Depletion of interfering antibodies in chronic
hepatitis C patients and vaccinated chimpanzees
reveals broad cross-genotype neutralizing activity
Pei Zhanga,1, Lilin Zhonga, Evi Budo Strublea, Hisayoshi Watanabeb, Alla Kachkob, Kathleen Mihalikb,
Maria Luisa Virata-Theimera, Harvey J. Alterc,1, Stephen Feinstoneb, and Marian Majorb,1
Divisions ofaHematology andbViral Products and Center for Biologics Evaluation and Research, United States Food and Drug Administration, Bethesda, MD
20892; andcDepartment of Transfusion Medicine, Warren Grant Magnuson Clinical Center, National Institutes of Health, Bethesda, MD 20892
Contributed by Harvey J. Alter, March 12, 2009 (sent for review February 4, 2009)
Using human immune globulins made from antihepatitis C virus
(HCV)-positive plasma, we recently identified two antibody
epitopes in the E2 protein at residues 412–426 (epitope I) and
434–446 (epitope II). Whereas epitope I is highly conserved among
genotypes, epitope II varies. We discovered that epitope I was
implicated in HCV neutralization whereas the binding of non-
neutralizing antibody to epitope II disrupted virus neutralization
mediated by antibody binding at epitope I. These findings sug-
gested that, if this interfering mechanism operates in vivo during
HCV infection, a neutralizing antibody against epitope I can be
restrained by an interfering antibody, which may account for the
persistence of HCV even in the presence of an abundance of
neutralizing antibodies. We tested this hypothesis by affinity
depletion and peptide-blocking of epitope-II-specific antibodies in
vaccinated chimpanzees. We demonstrate that, by removing the
restraints imposed by the interfering antibodies to epitope-II,
neutralizing activity can be revealed in plasma that previously
failed to neutralize viral stock in cell culture. Further, cross-geno-
type neutralization could be generated from monospecific plasma.
Our studies contribute to understanding the mechanisms of anti-
tical approach to the development of more potent and broadly
reactive hepatitis C immune globulins.
bodies (NAbs), develop chronic infections. These chronically
HCV-infected patients are at risk of developing cirrhosis and
liver cancer (1, 2). Although current standard treatment with
pegylated IFN and ribavirin results in cures in as many as 50%
of patients, neither antibody-based prophylaxis nor an effective
vaccine is available.
The mechanism by which HCV persists in the presence of
NAbs is unknown. Heterogeneity, a prominent feature of HCV,
has been considered important in immune escape. Previously we
identified an antigenic region in the E2 envelope glycoprotein of
hepatitis C virus that contains two important epitopes, i.e.,
others as an important neutralization site (3, 4). We showed that
antibody to epitope II interfered with antibody to epitope I,
inhibiting neutralization of the virus (4). In this study, we have
further characterized these epitopes and identified the amino
acid residues in epitope I important for antibody binding. By
absorbing out antibody to epitope II in plasma from a chronically
infected HCV patient, we show that neutralizing activity is not
only enhanced but also broadened to include additional geno-
types of the virus. Furthermore, by using plasma from 2 chim-
panzees that had been vaccinated with recombinant E1 and E2
envelope glycoproteins of a genotype 1a HCV, we demonstrate
that a monotypic immune response contained cross-neutralizing
capability that could be revealed only following depletion of the
antibodies to epitope II.
ost hepatitis C virus (HCV)-infected patients fail to clear
the virus and, despite the presence of neutralizing anti-
Amino Acid Specificities of Antibody Directed Against Epitope I.
