Hepatitis C Virus Hypervariable Region 1 Variants
Presented on Hepatitis B Virus Capsid-Like Particles
Induce Cross-Neutralizing Antibodies
Milena Lange1., Melanie Fiedler1., Dorothea Bankwitz2, William Osburn3, Sergei Viazov1,
Olena Brovko1, Abdel-Rahman Zekri4, Yury Khudyakov5, Michael Nassal6, Paul Pumpens7,
Thomas Pietschmann2, Jo ¨rg Timm1, Michael Roggendorf1, Andreas Walker1*
1Institute of Virology, University Hospital Essen, University of Duisburg-Essen, Essen, Germany, 2Division of Experimental Virology, TWINCORE, Hannover, Germany,
3Department of Medicine, Johns Hopkins University, Baltimore, Maryland, United States of America, 4Virology and Immunology Unit, National Cancer Institute, Cairo,
Egypt, 5Centers for Disease Control and Prevention, Atlanta, Georgia, United States of America, 6Department of Internal Medicine II, University Hospital Freiburg,
Freiburg, Germany, 7Department of Recombinant biotechnology, Latvian Biomedical Research and Study Centre, Riga, Latvia
Hepatitis C virus (HCV) infection is still a serious global health burden. Despite improved therapeutic options, a preventative
vaccine would be desirable especially in undeveloped countries. Traditionally, highly conserved epitopes are targets for
antibody-based prophylactic vaccines. In HCV-infected patients, however, neutralizing antibodies are primarily directed
against hypervariable region I (HVRI) in the envelope protein E2. HVRI is the most variable region of HCV, and this
heterogeneity contributes to viral persistence and has thus far prevented the development of an effective HVRI-based
vaccine. The primary goal of an antibody-based HCV vaccine should therefore be the induction of cross-reactive HVRI
antibodies. In this study we approached this problem by presenting selected cross-reactive HVRI variants in a highly
symmetric repeated array on capsid-like particles (CLPs). SplitCore CLPs, a novel particulate antigen presentation system
derived from the HBV core protein, were used to deliberately manipulate the orientation of HVRI and therefore enable the
presentation of conserved parts of HVRI. These HVRI-CLPs induced high titers of cross-reactive antibodies, including
neutralizing antibodies. The combination of only four HVRI CLPs was sufficient to induce antibodies cross-reactive with 81 of
326 (24.8%) naturally occurring HVRI peptides. Most importantly, HVRI CLPs with AS03 as an adjuvant induced antibodies
with a 10-fold increase in neutralizing capability. These antibodies were able to neutralize infectious HCVcc isolates and 4 of
19 (21%) patient-derived HCVpp isolates. Taken together, these results demonstrate that the induction of at least partially
cross-neutralizing antibodies is possible. This approach might be useful for the development of a prophylactic HCV vaccine
and should also be adaptable to other highly variable viruses.
Citation: Lange M, Fiedler M, Bankwitz D, Osburn W, Viazov S, et al. (2014) Hepatitis C Virus Hypervariable Region 1 Variants Presented on Hepatitis B Virus
Capsid-Like Particles Induce Cross-Neutralizing Antibodies. PLoS ONE 9(7): e102235. doi:10.1371/journal.pone.0102235
Editor: Ranjit Ray, Saint Louis University, United States of America
Received April 24, 2014; Accepted June 16, 2014; Published July 11, 2014
This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for
any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.
Funding: This work was supported by grants from the Deutsche Forschungsgemeinschaft (www.dfg.de; grant number Fi768/6-1 within the Priority program KFO
117/2) and the German federal ministry of education and research (www.bmbf.de; grant number EGY08/068). ML received a scholarship from the YEAL-Stiftung
(http://www.yael-stiftung.de/). 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 read the journal’s policy and have the following conflicts. Authors MN and AW are inventors of a patent application for
the SplitCore system [Ref: WO/2008/028535] entitled ‘‘Split-core-particles for the presentation of foreign molecules, especially for vaccine applications, and
method for their production’’ [Ref: EP 2059525 B1 and WO/2008/028535]. This does not alter the authors’ adherence to all PLOS ONE policies on sharing data and
* Email: Andreas.Walker@uni-due.de
. These authors contributed equally to this work.
At present, more than 180 million people worldwide are
chronically infected with the hepatitis C virus (HCV). Despite
many efforts (for review see ), there is still no vaccine against
HCV. Only 30% of infected patients can spontaneously resolve
the infection, and CD8+T cells are the key component for this
resolution . However, neutralizing antibodies are also impor-
tant in protecting people against HCV infection. Studies with
HCV pseudoparticles (HCVpp) and cell culture-derived HCV
(HCVcc) showed that neutralizing antibodies develop in sponta-
neous resolvers  and that rapid induction of neutralizing
antibodies is associated with viral control [4,5]. There is also
evidence that intravenous drug users (IDUs) who have previously
recovered from HCV infection are more likely than HCV-naı ¨ve
IDUs to resolve the infection. Again, this resolution is associated
with high titers of broadly neutralizing antibodies [6–8].
