Structural basis of hepatitis C virus neutralization bybroadly neutralizing antibody HCV1Leopold Konga, Erick Giangb, Justin B. Robbinsb, Robyn L. Stanfielda, Dennis R. Burtonb,c,d, Ian A. Wilsona,e,1,and Mansun Lawb,1

Departments of aMolecular Biology, bImmunology and Microbial Science, cInternational AIDS Vaccine Initiative Neutralizing Antibody Center, and eTheSkaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, CA 92037; and dThe Ragon Institute of MGH, MIT and Harvard University,Boston, MA 021199

Edited by Peter Palese, Mount Sinai School of Medicine, New York, NY, and approved April 25, 2012 (received for review February 17, 2012)

Hepatitis C virus (HCV) infectsmore than 2%of the global populationand is a leading cause of liver cirrhosis, hepatocellular carcinoma, andend-stage liver diseases. Circulating HCV is genetically diverse, andtherefore a broadly effective vaccinemust target conserved T- and B-cell epitopes of the virus. Human mAb HCV1 has broad neutralizingactivity against HCV isolates from at least four major genotypes andprotects in the chimpanzee model from primary HCV challenge. Theantibody targets a conserved antigenic site (residues 412–423) on thevirus E2 envelope glycoprotein. Two crystal structures ofHCV1 Fab incomplex with an epitope peptide at 1.8-Å resolution reveal that theepitope is a β-hairpin displaying a hydrophilic face anda hydrophobicface on opposing sides of the hairpin. The antibody predominantlyinteracts with E2 residues Leu413 and Trp420 on the hydrophobic faceof the epitope, thus providing an explanation for how HCV isolatesbearing mutations at Asn415 on the same binding face escape neu-tralization by this antibody. The results provide structural informa-tion for a neutralizing epitope on the HCV E2 glycoprotein andshouldhelpguide rational designofHCV immunogens to elicit similarbroadly neutralizing antibodies through vaccination.

neutralizing determinant | protective determinant | antigen-antibodycomplex | type I’ β-turn

Hepatitis C virus (HCV) infects >2% of the world population,with an estimated >500,000 new infections annually in the

highest endemic country, Egypt (1, 2). In the United States, therate of symptomatic HCV infection declined over the last decadeand began to level out at ∼4 million cases around 2005 (3).Alarmingly, however, in developed countries, new cases are oftenassociated with the younger age group (15-24 y) because of illegalinjection drug use (4). Although some HCV-infected individualscan resolve infection without drug treatment, ∼70% developchronic hepatitis and, over a period of 20–30 y, 20–30% willdevelop liver cirrhosis and 1–5% hepatocellular carcinoma (5).Furthermore, HCV infection is associated with several extrahe-patic manifestations, neuropathy, and autoimmune diseases in-cluding mixed cryoglobulinemia and Sjögren’s syndrome (6). Thestandard-of-care treatment for HCV infection uses a combinationof pegylated IFN-α and ribavirin, which is effective in approxi-mately 50% of treated patients but has many side effects. Twodirect-acting antiviral drugs targeting the virus protease NS3 haverecently been approved in the United States for triple therapywith IFN-α and ribavirin to improve success rates and to shortentreatment (7). To solve the global HCV problem and to eradicatethe virus, more effective, tolerable, and affordable drugs againstHCV, as well as a vaccine, are needed. Potent direct-acting an-tiviral drugs against additional viral targets are currently underdevelopment and show promise in IFN-free treatments (8). In thepast few years, progress has also been made in vaccine de-velopment for prophylaxis and therapeutic purposes (9, 10).A major challenge in vaccine design against HCV is the ex-

treme diversity of the virus. HCV is highly heterogeneous, with 6major genotypes (>30% overall nucleotide sequence difference)and more than 50 subtypes (10–25% difference in nucleotidesequence) (11). Genotypes 1 and 3 are the most widely distrib-uted in the world. Genotype 2 is also found worldwide, with

genotype 4 predominantly in Egypt, genotype 5 in South Africa,and genotype 6 in Southeast Asia. This great diversity of HCV isfueled by the poor fidelity of its RNA polymerase and rapidturnover of the virus, as evidenced by the estimate that an in-dividual produces as many as 1012 virions per day (12). Conse-quently, any given vaccine or drug effective against one isolatewill not necessarily be useful against more divergent isolates. Toovercome the challenge of viral diversity, a broadly effectivevaccine must target conserved immune epitopes of the virus.We, and others, reported previously that antibodies to the