Epitope I is now recognized as a major antibody neutralization
target (3–9). However, little is known about the antibody spec-
ificities that mediate neutralization. We mapped the key amino
acid residues responsible for antibody binding to epitope I by
screening a random peptide phage display library with eluate I,
derived by affinity purification of experimental immune globulin
IV made from anti-HCV-positive plasma (HCIGIV) with
epitope I peptide (Fig. 1A; see Materials and Methods). Eluate I
reacted with peptides, containing residues Q, L, S, and W, which
mimicked epitope I (Fig. 1A), suggesting that these residues are
sufficient for eluate I recognition regardless of their spatial
arrangement. ELISA analysis revealed that mutants bearing
QL413?AA or SW420?AA exhibited reduced antibody binding,
whereas HIN423?AAA did not affect antibody binding (Fig. 1 B
and C). When alanine was substituted at Q412, L413, S419, or W420
(Fig. 1 B and C), we discovered that L413and W420were the 2
discontinuous residues within epitope I indispensable for the
binding of NAb. This finding was corroborated by the fact that
both L413and W420are among the most conserved residues in all
HCV genotypes, suggesting that cross-genotype neutralization
might be achieved if antibody can effectively bind to epitope I.
Presence of Epitope-I- and Epitope-II-Specific Antibodies in Plasma
from Chronically HCV-Infected Patients and in HCIGIV Preparations.
We assayed the levels of epitope-I- and epitope-II-specific
antibodies in plasma from 9 chronically HCV-infected patients
by ELISA (Fig. 2A). Of these, 2 (H77 and no.1) had detectable
antibodies against epitope II (Fig. 2A). The appearance of
epitope-I-specific antibodies in these patients coincided with the
presence of high levels of epitope-II-specific antibodies. None of
these samples reacted with the epitope I mutant, SW420?AA,
indicative of the specificity of the antibody.
We then measured the levels of epitope-I- and epitope-II-
specific antibodies in six lots of HCIGIV by ELISA (Fig. 2B). As
predicted from the prevalence of antibodies in the patients’
plasma, all samples contained both antibodies, with the titer
higher for epitope II than for epitope I. The antibody ratio of
epitope-II-specific to epitope-I-specific for these HCIGIV lots
ranged from 2.0 to 3.8 (Fig. 2B).
We further examined the kinetics of epitope-I- and epitope-
Author contributions: P.Z., H.J.A., S.F., and M.M. designed research; P.Z., L.Z., E.B.S., H.W.,
new reagents/analytic tools; P.Z., L.Z., E.B.S., H.W., A.K., H.J.A., S.F., and M.M. analyzed
data; and P.Z., H.J.A., S.F., and M.M. wrote the paper.
The authors declare no conflict of interest.
Freely available online through the PNAS open access option.
nih.gov, or firstname.lastname@example.org.
May 5, 2009 ?
vol. 106 ?
no. 18 ?
chronic infection (Fig. 2C) (10). ELISA analysis revealed that
antibody to epitope II appeared within 51 days of infection. By
contrast, antibody to epitope I was not detectable until day 643.
At day 5,266, antibody responses to both epitopes were still
readily detectable. Notably, the appearance of epitope-I-specific
antibody coincided with the presence of elevated levels of
epitope-II-specific antibody only in the chronic phase of infec-
tion. This suggested that the lack of epitope-I-specific antibody
production during the early phase of infection might permit the
survival of HCV and that the co-existence of antibodies to
epitope II with antibodies to epitope I during the late phase
might hamper neutralization mediated by epitope-I-specific
antibodies, thereby contributing to HCV persistence.
