKN6.5.8). Values are plotted as the mean and standard error of three replicates.


The Epitopes for AP33 and 3/11 Are Sensitive toE1E2 Conformation. To examine the requirement forcorrect E2 conformation on the binding of MAb AP33and 3/11, we compared binding with native and dena-tured reduced E1E2. For comparison, the MAb ALP98,known to bind to a linear epitope in E2, and MAb H53,a well-characterized conformation-sensitive anti-E2 anti-body, were included in the analysis. After denaturation,MAb ALP98 binding was reduced by approximately10%, whereas both MAbs AP33 and 3/11 demonstrated a50% inhibition of binding (Fig. 7). Consistent with its

conformational nature, H53 binding was completely ab-rogated by denaturation. This indicates that, despite rec-ognizing a linear region of E2, optimal recognition byboth MAbs is affected by the conformation of E1E2.

Sequence Analysis of the VL and VH Regions ofMAb AP33 and 3/11. The deduced amino acid se-quences corresponding to the light and heavy chain vari-able regions of MAbs AP33 and 3/11 is presented in Fig.8. The most striking feature of this comparison was thatthe heavy chain CDR3 for MAb AP33 contained 10amino acids, whereas the MAb 3/11 heavy chain CDR3contained only three.

DiscussionWe have previously shown that the MAb AP33 can

potently neutralize HCVpp reconstituted with E1E2molecules from diverse genotypes of HCV. Similarly,MAb 3/11 has previously been shown to neutralizeHCVpp reconstituted with the autologous H77 E1E2protein. The current study highlights that two monoclo-nal antibodies directed to the same highly conserved re-gion of E2 have different neutralization potencies andbinding affinities. We show that MAb 3/11, althoughhaving a similar spectrum of activity, has a significantlylower affinity and lower neutralizing potency than MAbAP33.

Fig. 6. MAb AP33 is capable of potent neutralization of E1E2-mediated entry and HCV infectivity. (A) HCVpp reconstituted with func-tional E1E2 clones representative of diverse HCV genotypes were mixedwith MAb AP33 (solid) and 3/11 (open) to a final antibody concentrationof 50 �g/mL and allowed to infect Huh-7 cells. E1E2 clones wererepresentative of genotypes 1 (H77, UKN1A.14.38, UKN1A.20.8), 2a(UKN2A.2.4, UKN2A.1.2, JFH-1), 3a (UKN3A.1.28, UKN3A.13.6), 4(UKN4.11.1, UKN4.21.16), 5 (UKN5.15.11, UKN5.14.4), and 6(UKN6.5.8). Infectivity is expressed as a percentage of the infectivity ofthe HCVpp preparation in the absence of MAb. Data from repeatexperiments were consistent with those shown above. (B) JFH-1 virus wasmixed with phosphate-buffered saline, negative control Dengue type2-specific MAb 46D (50 �g/mL), MAb AP33 (50 �g/mL), and positivecontrol CD81-specific MAb JS-81 (2.5 �g/mL) and allowed to infectHuh-Lunet cells. Resulting infectivity is presented as mean and standarderror relative light units per well (RLU/well). pFK-Luc-JFH1�E1-E2 (DeltaE1E2) was also used in the analyses to control for non-specific trans-duction of reporter activity, and mock-infected cells were also included todetermine baseline luciferase activity.

Fig. 7. Monoclonal antibody AP33 and 3/11 binding is sensitive toE2 conformation. Native or denatured and reduced H77c E1E2 presentin clarified lysates from transfected 293FT cells were immobilized on GNAlectin microtiter plates and detected with MAb ALP98, AP33, 3/11, andH53. Binding is expressed as the percentage of binding observed fornative E1E2.


There was evidence that MAb recognition and neutral-ization varied across genotypes. However, such compari-sons should be treated with caution. First, differences inthe relative proportion of native and incorrectly foldedaggregates in the various E1E2 preparations will affectepitope exposure and reactivity. Similarly, differential in-corporation of E1E2 into HCVpp will result in alteredneutralization sensitivities. Therefore, KD and IC50 esti-mates were not attempted to avoid potentially misleadingcross-genotype comparisons.