CD81-receptor binding site (CD81bs) on E2 mediate cross-neu-tralization of diverse HCV isolates. These antibodies include themouse mAb AP33 (13), rat mAb 3/11 (14), and human mAbHCV1 (15), which block the interaction of E2 to CD81 by bindingto linear epitopes located within the highly conserved E2 anti-genic site, residues 412–423 (the standardized genome numberingof the HCV prototypic strain H77 will be used throughout).Other mAbs recognize discontinuous E2 epitopes overlappingwith the CD81bs on E2 involving residues 395–424, 425–447, and/or 523–540 (16–19). In addition, human polyclonal antibodiespurified from the sera of HCV patients using peptides spanningE2 residues 412–419 or 412–423 were also found to neutralizeHCV (20, 21). The fact that cross-neutralizing antibodies to theE2 antigenic site 412–423 have been isolated in multiple labo-ratories suggests that this conserved site is a prime target forHCV vaccine design. However, it has been reported recently thatonly ∼2% of chronic HCV patients are able to produce an an-tibody response to this antigenic site (21, 22), indicating that it isonly weakly immunogenic when presented on native virions.In this study, we determined two crystal structures of human

mAb HCV1 Fab in complex with the E2 peptide 412–423 at 1.8-Å resolution. The mAb HCV1 was isolated by MassBiologics(15) and has been shown to protect the chimpanzee model frominfection by an HCV-infected human serum inoculum (23). ThemAb is currently under evaluation in a phase II clinical trial toprevent recurrent HCV after liver transplantation in HCVpatients (ClinicalTrials.gov identifier NCT01121185). The mAbHCV1 cross-neutralizes HCV isolates from genotypes 1a, 1b, 2b,3a, and 4a by binding to its highly conserved linear epitope withnanomolar affinity. The residues critical for antibody bindinghave previously been mapped to Leu413 and Trp420 by alaninescanning mutagenesis (15). The present work reveals the struc-tural determinants of this interaction, allowing the precise design

Author contributions: M.L. designed research; L.K., E.G., J.B.R., and M.L. performed re-search; R.L.S. and D.R.B. contributed new reagents/analytic tools; L.K., R.L.S., I.A.W., andM.L. analyzed data; and L.K., I.A.W., and M.L. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

Data deposition: The atomic coordinates reported in this paper have been deposited inthe Protein Data Bank, www.pdb.org (PBD ID codes 4DGY and 4DGV).1To whom correspondence may be addressed. E-mail: wilson@scripps.edu or mlaw@scripps.edu.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1202924109/-/DCSupplemental.

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of immunogens as potential vaccine candidates to properlypresent this epitope.

ResultsExpression and Characterization of Recombinant mAb HCV1. Thevariable domains of heavy and light chains of mAb HCV1 (15)were inserted into a human IgG1 expression vector (19) to gen-erate full-length antibody. The biological activities of therecombinant mAb were verified by a series of assays. First, mAbHCV1 binds both native and reduced E1E2 antigens in ELISA,suggesting that it recognizes a continuous or linear epitope (Fig.1A). Second, HCV1 specifically recognizes a peptide containingthe mapped epitope (Fig. 1B). Third, HCV1 bound E1E2 with anapparent affinity of ∼3 nM, consistent with the affinity reportedpreviously for this mAb (15) (Fig. S1). Finally, as expected, HCV1was able to cross-neutralize HCV genotypes 1a and 1b (Fig. 1C).To determine the crystal structure of mAb HCV1 in complex

with the epitope, we produced the Fab HCV1 and investigatedpeptides for complex formation. The Fab fragment of mAbHCV1was generated by deleting the hinge, CH2, and CH3 sequences inthe pIgG1 expression vector, and then expressed and purified.Three peptides (18-mer, 15-mer, andR12-mer) were evaluated forcomplex formation. In the R12-mer, an arginine was added to theN terminus of the 12-aa epitope to improve peptide solubility.Interestingly, the antibody bound poorly to the shorter 15-mer andR12-mer compared with the 18-mer in ELISA when the peptideswere coated directly onto the microwells (Fig. 1D, Left). Never-theless, in competition ELISA all three peptides (in solution)blocked mAb HCV1 binding to the E1E2 antigen equally well

(Fig. 1D, Right). The shorter and more soluble R12 peptide (R-412-QLINTNGSWHIN-423) was selected for complex formationwith HCV1 Fab, and that complex was submitted for crystal-lization screening.