HCV persisted in these patients despite the presence of epitope-
I-specific antibodies exposed the inadequacy of such antibodies
to control HCV infection. Thus the question arose whether the
activity of epitope-I-specific antibodies was inhibited by epitope-
II-specific antibodies in these patients through antibody inter-
ference similar to that which we proposed for HCIGIV (4). We
examined neutralizing activity in plasma collected from pa-
tient-H on day 5,266 using an HCV cell culture system with a 2a
virus (HCVcc/2a) and a 1a/2a chimeric virus (HCVcc/1a; see
Materials and Methods). Both were constructed on the backbone
of JFH1 (genotype 2a). HCVcc/1a contained the envelope
proteins from strain H77, a genotype 1a virus isolated from
patient H (10), and HCVcc/2a contained envelope proteins from
strain J6, a genotype 2a virus (11, 12). Plasma 5,266, at a dilution
of 1:400, neutralized approximately 80% of HCVcc/1a, com-
pared with a mock-treated control (i.e., negative control [NC];
P ? 0.01; Fig. 3A). However, it did not significantly reduce the
infectivity of HCVcc/2a under the same experimental conditions
Depletion of Epitope-II-Specific Antibodies Reveals Cross-Genotype
Neutralization. Knowing that epitope I is highly conserved among
genotypes, the failure of the H77 plasma to cross-neutralize
HCVcc/2a indicated a potential disruption of epitope-I-specific
antibodies, possibly by epitope-II-specific antibodies. We thus
tested whether blocking epitope-II-specific antibodies would
lead to neutralization of HCVcc/2a. Indeed, when epitope-II-
specific antibodies in the plasma were blocked by an epitope-
II-specific peptide (Fig. 3A; see Materials and Methods), the
plasma exhibited detectable neutralizing activity against HCVcc/
2a. Approximately 50% reduction of infectivity was observed
(P ? 0.01; Fig. 3A). We determined the end-point titer that
would give 50% neutralization (ID50) (13) and found that the
ID50for untreated H77 plasma was 1:1,238 against HCVcc/1a
and ?1:200 against HCVcc/2a. After epitope-II-specific anti-
bodies were blocked, the ID50 increased to ?1:1,600 against
HCVcc/1a and to 1:531 against HCVcc/2a. These results dem-
onstrate that both genotype-specific neutralization and cross-
genotype neutralization could be enhanced or revealed by using
genotype-specific plasma in which interfering antibodies are
display library using a panning method (4). The key residues within both epitope I and peptidyl mimics are indicated (bold and underline). The numbers indicate
Eluate I at 1:50 dilution or HCIGIV at 1:2,000 dilution was used as the primary antibody in the ELISA. The x axis indicates individual peptides tested, and the y
axis shows corresponding absorbance at 450 nm (A450 nm) of each mutant relative to that of epitope I (in percent of control).
with chronic HCV infection. (A) Biotin-conjugated peptides encompassing
epitope II, epitope I, or the mutant of epitope I, SW?AA (Fig. 1 B and C) were
added to streptavidin-coated 96-well plates (200 ng/well), individually.
SW?AA was used as a NC for the assay. Human patient plasma samples, H77
primary antibody. Diluted HCIGIV lot A (1:2,000), eluate I, and eluate II, an
HCIGIV fraction affinity-purified by using epitope II peptide (4), were used at
1:50 dilution as controls. The y axis indicates A450 nm obtained in the ELISA,
HCIGIV lots (A–F) were diluted 1:2,000 and used as the primary antibodies in
the ELISA, in which biotin-conjugated peptides encompassing epitope II,
epitope I, or SW?AA were added to streptavidin-coated 96-well plates (200
ng/well). Peptide SW?AA was used as a NC. The ratios of antibody against
epitope II versus that against epitope I are presented. (C) Plasma samples
collected from patient H before and after HCV infection were diluted 1:100
except for the sample of d 5266, which was diluted at 1:400, and used as the
primary antibodies. Plasma levels of antibodies directed against epitope II,
epitope I, or SW?AA, as indicated by values of A450 nm, were determined in
the ELISA. SW?AA was used as a negative peptide control. Diluted HCIGIV lot
A at 1:2,000 and eluates I and II at 1:50 were used as controls for primary
Presence of HCV epitope-specific antibodies in plasma of patients
www.pnas.org?cgi?doi?10.1073?pnas.0902749106Zhang et al.