Given the importance of both cellular and humoral immune
responses for protection against chronic HCV infection, a
successful vaccine should be able to induce not only a vigorous
T-cell response but also high titers of neutralizing antibodies
capable of neutralizing various viral isolates. In HCV-infected
patients, most neutralizing antibodies are directed against hyper-
variable regions I through III (HVRI–HVRIII) in envelope
protein 2 (E2); therefore, these regions are a prime target antigen.
Unfortunately, HVRI is also the most variable region of HCV,
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and its constant evolution allows the virus to escape the existing
antibody response . That sequence evolution is indeed driven
by immune pressure is shown by the stability of HVRI in infected
individuals with agammaglobulinemia [10,11]. However, even in
HVRI the sequence flexibility is not unlimited, because this region
also contains highly conserved residues surrounded by mutational
hotspots . Furthermore, HVRI can be roughly divided into a
highly variable N-terminal domain, which may serve as an
immunological decoy , and a less variable C-terminal domain;
the higher conservation probably reflects the functional impor-
tance of HVRI for the interaction with the SR-BI receptor and
glycosaminoglycans and for shielding the CD81 binding site
[3,14–19]. Antibodies against this region are neutralizing ,
inhibit cell-to-cell spread in vitro , and protect chimpanzees
from HCV challenge in vivo . Recently, a phase I vaccination
trial in humans with E1 and E2 as antigens showed that patient
sera that were virus neutralizing contained high concentrations of
high-avidity HVRI-specific antibodies . However, in both
studies protection was restricted to viruses containing the same
HVRI sequences as those used for immunization. Hence,
sequence heterogeneity in the HVRI remains an important
challenge for HVRI-based vaccines.
One way of overcoming the problem of target heterogeneity is
immunization with a combination of several HVRIs. First, these
variants must be chosen in a way that enables them to induce a
broad, cross-reactive and neutralizing antibody response. Second,
the genuinely low immunogenicity of the HVRI peptides must be
boosted, e.g., by combination with an immune-enhancing carrier.
The first issue was recently addressed by the use of HVRI
mimotopes [24–26] designed to be cross-reactive with many
natural HVRI sequences and by selecting naturally occuring cross-
reactive HVRI sequences from patient isolates . Both types
induce cross-reactive but genotype-specific antibodies. The second
issue can be addressed by presentation of the peptides on capsid-
like particles (CLPs). Viral capsids combine several features that
make them attractive as antigen carriers for vaccine design .
The mammalian immune system is highly adapted to recognize
particles of viral size (20–200 nm)  and viral appearance (e.g.,
repetitive surface, incorporated nucleic acids). The 183 aa hepatitis
B virus (HBV) core protein (HBcAg) spontaneously assembles into
icosahedral 34-nm particles . Authentic nucleocapsids and E.
coli–expressed CLPs are highly stable, can package nucleic acids,
and are exceptionally immunogenic [31,32]. B-cell epitopes, such
as the immunodominant c/e1-epitope, are symmetrically arranged
in repetitive arrays on the HBV CLP surface. This arrangement
allows direct B-cell activation via B-cell receptor crosslinking .
In addition, HBV CLPs can induce both T cell–dependent and T
cell–independent immune responses . Finally, the much
higher stability of the packaged versus free nucleic acid promotes
its release only in late endosomes, where recognition via Toll-like
receptors (TLRs) efficiently activates the innate immune system
[34,35]. This exceptional immunogenicity of CLPs can be
transferred to foreign protein sequences, especially if they are
inserted into the surface-exposed but sequence-internal c/e1
epitope of the core protein [36,37]. In phase I trials, CLPs
displaying small epitopes were immunogenic and well tolerated
[38,39]. Because of the central location of the insertion site, only
small peptides or proteins with a compatible structure or size can
be presented on HBV CLPs without disturbing their structure and
self-assembling capability [40–42]. We recently overcame this
limitation by splitting the core protein inside the c/e1 loop into
two self-complementing fragments (CoreN and CoreC) that
associate into regularly shaped CLPs (SplitCore system). Foreign
sequences can now be fused to CoreN, CoreC, or both without
imposing conformational stress, largely regardless of their structure
and size . Importantly, the surface orientation of the antigen
can be deliberately chosen depending on whether it is fused to
CoreN or CoreC.
In the present study, we used this system with the goal of
showing that the presentation of HVRI variants on SplitCore
CLPs boosts the immune response and that a selected number of
HVRI variants can induce broadly neutralizing antibodies.