Crystal Structures of Fab HCV1 in Complex with Its Peptide Epitope.Crystal trials were conducted on purified protein complex sam-ples concentrated to 10 mg/mL at both 4 °C and 20 °C as de-scribed in Materials and Methods. The complex crystallized underseveral conditions containing PEG and two crystal forms (spacegroups C2 and P21) diffracted to 1.8-Å resolution (Table S1).Electron density for the peptide was well defined in the initialdifference maps in the antibody combining regions of bothstructures. In the P21 form, an initial concern was the possibilitythat the peptide structure might be affected by crystal contacts,because a symmetry-mate crowds the peptide-binding region(Fig. S2A). However, in the C2 form, the peptide-binding regionis exposed with no neighboring contacts, and the peptide struc-ture is very similar to that in the P21 form, with an rmsd of 0.38 Åfor all peptide backbone atoms (excluding the added terminalarginine) after aligning all peptide backbone atoms in common(Fig. S2B). The engineered arginine is ordered only in the P21form because of crystal contacts. Therefore, the peptide–Fabinteraction presented in these structures likely reflects how theantibody recognizes the peptide in solution and the epitope inthe intact E2 protein.The peptide forms an extensive β-hairpin loop with a type I’

β-turn at residues 416–419 (TNGS). The hairpin is straddledbetween the mAb CDR2 and CDR3 loops of the heavy chain

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Fig. 1. Biological activities of recombinantmAb HCV1. (A) Binding of mAb HCV1 to E1E2in ELISA. E1E2 antigens expressed in 293Tcells, at their native (closed symbols) or re-duced (open symbols) form, were capturedonto microwells by lectin. The control mAbsA4 and AR3A recognize a continuous E1 epi-tope (46) and a discontinuous E2 epitope (19),respectively. (B) HCV1 epitope. The specificityof mAb HCV1 was verified by binding tooverlapping peptides (250 ng per well) con-taining the sequence of the epitope (in boldtype). The mAb did not bind peptides withtruncated epitope sequences. Note that theamount of captured E1E2 is not equivalent tothe directly coated peptides. (C) Neutraliza-tion of HCV by mAb HCV1. The recombinantmAb neutralized HCV pseudotype virus par-ticles (HCVpp) displaying the genotype 1a or1b E1E2, but not the control envelope glyco-protein G from vesicular stomatitis virus(VSVpp). The mAbs AR3A and AR4A are con-trol neutralizing mAbs to E2 and the E1E2complex, respectively (39). (D) Peptide candi-dates for crystallography. The binding of mAbHCV1 to three candidate peptides (R12-mer,15-mer, and 18-mer) was first evaluated bydirect ELISA (Left). The mAb bound poorly tothe 15-mer and R12-mer when the peptideswere coated directly onto microwells. How-ever, when in solution, all three peptidesblocked the mAb binding to E1E2 at equiva-lent levels (Right). The epitope sequence ishighlighted in bold in the above peptides.

