A weak neutralizing antibody response to hepatitis C virus envelope glycoprotein enhances virus infection.

Abstract

We have completed a phase 1 safety and immunogenicity trial with hepatitis C virus (HCV) envelope glycoproteins, E1 and E2, with MF59 adjuvant as a candidate vaccine. Neutralizing activity to HCV genotype 1a was detected in approximately 25% of the vaccinee sera. In this study, we evaluated vaccinee sera from poor responders as a potential source of antibody dependent enhancement (ADE) of HCV infection. Sera with poor neutralizing activity enhanced cell culture grown HCV genotype 1a or 2a, and surrogate VSV/HCV pseudotype infection titer, in a dilution dependent manner. Surrogate pseudotypes generated from individual HCV glycoproteins suggested that antibody to the E2 glycoprotein; but not the E1 glycoprotein, was the principle target for enhancing infection. Antibody specific to FcRII expressed on the hepatic cell surface or to the Fc portion of Ig blocked enhancement of HCV infection by vaccinee sera. Together, the results from in vitro studies suggested that enhancement of viral infectivity may occur in the absence of a strong antibody response to HCV envelope glycoproteins.

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A Weak Neutralizing Antibody Response to Hepatitis C
Virus Envelope Glycoprotein Enhances Virus Infection
Keith Meyer1, Arup Banerjee1, Sharon E. Frey1, Robert B. Belshe1,2, Ranjit Ray1,2*
1Department of Internal Medicine, Saint Louis University, St. Louis, Missouri, United States of America, 2Molecular Microbiology & Immunology, Saint Louis University, St.
Louis, Missouri, United States of America
Abstract
We have completed a phase 1 safety and immunogenicity trial with hepatitis C virus (HCV) envelope glycoproteins, E1 and
E2, with MF59 adjuvant as a candidate vaccine. Neutralizing activity to HCV genotype 1a was detected in approximately
25% of the vaccinee sera. In this study, we evaluated vaccinee sera from poor responders as a potential source of antibody
dependent enhancement (ADE) of HCV infection. Sera with poor neutralizing activity enhanced cell culture grown HCV
genotype 1a or 2a, and surrogate VSV/HCV pseudotype infection titer, in a dilution dependent manner. Surrogate
pseudotypes generated from individual HCV glycoproteins suggested that antibody to the E2 glycoprotein; but not the E1
glycoprotein, was the principle target for enhancing infection. Antibody specific to FcRII expressed on the hepatic cell
surface or to the Fc portion of Ig blocked enhancement of HCV infection by vaccinee sera. Together, the results from in vitro
studies suggested that enhancement of viral infectivity may occur in the absence of a strong antibody response to HCV
envelope glycoproteins.
Citation: Meyer K, Banerjee A, Frey SE, Belshe RB, Ray R (2011) A Weak Neutralizing Antibody Response to Hepatitis C Virus Envelope Glycoprotein Enhances Virus
Infection. PLoS ONE 6(8): e23699. doi:10.1371/journal.pone.0023699
Editor: Sujit Basu, Ohio State University, United States of America
Received June 26, 2011; Accepted July 22, 2011; Published August 22, 2011
Copyright: ? 2011 Meyer et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This research was supported by grant AI068769 and contract N01-AI-25464 from the National Institutes of Health. The funders had no role in study
design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: rayr@slu.edu
Introduction
HCV continues to be a major global public health problem
despite significant advances in interferon based treatment. New
generation of specific antivirals are entering clinical trials; and the
use of combination therapy is likely to diminish the development
of resistant variants, and provide effective virus control and
eradication. The use of cell culture grown HCV and VSV/HCV
pseudotype as a surrogate model have proven to be important
tools in understanding the role of virus envelope glycoproteins for
interactions with cell surface proteins [1,2,3,4,5]. The entry stage
of viral replication constitutes a target for neutralizing antibodies
as well as pharmacologic agents. Several lines of evidence suggest
important contributions for the two HCV envelope glycoproteins
(E1 and E2) in HCV entry using pseudotype models and include:
(i) Initial contact of HCV partly depends on sulfated polysaccha-
rides present on mammalian cells and this contact appears to be
stronger with the E2 glycoprotein [2,3,5,6,7,8,9]. (ii) CD81 may
play a role in virus infectivity through interaction with E2
[2,3,10,11]. (iii) Virus titer decreases with interruption of LDL-R
activity and this may be mediated via an E1 specific interaction
[2,3]. These observations suggest that the two different forms of
recombinant HCV envelope glycoproteins (chimeric E1-G/E2-G
used in VSV pseudotype generation or unmodified E1–E2 used in
HIV or MuLV derived pseudotype) display similar functional
profiles, and that more than one cellular protein may be
responsible for binding and entry of virus particles into
hepatocytes. Claudin-1 (CLDN1) has been shown to act at a
post-binding stage of HCV [12], although the precise function of
CLDN1 in the orchestration of HCV entry is under investigation.