Previous mapping studies have shown that the epitopesfor both antibodies lie within a region of E2 encompassingamino acids 412-423.21,22 Using random peptide phage li-brary enrichment and antibody probing of a panel of 412-423 point mutated E1E2 glycoproteins, we have identifiedfour key residues critical for MAb AP33. These residues werediscontinuous, spanning an 8–amino acid region of the pu-tative AP33 epitope. Probing the same panel of point mu-tated E1E2 clones with MAb 3/11 identified three residuescritical for binding, two of which were also critical for AP33binding. When MAb 3/11 was used to selectively enrich a12mer random peptide phage display library, the diversityobserved in the amino acid sequences of the selected phagewas greater than that observed for AP33. However, all butone of the reactive phage contained one or more of the threeamino acids identified as being critical for MAb 3/11 bind-ing when probing the panel of mutant E1E2s. These analysesindicate that MAbs AP33 and 3/11 recognize the same re-gion of E2, but their epitopes are distinct. In addition, thesedata also suggest that AP33 binding to E2 is highly depen-dent on all of the four critical residues being present in bothE2 and the reactive phage peptides. By contrast, binding of

MAb 3/11 to phage enriched by this antibody was not de-pendent on all of the residues identified in the mutant E1E2analysis being present. Clustering and overlap of neutralizingepitopes has been reported for other viruses, most notablyHIV, where a membrane proximal region in gp41 has beenshown to elicit human antibodies with different specificitiesand varying degrees of neutralizing potency.47 Similarly, weshow that MAbs AP33 and 3/11 also have very distinct phe-notypes. A better understanding, at the molecular level, ofthe mechanism of binding by each antibody will help eluci-date the reasons for these differences, as well as determinewhether the residues identified as being important forbinding are contact residues or provide a molecular environ-ment suitable for binding. Surface plasmon resonance andcrystallography experiments are underway to address theseissues.

Alanine replacement of the glutamine residue at posi-tion 412 enhanced AP33 binding. This amino acid liesimmediately upstream of the asparagine residue criticalfor AP33 binding. Interestingly, this replacement had noeffect on 3/11 binding, even though mutagenesis experi-ments implicated this residue in MAb 3/11 binding. Sim-ilarly, alanine replacement of the isoleucine residue at 423enhanced MAb 3/11 binding yet had no effect on AP33binding. Why these mutations have a differential enhanc-ing effect is unclear. Enhanced binding might resultthrough abrogation of a potentially inhibitory effect of acharged or polar residue or removal of steric hindrancecaused by a relatively more bulky side chain. These sub-stitutions also might lead to a localized conformationalchange, improving the exposure of the contact residues.We also showed, using protein denaturation experiments,

Fig. 8. Aligned amino acid se-quences of complementarity deter-mining (CDR) and framework (FWR)regions of light and heavy chains ofMAb AP33 and 3/11. CDR and FWRwere assigned using the Web Anti-body Modelling package with man-ual adjustment. The heavy chainarginine and aspartate residues cor-responding to positions 94 and 101(Kabat numbering system), respec-tively, are underlined. Shaded textindicates primer-derived sequences.*, sequence conservation betweenMAb AP33 and 3/11 sequences.


that MAb binding is sensitive to conformational changes,at least in the context of the E1E2 complex.

Region 412-423 contains one potential N-glycosyla-tion site at position 417, which appears to be used.48

Alanine substitution at this site resulted in reduced MAbAP33 binding. What effect glycosylation via this site hason antibody binding or its effects on immunogenicity ofthis region is unclear. Glycosylation may result in im-mune shielding, yet the ability of MAbs AP33 and 3/11 tobind to E2 suggests that even when glycosylated theirepitopes are still accessible. In addition, reactivity to pep-tides suggests that glycosylation might not be necessaryfor induction of AP33-like antibodies. These findings areof significant relevance if this epitope is to be included infuture vaccine design.