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and the CDR3 loop of the light chain (Fig. 2A). Although theantiparallel β-hairpin of the peptide is mostly in contact with VH,the β-turn region interacts with VL. The low crystallographic B-values of the side chains on the peptide facing the antibody (36.0Å2 in the C2 structure and 19.1 Å2 in the P21 structure) com-pared with those facing toward solvent (55.5 Å2 in the C2structure and 32.8 Å2 in the P21 structure), and the corre-spondingly low B-values of Fab residues contacting the peptidecompared with surrounding residues, indicate a highly orderedand stable interaction (Fig. 2B). The interaction is primarilyhydrophobic, as shown by the electrostatic surface potential mapof the epitope and the paratope (Fig. 2C), as well as at least 88van der Waals interactions (Table S2). As expected, the sidechains of residues on the peptide facing away from the in-teraction site are more flexible, as observed when the peptidesfrom each structure are superimposed on each other (Fig. 2D).The Fab-facing residues of the peptide (Leu413 in the firstβ-strand, and Trp420 and Ile422 in the second β-strand) are pre-dominantly hydrophobic and fit into a deep hydrophobic de-pression (Table S2). This depression is formed by aliphatic andaromatic residues from the base of CDR H2 and CDR H3, VHframework regions (FRs) 2 and 3, CDR L3, and VL FR3 (Fig.3A). Hydrogen bonds also stabilize the interaction: one at the tipof the turn and two on the edges of the sheet for the P21structure (Fig. 3B), with three more hydrogen bonds to the edgein the C2 structure (Table S3). Altogether, the interactions aremainly focused on four residues of the peptide: Leu413, Asn415,and Trp420 on the β-strands and Gly418 at the tip of the β-turn.The peptide hairpin loop itself is stabilized by a number of

backbone hydrogen bonds (Fig. 2E), and this structural motif isin good agreement with previously published secondary structurepredictions based on the amino acid sequence (24, 25). The twoasparagine residues on the peptide facing away from the Fab(Asn417 and Asn423) are parts of N-linked glycosylation sequonsthat are likely glycosylated in the native protein (26) (Fig. S3).Another interesting feature of the interaction is that the para-tope contains residues near the base of the CDR loops and FR2,

which are generally more conserved than those at the tips of theCDR loops (Fig. 3 A and B). Some of these residues are highlyconserved across human antibodies for structural reasons, suchas Trp47 on VH FR2, which is 97.5% conserved, and Tyr58 onCDR H2, which is 45.6% conserved, across 16,946 distinctantibodies (Abysis Database: www.bioinf.org.uk/abysis) (27)(Fig. S4). Although other contacting residues, such as VH Val50,Phe100C and Ile100D, and VL Asn93 are less than 15% conserved,more than 50% of their alternative substitutions have similarchemical properties (hydrophobicity or polarity) (Fig. S4).

Structural Explanation for Broad Neutralization of HCV by mAb HCV1and Virus Escape. The basis for the broad neutralizing activity ofmAb HCV1 is the high sequence conservation of the targetepitope (Fig. 3C and Table S4) and the apparent solvent ac-cessibility of the binding face of the peptide in the intact E2protein. The peptide residues facing the antibody are highlyconserved (Leu413, Asn415, Trp420, and Ile422 in 99.9%, 97.2%,99.9%, and 96.9%, respectively, of 2,161 HCV E2 sequences inthe Virus Pathogen Resource Database, www.viprbrc.org),whereas some of the residues facing away from the binding siteare slightly more variable (Gln412, Ile414, and Thr416 in 93.5%,67.6%, and 85.1% of E2 sequences, respectively). Several resi-dues on the peptide are likely conserved because of structuralconstraints. For example, residues in the turn are conserved inline with β-turn propensities (28). Another structurally importantresidue is Asn415 because it makes a hydrogen bond to a back-bone amide of Gly418, a residue at the tip of the turn (Fig. S5).Incidentally, this is the only residue facing the hydrophobic de-pression of the Fab that is not hydrophobic.Previous alanine scanning mapping experiments using bacte-

rially expressed fusion proteins containing the epitope showedthat Leu413 and Trp420 are critical for the binding of mAb HCV1(15). These results agree well with the crystal structures that showthese two residues to be the most stabilized and buried within thebinding site (Fig. 3C). However, the same study also showed thatalanine substitution of Asn415 or Gly418 did not affect mAb