Specific entry factors of HCV, like CD81 or SR-B1, may associate
with CLDN1 on the basolateral surface of polarized hepatocytes
and facilitate HCV cell to cell spread [13,14]. A recent report
suggests that tyrosine kinases mediate HCV entry by regulating
CD81-claudin-1 co-receptor associations and HCV glycoprotein
dependent membrane fusion [15]. Thus, multiple cellular proteins
and cell surface receptors may be involved in an interaction
between HCV and host cells for virus entry.
HCV patient derived glycoproteins exhibit marked differences
in susceptibility to serum neutralizing antibodies [16]. HCV may
exist in the blood as free virus or complexed with antibodies. A
large percentage of sera from chronically HCV infected patients or
vaccinee sera bind to HCV envelope glycoproteins, but fail to
efficiently neutralize infection, and some of these serum antibodies
as well as human monoclonal antibodies enhance pseudotype
infectious titer [17,18,19,20,21]. This enhancement may be due to
the presence of non-neutralizing antibodies and/or antibodies of
low affinity.
Antibody-dependent enhancement of infection has been
observed in vivo in animal models and among individuals
vaccinated against certain viruses, such as flavivirus (yellow fever,
dengue), HIV-1, Ebola virus, and Hantavirus [22,23]. Increased
infection occur both through interactions with Fc receptors, and
receptorsforcomplement in
[24,25,26,27,28]. The ability of sera to enhance HIV-1 infection
in the presence of complement is associated with a progression
towards AIDS [23,24,25,29], and an in vivo correlate of increased
viral burden and antigenemia in a SIV/macaque model [30].
Viruses elicit antibodies that enhance infectivity through the
differenthumancelllines
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binding of virus-antibody complexes to cellular Fc receptors via
the Fc portion of the antibodies; leading to an increase in viral
uptake, with an accompanying increase in replication and higher
viral titer [31,32]. Understanding the nature of antigen-antibody
interactions, along with the role of the complement system in
HCV infection may help to clarify the poor performance of anti-
HCV specific immunoglobulins in disease progression.
In this study, we evaluated the modulation of cell culture grown
HCV and VSV derived pseudotype infectivity by antibodies
obtained from volunteers vaccinated against HCV in the presence
or absence of complement. The results indicate that enhancement
of viral infectivity may occur in the absence of a strong antibody
response to HCV envelope glycoproteins.