Design and expression of recombinant HVRI core fusion
Because of the huge intragenotype differences, we used HVRI
variants that were most similar to genotype (GT) 1b for proof of
principle that HVRI-displaying CLPs can induce neutralizing
antibodies. The sequence of the four 27 aa long HVRI variants is
depicted in Figure 1. Variants YK5807 and YK5829 are naturally
occurring cross-reactive HVRI variants derived from patients
infected with HCV GT1b (Fig. 1A). Variants R9 and G31 are
artificially selected HVRI mimotopes generated by Puntoriero et
al. . The mimotope R9 is similar to Gt1b and was reported to
react with 75% of patient sera. Although the mimotope G31 is
similar to GT3a, it was included because of its high cross-reactivity
(79%) and because we wished to determine whether an HVRI
variant from a different genotype can broaden the response.
Fusion of the HVRI sequences to the SplitCore CoreC
fragment (Fig. 1B) would mimic the natural orientation of the
HVRI in the E2 protein (exposed HVRI N-terminus). However,
because the neutralizing determinants are believed to be located
primarily in the C terminus [45,46], we decided to fuse the HVRI
variants to the CoreN fragment to maximize its exposure to the
The various fusion proteins were highly expressed in E. coli (as
high as 10–15 mg/L of culture) and soluble. For the SplitCore
HVRI R9 fusion protein, the CoreN HVRI and the CoreC
fragment co-sedimented into particle-typical center fractions in
sucrose gradients (fractions 5–8 of 14) (Fig. 1C), indicating that
SplitCore HVRI R9 can form CLPs. In the same gradient
fractions, assembled particles were detected by native agarose gel
electrophoresis (NAGE), where particles with their low diffusion
coefficient migrate as distinct bands (Fig. 1D NAGE-CB).
Immunostaining with the HBcAg particle-specific monoclonal
antibody (mAb) 3120 further confirmed the formation of particles
(Fig. 1D NAGE-WB). Comparable data were obtained for the
other three fusion proteins (not shown). Finally, electron micro-
scopic inspection of all four HVRI CLPs showed abundant
spherical particles (Fig. 1E). As expected from the relatively small
size of the fused HVRI sequences, no morphological differences
were detectable between HVRI CLPs and wild-type (wt) CLPs.
To determine whether immunization with a single HVRI
variant is sufficient to induce neutralizing antibodies, we first used
each purified HVRI CLP preparation separately for immuniza-
tion. C57BL/6 mice were immunized subcutaneously on days 0,
14, 28, and 56 with 20 mg of SplitCore fusion proteins HVRI R9,
HVRI G31, HVRI YK5809, or HVRI YK5829. Antibody titers
were determined on days 28, 56, and 84.
After two immunizations, all constructs induced high titers of
HVRI-specific antibodies (endpoint titers in the range of 1:15,000
to 1:24,000; Fig. 2A). After four immunizations, all HVRI CLPs
induced final titers higher than 1:24,000. As expected, all sera also
contained high titers of carrier-specific antibodies (anti-HBc;
Fig. 2B), demonstrating the excellent immunogenicity of the core
protein. The antigenic cross-reactivity between the HVRI variants
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was determined in antisera from day 84 (Fig. 2C); the
corresponding peptides were used as targets. A monoclonal
antibody directed against the R9 sequence, which was used as a
control, reacted exclusively with the R9 peptide. As expected, all
CLP-induced sera reacted strongly to the HVRI variant used for
immunization (highest dilution tested, 1:24,000; Fig. 2C, shaded in
grey); however, some of the heterologous peptides were also well
recognized, even though none of the sera reacted with all test
peptides. Hence, the HVRI CLPs induced at least a partially cross-
reactive antibody response.
Anti-HVRI antibodies cross-neutralize chimeric HCVpp
To analyze the neutralization ability of our antisera, we
generated chimeric HCV pseudoparticles (HCVpp) in which the
HVRI-encoding region of the E2 protein from the HCV single-
source outbreak AD78 isolate (, Gt1b) was swapped for the
correspondingR9, G31, YK5807,
(HCVpp-R9, -G31, -YK5807, and -YK5829, respectively).
or YK5829 sequences
Replacement of the HVRI had little effect on HCVpp formation,
and all chimeric HCVpp formed infectious particles. As expected,
all antisera were able to neutralize the HCVpp containing the
HVRI variant used for immunization in a dose-dependent manner
(neutralization at dilution, 1:200: R9, 70%; G31, 41%; YK5807,
67%; and YK5829, 60%; Fig. 2D–G). Cross-neutralization of the
other HCVpp was detectable but clearly weaker (Fig. 2D–G).
Interestingly, although all antisera efficiently neutralized HCVpp
containing the natural HVRI variants (YK5807 and YK5829),
HCVpp with HVRI mimotopes (HCVpp-R9 and HCVpp-G31)
were neutralized only by R9 antisera or G31 antisera, respectively.
