Previous Article
J Virol, May 1998, p. 4541-4545, Vol. 72, No. 5
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Identification of Hepatitis G Virus Particles in
Human Serum by E2-Specific Monoclonal Antibodies Generated by
DNA Immunization
Susanne
Schmolke,
Michael
Tacke,
Urban
Schmitt,
Alfred M.
Engel,* and
Beatus
Ofenloch-Haehnle
Boehringer Mannheim GmbH, R&D Infectious
Diseases, 82377 Penzberg, Germany
Received 10 November 1997/Accepted 3 February 1998
 |
ABSTRACT |
In order to elucidate the structure and morphology of hepatitis G
virus (HGV), a recently isolated flavivirus, we generated a panel of
eight monoclonal antibodies (MAbs) against the putative second envelope
protein (E2) following DNA immunization. The MAbs were shown to be
specific for four different epitopes on recombinant E2. MAb Mc6 was the
only antibody able to detect the linear epitope LTGGFYEPL. In addition,
Mc6 was able to immunoprecipitate viral particles in human blood
samples as detected by reverse transcription-PCR amplification
of HGV RNA. This precipitation could be competed by addition of
saturating amounts of the linear peptide or abolished by addition of
Nonidet P-40. We conclude that, albeit lacking the N-terminal sequence
of a functional core protein, HGV builds classical viral
particles displaying E2 envelope protein on their outer surfaces.
| |
TEXT |
Introduction.
Recently, two
groups reported independently on the isolation of new positive-strand
RNA viruses, designated hepatitis G virus (HGV) (14) and GB
virus C (GBV-C) (12). Sequence analysis revealed that both
genomes are different isolates of the same virus and show ~85%
nucleotide sequence identity, including a single, continuous open
reading frame encoding 2,873 amino acids with a number of motifs
characteristic for members of the Flaviviridae family
(2). The genetic organization of HGV resembles that of HCV,
but the lack of a sequence coding for a functional core-like protein
raises important questions with regard to the morphology of the virus
(22). HGV is transmitted parenterally and is therefore commonly distributed among risk groups, such as intravenous drug users,
hemophiliacs, and patients who receive multiple transfusions (14,
15, 23). Among apparently healthy blood donors, an HGV RNA
prevalence of 0.9 to 3% has been reported (14, 15, 23). HGV
can cause acute and persistent infection, but the clinical significance is still unclear. Based on the cloning sources, HGV was initially discussed as another potential causative agent of acute
and chronic hepatitis, but studies so far have been unable to prove the
link between HGV and liver disease (1).
Two different tools for HGV diagnosis in human blood specimens were
available until now, reverse transcription-PCR (RT-PCR) detection
of HGV RNA (20) and immunoassays for the detection of
specific antibodies against the putative envelope protein E2 (anti-E2)
(5, 17, 24, 25). The glycoprotein E2 features a
C-terminal transmembrane anchor domain, three potential
glycosylation sites, and 18 cysteine residues which might be involved
in disulfide bonds. In analogy to other flaviviruses, E2 is presumed to
play an important role in binding of the virus to target cells. In contrast to HCV, sequence variability of E2 is very low among isolates
collected worldwide and the appearance of antibodies to E2 is normally
associated with recovery from HGV viremia (5, 13, 24).
Obviously, a high proportion of immunocompetent individuals infected
with HGV are able to clear the virus, although viremia has been shown
to persist in some patients (25). The present work describes
the generation of monoclonal antibodies (MAbs) to E2 which share
epitopes with antibodies present in sera of HGV-infected individuals.
They provide tools for the characterization of HGV particles and the
establishment of immunoassays for the detection of viral antigen in
human sera.
DNA immunization and generation of E2-specific MAbs.
Immunization by intramuscular injection of plasmid DNA encoding the
antigen seems to be advantageous over classic immunization with
purified antigen, especially if the antigen is difficult to synthesize
and/or to purify (28). In addition, the method allows host
processing of newly synthesized proteins, correct glycosylation, and
proteolytic processing. This method has recently been shown to induce
both humoral and cellular immune responses against a number of
infectious agents, including HBV surface antigen (3),
influenza virus nucleoprotein (16), and HCV E2
(26).