We asked whether removal, instead of blocking, of epitope-
II-specific antibodies from the plasma could also reveal the
neutralization of HCVcc/2a. Epitope-II-specific antibodies were
thus depleted from plasma 5266 (Fig. 2B; see Materials and
Methods). As expected, the plasma with a reduced level of
epitope-II-specific antibodies neutralized HCVcc/2a (P ? 0.05;
Fig. 3C). Therefore, cross-neutralization of the 2a virus could be
realized when interfering antibodies were removed from a
genotype 1a-specific plasma, even when the removal was less
This conclusion was further substantiated by generation of
cross-genotype neutralizing activity in the plasma of vaccinated
chimpanzees (Fig. 4). Plasma from 2 chimpanzees, Ch1587 and
Ch1601, was collected 2 weeks after vaccination with rE1E2
protein (14). The sequence of rE1E2 used for vaccination was
identical to that of HCVcc/1a. We found that plasma from
Ch1587 contained antibodies capable of reducing the infectivity
of HCVcc/1a to approximately 40% of the NC at a 1:400 dilution
(Fig. 4A). By contrast, the plasma showed enhanced neutralizing
activity when epitope-II-specific antibody was blocked, reducing
virus infectivity to 10% of the NC. Similarly, only after epitope-
II-specific antibodies were blocked by peptide did the plasma
show neutralizing activity against a second chimeric virus
HCVcc/1b, which was constructed on the backbone of JFH1
containing the core and envelope regions of a 1b isolate. Such
a cross-genotype neutralization was observed when HCVcc/2a
was investigated under the same experimental condition (Fig.
4A). These findings were confirmed when the plasma from the
second vaccinated chimpanzee, Ch1601, was studied for its
ability to neutralize the infectivity of HCVcc/1a and 2a (Fig. 4B).
These data demonstrated that cross-genotype neutralizing ac-
tivity was induced following vaccination with a monotypic anti-
gen, which not only represents a single genotype, but also
consists of a single protein sequence. However, the cross-
genotype neutralizing ability was revealed only when interfering
antibodies directed against epitope II were blocked.
Our data provide evidence of a general concept that survival of
virus by escaping from antibody neutralization does not neces-
sarily require mutations occurring at a neutralization epitope.
Instead, there appears to be an additional escape mechanism
whereby non-neutralizing antibody binding to virus interferes
with the binding of antibodies with neutralizing capability.
Interestingly, the epitope I region is reportedly recognized by at
least 3 mAbs (Ap33, 3/11, and e137) that have been shown to
neutralize HCV in vitro (7, 8, 15, 16). However, these mAbs
appear to contact different sets of amino acids, including those
plasma. (A) epitope-II-specific antibodies in plasma 5266 were specifically
blocked by epitope II peptide or left untreated as a control. These samples
were then tested for their abilities to neutralize genotype 1a/2a or 2a virus in
HCV cell culture. A normal IGIV at 1:400 dilution was used as NC. The x axis
indicates the plasma sample used in this assay at 1:400 dilution. The y axis
indicates the infectivity expressed as percentage of NC. Statistical significance
of difference in infectivity is indicated. (B) Biotin-conjugated epitope II and
epitope I peptides were added to streptavidin-coated 96-well plates (200
ng/well). Plasma 5266, before and after 3 rounds of absorption with epitope
II peptide, was diluted at 1:800 and used as primary antibody in ELISA. HCIGIV
lot A, at 1:2,000 dilution, was used as the positive control. The y axis indicates
to each individual peptide. (C) Plasma 5266 was absorbed with epitope II
peptide to deplete epitope-II-specific antibodies. These absorbed samples,
along with those left untreated, were diluted at 1:400 and assayed for their
abilities to neutralize the 2a virus in HCV cell culture. A normal IGIV at 1:400
infectivity of the virus, i.e., percent of NC. Statistical significance of difference
in infectivity is indicated.
Recovery of cross-genotype neutralizing activity from patient H
rE1/E2 vaccinated chimpanzees. Epitope II peptide (500 ng/mL) was incubated
with the plasma of 2 vaccinated chimpanzees, Ch1587 and Ch1601, to block
epitope-II-specific antibodies wherein. These samples, at 1:400 dilution, were
then tested for their abilities to neutralize the 1a/2a, 1b/2a, or 2a virus in HCV
cell culture. (A) Neutralization with plasma from Ch1587 and (B) neutraliza-
tion with plasma from Ch1601. For comparisons, control plasma samples and
relative infectivity of the virus, i.e., percent of NC. Statistical significance of
difference in infectivity is indicated.