We have previously shown that the 412-423 region ofE2 is highly conserved across all genotypes.33 Re-analysisof sequences available from the Los Alamos HCV se-quence database shows that the residues critical for MAbAP33 and 3/11 binding are conserved to an even greaterdegree, with sequence variation of less than 0.5% to 2.5%at these sites (data not shown). Such high conservationsuggests that these residues might be important in main-taining envelope protein conformation or function. Sim-ilarly, it would also suggest that these sites are not targetedby neutralizing antibodies in vivo. Indeed, we have re-cently shown that the prevalence of antibodies reactive toregion 412-423 is low in natural infection (Tarr et al.,manuscript in preparation). Focussing the human im-mune response on the exact epitope recognized by MAbAP33 may be important in vaccine design. In this context,the availability of phage mimotopes may be beneficial, asprevious studies have shown that such mimotopes arecapable of inducing antibodies of similar phenotype to theselecting antibody.49

Finally, we compared the sequence of the MAb AP33and 3/11 VH and VL chains. The striking difference be-tween the two antibodies was in the length of the heavychain CDR3. The heavy chain CDR3 region is thoughtto have a critical role in antigen binding and demonstrateshigher levels of sequence and structural variability thanthe other variable loops. MAb 3/11 contained arginineand aspartate at positions 94 and 101, respectively, whichare capable of forming a salt bridge and subsequentbulged50 or kinked51 structure. However, MAb 3/11 has avery large deletion within this region and is therefore un-likely to adopt a complex conformation. MAb AP33 lacksthe arginine at position 94 and is therefore likely to takeon a non-bulged structure.51 Given the large differencebetween the two MAbs at the sequence level, a betterunderstanding of the molecular interactions between the

antibodies and their epitope will help explain the under-lying reasons for their phenotypical differences.

In summary, these data show that the region of E2encompassing residues 412-423 contains more than oneoverlapping and distinct neutralizing epitope. We alsoshow that antibodies recognizing this region may differsignificantly in their ability to bind to E2 and also in theirability to neutralize HCV infection; an important findingif this region is to be the focus of future vaccine candi-dates.

Acknowledgment: The authors thank Jane McKeat-ing and Jean Dubuisson for the gift of MAbs 3/11 and forMAb H53 and the MLV luciferase construct, respec-tively. Takaji Wakita and Francois-Loic Cosset for provi-sion of the JFH-1 clone and retroviral pseudotypeconstructs, respectively.

References1. Anonymous. Global surveillance and control of hepatitis C. J Viral Hepa-

tol 1999;6:35-47.2. Bartosch B, Dubuisson J, Cosset FL. Infectious hepatitis C virus pseudo-

particles containing functional E1–E2 envelope protein complexes. J ExpMed 2003;197:633-642.

3. Hsu M, Zhang J, Flint M, Logvinoff C, Cheng-Mayer C, Rice CM, et al.Hepatitis C virus glycoproteins mediate pH-dependent cell entry ofpseudotyped retroviral particles. Proc Natl Acad Sci U S A 2003;100:7271-7276.

4. Zhong J, Gastaminza P, Cheng G, Kapadia S, Kato T, Burton DR, et al.Robust hepatitis C virus infection in vitro. Proc Natl Acad Sci U S A2005:102:9294-9299.

5. Wakita T, Pietschmann T, Kato T, Date T, Miyamoto M, Zhao ZJ, et al.Production of infectious hepatitis C virus in tissue culture from a clonedviral genome. Nature Med 2005;11:791-796.

6. Lindenbach BD, Evans MJ, Syder AJ, Wolk B, Tellinghuisen TL, Liu CC,et al. Complete replication of hepatitis C virus in cell culture. Science2005:309:623-626.

