Toward a Hepatitis C Virus Vaccine: the Structural Basis of Hepatitis
C Virus Neutralization by AP33, a Broadly Neutralizing Antibody
Jane A. Potter,aAnia M. Owsianka,bNathan Jeffery,bDavid J. Matthews,cZhen-Yong Keck,dPatrick Lau,dSteven K. H. Foung,d
Garry L. Taylor,aand Arvind H. Patelb
Biomedical Sciences Research Complex, University of St Andrews, St Andrews, Fife, United Kingdoma; MRC-University of Glasgow Centre for Virus Research, Glasgow,
United Kingdomb; MRC Technology, London, United Kingdomc; and Department of Pathology, Stanford University School of Medicine, Stanford, California, USAd
disease. The standard of care for chronic infection—a combina-
tion of pegylated alpha interferon and ribavirin—is effective in
only 50% of patients infected with genotype 1 and is further lim-
ited by significant side effects, resistance, and high costs. This
treatment has recently been updated to include two new direct-
acting antivirals (DAAs), boceprevir (30) and telaprevir (36). A
ribavirin has become the new standard therapy for patients with
HCV genotype 1 infections. This approach to treatment, while
improving the sustained virological response (SVR) rate com-
pared to pegylated alpha interferon and ribavirin alone, still suf-
fers a number of drawbacks: the regimen is restricted to patients
with genotype 1 HCV infection, and there is an increased rate of
adverse effects. Additionally, since the DAA treatment still re-
quires coadministration of pegylated alpha interferon and ribavi-
rin to reduce the risk of selecting for resistant strains (45), the
problems of high cost and low tolerance associated with these
drugs remain. There is therefore a pressing need to develop
alternative anti-HCV therapies, particularly in the arena of pre-
ventative or therapeutic vaccines. The observation that some in-
dividuals are able to spontaneously clear HCV infection with vi-
rus-specific immune responses (37) has spurred interest in the
particular the considerable genetic diversity of HCV.
HCV, a member of the Flaviviridae family of positive-strand
RNA viruses, is composed of a nucleocapsid core enveloped by a
anchored. E1 and E2 exist as heterodimers and play an essential
role in viral entry into target cells (11). The entry process, while
epatitis C virus (HCV) infects an estimated 2 to 3% of the
not fully understood, is known to involve a number of host cell
surface entry factors, including CD81, scavenger receptor class B
type I (SR-BI), and the tight junction proteins Claudin 1 and Oc-
ies and contains hypervariable region 1 (HVR1), which is immu-
nodominant and highly variable in sequence (22). Consequently,
while antibodies to HVR1 can be neutralizing, they tend to be
isolate specific and are unable to recognize E2 from other geno-
types or isolates (14, 49). While more broadly neutralizing anti-
bodies exist, the majority of these recognize conformational
epitopes on E2 that are noncontiguous and therefore extremely
challenging to mimic in a potential vaccine (1, 3, 18, 19).
antibodies (NAbs) that are directed against conserved, linear
epitopes. AP33 is a mouse monoclonal antibody (MAb) that can
strongly inhibit the interaction between E2 (in various forms, in-
cluding soluble E2, E1E2, and virus-like particles) and CD81 (8,
41, 42). The AP33 epitope, which spans residues 412 to 423 of
HCV E2, is linear and highly conserved and encompasses a tryp-
tophan residue that plays a critical role in CD81 recognition. In-
deed, the antibody has been shown to be capable of potently neu-
tralizing infection across all the major genotypes (20, 42). The
Received 7 August 2012 Accepted 12 September 2012
Published ahead of print 19 September 2012
Address correspondence to Arvind H. Patel, firstname.lastname@example.org, or
Garry L. Taylor, email@example.com.
Copyright © 2012, American Society for Microbiology. All Rights Reserved.
December 2012 Volume 86 Number 23Journal of Virology p. 12923–12932jvi.asm.org
AP33 epitope is also recognized by several other MAbs, including
HCV1, 95-2, and 3/11 (6, 15).