Recovery of cross-genotype neutralizing activity from plasma of
Zhang et al.PNAS ?
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H421were mapped for AP33 binding; T416, W420, W529, and G530
for 3/11; and T416, W420, W529, G530, and D535for e137. In
addition, a recent study revealed a conformational neutraliza-
tion epitope that contains at least 3 segments at residues
396–424, 436–447, and 523–540 (8). Notably, the first 2
segments overlap with epitope I and epitope II, respectively (5,
6, 17–19). Recognition of the difference in antibody specificity
raises the possibility that the approach used for obtaining the
NAb, i.e., either direct purification from the plasma of infected
patients or immunization with recombinant proteins, affects
the breadth of neutralization. In this connection, antibodies
obtained directly by elution after absorption of HIV-1-positive
sera with specific viral antigen can neutralize diverse strains of
HIV-1 that are partially or fully resistant to mAbs (20).
Because epitope II can be relatively heterogeneous, differ-
ent binding capacities of antibodies to epitope II are expected
in patients infected with distinct HCV genotypes. In essence,
a difference of this nature can be viewed as an acquired
characteristic of the virus to tune the inhibition of neutraliza-
tion by the interfering antibody, whereby the state of disease
could be modified to an extent depending on both the geno-
type(s) of the virus and the binding strength of the interfering
Indeed, by changing the ratio of interfering/NAbs in plasma of
we were able to recover otherwise undetectable, cross-genotype
neutralizing activity. Based on these findings, we propose that
both the successful generation of an HCIGIV product and the
development of an effective HCV vaccine may be feasible by
eliciting strong NAbs against epitope I while avoiding the
production of antibodies against epitope II.
Finally, the operation of a mechanism of viral escape
mediated by interfering antibody should not be thought of as
limited to HCV. It should be considered in the development
of strategies for prophylaxis and treatment of other infections
in humans, especially those with highly heterogeneous viruses
such as HIV.
Materials and Methods
Immune Globulins. Several independent lots of HCIGIV (A-F) were made from
requirements for normal plasma donations, i.e., negative for both anti-HIV
and hepatitis B surface antigen and without elevated levels of alanine ami-
notransferase. These preparations had been treated by a solvent–detergent
process to inactivate potential contaminating viruses. Neutralization of HCV
infection by HCIGIV lot A was demonstrated previously in both a pseudo-
particle system and a chimpanzee model (3). A commercial 5% immune
globulin IV (IGIV) solution, which was manufactured from anti-HCV (EIA-2)-
negative plasma donations, was used as NC. This IGIV preparation was also
virally inactivated by a solvent–detergent treatment.
Plasma Samples. Nine human plasma samples were obtained at the National
Institutes of Health (NIH) Clinical Center from patients, including patient H,
All samples were collected according to protocols approved by the NIH insti-
tutional review board. Chimpanzee plasma samples were collected from 2
(Ch1587), both of which were vaccinated with recombinant envelope glyco-
High ELISA antibody titers to E1E2 after immunization had been previously
Phage Display. Selection of peptides from a random peptide phage display
library (PhD-12; New England Biolabs,) was described previously (21). Briefly,
approximately 1010phages were incubated with an individual Ig fraction/
protein-G mixture for 20 min at room temperature. After 8 washings with 10
mM Tris-HCl buffer (pH 7.5) containing 0.02% Tween-20, the phages were
eluted from the complex with 0.1 M HCl for 8 min at room temperature. The
eluted phages were then amplified in the host strain ER2738. Amplified
phages were subjected to 3 additional rounds of selection by the same Ig
from each single phage plaque was sequenced, and the corresponding pep-
tide sequence was then deduced from the DNA sequence.