7. Gardner JP, Durso RJ, Arrigale RR, Donovan GP, Maddon PJ, Dragic T,et al. L-SIGN (CD 209L) is a liver-specific capture receptor for hepatitis Cvirus. Proc Natl Acad Sci U S A 2003;100:4498-4503.

8. Pohlmann S, Zhang J, Baribaud F, Chen Z, Leslie GJ, Lin G, et al. Hep-atitis C virus glycoproteins interact with DC-SIGN and DC-SIGNR.J Virol 2003;77:4070-4080.

9. Scarselli E, Ansuini H, Cerino R, Roccasecca RM, Acali S, Filocamo G, etal. The human scavenger receptor class B type I is a novel candidate recep-tor for the hepatitis C virus. EMBO J 2002;21:5017-5025.

10. Pileri P, Uematsu Y, Campagnoli S, Galli G, Falugi F, Petracca R, et al.Binding of hepatitis C virus to CD81. Science 1998;282:938-941.

11. Barth H, Cerino R, Arcuri M, Hoffmann M, Schurmann P, Adah MI, etal. Scavenger receptor class B type I and hepatitis C virus infection ofprimary tupaia hepatocytes. J Virol 2005;79:5774-5785.

12. Bartosch B, Verney G, Dreux M, Donot P, Morice Y, Penin F, et al. Aninterplay between hypervariable region 1 of the hepatitis C virus E2 glyco-protein, the scavenger receptor BI, and high-density lipoprotein promotesboth enhancement of infection and protection against neutralizing anti-bodies. J Virol 2005;79:8217-8229.

13. Lavillette D, Tarr AW, Voisset C, Donot P, Bartosch B, Bain C, et al.Characterization of host-range and cell entry properties of the majorgenotypes and subtypes of hepatitis C virus. HEPATOLOGY 2005;41:265-274.

14. Voisset C, Callens N, Blanchard E, Op De Beeck A, Dubuisson J,Vu-Dac N. High density lipoproteins facilitate hepatitis C virus entry


through the scavenger receptor class B type I. J Biol Chem 2005;280:7793-7799.

15. Bukh J, Miller RH, Purcell RH. Genetic heterogeneity of hepatitis C virus:quasispecies and genotypes. Semin Liver Dis 1995;15:41-63.

16. Simmonds P. The origin and evolution of hepatitis viruses in humans.J Gen Virol 2001;82:693-712.

17. Abrignani S, Rosa D. Perspectives for a hepatitis C virus vaccine. ClinDiagn Virol 1998;10:181-185.

18. Allander T, Drakenberg K, Beyene A, Rosa D, Abrignani S, Houghton M, etal. Recombinant human monoclonal antibodies against different conforma-tional epitopes of the E2 envelope glycoprotein of hepatitis C virus that inhibitits interaction with CD81. J Gen Virol 2000;81:2451-2459.

19. Hadlock KG, Lanford RE, Perkins S, Rowe J, Yang Q, Levy S, et al.Human monoclonal antibodies that inhibit binding of hepatitis C virus E2protein to CD81 and recognize conserved conformational epitopes. J Virol2000;74:10407-10416.

20. Shang D, Zhai W, Allain JP. Broadly cross-reactive, high-affinity antibodyto hypervariable region 1 of the hepatitis C virus in rabbits. Virology1999;258:396-405.

21. Flint M, Maidens C, Loomis-Price LD, Shotton C, Dubuisson J, Monk P,et al. Characterization of hepatitis C virus E2 glycoprotein interaction witha putative cellular receptor, CD81. J Virol 1999;73:6235-6244.

22. Owsianka A, Clayton RF, Loomis-Price LD, McKeating JA, Patel AH.Functional analysis of hepatitis C virus E2 glycoproteins and virus-likeparticles reveals structural dissimilarities between different forms of E2.J Gen Virol 2001;82:1877-1883.