The rational development of immunogens that might mimic
such epitopes and elicit AP33-like antibodies has been stymied by
the lack of detailed structural information available for the viral
glycoproteins. To further understand the mechanism by which
AP33 neutralizes HCV infection and to aid the development of a
potential epitope vaccine, the X-ray crystal structure of the Fab
portion of AP33 in complex with its epitope peptide has been
determined to 1.8Å. The structure is compared to the recently
described complex of a different MAb, HCV1, with a similar pep-
tide (26). Additionally, the interaction between AP33 and E2 has
been further characterized by cross-competition analyses, ala-
MATERIALS AND METHODS
Crystallization of AP33 Fab and Fab-peptide complexes. Purified AP33
trials. Trials were carried out at 293 K using a nanodrop crystallization
robot (CartesianHoneybee; Genomic Solutions). An initial hit was ob-
tained in condition 47 of the PACT Suite crystallization screen (Qiagen).
glycol 6000 (PEG 6K), 0.1 M Tris-HCl (pH 8), and 0.2 M CaCl2, and the
to soak the Fab crystals with peptide proved unsuccessful, cocrystalliza-
tion was performed by screening around the unliganded Fab crystalliza-
tion conditions. One millimolar synthesized 14-mer 8834 (IQLINTNG-
SWHINR) or 12-mer 8741 (GlpLINTNGSWHVN) (Enzo Life Sciences)
was premixed with 4 mg ml?1Fab in 20 mM Tris-HCl (pH 7.2). Opti-
mized crystals for each peptide complex grew in 20% PEG 6K, 0.1 M
Tris-HCl (pH 8.0), and 0.3 M CaCl2.
Data collection, structure determination, and refinement of AP33
Fab. AP33 Fab crystals were soaked briefly in cryoprotectant containing
crystallant plus 20% glycerol before flash-freezing in a nitrogen stream at
100 K. X-ray data were collected to 2.65 Å on an in-house Rigaku Micro-
Max-007HF rotating-anode X-ray generator and Saturn 944 charge-cou-
pled-device (CCD) detector. Crystals belonged to space group I4122 with
unit cell dimensions a ? b ? 90.8 Å, c ? 458.7 Å, ? ? ? ? ? ? 90o. Data
(35). In order to sidestep the problem of an unknown elbow angle, three
ensembles that encompassed the CHCL, VH, and VLregions, respectively,
were used as the molecular replacement model in Phaser. These ensem-
bles were selected on the basis of highest sequence homology to the cor-
responding AP33 Fab subdomain: CHCLfrom Fab HGR-2 F6 (PDB ac-
cession code 1DQD), VHof Fab B2B4 (3KS0), and VLfrom BION-1
(1EGJ). The top Phaser solution (translation function Z-score [TFZ
unit. Each Fab was composed of four canonical immunoglobulin folds.
The model was refined to an Rcrystof 24.7% (Rfree? 31.9%) at 2.65 Å
refinement using the program Refmac (12, 39).
complex with peptide 8741. Crystals were soaked briefly in cryopro-
tectant containing crystallant plus 20% glycerol before flash-freezing in a
nitrogen stream. Data were collected to 1.8 Å on an in-house Rigaku
MicroMax-007HF rotating-anode X-ray generator. Crystals belonged to
space group C2 with the following unit cell dimensions: a ? 127.6 Å, b ?
56.9 Å, c ? 81.8 Å, ? ? ? ? 90o, and ? ? 113.9o. Data were processed
using HKL-2000 (40), and the structure was determined by molecular
Fab structure as the search model. The Phaser solution contained one
AP33 Fab molecule in the asymmetric unit (TFZ score ? 20.1; LLG ?
in Coot (12), the peptide could be built unambiguously into the electron
density in the antigen binding site. Rcrystand Rfreevalues for the refined
model were 17.8% and 21.0%, respectively.
Data collection, structure determination, and refinement of AP33
Fab in complex with peptide 8834. Crystals belonged to space group C2
Å, ? ? ? ? 90o, and ? ? 112.1o. Data collected in-house were processed
using the software program Mosflm (33), and the structure was deter-
AP33 Fab molecule in the asymmetric unit (TFZ score ? 37.2; LLG ?
ing in Coot (12), the peptide could be built unambiguously into the elec-
tron density in the antigen binding site. Further refinement and addition
of waters yielded a final model with an Rcrystvalue of 24.4% (Rfree?
Statistics of the data processing and refinement of unliganded and
peptide-bound AP33 Fab are detailed in Table 1.
Cells. AP33 hybridoma cells were grown in Dulbecco’s modified Ea-
gle’s medium (Gibco) supplemented with 10% ultra-low-IgG fetal calf
ing to the manufacturer’s instructions. Human epithelial kidney cells
Antibodies. The anti-E2 human monoclonal antibodies (hMAbs)
CBH-4B, CBH-4D, CBH-5, CBH-7, CBH-17, HC-1, and HC-11 and an
Fab fragment of AP33 was made by digesting the MAb for 7 h with im-
mobilized papain, followed by purification through a protein A column
according to the manufacturer’s protocol (Pierce). It was further purified
for crystallization by anion exchange on a Mono Q 5/50 GL column (GE
for elution. Biotinylation was carried out using the Immunoprobe bioti-
Wild-type (WT) and mutant chimeric AP33 MAbs were produced by
cotransfection, using the calcium phosphate method, of 1.5 ? 106sub-
confluent HEK-293T cells with 8 ?g each of the heavy- and light-chain
expression plasmids pGID200 and pKN100. Two days after transfection,
the cells were subcultured into a larger flask and allowed to grow for
another 3 days, and the supernatant, which contained about 0.3 ?g/ml of
IgG, was harvested. The concentration of IgG was measured by enzyme-
linked immunosorbent assay (ELISA).