The expression construct CHO-E2-TM8 used for plasmid DNA immunization
was proven to correctly express glycosylated FLAG-E2
fusion protein
in Chinese hamster ovary (CHO) cells (
25). Viral
E2 is
expressed as part of a polyprotein, and therefore the construct
features a heterologous signal sequence besides an N-terminal
FLAG epitope (
9) and the E2-coding sequence containing its
C-terminal membrane anchor (
25). Earlier reports claim
higher
efficiency of DNA uptake in regenerating muscle cells
(
3).
Therefore, 80 µl of 10 µM cardiotoxin (Latoxan;
Rosans) was injected
into tibialis anterior muscles of five female
15-week-old BALB/c
mice. Five days later, 50 µl of phosphate-buffered
saline (PBS)
containing plasmid DNA (1 µg/µl) was injected into
each muscle.
This was repeated after another 5, 10, 11, and 12 weeks.
Serum
samples collected after the second and the fifth immunizations
were tested for E2-specific antibodies in a whole-cell enzyme-linked
immunosorbent assay (ELISA): CHO cells displaying membrane-bound
FLAG-E2 (
25) were seeded overnight in 96-well tissue
culture
plates (4 × 10
4 cells/well). The next day,
cells were first incubated for 2 h
with medium containing 1% Byco
C to block unspecific binding sites.
Serum samples were added and
incubated for another hour. After
being washed with PBS-0.02% Tween
20, cells were incubated with
horseradish peroxidase-anti-mouse
immunoglobulin G (IgG)-Fab conjugate
(50 mU/ml) for 1 h. The
wells were washed, and an enzymatic color
reaction was developed by
adding the substrate solution, 1.9 mM
2,2'-azino-di(3-ethylbenzthiazolinesulfonate) diammonium salt
(ABTS) in
100 mM phosphate citrate buffer (pH 4.4)-3.2 mM hydrogen
peroxide (as
sodium perborate). Adsorbance at 422 nm was read
after 1 h. To
check for unspecific binding, all supernatants were
also tested on CHO
cells expressing the human urokinase receptor
with an N-terminal FLAG
peptide.
After the second immunization, no significant titer was detectable.
However, after the fifth immunization, three of five mice
had developed
an E2-specific titer over 1:1,000. One mouse was
selected and given one
booster by intravenous injection of 10
7 CHO cells
expressing FLAG-E2 prior to fusion of spleen cells
with the nonproducer
cell line P3X63-Ag8.653 (ATCC CRL 1580) using
polyethylene glycol
(molecular weight, 4,000) (
7). Supernatants
of
hybridomas obtained after selection with hypoxanthine
aminopterine
thymidine-containing medium were tested for
E2-specific antibodies,
and finally, eight cell lines producing IgG
antibodies to HGV
E2 were recloned by fluorescence-activated cell
sorting. MAbs
designated Mc3, Mc5, Mc6, Mc11, Mc13, Mc17, Mc19, and
Mc30 were
further purified and conjugated with biotin and digoxigenin
(Table
1).
Antibody isotyping revealed that two of eight cell lines produced IgG1,
while one of eight and five of eight produced IgG2a
and IgG2b,
respectively (Table
1). This is in contrast to former
studies reporting
a preponderance of IgG2a antibodies after intramuscular
inoculation
(
6,
16,
18). These reports used polyclonal
sera for
subtyping, whereas the present report describes the generation
of MAbs
after multiple injections of plasmid DNA followed by a
final boost with
cells expressing antigen. Cross-boosting experiments
imply that the
type of immune response is determined by the initial
immunization;
subsequent booster immunizations by an alternative
method do not alter
the established IgG isotype profile (
6,
16). Therefore, the
final boost with cells expressing antigen
is unlikely to be the reason
for the preponderance of subtype
IgG2b. A selection for the subtype
IgG2b antibodies during the
screening of the hybridomas is also
unlikely, since the polyclonal
antibody which was used for the first
screening was generated
against mouse Fc
. It recognizes the
different mouse IgG subclasses
with relative affinities in the
following order (from greatest
to least): IgG1, IgG2a,
IgG2b, IgG3. Differences in the type or
form of the antigen probably
account for the different subtypes
found, as membrane-bound, cytosolic,
and secreted proteins may
elicit different types of antibody isotype
responses.