Fig. 2. Structure of mAb HCV1 in complex with its HCV E2 peptide epitope. Two X-ray structures of a broadly neutralizing antibody in complex with its epitopefrom two crystals forms, P21 and C2, were solved and refined to1.8-Å resolution. (A) The structure from the P21 crystal is shown in cartoon representation. Thepeptide epitope (red) is inserted between the heavy chain (blue) CDR2 and CDR3 loops and makes contact with the light chain (green) CDR3 loop. Arg1 in thepeptide, which was introduced to enhance the solubility of the peptide and is therefore not part of the HCV antigenic region, is colored gray. (B) B-values ofthe crystal structure were mapped onto the molecular surface of the paratope (Left) and a stick representation of the peptide (Right) by temperature gradientcoloring from 9.8 Å2 (deep blue) to 96.5 Å2 (red). The paratope is shown in gray to highlight the peptide at Right. (C) The adaptive Poisson-Boltzmann solver wasused to calculate the surface potential across the solvent-accessible surfaces of both the paratope and the peptide [−3 kT/e (red) to 3 kT/e (blue)]. For thepeptide, the surface potential is shown looking from above the antibody (Upper) and from the paratope toward the binding surface, which is a 180° rotation(Lower). The engineered arginine was included for the calculation and is shown here boxed in gray. (D) A comparison of the peptides between the two crystalstructures. The peptides are superimposed on each other, with the P21 structure in red and the C2 structure in blue. The two strands of the peptide are separatedand reoriented to better visualize the differences in side chains between the structures. The dots indicate where the two strands meet at the turn. Overall, theside chains pointing away from the antibody have greater differences between the structures than those pointing into the binding site. The main chains mostlyoverlap each other. (E) The peptide forms a β-hairpin, and the backbone hydrogen bonding that stabilizes this structure is indicated.

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binding. This result is surprising because the crystal structureshows that Asn415 hydrogen bonds to the tip of the β-turn, andGly418 is a highly preferred amino acid at this position for thistype of β-turn. Therefore, we repeated the analysis using alanine-scanning mutants of E1E2 (Fig. 4A). The results demonstratedthat replacement of Asn415 or Gly418 by alanine abolished anti-body–antigen interaction, suggesting that proper folding of the tipof the β-turn, in addition to the hydrophobic residues Leu413 andTrp420, is important for the antibody recognition of this epitope.Interestingly, alanine substitution of E2 Gln412, Ile414, Asn417,Ser419, and Ile422 reduced binding of the mAb to varying extents,indicating that these substitutions can perturb the optimal con-formation of the epitope for antibody binding.At a low incidence, some alternative substitutions are found in

natural viruses for the antibody facing residues, and these mayrepresent virus variants that could escape neutralization by thisparticular antibody (Table S4). We were particularly interested insubstitutions of Asn415 because this residue may be involved instabilizing the β-turn and binds within a less tightly packed regionof the hydrophobic depression of the mAb. In steric clash analysisof different rotamers modeled into position 415, the hydrophobicdepression will not accommodate residues with bulky side chainsor side chains with carbons beyond Cγ (e.g., lysine, histidine, ty-rosine, glutamate, and glutamine) without significant movementor conformational change in the peptide and/or the antibodyparatope (Fig. S5). To evaluate whether this analysis successfullypredicted virus escape from the mAb, Asn415 was replaced withthese low-frequency variants found in nature or by a glutamine,which is similar in polarity but has a side chain that extends be-yond the Cγ position. Although all of the seven mutated E1E2antigens expressed at a similar level in transient transfectionexperiments, they all bound mAb HCV1 at a lower level com-pared with the wild-type E1E2 (Fig. 4B). The loss of antibodybinding to the bulky lysine, glutamine, histidine, and tyrosinemutants is consistent with the clash analysis results (Fig. S5). Theanalysis also predicted that the aspartate and serine mutants couldstill fit the hydrophobic cavity, but antibody binding was abolishedfor the aspartate mutant, which would introduce a buried negativecharge, and reduced significantly for the serine mutant, whichwould no longer stabilize the β-turn with a hydrogen bond to thebackbone amide of the glycine at the tip. Interestingly, the glu-tamine mutant bound the antibody at a respectable level despitepredicted steric clashes. The results suggest that some minorconformational rearrangements must take place in the binding siteto accommodate the larger glutamine residue.In terms of biological function, substitution of Asn415 with as-

partate, histidine, tyrosine, or serine abolished most virus infectivity

in the pseudotype virus system (Fig. 4C). The glutamine and glu-tamate substitutions did not have a significant effect on virus in-fectivity, whereas the lysine substitution increased virus infectivityby two- to fourfold. These results are surprising because one wouldexpect that viral quasispecies harboring these mutations would beless fit because they are rarely observed in nature (combined<1.5% of 2,161 E2 sequences in the National Institute of Allergyand Infectious Diseases Virus Pathogen Database and AnalysisResource (ViPR) database; Table S4). In neutralization experi-ments, the N415K substitution enabled the virus to escape mAbHCV1 entirely, whereas the N415Q and N415E substitutions in-creased virus resistance to the mAb (Fig. 4C). Interestingly, theN415Q substitution is not found in the HCV sequence data-base, although the mutagenesis data suggest that the virus couldaccommodate this mutation. Altogether, these results suggestthat the lysine, glutamine, and glutamate substitutions may re-place asparagine in stabilizing the β-hairpin fold in E2, and todifferent extents facilitate virus escape of the mAb.