Plasmid constructs and mutagenesis. The variable domains of the
heavy and light chains of mouse MAb AP33 were grafted onto a human
expression vectors pGID200 and pKN100 (a kind gift from MRC Tech-
nology, London, United Kingdom), respectively. Selected amino acids
within the complementarity-determining regions (CDRs) were changed
to alanine by site-directed mutagenesis, and the mutations were con-
firmed by nucleotide sequencing. The mutagenesis was carried out on a
human-mouse chimeric MAb because this facilitated use of the mutants
in other related studies.
Alanine substitution mutants of genotype 1a strain H77c E1E2
(GenBank accession no. AF009606) were constructed as previously de-
night with anti-human IgG (Fab-specific) antibody produced in goat (I-
9010, 1:10,000; Sigma) and blocked with 4% skimmed milk– 0.05%
Potter et al.
jvi.asm.orgJournal of Virology
Tween 20 in phosphate-buffered saline (PBS). Serially diluted IgG-con-
taining medium was added, with an appropriate IgG standard alongside.
The captured MAb was detected with anti-human IgG (Fc-specific) per-
oxidase conjugate (A-0170, 1:10,000; Sigma), followed by TMB (3,3=,
5,5=-tetramethylbenzidine, Invitrogen) substrate. Absorbance was mea-
sured at 450 nm.
performed essentially as described previously (44). Soluble genotype 1a
E2 was made by infecting BHK cells at 5 PFU/cell with recombinant vac-
cinia virus expressing amino acids (aa) 384 to 660 of genotype 1a strain
H77c E2 (41). Four days after infection, the cells were harvested, washed
in PBS, and resuspended in lysis buffer (40 mM Tris [pH 7.5], 1 mM
EDTA, 150 mM NaCl, 1% Igepal CA-630, 20 mM iodoacetamide, and
complete protease inhibitor cocktail [Roche]). Nuclei were pelleted by
was stored in aliquots at ?20°C.
HEK-293T cells, using the calcium phosphate method, with the plasmids
and the cytoplasmic extracts were prepared as described above.
to capture E2 glycoproteins from cell lysates. Serially diluted MAbs were
added and incubated for 90 min. After washing, the bound antibodies
and measurement of absorbance at 450 nm.
Competition assay. Competition analysis was performed by modify-
ing the GNA-E2 ELISA essentially as described previously (24). Briefly,
genotype 1a strain H E1E2 glycoproteins from transiently transfected
HEK-293T cell lysates were captured on Immulon II microtiter plates
precoated with GNA. After overnight incubation, the plates were washed
added at four times the half-maximal effective concentration (EC50), so
that the final concentration of biotinylated antibody was twice the EC50,
and that of competitor was 20 ?g/ml. Under these conditions, the signal
obtained is closely proportional to the concentration of biotinylated an-
tibody bound to the plate. The plates were incubated for 90 min and
washed, and 100 ?l of streptavidin-HRP polymer was added per well
(S2438, 1:20,000; Sigma). This was followed by TMB substrate and mea-
surement of absorbance at 450 nm.
each biotinylated antibody in the presence of competing antibody was
4gaj, and 4gay.
sponding to residues 411 to 424 of HCV E2 (IQLINTNGSWHI
Fab as a molecular replacement model. However, better-quality
(GlpLINTNGSWHVN), which represents residues 412 to 423 of
the genotype 1a Glasgow strain (44). This peptide differs slightly
from 8834 in that Q412 is a pyroglutamic acid (Glp) and I422 is a
valine in 8741. Data collection and refinement statistics are de-
tailed in Table 1. Structural comparison of the two complexes
confirms that the backbones of the differing residues are similarly
placed and the side chains of these residues are solvent exposed
and do not make any direct interactions with AP33 Fab (data not
TABLE 1 X-ray data collection and refinement statistics for unliganded and peptide-bound AP33 Fab
Value for AP33 Faba
AP33 FabAP33 Fab complex with peptide 8834AP33 Fab complex with peptide 8741
Unit cell dimensions (Å)/(o)
I 41 2 2
a ? 90.9, b ? 90.9, c ? 459.1
? ? ? ? ? ? 90
a ? 171.9, b ? 40.7, c ? 73.8
? ? ? ? 90, ? ? 112.1
a ? 127.6, b ? 56.9, c ? 81.8
? ? ? ? 90, ? ? 113.9
Resolution range (Å)
No. of unique observations
No. of Fab atoms
No. of peptide atoms
Avg B factors (Å2)
RMSD bond distance (Å)
RMSD bond angle (°)
aNumbers in parentheses refer to the highest-resolution shell.