Methods
Vaccinee sera
Arandomized,
(DMID 01–012) was completed after approval by the Saint Louis
University Institutional Review Board. All subjects provided
informed consent, and the work was conducted with Institutional
Approval (IRB # 15719). Immunizations included 4 doses of
purified Chinese hamster ovarian cell–derived full-length recom-
binant E1/E2 glycoproteins of HCV genotype 1a with the MF59
adjuvant (Novartis) in 3 different volunteer groups. The volunteers
in each group were immunized 4 times (at 0, 4, 24, and 48 weeks)
with 4, 20, or 100 mg of E1/E2 per vaccine dose and MF59
adjuvant. Sixteen volunteers in each group received vaccine using
one of the dosage levels and 4 received a placebo control in each
group. Serum samples were collected from the volunteers at
different time points (0, 6, 26, 50, and 64 weeks) for testing, heat
inactivated, and the nature of the antibody responses were
characterized [21,33]. For this study; serum drawn at week 64
(16 weeks after the fourth immunization) was used to evaluate the
potential of non-neutralizing sera to affect HCV titer.
double-blinded,placebo-controlledstudy
Generation of HCV in cell culture
HCV genotype 1a (clone H77) or HCV genotype 2a (clone
JFH1) were grown in immortalized human hepatocytes (IHH) as
previously described [4]. Virus was harvested from cells and
clarified by low speed centrifugation, followed by filtration through
a 0.45-mm cellulose acetate membrane (Nalgene, Rochester, NY).
Cell culture grown HCV (HCVcc) were aliquoted and stored at
270uC for single use. RNA (IU/ml) was quantified by real-time
polymerase chain reaction (Prism 7500 real-time thermocycler;
ABI) with the use of HCV analyte-specific reagents (ASR, Abbott)
from the Pathology Clinical Laboratory at Saint Louis University.
Virus titer was measured from cell culture supernatant by
fluorescent-focus formation (FFU) assay using HCV specific
reagents. HCV titer was typically calculated between 104–106
IU/ml and 104–105FFU/ml. IU/ml values were normally .10
fold as compared to FFU/ml for HCV genotype 1a, and this
difference was higher with HCV genotype 2a generated in cell
culture.
Assay for HCVcc infectivity
To quantify antibody dependent enhancement (ADE) of HCV
infection by antibody from viral RNA estimation, a predetermined
IU of cell culture grown HCV was exposed to serial dilutions of
vaccinee sera and incubated for 1 h at 37uC prior to addition to
Huh7 cells. Virus containing supernates were incubated with cells
for 3 h at 37oC. Cells were rinsed twice and incubated at 37uC for
96 h. Cells were rinsed, lysed, and RNA was isolated using the
RNAeasy Miniprep kit (Qiagen). cDNA was generated using a
Superscript III kit (Invitrogen), followed by real-time PCR to
quantify viral RNA titer [34]. A curve of virus dilutions was used
as a comparative control, and 18S RNA was used as an internal
control. The reciprocal serum dilution is given as X, where X is 1/
X.
Further, HCV (clone JFH1) was used to infect Huh7.5 cells
using a predetermined (FFU) number of viral particles as
determined by FFU. Briefly, virus supernates were diluted as a
pool to cover the entire experiment, separated, and treated
individually with vaccinee sera for one hour prior to infection of
Huh7.5 cells. Infected cells were incubated for 72 hours at 37oC.
Cells were washed three times with phosphate-buffered saline
(PBS) and stained with antibody specific to HCV core (C750,
Thermo Fischer, IL) at a dilution of 1/400 or NS5A (kindly
provided by Chen Liu, University of Florida) at a dilution of 1/
100. Cells were washed three times with PBS, stained with anti-
mouse immunoglobulin (Ig) conjugated with Alexa 568 (Molecular
Probes, Eugene, OR), and mounted for fluorescence microscopy.
Figure 1. Screening for cell culture grown HCV genotype 1a
(clone H77) infectivity in the presence of vaccine sera. The bar
diagrams represent the overall outcome of the vaccinee sera to
interfere with virus infectivity. Enhancing sera are defined as those
which display a .2 fold increase in FFU, while not displaying
neutralization at dilutions between 1:10 and 1:160. Neutralizing sera
are those which display $50% decrease in FFU at a dilution of $1:20
(panel A). Enhancement of cell culture grown HCV genotype 1a
infectivity using serial dilutions of selected vaccinee sera are shown
(panel B). Fold increase in real-time PCR values of HCV RNA with three
representative sera at different dilutions, and a decrease in HCV RNA in
response to a different vaccinee serum are shown.