23. Rosa D, Campagnoli S, Moretto C, Guenzi E, Cousens L, Chin M, et al.A quantitative test to estimate neutralizing antibodies to the hepatitis Cvirus: Cytofluorimetric assessment of envelope glycoprotein 2 binding totarget cells. Proc Natl Acad Sci U S A 1996;93:1759-1763.

24. Shimizu YK, Hijikata M, Iwamoto A, Alter HJ, Purcell RH, Yoshikura H.Neutralizing antibodies against hepatitis C virus and the emergence ofneutralization escape mutant viruses. J Virol 1994;68:1494-1500.

25. Farci P, Alter HJ, Wong DC, Miller RH, Govindarajan S, Engle R, et al.Prevention of hepatitis C virus infection in chimpanzees after antibody- medi-ated in vitro neutralization. Proc Natl Acad Sci U S A 1994;91:7792-7796.

26. Bartosch B, Bukh J, Meunier JC, Granier C, Engle RE, Blackwelder WC,et al. In vitro assay for neutralizing antibody to hepatitis C virus: evidencefor broadly conserved neutralization epitopes. Proc Natl Acad Sci U S A2003;100:14199-14204.

27. Allander T, Drakenberg K, Beyene A, Rosa D, Abrignani S, Houghton M, etal. Recombinant human monoclonal antibodies against different conforma-tional epitopes of the E2 envelope glycoprotein of hepatitis C virus that inhibitits interaction with CD81. J Gen Virol 2000;81:2451-2459.

28. Habersetzer F, Fournillier A, Dubuisson J, Rosa D, Abrignani S,Wychowski C, et al. Characterization of human monoclonal antibodiesspecific to the hepatitis C virus glycoprotein E2 with in vitro bindingneutralization properties. Virology 1998;249:32-41.

29. Ishii K, Rosa D, Watanabe Y, Katayama T, Harada H, Wyatt C, et al. Hightiters of antibodies inhibiting the binding of envelope to human cellscorrelate with natural resolution of chronic hepatitis C. HEPATOLOGY

1998;28:1117-1120.30. Bugli F, Mancini N, Kang C-Y, Di Campli C, Grieco A, Manzin A, et al.

Mapping B-cell epitopes of hepatitis C virus E2 glycoprotein using humanmonoclonal antibodies from phage display libraries. J Virol 2001;75:9986-9990.

31. Hadlock KG, Lanford RE, Perkins S, Rowe J, Yang Q, Levy S, et al.Human monoclonal antibodies that inhibit binding of hepatitis C virus E2protein to CD81 and recognize conserved conformational epitopes. J Virol2000;74:10407-10416.

32. Puig M, Major ME, Mihalik K, Feinstone SM. Immunization of chim-panzees with an envelope protein-based vaccine enhances specific humoraland cellular immune responses that delay hepatitis C virus infection. Vac-cine 2004;22:991-1000.

33. Owsianka A, Tarr AW, Juttla VS, Lavillette D, Bartosch B, Cosset F-L, et al.Monoclonal antibody AP33 defines a broadly neutralizing epitope on thehepatitis C virus E2 envelope glycoprotein. J Virol 2005;79:11095-11104.

34. Triyatni M, Vergalla J, Davis AR, Hadlock KG, Foung SKH, Liang TJ.Structural features of envelope proteins on hepatitis C virus-like particles asdetermined by anti-envelope monoclonal antibodies and CD81 binding.Virology 2002;298:124-132.

35. Flint M, Dubuisson J, Maidens C, Harrop R, Guile GR, Borrow P, et al.Functional characterization of intracellular and secreted forms of a trun-cated hepatitis C virus E2 glycoprotein. J Virol 2000;74:702-709.

36. Patel AH, Wood J, Penin F, Dubuisson J, McKeating JA. Constructionand characterization of chimeric hepatitis C virus E2 glycoproteins: anal-ysis of regions critical for glycoprotein aggregation and CD81 binding.J Gen Virol 2000;81:2873-2883.