bRmerge? ?hkl?i| Ihkl, i? ?Ihkl?|/?hkl?Ihkl?, where ?Ihkl? is the average of symmetry-related observations of a unique reflection.
cRcryst? (? | |Fo| ? |Fc| |)/(? |Fo|).
dTest set comprised 5% of reflections.
eValue for Fab.
Structural Basis of HCV Neutralization by AP33
December 2012 Volume 86 Number 23jvi.asm.org 12925
shown). In view of this similarity and the more ordered composi-
tion of the Fab component of the higher-resolution complex, the
structural analysis presented here will refer to the complex with
The Fab is composed of four canonical immunoglobulin folds
(Fig. 1A), and when it is in complex with peptide 8741, clear elec-
tron density is visible for residues 1 to 214 of the light chain and
127 to 132 of the heavy chain, which are disordered. The elbow
angle is 142o, which is within the range observed for Kappa Fab
structures (50). In the unliganded Fab structure, the elbow angles
for the two Fab molecules in the asymmetric unit are 131oand
metry interactions in the crystal. The elbow angle of the Fab in
strated that it is not unusual to observe variation in elbow angles
ded states (50). There are two residues, RL68 and AL51 (the sub-
script L or H denotes the light or heavy chain), in disallowed
regions of the Ramachandran plot. The side chain of RL68 is dis-
ordered, but the main chain has clear electron density. AL51,
which is clearly defined in the electron density map, is commonly
observed to possess disallowed dihedral angles in Fab structures.
CDR classification. Despite their sequence variability, five of
the six CDR loops of antibodies usually adopt one of a small rep-
ertoire of canonical structures (7). In AP33, the light-chain CDRs
L1, L2, and L3, and the heavy-chain CDRs H1 and H2 belong to
canonical classes 5,1,1,1 and 1, respectively (2, 34). The third hy-
pervariable loop of the heavy chain of antibodies, H3, is too vari-
able to be classified into canonical structures but does tend to
the loop. In AP33, the heavy-chain CDR H3 exhibits the more
tween the nitrogen of WH103 and the carbonyl oxygen of
E2 peptide conformation. The peptide adopts a ?-hairpin
structure within a pocket formed by the antibody combining site
(Fig. 1A). All 12 residues of the peptide are clearly defined in the
electron density (Fig. 1B) with the exception of the side chains of
N417 and H421, which are partially disordered. The ?-hairpin
conformation of the peptide is maintained by a pair of backbone
hydrogen bonds between I414 and H421 and another pair be-
conformation is provided by a hydrogen bond formed by O?1 of
N415 to the amide nitrogen of G418 (distance, 3.05 Å) and by a
bridging water molecule positioned between the backbone oxy-
gens of pyroglutamic acid residue 412 and V422.
Overview of peptide binding by AP33. The antigen binding
lining the combining site. Two asparagine residues of the light-
chain CDR L3 contribute some polar character and improve the
The CCP4 software program SC (32) confirms the high shape
complementarity at the Fab-peptide interface, giving a shape cor-
relation (SC) value of 0.81. Residues from CDRs L1, L3, H1, H2,
and H3 directly contact the peptide. CDR L2, which generally
bound in the AP33 Fab combining site. The Fab heavy and light chains are
colored dark blue and cyan, respectively. The positions of the CDR loops are
labeled. The peptide is colored magenta. (B) Stereoview of the Fo-Fc electron
density map (green), contoured at 3?, superimposed on the peptide. Peptide
residues are numbered according to the corresponding residues in the E2
glycoprotein. (C) Stereoview of the surface of AP33 Fab (colored by electro-
atoms. The figures were produced using the software program PyMOL
TABLE 2 Inter- and intramolecular hydrogen bonds formed by peptide
8741 in its complex with AP33 Faba
Bridges to peptide (V422)
Bridges to YH33
Peptide ?-sheet interaction
Peptide ?-sheet interaction
Bridges to SH54 via water 163
Bridges to CDR H2 via waters
Peptide ?-sheet interaction
Peptide ?-sheet interaction
Bridges to DL94 via water 97
2.8Bridges to peptide (Glp412)
aDirect hydrogen bonds connecting the peptide to the Fab are highlighted in boldface.