DiscussionAmajor challenge in the design of an HCV vaccine is to maximizethe cross-reactive immune response to protect against the hugediversity of HCV isolates found in nature (>30% genetic differ-ence). In vaccine research, both cellular (29, 30) and humoralimmunity (31, 32) have been shown to protect in the chimpanzeemodel from challenge with homologous or closely related HCV.Choo and colleagues (31, 33) demonstrated the possibility ofeliciting sterilizing immunity against low-dose homologous viruschallenge by vaccination of chimpanzees with recombinant E1E2proteins, and protection was associated with high antibody titers toE2 and the ability of the antibodies to inhibit E2 binding to cellsexpressing HCV receptors. However, when the vaccinated chim-panzees were challenged with a heterologous HCV isolate, noneof the animals were protected from acute infection, although themajority (90% vs. 40% in the control group) resolved acute in-fection and did not progress to the carrier state (9).Theseexperiments support the notion that vaccination with E1E2 pro-teins may protect infected humans from progressing to chronicinfection, which is a major risk factor for cirrhosis and hepato-cellular carcinoma. However, the lower level of protection againstheterologous challenge is consistent with observations that anti-body responses to E1E2 immunization are biased to isolate-spe-cific epitopes and not effective against divergent HCV genotypes.This conclusion is supported by the observations that both passiveantibodies against the hypervariable 1 region (HVR1) of E2 (32)and immunization with a mixture of recombinant E1, E2 proteinsand multiple HVR1 peptides (34) were unable to protect against

Fig. 3. Molecular details of antibody binding to the HCV peptide. (A) The Leu413and Trp420 residues on the peptide (red) are shown buried in a hydrophobicdepression formed by LC CDR3 residues (green) and by heavy chain FR2, CDR2, and CDR3 residues (blue). (B) Three hydrogen bonds between peptide andantibody also stabilize the interaction, as depicted in wall-eye stereo. Bonds and distances are labeled in black. (C) To further analyze the peptide binding,HCV sequence conservation across 2,161 isolates for this region, crystallographic B-value, rmsd between the two structures, and surface burial by antibody onthe peptide are shown. Sequence conservation was taken from the ViPR database (Table S4), whereas the B-values were extracted from the structure and thebinding data from ref. 15. The rmsd was calculated on a residue-by-residue basis in PyMOL. Surface burial corresponds to the accessible surface area of eachresidue on the peptide in the bound structure (P21 structure) normalized by the surface area calculated after the Fab is removed.

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heterologous HCV challenge in chimpanzees. These early studieshighlight that a broadly effective HCV vaccine must target con-served immune epitopes.In this study, we determined crystal structures of a B-cell

epitope located within the conserved antigenic site 412–423 ofthe E2 envelope glycoprotein that is also involved in binding tothe coreceptor CD81. The mAb HCV1 is one of the antibodiesknown to recognize this antigenic site and is currently underdevelopment to treat recurrent HCV in liver transplant patients(15, 23). The mAb has broad activity against diverse HCV iso-lates and protected in the chimpanzee model from challenge byan HCV-infected human serum inoculum (15, 23). This antigenicsite is poorly immunogenic on virus: only ∼2% of chronic HCVpatients seem to generate antibodies to this site (21, 22). Inpatients who make such antibodies, the quantities do not seem tobe sufficient to mediate virus neutralization or select for escapevirus (22). The structural studies here reveal that the conservedE2 antigenic site 412–423 forms an extended β-hairpin structure,and mAb HCV1 neutralizes HCV by contacting residues on thehydrophobic face of the β-hairpin that are nearly 100% con-served (Fig. 3 and Table S4). Such high conservation of thecontacting residues on the virus likely explains the broad activityof this mAb. The antibody–epitope interaction is dependent oninserting residues from the hydrophobic face of the epitope intoa hydrophobic depression on the antibody combining site. Re-placement with bulky, polar, and charged residues can weaken or