To rule out the possibility that the observed cross-reactivity is
biased by our choice of the four HVRI variants, we further
analyzed neutralization of HCVpp bearing the H77c (, Gt1a),
AD78 (, Gt1b), or J6 (, Gt2a) sequence (HCVpp-H77, -
Ad78, and -J6, respectively) (Fig. 2H–J). At a dilution of 1:100 all
antisera were able to neutralize HCVpp-H77 with 50% to 80%
efficiency. Neutralization was still detectable at a dilution of 1:200
Figure 1. Efficient particle formation of HVRI SplitCore fusion proteins. A X-ray structures of HBV core protein. Side and top views of the
HBc monomer (PDB, 1QGT; aa 1–143). The insertion site for HVRI variants lies in the c/e1 epitope connecting helix a-3 and helix a-4. In SplitCore
constructs the core protein was separated between residues Pro-79 and Ala-80 (green) spheres, indicating C-positions. Below: aa sequence of the
HVRI variants used. The C-terminal part is marked yellow. B Schematic view. The antigen orientation on the particle surface can be deliberately
chosen depending on the fragment to which the HVRI variants are fused. Fusion of HVRI variants to the CoreN fragment exposes the more conserved
C-terminus of the HVRI. Linker sequences (G4S) are not shown. C Expression and particle formation of CoreN-HVRI-R9. Crude lysate from the bacteria-
expressing CoreN-HVRI-R9 fusion protein was sedimented through a preparative 10% to 60% sucrose step gradient; 14 fractions of 860 ml each were
harvested from the top. Aliquots of 8 ml each were analyzed by SDS-PAGE and Coomassie Blue (CB) staining; marker proteins with their molecular
masses (in kDa) are indicated on the left. Both fragments, CoreN (arrow up) and CoreC (arrow down), peaked around fractions 5 to 8, as is typical for
intact CLPs . D Native agarose gel electrophoresis (NAGE). Aliquots of the gradient shown in C were run in 1% agarose gels; they were either
stained with CB or the gel content was blotted onto polyvinylidene difluoride (PVDF) membranes and particles were detected with the particle-
specific mAb 3120  E Electron microscopy. Aliquots of the fusion proteins were negatively stained with uranyl acetate.
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in case of anti-YK5907 and anti-G31 antibodies (Fig. 2H).
HCVpp-Ad78 was neutralized by anti-YK5829 antibodies up to
a dilution of 1:100 but not by R9-, G31-, or YK5807-specific
antibodies (Fig. 2I). Interestingly, HCVpp-J6, containing a
genotype Gt2a HVRI, was poorly neutralized by all antisera,
and G31 anti-serum completely failed to react with HCVpp-J6 in
several independent experiments (Fig. 2J). In conclusion, immu-
nization with even one HVRI variant induced strong and partially
Combination of HVRI CLPs induces cross-reactive
Aiming to further improve the neutralization capability, we
vaccinated mice with a mixture of all four HVRI CLPs
simultaneously (5 mg per construct, termed HVRI mix). Immuni-
zation with HVRI mix induced a strong HVRI-specific antibody
response (Fig. 3 A, left, endpoint titer of 1:14,000 to 1:36,000),
cross-reacting with peptides corresponding to all four HVRIs used
for immunization. Anti-HBc-specific antibody titers were compa-
rable to those detected after immunization with the single HVRI
CLPs (Fig. 3 A, right).
To analyze the cross-reactivity with naturally occurring HVRI
sequences, we used ELISA to test the binding of HVRI mix sera to
a panel of 326 patient-derived HVRI peptides from various
genotypes. For comparison we used pooled sera from immuniza-
tion with single HVRI CLPs (termed pooled sera). The HVRI mix
sera reacted with 81 of 326 peptides (24.8%) while the pooled sera
reacted with 62 of the 326 peptides (19%) (Fig. 3B). Compared to
pooled sera, the HVR mix sera also showed a stronger binding of
individual peptides (Fig. 3B).
The increased cross-reactivity after immunization with HVRI
mix was also reflected by enhanced neutralization of HCVpp.
HVRI mix–specific antibodies neutralized HCVpp-R9, -YK5807,
-YK5829, and HCVpp-H77 up to a dilution of 1:200 (Fig. 3C).
Interestingly, HVRI mix antisera were also poorly able to
neutralize HCVpp-J6 (Fig. 3D, black triangles), a finding
indicating that immunization with HVRI mix induced a strong
and broad antibody response but was not cross-reactive with
Figure 2. HVRI CLPs are highly immunogenic in mice and induce neutralizing antibodies. Groups of mice (6 per group) were immunized
subcutaneously with the four HVRI particles (20 mg per variant) in IFA at days 0, 14, 28, and 56. Mice were bled on days 0, 28, 56, and 84. Serum was
pooled and tested for HVRI peptide and HBV core-specific antibodies by ELISA. Bars represent the mean of 3 replicate values; error bars represent
standard error of the mean (SEM). A Antibody response against the HVRI variant used for immunization. B Antibody response against HBcAg. C
Cross-reactivity between the various HVRI variants. A monoclonal antibody against R9 (concentration, 1.2 mg/ml) was used as a control. D–J HCVpp
encoding a luciferase reporter were preincubated with pre-sera or with day 84 sera for 2 h before infection of Huh-7.5 cells. Luciferase activity was
measured 72 h after infection. Neutralization efficacy was calculated by comparing the infectivity in the presence of preimmune serum to the
infectivity in the presence of postimmune serum. Values higher than 50% were scored as neutralization (continuous black line).