Potter et al.
jvi.asm.orgJournal of Virology
contributes few or no interactions in other peptide-bound Fabs
(53), is too distant to interact directly with the peptide.
Interactions between AP33 and the peptide. The Fab forms
of the peptide also appears to play a role in recognition by the
antibody, being within hydrogen bonding distance of nine water
molecules, of which six bridge either directly or via another water
molecule to residues of the antibody CDRs (Table 2). The E2
is involved in extensive van der Waals interactions with YH33,
bic nature of the pocket. Additionally, the backbone amide and
side chain Nε1 of W420 are within hydrogen-bonding distance of
NL91 O and NL92 O?1, respectively. The side chain of the E2
residue N415 is 94% buried in the complex and forms hydrogen
bonds to YH33 and YH50. Peptide residue L413 is 82% buried in
the complex, while G418 is 100% buried. The main-chain amide
of L413 hydrogen bonds to the side-chain hydroxyl of YH100,
while the carbonyl of G418 forms a hydrogen bond with WL100
Nε1. Peptide residues Glp412, I414, T416, N417, S419, H421,
V422, and N423 are solvent exposed and make no direct interac-
tions with AP33 Fab, although the T416 side chain links to a net-
work of waters that reaches CDR H2, and S419 is indirectly con-
nected to DL94 via two bridging water molecules.
Comparison with MAb HCV1 bound to a similar epitope.
with a different neutralizing antibody Fab, HCV1, was reported
(26). Two crystal forms (P21and C2) of HCV1 in complex with
the epitope were described. This antibody shares little sequence
homology with AP33 in the CDR regions (Fig. 2A) and conse-
quently forms a completely different set of interactions with the
epitope peptide. In the AP33 Fab structure, the peptide runs ap-
chain CDRs, while in the HCV1 Fab structure the peptide lies
across this interface (Fig. 2B). Interestingly, the peptide itself
ent in the central portion of the hairpin: the backbone root mean
C2 forms, respectively), reducing to 0.90 Å or 0.79 Å for residues
413 to 422. Consequently, the paratopes share some similarities
compositions of the antigen-binding residues. Residues that are
G418, and W420, are also buried in the HCV1 Fab-peptide com-
plex (Fig. 2D). Residues 413 to 422 bury a larger area on the sur-
face of AP33 (559 Å2) than on the two HCV1 structures (476 Å2
and 488 Å2for the P21and C2 forms, respectively), suggesting a
slightly more intimate association between the peptide and AP33
relative to the complex with HCV1.
AP33 mutagenesis. To experimentally determine the impor-
tance of individual antibody residues for E2 binding, 15 AP33 resi-
dues in close proximity to the E2 epitope peptide (Table 3) were
FIG 2 AP33 and HCV1 epitopes adopt a similar conformation. (A) Sequence
alignment of AP33 and HCV1 CDRs. The residues of each Fab that interact
AP33 (this study) and HCV1 (26) Fab combining sites. Residues of the heavy-
chain CDRs are colored blue, and those of the light-chain CDRs are shown in
cyan. The peptides are shown as sticks with yellow carbon atoms. (C) Super-
position of E2 epitope peptides from complexes with AP33 (pale green) and
HCV1 (magenta). Backbone atoms of peptide residues 412 to 422 are shown.
(D) Percentage surface area of each peptide residue buried upon binding to
AP33 and HCV1, calculated by the software program PISA (28).
TABLE 3 AP33 residues in close proximity to E2 peptide 8741
No. of contacts
with peptide 8741a
Buried surface area (%)b
aAtomic intermolecular contacts between AP33 Fab residues and the E2 epitope
peptide 8741 were determined using the software program CONTACT, as implemented
in the CCP4 suite (54). Atoms within 4.5 Å of each other were considered to be in
bPercent surface area of the AP33 residue that becomes buried upon complex
formation with the peptide. Buried surface areas were calculated by the software
program PISA (33).
Structural Basis of HCV Neutralization by AP33
December 2012 Volume 86 Number 23jvi.asm.org 12927
E2 binding was measured by ELISA. The mutants were named ac-
position 33 in the heavy chain was changed to alanine (Table 4).
Expression vectors encoding the appropriate antibody heavy- and
light-chain combinations were cotransfected into HEK-293T cells.