Epitope mapping by competition ELISA.
In order to analyze the
binding sites of the MAbs on recombinant E2, a competitive binding
inhibition ELISA was developed using the automated ES 300 serum
analyzer system (Boehringer Mannheim [BM]): pairs of MAb-biotin
conjugates for capturing by streptavidin and
MAb-digoxigenin conjugates for detection (3 µg/ml each) were incubated with crude lysates of CHO cells expressing FLAG-E2
(25) for 3 h in streptavidin-coated test tubes. Some
MAbs showed slight differences depending on their use as capture or
detection antibody, probably due to differences in their binding
affinities. Detection of digoxigenin-conjugated antibody complexes was
done by incubation with horseradish peroxidase-antidigoxigenin
conjugate (50 mU/ml) for 3 h. After the cells were washed,
enzymatic color reactions were developed by adding ABTS substrate
solution.
Simultaneous use of each of the E2-specific MAbs for capturing and
detection resulted in strong inhibition in all eight cases
(diagonal of
Table
1), indicating that E2 is present as monovalent
antigen in CHO E2
lysates. Different MAb combinations for capturing
and detection
indicated the presence of several MAb groups specific
for epitope
clusters (Table
1): Mc5 and Mc17 completely inhibited
each other. They
obviously bind to the same or correlated epitopes
and thus constitute
MAb group I. The same holds true for Mc6 and
Mc11 (group II) and Mc13
and Mc19 (group III). MAbs of groups
II and III slightly inhibited each
other. Although the overall
reactivity of Mc3 was relatively weak
compared to that of the
other MAb, Mc3 interfered with MAbs of groups
II and III. Mc30
could be competed only by itself and is therefore
specific for
another epitope.
Pepscan.
All E2-specific MAbs were tested for their reactivity
against synthetic 13-mer peptides covering the 385-amino-acid putative primary sequence of mature HGV-E2 from APASVL to PAVEAA. Each peptide
overlapped the adjacent peptide by nine residues and carried an
N-terminal biotin tag for capturing followed by
-lysyl-
-alanyl--aminocaproyl--alanine as a spacer
module. Peptides (200 ng/ml) were incubated with MAb-digoxigenin conjugates (1 µg/ml) and bound to streptavidin-coated
test tubes for 3 h. Detection of digoxigenin-conjugated antibody
complexes was done as described above. Mc6 strongly reacted with two
peptides, 277 (biotin-spacer-GGAGLTGGFYEPL) and 281 (biotin-spacer-LTGGFYEPLVRRC), sharing residues 281 to
289 (LTGGFYEPL) (Fig. 1). This linear epitope is located near the C terminus of the extracellular domain of
E2, assuming that the membrane anchor region starts around residue 340. Neither Mc11, which can be competed by Mc6, nor any other MAb showed
reactivity with any of the peptides. These data imply that seven of
eight MAbs are specific for conformational epitopes. This correlates
with previous observations that no antigenic epitopes could be
determined by screening the HGV genome for linear epitopes
(20a).
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FIG. 1.
Reaction profile of MAb Mc6 with synthetic overlapping
peptides. Eleven peptides, spanning residues 253 to 305 of HGV E2, are
shown. The amino acid residue numbers indicate the position of the
first residue from each 13-mer; numbering starts with the first residue
of the putative mature E2 protein. Only peptides 277 and 281, which
share amino acids 281 to 289 (LTGGFYEPL), were recognized by Mc6.
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Immunoprecipitation of viral particles.
An immunoprecipitation
assay was developed in order to test whether MAbs generated
against recombinant FLAG-E2 are able to bind native E2 present on viral
particles. Serum samples were obtained from volunteer blood donors of
the on-site medical center (sample designations starting with BM) and
the blood bank of Salzburg (sample designations starting with SB).
All sera tested negative for HBV, HCV, and human
immunodeficiency virus markers and were preabsorbed with protein
G-agarose to reduce unspecific IgG binding.