abolish the hydrophobic interactions between the antibody andantigen by steric clash or charge burial, thus resulting in virusescape. However, these hydrophobic residues are hardly eversubstituted in natural viruses, indicating that these residues arerequired for the function of the virus. However, the polar Asn415,which also points toward the hydrophobic depression but is notas intimately involved as Leu413 and Trp420, seems to be requiredfor formation of the β-hairpin structure. Consequently, mostmutations at Asn415 likely destabilize the β-hairpin, thus per-turbing E2 function, but a limited number of residues (lysine,glutamine, or glutamate) can still generate equally or more in-fectious virus in vitro (35) (Fig. 4C). Interestingly, these muta-tions are rarely observed in circulating HCV, suggesting that theydo not improve viral fitness when other host factors (e.g.,adaptive immunity) are present. This suggestion is also sup-ported by another study in which HCV carrying the mutationN415D, T416A, N417S, or I422L was more sensitive to anti-bodies targeting other neutralizing epitopes (36). Nevertheless,the possibility of virus escape through mutations in this antigenicsite should be considered in the design and evaluation ofimmunogens.The structure of the HCV peptide that we derived from its

complex with a broadly neutralizing antibody has some in-teresting implications on how the E2 protein is folded in thatregion. The side of the β-hairpin that binds to the antibody ishighly hydrophobic and, therefore, one would predict and expectthat it would most likely face the protein core. In fact, whena full-length primary sequence of the E2 protein was submittedto ab initio folding on the Robetta server, only structures wherethat face of the peptide was covered by protein were predicted(37). However, from the crystal structure, it is clearly accessibleto the antibody and, therefore, should be solvent exposed in thenative protein on the virus. On the other hand, the nonbindingface of the β-hairpin should also be exposed to accommodate thetwo putative N-linked glycans in that region (Fig. S3).Taken together, the results impose several constraints on the

structure of this antigenic site in the native context: (i) it is a β-hairpin, and disruption of the hairpin reduces viral fitness; (ii) itis solvent exposed on both sides, despite its hydrophobic char-acter, but perhaps masked to some extent by the hydrophilic N-linked glycans at Asn417and Asn423 (26); and (iii) it extends awayfrom the rest of the protein because otherwise there would besteric clashes with the antibody to the rest of the protein. Thisantigenic site has previously been predicted to fold into anti-parallel β-strands (24, 25), and Krey et al. (24) proposed that itforms part of “domain I” in class II viral fusion proteins. Withindomain I, a succession of β-hairpins forms a β-sandwich, in whichthe nonpolar sides of the β-hairpins face toward mainly the hy-drophobic core of the sandwich, and the opposite sides are ex-posed to solvent. The crystal structures here show that thehydrophobic side of this hairpin is accessible to the antibody,whereas the N-linked glycan(s) on the opposite side will likelyprevent it from participating in the proposed β-sandwich core(displayed in Fig. S3). Thus, our data suggest that this particularhairpin folds into a flap-like structure on E2 and is not part ofdomain I as predicted previously (24).In conclusion, the structures of the mAb HCV1 epitope pre-

sented here provide a structural basis for how this mAb neu-tralizes diverse HCV strains. The structures also suggesta limited number of virus mutations that may escape this mAb,but fortunately have not yet been found to any extent in knownHCV viruses. Vaccination is the most cost-effective solution tomany infectious diseases, but there is no prophylactic or thera-peutic vaccine available for HCV. The structure of this con-served HCV-neutralizing epitope can be used as a template forthe design of biomimetic antigens to direct the immune system toelicit similar cross-NAbs in vaccination (38). Clearly, the long-term goal is to determine the crystal structures of E2 and E1E2in complex with other broadly neutralizing mAbs (19, 39) toreveal additional conserved B-cell epitopes, as has been achievedso effectively for HIV-1 and influenza virus (40–44). This will