Center for Biologics Evaluation and Research at the US Food and Drug Ad-
ministration with an Applied Biosystems model 433A peptide synthesizer
carried out with Fmoc-Lys (Biotin-LC)-Wang resin (AnaSpec). The crude pep-
purified by RP HPLC on a DeltaPak C-18 reversed-phase column (Waters), and
eter (PE Biosystems).
ELISA. Streptavidin-coated 96-well plates were used for ELISA according to
the manufacturer’s instructions (Pierce). Biotinylated peptides (200 ng/
well) were added to streptavidin-coated wells and incubated at room
temperature for 30 min in SuperBlocker blocking buffer (Thermo Scien-
tific). The wells were then blocked with SuperBlocker at 37 °C for 1 h. After
washings with PBS buffer containing 0.05% Tween-20, antibodies were
added to the wells and incubated for 1 h at 37 °C. After removal of
20, a goat anti-human peroxidase-conjugated IgG (Sigma-Aldrich) at
1:5,000 dilution was added to the wells and incubated at 37 °C for 30 min.
After washings, the plates were incubated in the dark for 10 min with 100
?L/well of 1-Step Ultra TMB-ELISA (Thermo Scientific). The reaction was
stopped by adding 100 ?L/well 4 N H2SO4. The absorbance of each well was
measured at 450 nm with a microtiter plate reader (Optimax; Molecular
Peptide-Blocking and Affinity Depletion of Epitope-II-Specific Antibodies. For
peptide-blocking of epitope-II-specific antibodies in the plasma, epitope II
peptide corresponding to the amino acid sequence of HCV genotype 1a (H77)
was synthesized. One microgram of peptide was added to the diluted plasma
the neutralization assay. For affinity depletion of epitope-II-specific antibod-
ies, biotinylated epitope II peptide (500 ng/well) was added to streptavidin-
coated wells and incubated at room temperature for 30 min in Tris-HCl buffer
(pH 7.5) containing 0.02% Tween-20. A diluted plasma was added to the well
and incubated for 30 min at room temperature for absorption. The unbound
portion was collected. ELISA analysis was performed to monitor the levels of
the antibodies during affinity depletion.
Neutralization Assay. Virus stock was prepared by transfecting HCV RNA
derived from genotype 1a/2a or 1b/2a chimeras or 2a (J6/JFH1) into Huh 7.5
Huh 7.5 cells were seeded at a density of 4–5 ? 103cells/well in 96-well plates
to obtain 50%–60% confluence in 24 h. Virus stock was diluted in DMEM
supplemented with 10% FBS/1% penicillin/streptomycin/2 mM glutamine to
yield approximately 100 infected foci per well in the absence of NAbs. To test
neutralization capacity, an antibody, in parallel with a positive (HCIGIV) and
an NC, was mixed with the virus stock before addition to the cells. After
incubation at 37 °C for 1 h, the supernatants containing the virus/antibody
added to each well. The cells were continuously cultured in DMEM for 3 d. To
count infected foci, the cells were fixed with cold methanol and stained with
a monoclonal antibody for the HCV core antigen that recognized both the 1a
were visualized with diaminobenzidine tetra-hydrochloride. Positive foci
were counted. Infectivity was expressed as percent of NC, i.e., numbers of
infected foci with a given antibody divided by numbers of foci with a NC
antibody, and the quotient multiplied by 100%.
Statistical Analysis. JMP software (v.7.0; SAS Institute) was used for analyzing
data. Means were compared between 2 samples by using the Student t test.
For an overall comparison of means, the Tukey-Kramer HSD test was used.
Statistical significance was set at an ? of 0.05. A positive test value generated
between 2 means is indicative of a significant difference.
ACKNOWLEDGMENTS. We thank Drs. John Finlayson and Mahmood Farshid
for comments on the manuscript; Dr. Basil Golding for interest and support;
the Core Laboratory of the Center for Biologics Evaluation and Research for
peptide synthesis and DNA sequencing; and Dr. Mei-ying Yu and Nabi Bio-
pharmaceuticals for providing experimental HCIGIV preparations for this
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