Antibody concentrations in the culture medium were measured by
IgG capture ELISA, and the medium was diluted as necessary to
equalize the concentrations of IgG. The WT and mutant antibodies
were then tested by GNA-capture ELISA for reactivity with soluble
4, expressed as a percentage of binding by WT AP33. Of the 15 mu-
tations tested, YH33A, YH50A, YH58A, IH95A YH100A, FL32A,
NL91A, and WL96A resulted in a greater than 90% reduction of E2
while the remainder exhibited little or no reduction in E2 binding.
AP33 cross-competition analysis. The major element of the
AP33 epitope clearly consists of the conserved linear sequence
perimental data show that AP33 binds more weakly to denatured
than to native E2, indicating that optimal recognition is affected
due to denaturation of the ?-hairpin structure of the epitope or
could indicate that the MAb has some contact residue(s) beyond
between AP33 and a panel of well-characterized E2 human MAbs
(hMAbs) was performed.
The hMAbs CBH-4B, CBH-5, CBH-7, HC-1, and HC-11 all
recognize conserved, conformational epitopes on E2 and have
been extensively analyzed by cross-competition analysis and by
and HC-11 bind to overlapping epitopes in antigenic domain B,
C hMAbs inhibit E2 binding to CD81 and neutralize HCV infec-
tion, while antigenic domain A hMAbs are nonneutralizing. The
binding of each biotinylated hMAb to E2 was measured in the
AP33 was measured in the presence of an excess of each hMAb.
The effectiveness of the assay was demonstrated by testing each
the results showed a marked (?85%) reduction of antigenic do-
main B antibody binding in the presence of excess AP33 but no
of any of the hMAbs (Table 5). The same one-way competition
was seen with the Fab fragment of AP33 as with the whole AP33
MAb (Table 5), indicating that the AP33 epitope may overlap or
be very close to antigenic domain B.
E2 alanine-scanning mutagenesis. To further investigate the
possibility that there may be contact residues outside the main
epitope that participate in AP33 binding, alanine substitution
studies were performed in which each residue of E2 from 410 to
446, 526 to 540, 611 to 619, and 649 to 655 was individually mu-
tated to alanine. These regions encompass residues involved in
CD81 binding, recognition by broadly neutralizing antibodies,
and also a region associated with an AP33-resistant virus escape
mutant (10, 16). HEK-293T cells were transfected with plasmids
were recovered by cell lysis. The lysates were first normalized for
E2 on the basis of reactivity with CBH-17, an antibody to an un-
to AP33 and antigenic domain A, B, and C hMAbs. As expected
from the crystal structure, alanine substitution at L413, N415,
4). Greater than 40% reduction in AP33 binding to E2 with sub-
stitutions Y611A, R614A, or C652A was also observed. However,
the Y611A and R614A substitutions also prevented binding by all
three conformation-sensitive antibodies, and C652A reduced
B, and C hMAbs bind to nonoverlapping epitopes and do not
TABLE 4 Reactivity of E2 with AP33 alanine substitution mutants
Relative strength of binding to E2
aBinding strength of each AP33 mutant, expressed as a percentage of WT AP33 binding
to soluble genotype 1a E2 in GNA ELISA. Values shown are the means and standard
deviations of data from three replicate assays. Alanine substitutions that reduced E2
binding by 90% or more are displayed in boldface.
?90% are colored purple. NL92A, which reduced binding by 40%, is in pink.
Positions of mutations that had little or no effect on E2 binding are colored
cyan. The peptide is displayed as sticks with yellow carbon atoms.
Potter et al.
jvi.asm.orgJournal of Virology
hMAb is acting by disrupting E2 conformation. The observed re-
duction in binding of AP33 to these mutants does not therefore
indicate that they are contact residues but agrees with previous
data which show that AP33 binds less well to misfolded E2. In-
stead, alanine mutation at L654 did not alter binding to antigenic
this might be a common contact residue shared by AP33 and the
antigenic domain B hMAbs.
This region encompasses the highly conserved E2 residue W420,
which plays a critical role in CD81 recognition and serves as an
important contact residue for several broadly neutralizing anti-
bodies, including AP33 and HCV1 (reviewed by Angus and Patel
and Di Lorenzo et al. [3, 10]). Antibodies to this region are rela-
is poorly immunogenic (51).
The current study reveals the structural details of AP33 Fab in
lows comparison with the recently described structure of a differ-
ent antibody (HCV1) in complex with a highly similar peptide
(R-QLINTNGSWHIN). It is notable that the ?-hairpin confor-
mations of the peptides are very similar, despite the very different
compositions of the antibody CDRs in AP33 and HCV1. This is a
strong indication that the ?-hairpin structure represents the con-
formation of this region in intact E2. Each E2 residue is buried or
accessible to a similar extent whether bound to AP33 or HCV1,
therefore likely to be solvent exposed, despite its hydrophobic
nature. However, surface-exposed tryptophans are occasionally
found, particularly in proteins involved in protein-protein inter-
actions (43, 48).