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AS03 adjuvant further boosts the immunogenicity of
The previous immunizations were performed with water-in-oil
adjuvant IFA as an adjuvant. To determine whether an oil-in-
water adjuvant formulation could further improve immunogenic-
ity, a mixture of all four HVRI CLPs were administered in AS03
in a 14-day immunization scheme (termed AS03-M). Although
anti-HBc–specific antibody titers were comparable to those
achieved in the previous immunizations, AS03-M induced a
much stronger HVRI-specific antibody response than did HVR
CLPs in IFA (Fig. 4 A; endpoint titer of 1:32,000 to 1:80,000).
Furthermore, the neutralization capability of AS03-M antisera was
dramatically increased, especially against HCVpp-R9, -G31, -
YK5807, and HCVpp-YK5829, all of which were neutralized up
to a dilution of 1:3,200 (Fig. 4B). The neutralization of HCVpp-
H77 and HCVpp-AD78 was also enhanced (Fig. 4C). However, in
line with previous findings, J6-HCVpp was not neutralized
(Fig. 4C, black triangles).
HVRI CLP–induced antibodies can neutralize patient
For proof of principle that antibodies induced by AS03-M are
broadly neutralizing within one HCV genotype, we tested the
neutralization of 19 additional HCVpps from genotypes 1a and 1b
. When the cutoff for neutralization was set to 50% the AS03-M
serum was able to neutralized 4 of 19 (21%) HCVpp (Figure 4D).
Interestingly, when the cutoff is slightly reduced to 45% the AS03-
M serum was able to neutralize 8 of 19 (42%) HCVpp (Fig. 4D),
indicating that with slight lower dilutions more HCVpp could be
neutralized. As expected, HCVpp from GT1b were neutralized
more efficiently; however, selected HCVpp from GT1a were also
neutralized. Interestingly, the only sequence similarities between
the neutralized HCVpp are aa 20–24 in the C-terminal part of the
HVRI (Table S1).
Finally, we analyzed the ability of the anti-HVRI antibodies to
neutralize infectious JC1-HCVcc virions (a genotype 2a chimera,
). No antiserum was able to neutralize JC1. This finding was
expected because neither the J6-HVRI peptide nor the HCVpp-J6
(both represent the HVRI present in JC1) was recognized by our
HVRI-specific antibodies. Therefore, we replaced the J6-HVRI in
the JC1 virus with the naturally occurring HVRI variant YK5829.
Incubation of JC1-YK5829 with AS03-M sera efficiently blocked
the infection of Huh7.5 cells in a dose-dependent matter (Fig. 4E),
whereas pre-serum or serum from mice immunized with empty wt
CLPs did not. A JC1 variant lacking the HVRI region was not
neutralized (not shown), showing that neutralization was HVRI
Taken together, we could show that immunization with 4
designed HVRI variants presented on CLPs induces high titers of
cross-reactive antibodies that can neutralize many circulating
GT1a and GT1b isolates.
HCV infection is still a fundamental health problem, especially
in developing countries. Although new antiviral therapies are
rapidly developed and promise to effectively cure most of the
infected patients in the developed world, a protective vaccine
would be needed for the eradication of HCV. An effective vaccine
Figure 3. Immunization with pooled HVRI CLPs enhances cross-reactivity. A Mice were immunized subcutaneously with a mixture of HVRI
CLPs (5 mg per variant) in IFA, and sera were analyzed as described in Fig. 2. B Reactivity with 326 patient-derived HVRI peptides. Sera from day 84
after immunization with mixture HVRI CLPs (HVRI mix) and pooled sera from a single immunization were tested with ELISA at a dilution of 1:800 for
binding to 326 naturally occurring patient-derived HVRI peptides. Peptides yielding S/Co .1 were scored as positive. The cut-off value was the
optical density at l=495 nm+3.5*StDev obtained with unrelated peptides. C Neutralization of HCVpp bearing HVRI variants used for immunization.
D Neutralization of unrelated heterologous HCVpp. For details see Fig. 2.
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should elicit broad immune responses conferring antibody- and
cell-mediated protection [4,6,20]. In particular, the induction of
strong and broadly neutralizing antibody responses against HCV
is a challenging task. Vaccination trials in humans with
recombinant E1 and E2 showed that a key determinant in
neutralizing serum samples is the presence of high-affinity HVRI-
specific antibodies . Previous vaccination with various HVRI
peptides induced HVRI-specific binding antibodies [24,25,44];
however, their neutralizing potential was not addressed. Our
current findings showed that immunization with CLPs presenting
HVRI variants can indeed induce neutralizing antibodies. We
further showed that immunogenicity is improved by using
SplitCore CLPs as particulate antigen carriers and by deliberate
exposure of the conserved parts of HVRI on the particle surface.