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Primary antibodies and secondary antibody-fluorochrome conju-
gates were titrated for use of optimum dilutions where there was
no background fluorescence. DAPI was used to enhance focus by
nuclear staining prior to imaging. Fluorescence positive infected
cells were observed by microscopy, and counted. A human
monoclonal antibody to HCV E2 glycoprotein (CBH5) was used
as a neutralizing positive control, and HCV negative sera was used
as a further control.
Blocking FcR interactions
A Fab fragment recognizing the Fc region of immunoglobulin
(Sigma, St. Louis, MO) was examined for blocking HCV
infectivity. The Fc-specific Fab fragment was added at the time
of pseudotype/antibody incubation at a concentration of 1ug/ml.
Control virus for this experiment included a duplicate treated with
the Fc specific Fab fragment, with no change in virus titer. The
remainder of the experiment was carried out as described above.
Cell culture grown HCV was diluted to a predetermined FFU for
estimation of IU, and exposed to vaccinee sera in the presence or
absence of an Fc specific Fab fragment and incubated for 1 h at
37oC. Virus containing supernates were added to cells for 3 h at
37oC. Cells were rinsed and incubated at 37uC for 96 h. RNA
from infected cells was isolated using a RNAeasy Miniprep kit
(Qiagen). cDNA was prepared using a Superscript III kit, followed
by real-time PCR to quantify enhancement of virus titer. A curve
for virus dilutions was used as a comparative control, and 18S
RNA was used as an internal control.
Figure 2. Enhancement of HCVgenotype 2a (clone JFH1) infectivity by serial dilutions of the selected vaccinee sera. A predetermined
HCV titer was incubated with vaccinee sera for 1 h prior to addition to an Huh-7.5 cell monolayer for infectivity. Fold increase of HCV RNA by real-time
PCR is shown (panels A). Sera were also analyzed for ADE by immunofluorescence, and photomicrographs were captured at 200X magnification
(panel B). A predetermined number of FFU were used to infect Huh-7.5 cells in the presence or absence of vaccinee sera at a 1:80 dilution, followed
by 72 h incubation. HCV protein expression was detected using an antibody specific to the HCV core protein, followed by anti-mouse
immunoglobulin (Ig) conjugated with Alexa 568 (Molecular Probes, Eugene, OR). Cells infected with virus prior treated with vaccinee sera displayed a
greater degree of infection as compared to infected controls in the absence of sera.
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For determining the role of specific FcR, cells (16105) were
treated with antibodies directed to respective cellular FcRs (Ancell,
Bay Port, MN) at a concentration of 2ug/ml. Cells were incubated
for 15 minutes in the presence of 1 ug of each individual FcR-
inhibiting antibody prior to adsorption of the virus-antibody
mixture. Viral RNA was quantified by real-time PCR (as above)
and compared to those of control ADE infections in the absence of
an antibody directed specifically against FcR.
Flowcytometry
Cell surface expression of Fc receptors was quantified by flow
cytometry. IHH or Huh7 cells were grown on plastic surface and
detached by treatment with Accutase (Sigma), washed with
phosphate-buffered saline (PBS), and incubated with antibodies
to Fc receptors (CD16, CD32, and CD64 - procured from Ancell,
Bayport, MN) at a concentration of 4 ug/ml, followed by the
addition of a FITC conjugated secondary antibody for staining.
Cells were rinsed and resuspended in methanol free 1%
paraformaldehyde in PBS and gated according to their size
(forward light scatter) and granularity (side light scatter) using a
Becton Dickinson flow cytometer. Surface marker expression on
gated cells was analyzed using FlowJo (Tree Star) and CellQuest
(BD Immunocytometry Systems) software. The Statistica program
was used for analyses of variations.