TABLE 5 Competition matrix
Bindingaof biotinylated test antibodybto E2
CBH-4BCBH-5 HC-1 HC-11CBH-7AP33 AP33 Fab
aThe binding of each biotinylated antibody to E2 in the presence of competing antibody is given as a percentage of binding in the absence of competing antibody.
bThe concentration of each biotinylated test antibody was twice the EC50(range, 0.05 to 1.0 ?g/ml).
cLetters in parentheses denote antigenic domain A, B, or C.
dIsotype-matched control antibody to a cytomegalovirus protein.
FIG 4 Epitope mapping of AP33 using alanine-scanning mutagenesis of E2. WT genotype 1a H77c E2 and alanine substitution mutants were expressed in
HEK-293T cells. In each mutant, one amino acid residue within four selected regions of E2 (410 to 446, 526 to 540, 611 to 619, and 649 to 655) was replaced by
alanine (or glycine, where alanine is the WT residue). The cell lysates were first normalized for E2 on the basis of reactivity to CBH-17, an hMAb to a linear E2
epitope. The normalized lysates were then used in GNA capture ELISA to test the binding of AP33 alongside that of the hMAbs CBH-4D, HC-11, and CBH-7,
which recognize nonoverlapping conformational E2 epitopes in antigenic domains A, B, and C, respectively. Antibody binding to each mutant is expressed as a
percentage of binding to WT E2. Red indicates 0 to 20%, orange 21 to 40%, yellow 41 to 60%, white 61 to 90%, and green ?90% of WT binding.
Structural Basis of HCV Neutralization by AP33
December 2012 Volume 86 Number 23jvi.asm.org 12929
insights into the positioning of the epitope within the full-length
glycoprotein. No high-resolution structural data currently exist
for HCV E2, but Krey et al. have constructed a homology model
based on related class II fusion proteins using the positions of the
protein’s disulfide bridges together with functional data and sec-
ondary structure predictions (27). In the homology model, the
AP33/HCV1 epitope falls within the central domain (domain I),
an eight-stranded ?-sandwich structure with up-and-down to-
to note that the epitope is predicted to encompass the majority of
AP33 and HCV1 Fab-peptide complexes, in which the peptide
forms a ?-hairpin, with G418 positioned at the turn. In the AP33
and HCV1 complexes, the side of the ?-hairpin that lies opposite
wich, the antibody-binding face of the peptide, which is deeply
buried when complexed with both AP33 and HCV1, would be
inaccessible to antibody or CD81. In this situation, the AP33 and
HCV1 antibodies would need to induce a conformational change
that AP33 and HCV1 are both able to bind E2 with high affinity
(26, 52; unpublished data) lends strong support to the view that
In the current study, we investigated the effects of mutations
within the antibody combining site. Antibody residues that were
identified from the crystal structure as being in close proximity to
mutations that had the greatest effect on E2 binding were clus-
tered around the central portion of the binding pocket, while
to epitope recognition is minimal. It is noteworthy that the light-
chain residue YL28A did not appear to affect E2 binding, even
with residues toward both the N and C termini of the peptide.
These results confirm that the peptide structure reflects the true
that the key determinants for epitope recognition are those that
anchor the central and turn regions of the ?-hairpin.
The structural details of the Fab-peptide complex provide fur-
ther confirmation of previous data that indicated that the linear
sequence QLINTNGSWHIN (E2 residues 412 to 423) comprises
the major element of the AP33 epitope. This was originally estab-
lished by peptide mapping (41), and the contact residues were
identified by phage display and site-directed mutagenesis (52). It
cannot be discounted, however, that recognition by AP33 may
also involve additional E2 residues beyond those represented by
the linear epitope. The observation that AP33 exhibits slightly
reduced binding to denatured E1E2 suggests that there may be a
modest conformational component to the interaction (42, 52).