The first important observation was that HVRI SplitCore fusion
proteins formed intact CLPs. Genetic insertion of heterologous
sequences into surface-exposed loops of viral capsid proteins often
suffers from structural incompatibility and inaccessibility of
neutralizing epitopes. The HBc SplitCore approach overcomes
this restriction by providing surface-exposed N- and C-termini,
which leads to the successful presentation of HVRI variants on
intact particles. Structural integrity of the HVRI CLPs is crucial
because enhanced immunogenicity is intimately linked to intact
particles . Although large aggregates can also induce a strong
immune response , intact particles induce higher titers and, in
particular, more neutralizing antibodies . Our HVRI CLPs
induced high titers of antibodies comparable to those induced by
multiple antigenic HVRI peptides , and our antibodies were
highly neutralizing. Taken together, the findings of the present
study corroborated the observation that the presentation of small
peptides on a large antigen carrier enormously enhances their
Another advantage of SplitCore CLPs is the expression in
bacteria. Bacterial expressed CLPs contain only bacterial mRNA
, avoiding any potential regulatory concern about eukaryotic
or viral DNA content in the CLPs. However, during assembly the
CLPs can package E.coli proteins and crude preparations usually
contain high concentrations of endotoxin. The later issue has been
solved by repeated TX114 phase separation reducing the
endotoxin concentration .1 EU/mg . To purify CLPs from
packed protein, CLPs can be disassembled into dimers, purified
and later reassembled into CLPs .
It may be noteworthy that HBcAg is derived from a human
pathogen and that worldwide approximately 2 billion people are
anti-HBcAg positive. The role of preexisting immunity to a carrier
is still controversial, with examples in favor of [54,55] and against
 carrier-mediated suppression . Although repeated vacci-
nations with HVRI-CLPs did not lead to carrier-mediated
suppression of the anti-HVRI response, despite the strong anti-
carrier response, the use of HBcAg as a carrier may therefore be
problematic. This potential shortcoming can be overcome by
substituting HBcAg with other hepadnaviral core proteins
[43,55,57,58]. Woodchuck hepatitis core protein (WHc), for
instance, is not cross-reactive with HBcAg-specific antibodies.
Indeed, the mimotope R9 fused to a split woodchuck core variant
(SplitWHc-R9) efficiently formed CLPs (Fig. S1) In previous
studies, WHc CLPs and HBc CLPs were comparable in
Figure 4. The water-in-oil adjuvant AS03 increases antibody and neutralization titers. A Mice were immunized intramuscularly with
pooled HVRI CLPs (5 mg per variant) in AS03 on days 0, 14, and 28. Mice were bled on days 0, 14, 28, and 42. Sera were analyzed as described in Fig. 2
B Neutralization of homologous HCVpp. C Neutralization of heterologous HCVpp. For details see Fig. 2. D Neutralization of patient-derived HCVpp.
Mice sera from day 42 were tested at a dilution of 1:50 for the ability to neutralize patient-derived HCVpp from GT1a isolates (white bars) and GT1b
isolates (black bars). Continuous line: 50% neutralization. HVRI sequences of the HCVpp are depicted in Table S1. E HVRI CLPs can induce HCVcc-
neutralizing antibodies. Huh7.5 cells were inoculated with JC1-YK5829 viruses in the presence of decreasing amounts of serum from day 42. Pre-
serum and serum from mice immunized with wild-type CLPs served as a control. Neutralization efficiency was determined by luciferase assays 72 h
after inoculation. Data are expressed as means and standard deviations of triplicate measurements. One of three representative experiments is
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Because of the enormous variability of HCV, the induction of
highly cross-reactive antibodies is very important for an effective
vaccine. Although we used primarily HVRI variants similar to
GT1b for immunization, the HVRI mix antibodies were cross-
reactive with 25% of patient-derived HVRI peptides from all
genotypes. In the HCVpp neutralization assay we observed
neutralization of Gt1b HCVpp and also of GT1a HCVpp.
Notably, the only sequence similarity between the neutralized
HCVpps are 4 aa located in the C-terminal part of HVRI. This
finding agrees with those of a previous study  and highlights
the importance of the correct antigen orientation on a carrier .
Rey et al showed that high-affinity HVRI-specific antibodies are
a key determinant of neutralization . Therefore, one of the
most important observations was that immunization by HVRI
CLPs with AS03 adjuvants induced antibodies with drastically
enhanced neutralization efficiency. As shown previously, the TH1
response induced by AS03 may have led to the production of
superior neutralizing antibodies . The enhanced stability of the
particles in the oil-in-water emulsion AS03 may be another
explanation for the enhanced neutralization. Intact particles are
crucial for efficient B-cell activation and maturation, and both the
core protein and the assembled particle structure are stabilized by
hydrophobic interactions  and may therefore be more stable in
The antibodies were also able to inhibit the infection of Huh7.5
cells with HCVcc-derived viruses but only when the GT2a HVRI
was swapped with a GT1 HVRI. This finding was expected
because we used GT1b HVRI sequences for immunization, and
the antibodies were not able to neutralize GT2a-derived HCVpp
and HCVcc (HCVpp-J6 and HCVcc, respectively). Interestingly,
Meunier et al.  also observed good cross-reactivity within GT1
and a lack of cross-reactivity with GT2 and GT3 when they
immunized chimpanzees with complete E1/E2 from GT1a. This
finding implies that HVRI variants from all genotypes must be
included in a vaccine cocktail. And, indeed, preliminary findings
suggest that immunization with a number of selected HVRI
peptides induces cross-reactive antibodies that react with more
than 95% of our peptide library . Using twenty or more
related antigens in a vaccine cocktail is a feasible approach, as
shown by the commercial 23-valent polysaccharide vaccine, which
has been used for 35 years in the vaccine against Streptococcus
The exact nature of protective HCV immunity is not clear.