37. Clayton RF, Owsianka A, Aitken J, Graham S, Bhella D, Patel AH. Anal-ysis of antigenicity and topology of E2 glycoprotein present on recombi-nant hepatitis C virus-like particles. J Virol 2002;76:7672-7682.

38. Dubuisson J, Hsu HH, Cheung RC, Greenberg HB, Russell DG, RiceCM. Formation and intracellular localization of hepatitis C virus envelopeglycoprotein complexes expressed by recombinant vaccinia and sindbisviruses. J Virol 1994;68:6147-6160.

39. Nakabayashi H, Taketa K, Miyano K, Yamane T, Sato J. Growth ofhuman hepatoma cells lines with differentiated functions in chemicallydefined medium. Cancer Res 1982;42:3858-3863.

40. Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG. TheCLUSTAL_X windows interface: flexible strategies for multiple sequencealignment aided by quality analysis tools. Nucleic Acids Res 1997;25:4876-4882.

41. Moore JP, Thali M, Jameson BA, Vignaux F, Lewis GK, Poon SW, et al.Immunochemical analysis of the gp120 surface glycoprotein of humanimmunodeficiency virus type 1: probing the structure of the C4 and V4domains and the interaction of the C4 domain with the V3 loop. J Virol1993;67:4785-4796.

42. Yanagi M, Purcell RH, Emerson SU, Bukh J. Transcripts from a singlefull-length cDNA clone of hepatitis C virus are infectious when directlytransfected into the liver of a chimpanzee. Proc Natl Acad Sci U S A1997;94:8738-8743.

43. Lindenbach BD, Evans MJ, Syder AJ, Wolk B, Tellinghuisen TL, Liu CC,et al. Complete replication of hepatitis C virus in cell culture. Science2005;309:623-626.

44. Friebe P, Boudet J, Simorre JP, Bartenschlager R. Kissing-loop interactionin the 3� end of the hepatitis C virus genome essential for RNA replication.J Virol 2005;79:380-392.

45. McCafferty J, Johnson KS. Construction and screening of antibody displaylibraries. In: Kay BK, Winter J, McCafferty J, eds. Phage Display of Pep-tides and Proteins: A Laboratory Manual. San Diego, CA: Academic Press,1996:79-111.

46. Wang ZD, Raifu M, Howard M, Smith L, Hansen D, Goldsby R, et al.Universal PCR amplification of mouse immunoglobulin gene variable re-gions: the design of degenerate primers and an assessment of the effect ofDNA polymerase 3� to 5� exonuclease activity. J Immunol Methods 2000;233:167-177.

47. Zwick MB, Labrijn AF, Wang M, Spenlehauer C, Saphire EO, Binley JM,et al. Broadly neutralizing antibodies targeted to the membrane-proximalexternal region of human immunodeficiency virus type 1 glycoproteingp41. J Virol 2001;75:10892-10905.

48. Goffard A, Callens N, Bartosch B, Wychowski C, Cosset F-L, MontpellierC, et al. Role of N-linked glycans in the functions of hepatitis C virusenvelope glycoproteins. J Virol 2005;79:8400-8409.

49. Melzer H, Fortugno P, Mansouri E, Felici F, Marinets A, Wiedermann G,et al. Antigenicity and immunogenicity of phage library-selected peptidemimics of the major surface proteophosphoglycan antigens of Entamoebahistolytica. Parasite Immunology 2002;24:321-328.

50. Morea V, Tramontano A, Rustici M, Chothia C, Lesk AM. Conforma-tions of the third hypervariable region in the VH domain of immuno-globulins. J Mol Biol 1998;275:269-294.

51. Shirai H, Kidera A, Nakamura H. Structural classification of CDR-H3 inantibodies. FEBS Lett 1996;399:1-8.


Characterization of the Hepatitis C Virus E2 Epitope Defined by the Broadly Neutralizing Monoclonal...

Documents

Transcript of Characterization of the Hepatitis C Virus E2 Epitope Defined by the Broadly Neutralizing Monoclonal...