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Fig. 4. Viral escape through substitutions at E2 residue 415. (A) Alanine scan-ning mapping of the HCV1 epitope. (B) Binding of mAb HCV1 to E1E2 variantswith substitutions at position 415, including naturally occurring substitutionsandGln. (A andB) Left: Expressionof the variants confirmedbymAbAR2A (1 μg/mL) (19). Right: Binding ofmAbHCV to the variants. (C) Escape ofmAbHCV1 bysubstitution at E2 Asn415. HCVpp bearing the specific substitutions were gen-erated as described previously (19). The infectivity of the variant panel wascompared according to the activity of the reporter gene luciferase (relative lightunit, RLU) (Left). Only theK,Q, andE variants producedHCVppwith significantlyhigher signals (>10-fold) than the control pseudotype virus generated withoutE1E2. The sensitivity of these variants to mAb HCV1 was determined by in-cubating them with serially diluted mAb (Right). The results shown are themeans ± SD of two independent experiments of triplicate measurement.

Kong et al. PNAS | June 12, 2012 | vol. 109 | no. 24 | 9503

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greatly aid the rational design of a broadly effective vaccine toeradicate hepatitis C.

Materials and MethodsExpression of Recombinant mAb HCV1. The variable domains of heavy and lightchains of mAb HCV1 (15) were cloned into the pIgG1 vector (19) for expressionas a full-length human IgG1 in stably transfected CHO-K1 cells. Fab fragmentsfor crystallization were prepared by deleting the hinge, CH2, and CH3sequences of the mAb expression vector and transient transfection of Free-Style 293-F cells. The purified Fab was concentrated to 10 mg/mL in 20 mMTris·HCl and 140 mM NaCl buffer (pH 7) and allowed to form a complex withthe R12-mer peptide at a 1:10 (protein:peptide) molar ratio overnight at 4 °C.

Protein Crystallization and Structure Determination. The antibody–antigencomplex was screened for crystallization using the International AIDS Vac-cine Initiative (IAVI)-Joint Center for Structural Genomics (JCSG)-The ScrippsResearch Institute (TSRI) CrystalMation robot (Rigaku) (45). Multiple crystalswere obtained, and datasets were collected from two crystals at the Ad-vanced Photon Source (APS) beamline 23ID-D. The C2 form crystal wasgrown in 25% (wt/vol) PEG 4000, 0.2 M ammonium sulfate, and 0.1 M so-dium acetate (pH 4.6), and the P21 form crystal in 40 mM potassium dihy-drogen phosphate, 20% (vol/vol) glycerol, and 16% (wt/vol) PEG 8000. Bothcrystals diffracted to 1.8-Å resolution. The structures were determined by

the molecular replacement method using an unrelated anti-CD20 Fabstructure (Protein Data Bank ID code 3GIZ). All structural visualizations weregenerated with PyMOL (PyMOL Molecular Graphics System, version 1.2r3pre;Schrödinger, LLC). Electron density for the peptide epitope was clear in bothcrystal forms (Fig. S6).

Detailed methods and the associated references can be found in SIMaterials and Methods.

ACKNOWLEDGMENTS. We thank MassBiologics for discovering mAb HCV1for the HCV field, Jonathan Ball and François-Loïc Cosset for generous shar-ing of reagents, Travis Nieusma for excellent technical support with antibodyproduction, Henry Tien for setting up crystal screens on the CrystalMationrobot, Tinashe Ruwona for comments on the manuscript, and Alex Tarr forvaluable discussions on the immunogenicity of the E2 antigenic site. Weacknowledge the ViPR database which is funded by the National Institutesof Health (NIH) Contract HHSN272200900041C. This work is supported byNIH Grants AI80916 and AI79031 (to M.L.), AI71084 (to D.R.B), andAI84817 (to I.A.W.), and by The Skaggs Institute (I.A.W.). The GM/CA CAT23-ID-B beamline has been funded in whole or in part with federal fundsfrom National Cancer Institute (Y1-CO-1020) and National Institute of Gen-eral Medical Sciences (NIGMS) (Y1-GM-1104). Use of the Advanced PhotonSource (APS) was supported by the US Department of Energy, Basic EnergySciences, and Office of Science under contract no. DE-AC02-06CH11357. Thisis The Scripps Research Institute manuscript number 21617.

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