Compelling evidence suggests that self-limiting infection is
associated with an early anti-HCV antibody response [4,6,20].
However, the role of neutralizing antibodies in the late phase of
infection remains elusive, because most chronically infected
patients cannot clear the infection despite the presence of
neutralizing antibodies [3,63–65]. Interestingly, immunizing
chimpanzees with complete E1/E2 protected 4 out of 5 animals.
The one animal with viral breakthrough developed an acute self-
limiting infection . These findings lead us to speculate that
neutralizing antibodies in the early phase attenuate the HCV
infection. Because of the enormous number of HCV quasispecies,
it is unlikely that an HVRI-based vaccine will offer sterile
protection. Rather, such antibodies may blunt the HCV infection
to facilitate the development of protective cellular immune
responses . Therefore, the most promising vaccine should be
a combination of HVRI CLPs and vaccines that induce robust T-
cell responses, such as transgenic adenoviruses or MVA . For
example, Barnes et al.  have recently shown that immuniza-
tion with adenoviral vectors encoding the HCV NS genes induces
strong, broad, and long-lasting T-cell responses in humans. The
authors suggest that such a vaccine should accelerate the
generation of HCV immunity after infection. At the same time,
Barouch et al. showed in the SIV model that immunization with
an adenovirus primer and a MVA boost is partially protective;
however, a substantial reduction in the risk of infection requires
the inclusion of envelope proteins in the vaccine .
In conclusion, by presentation of HVRI-peptides in a top-side
down orientation on SplitCore CLPs we were able to induce cross-
protective antibody in mice. This approach might be useful for the
development of a HCV vaccine, however could also be adapted
for other highly variable pathogens.
Materials and Methods
Mice entered experiments when they were between 8 and 10
weeks of age and treated in accordance with the 8th Edition Guide
for the Care and Use of Laboratory Animals and the institutional
guidelines of the University Hospital Essen, Germany. The study
was approved by the Northrhine-Westphalia State Office for
Nature, Environment and Consumer Protection (LANUV NRW)
and was carried out on the project license numbers G1088/09 and
G815/05 issued by the same state office. The animals were housed
under specific pathogen free [SPF] conditions in ICV mouse racks.
All experiments were performed under isoflurane anesthesia. Mice
were sacrificed by cervical dislocation under isoflurane anesthesia
and all efforts were made to minimize suffering.
All fusion protein constructs were based on the parental
plasmids pET28a2_HBc_c1-79_80-149H6  and featured a
T7 promoter–controlled synthetic  HBV SplitCore protein
(HBV genotype D, serotype ayw; accession no.: CAA24706)
truncated after aa149 and containing a C-terminal His6 tag.
HVRI variants were introduced by standard PCR mutagenesis
using long primers encoding the complete HVRI variants
including a G4S linker. Plasmid pNL4-3.luc.R-E and phCMV-
IRES-E1-E2 (H77) were kindly provided by Jane McKeating.
Plasmid phCMV-IRES-E1-E2 (Ad78) was kindly provided by
Thomas Baumert. HCVpp derivatives were generated by intro-
ducing the corresponding HVRI sequence by standard PCR
mutagenesis into plasmid phCMV-IRES-E1-E2 (Ad78). All
constructs were verified by DNA sequencing.
Protein expression and purification
E. coli BL21(DE3) Codonplus cells (Stratagene) were used
throughout as described [43,71,72]. Expression and purification
were performed as previously described . In brief, crude E. coli
lysates were loaded onto 10% to 60% sucrose step gradients and
sedimented at 20uC. Depending on the sample volume, we used
SW28 rotor (3:45 h at 28,000 rpm) or SW40 rotor (2 h at
40,000 rpm). The gradients were harvested in 14 equal fractions
from the top (2.6 ml per fraction for SW28; 820 ml per fraction for
SW40). For further purification, the pooled center gradient
fractions were dialyzed against TN150 buffer (25 mM Tris-Cl
[pH 7.5], 150 mM NaCl), concentrated, and subjected to a
second gradient sedimentation or size exclusion chromatography
on Superose 6 (GE Healthcare).