To investigate this possibility, a cross-competition analysis
with well-characterized conformation-sensitive hMAbs was per-
formed. It was surprising to note that while AP33 competed with
the antigenic domain B hMAbs CBH-5, HC-1, and HC-11, there
was no reduction of AP33 binding in the presence of an excess of
any of the hMAbs. There are several possible interpretations of
these findings. The first is that they reflect a difference in binding
strengths. An antibody with high affinity will compete effectively
epitopes is observed if their affinities for the antigen are markedly
different. AP33 binds strongly, with an EC50of about 0.23 nM, to
genotype 1a E2 (52; unpublished data), while the apparent affin-
ities of HC-1, HC-11, and CBH-5 are lower, with EC50values of
1.3 nM, 2.4 nM, and 220 nM, respectively (23). Alternatively, if
be an indication of close proximity, rather than overlap, of
epitopes (24, 38). We know that the HC-11 epitope includes E2
in this region (25). A third possibility is that AP33, although di-
rected primarily at the linear epitope between residues 412 and
a minor component to its epitope and are shared contact residues
with antigenic domain B hMAbs. Given the conformational na-
ture of the antigenic domain B hMAb epitopes, the availability of
all discontinuous contact points may be required for binding by
these antibodies, and competition for even one shared contact
residue could more easily displace them. Finally, an interesting
interpretation is that AP33 binding induces a conformational
antigenic domain B hMAbs sufficiently to prevent them from
binding. However, this seems doubtful, since the entropic cost of
such a rearrangement is likely to be prohibitively high.
tested for reactivity to AP33 and to the conformational hMAbs
CBH-4D, HC-11, and CBH-7, which bind to antigenic domains
A, B, and C, respectively. The results showed that E2 residues
L413, N415, G418, and W420 are critical for binding, as demon-
strated by a more than 73% reduction in AP33 binding of mutant
compared to wild-type E2 when these residues were individually
mutated to alanine. These findings correlate well with the X-ray
are the most intimately associated with the antibody, with buried
surfaces of 82%, 94%, 100%, and 99% for L413, N415, G418, and
W420, respectively. Significant (?40%) reductions in binding
these mutations also affected binding by two or three of the con-
formation-sensitive hMAbs, indicating that they disrupted the
tertiary structure of E2. The reduction of AP33 binding by these
mutations agrees with the observation that there is a conforma-
tional element to the AP33-E2 interaction. Interestingly, alanine
A or domain C antibody binding, reduced binding of AP33 and
HC-11 by 40%. This is the only indication of a possible contact
consistent with the observation that the adjacent residue, E655,
has previously been implicated in AP33 recognition (16). We in-
cluded an E655A mutant in the analysis and saw no significant
critical residues L413, N415, G418, and W420, the reduction of
fore this is not strong evidence of a contact residue outside the
linear epitope. That said, our analysis was limited to selected re-
analysis to definitively settle this question.
Potter et al.
jvi.asm.orgJournal of Virology
Residues N417 and N423 of E2 are glycosylated (17). The ex-
posed nature of these two asparagine side chains in the Fab-pep-
tide complex is consistent with the observation that glycosylation
at these sites does not prevent AP33 binding (52). The structural
information also sheds further light on our previous analyses of
four cell culture-adaptive E2 variants that arose during extensive
passaging of infected cells and exhibit enhanced in vitro replica-
tion (9). These mutations (N415D, T416A, N417S, and I422L)
to WT E2, the N415D and N417S variants were completely resis-
tant to neutralization by AP33 and showed greatly reduced bind-
and it is not unexpected that mutation of this residue in a highly
interaction. N417, by contrast, is exposed in the complex, and its
side chain makes no contact with the antibody. However, muta-
tion of N417 to serine is likely to introduce a new potential glyco-
in recognition by AP33, glycosylation at N415 would certainly be
expected to prevent AP33 binding. T416A and I422L remained
no direct contact with AP33 and would not be expected to greatly
affect the interaction. A further mutation, G418D, was generated
mutant proved resistant to neutralization by AP33. G418, present
at the turn of the ?-hairpin, becomes buried upon AP33 binding,
and the structure suggests that the interaction could not accom-
modate a larger side chain at this position, particularly as this
would disrupt the ?-hairpin.
The data presented here provide structural details of the AP33
HCV E2 epitope in its AP33-bound form and suggest that the key
determinants for epitope recognition are those that anchor the
central and turn regions of the ?-hairpin. That the peptide dis-
plays a similar conformation whether bound to AP33 or HCV1
strongly suggests that this region does indeed form a ?-hairpin in
the intact glycoprotein, which is essential information for poten-
tial vaccine design. In terms of vaccine potential, the hairpin con-
the peptide that may stabilize its secondary structure in solution.
of eliciting AP33-like antibodies is ongoing.
This work was supported by the Medical Research Council and the Uni-
versity of St Andrews, St Andrews, United Kingdom. Resources of the
Scottish Structural Proteomics Facility, funded by the Scottish Funding
Council, were used in the crystallization experiments.
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