Pseudotype virus
VSV derived pseudotypes were generated from BHK cells
expressing HCV envelope glycoproteins (E1 and/or E2), with control
virus being derived from cells expressing VSV-G protein as previously
described[3].TreatmentofVSV/HCVpseudotypewithanantiserum
to VSV G did not alter virus titer, suggesting the absence of revertant
VSV G in pseudotype preparation(s), while positive-control virus
incorporating native VSV G exhibited pseudotype neutralization as
previously reported. NMSO3 (1.25 mg/ml) was used for an additional
safeguard from contaminating VSV G glycoprotein reconstitution on
pseudotype virus [3]. The assay for pseudotype infectivity in the
presence or absence of antibody was performed in Huh7 cells as
described earlier [20]. Parental VSV was exposed to the same
experimental conditions for use as a control against nonspecific ADE.
Results
Enhancement of HCVcc infection by vaccinee sera
Vaccination stimulated low levels of antibodies in subjects [21].
Results obtained from experiments examining the neutralization
of cell culture grown HCV (HCVcc) by immunofluorescence
(FFU) displayed the number of neutralizing, non-reactive, or
enhancing vaccine sera as summarized (Fig. 1, panel A). To
further define the ability of non-neutralizing sera to enhance HCV
infectivity, we infected IHH with HCV genotype 1a (clone H77) in
the presence or absence of heat inactivated HCV specific vaccinee
sera. Subsequently, we used a sensitive real-time PCR assay to
quantitate HCV genotype 1a genomic RNA copies to demonstrate
increased levels of virus after antibody mediated enhancement of
HCV infection. The results from three representative sera
displaying at least 3 fold increase in HCV RNA at different
serum dilutions is shown (Fig. 1, panel B). A separate vaccine sera
observed to neutralize both HCVcc and pseudotype was used at
the same dilutions and was observed to strongly neutralize virus
infectivity at all dilutions tested. Sera from pre-vaccinated subjects
did not display any significant reactivity to cell culture grown
HCV in these experiments.
The use of cell culture grown HCV JFH1 has become widely
accepted virus of choice in understanding diverse aspects of HCV.
In our hands, we have observed that the infectious titer of this
virus is much higher and the readout is therefore more convenient.
Higher growth of the HCV2a clone is likely due to a stronger
polymerase activity of the virus [35]. We also examined how these
vaccinee sera react with cell culture grown HCV genotype 2a
(Fig. 2, panel A). Interestingly, vaccinee sera exhibited a higher
level of viral RNA detected upon dilution to 1:80, which could be
attributed to genotype differences. Together, these results display
an increased HCV titer or quantity of genomic RNA in
hepatocytes in the presence of some of the vaccinee sera,
suggesting ADE of HCV infection. A separate vaccine sera
observed to neutralize both HCVcc and pseudotype virus was used
at the same dilutions and was observed to neutralize virus to
higher dilutions (1:320).
Immunofluorescence to detect the level of FFU(Focus Forming
Units) in infected cells was performed to further verify serum
generating ADE of HCV JFH-1 infection. Huh7.5 cells infected with
virus treated with vaccinee sera prior to cell overlay displayed a
distinctly higher level of infection, as evidenced from FFU in all fields
analyzed.Arepresentative seriesof imagesareshown(Fig.2,panelB).
Enhancement of VSV/HCV pseudotype infection by
vaccinee sera
In earlier studies, we have observed a rise in pseudotype plaque
number when serum dilutions fell well below a neutralizing
Figure 3. Weak neutralizing activity of vaccinee sera correlates
with ADE. Vaccinee sera were selected by their ability to enhance or
neutralize of VSV/HCV pseudotype. Sera were serially diluted and
incubated for 1h at 37uC prior to infection of Huh-7.5 cells. Sera with
neutralizing titers (.1/20) did not exhibit ADE at the dilutions tested.
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