Endotoxin was depleted by Triton X-114 phase separation and
repeated dialysis against excess PBS at 4uC, as described
previously . Endotoxin contents dropped from approximately
104EU/mg protein in the crude lysates to less than 1 EU/mg, as
Induction of Cross-Neutralizing Antibodies against HCV
PLOS ONE | www.plosone.org7July 2014 | Volume 9 | Issue 7 | e102235
determined by the pyrochrome assay (Associates of Cape Cod,
Native agarose gel electrophoresis (NAGE) and
NAGE was performed in 1% agarose gels, as previously
described . For immunological detection, NAGE or SDS-
PAGE gels were blotted to PVDF membranes, which were probed
with specific primary antibodies and peroxidase-conjugated
secondary antibodies plus chemiluminescent substrates. We used
the monoclonal anti-HBc antibodies 10E11 and 10F10 (anti-
coreN and anti-coreC, respectively)  and the particle-specific
Negative staining electron microscopy (EM) using 2% uranyl
acetate was kindly performed by G. Lattwig (Pathology, University
Hospital Essen), as previously described .
Immunization experiments in mice
Groups of 6 to 10 female C57BL/6 mice (Harlan Laboratories,
Germany) housed under specific pathogen–free (SPF) conditions
were immunized with HVRI CLPs either intramuscularly (i.m.)
with AS03 or subcutaneously (s.c.) with incomplete Freund’s
adjuvant (IFA). Mice were immunized with either 20 mg of single
HVRI CLPs or 20 mg of HVRI mixture (5 mg per variant) as
indicated. Mice were bled either at days 28, 56, and 84 or at days
14, 28, and 42. Sera were pooled, and anti-HBc or anti-HVRI
antibodies were measured by indirect solid-phase ELISA using
solid-phase wt HBcAg (50 ng per well) or HVRI peptides (0.5 mg
per well). Goat anti-mouse IgG (or IgG isotype-specific) antibodies
were used as secondary antibodies. Synthetic peptides were
synthesized by the CDC peptide synthesis facility (Atlanta,
Georgia, US). Peptide sequences used for cross-reactivity analysis
can be obtained upon request.
HCV pseudoparticles (HCVpp) and HCVcc neutralization
Human immunodeficiency virus (HIV)-HCV pseudotypes were
generated as previously described . Briefly, we used the
CalPhos mammalian transfection kit (Clontech) to co-transfect
2.56106293T cells with plasmid pNL4-3.luc.R-E  encoding
HIV DEnv and expressing luciferase or with vector phCMV-
IRES-E1-E2  encoding the HCV envelope glycoproteins.
Sixteen hours after transfection, the medium was replaced.
Supernatants were harvested at 48 h and 72 h, pooled, and
clarified by centrifugation. The resulting supernatants were
immediately used for infectivity and neutralization assays.
To investigate pseudotype virus infectivity, we seeded 56105
Huh7.5 cells in 96-well plates on the day before infection. The
infection medium was removed, and HCVpp diluted in DMEM
supplemented with 6% FCS were added. For neutralization,
serum samples were mixed with the HCVpp at various dilutions
and were incubated at 37uC for 2 h before to infection of the cells.
After 72 h of incubation, the medium was removed, and cells were
lysed with Bright Glo lysis buffer (Promega) for 2 h at 220uC.
Luciferase activity was measured 10 min after the addition of the
Bright Glo Luciferase Assay buffer (Promega) in a luminometer
(Glomax Multi Detection System, Promega). Cell lysates were
measured in triplicate; data are expressed as the mean of the
results from at least two independent experiments. HCVcc
neutralization experiments were performed as previously de-
SplitWHC-R9 fusion protein was sedimented through a prepar-
ative 10% to 60% sucrose step gradient; 14 fractions of 860 ml
each were harvested from the top. Aliquots of 8 ml each were
analyzed by SDS-PAGE and Coomassie Blue (CB) staining;
marker proteins with their molecular masses (in kDa) are indicated
on the left. Both fragments, CoreC (arrow up) and CoreN-R9
(arrow down), peaked in the center fractions. B Native agarose gel
electrophoresis (NAGE). Aliquots of the gradient shown in A were
run in 1% agarose gels; they were either stained with CB or their
gel content was blotted onto polyvinylidene difluoride (PVDF)
membranes and detected with the monoclonal antibody 10E11. C
Electron microscopy. Aliquots of the fusion proteins were
negatively stained with uranyl acetate.
Alignment of HVRI sequences used for HCVpp
We thank Kerstin Uhde-Holzem and Vadim Bichko for monoclonal
antibodies and Gabi Lattwig for negative-staining electron microscopy.
Conceived and designed the experiments: MF SV ARZ YK MN PP TP JT
MR AW. Performed the experiments: ML MF DB WO OB YK AW.
Analyzed the data: ML MF DB WO SV ARZ YK TP JT MR AW.
Contributed reagents/materials/analysis tools: MN PP ARZ. Wrote the
paper: MF MN JT MR AW.
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