In a pilot experiment, 100 µl of a 10
2 dilution (in
Dulbecco's PBS) of serum BM7822 (HGV RNA positive, anti-E2 negative),
corresponding
to a final concentration of 10
5 GE/ml, was
incubated for 2 h at 4°C under continuous shaking
with 100 µl
of a 10
2 dilution (in PBS) of serum SB9700575 (anti-E2
positive, HGV RNA
negative) or SB315 (negative for both markers).
Protein G-agarose
was added, and antibody binding was allowed for
2 h. Immunoprecipitates
were collected and extensively washed
three times with PBS-1%
bovine serum albumin, by using spin modules
(Bio 101, Vista, Calif.).
RNA was isolated using the High Pure RNA
Isolation Kit (BM). For
qualitative RT-PCR amplification of HGV RNA, we
used primers derived
from the 5' noncoding region,
5'-CGGCCAAAAGGTGGTGGATG-3' (forward)
and
5'-biotin-CGACGAGCCTGACGTCGGG-3' (reverse), in combination
with the Titan One Tube RT-PCR Kit (BM). Automated detection of
amplicons on a modified ELECSYS 1010 analyzer (BM) via
electrochemiluminescence
was performed as follows. After
denaturation and addition of a
ruthenium-labeled hybridization
probe,
5'-CCACTATAGGTGGGTCTT-Ru(bpy)
32+-3',
amplicons were captured onto streptavidin-coated microparticles
via the biotinylated reverse primer and detected in an electrochemical
reaction on the surface of an electrode. The luminescence at a
wavelength of 620 nm was measured as arbitrary electrochemiluminescence
counts (
11).
A significant amount of HGV RNA could be detected following
precipitation with anti-E2-positive serum SB9700575, but not with
anti-E2-negative serum SB315 (Fig.
2a).
Precipitation of viral
particles was not quantitative, since HGV RNA
could also be detected
in the supernatant (unbound fraction [data not
shown]). Subsequently,
all eight recombinant E2-specific MAbs were
used instead of anti-E2-positive
serum. As negative controls, MAbs
specific for the FLAG epitope
(M1, isotype IgG2b) and
-galactosidase (anti-
-Gal, isotype IgG2b)
were included.
Out of 10 MAbs used, only Mc6 was able to precipitate
a considerable
amount of viral particles from HGV RNA-positive
serum BM7822
(Fig.
2a). To verify that immunoprecipitation was
not
only confined to serum BM7822, we examined two additional
HGV
RNA-positive human sera, SB373 (final concentration, 1 × 10
4 GE/ml) and BM388 (final concentration, 5 × 10
4 GE/ml), in combination with anti-E2-positive SB9700575,
anti-E2-negative
SB315, E2-specific Mc6, and FLAG-specific M1 (Fig.
2b). Again,
only SB9700575 as well as Mc6 was able to precipitate
significant
amounts of viral particles (Fig.
2b). The results indicate
that
Mc6 can be used for immunoprecipitation of HGV RNA-containing
particles present in different HGV PCR-positive human sera.
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FIG. 2.
Immunoprecipitation of viral particles. HGV
RNA-positive sera were incubated with either MAbs or human sera.
Following precipitation by protein G-agarose, RNA was isolated and
detected by RT-PCR. Shown are representative examples of three
independent experiments. (a) HGV RNA-positive serum BM7822 was
incubated with eight murine E2-specific MAbs, anti--Gal,
FLAG-specific M1 (final concentration, 5 µg/ml), anti-E2-positive
serum SB9700575, and anti-E2-negative serum SB315. Only Mc6 and
SB9700575 were able to immunoprecipitate HGV. (b) HGV RNA-positive
sera SB373, SB388, and BM7822 were incubated with anti-E2-positive
serum SB9700575, anti-E2-negative serum SB315, E2-specific Mc6, and
FLAG-specific M1. SB9700575 and Mc6 were able to immunoprecipitate HGV
from all sera. (c) BM7822 was incubated with Mc6 in the presence of
peptides 229, 273, 277, and 281 (final concentration, 1 µg/ml). For
comparison, immunoprecipitation was performed with Mc6 without addition
of a peptide and with anti--Gal. Peptides 277 and 281 competed for
binding sites on viral E2. (d) BM7822 was incubated with Mc6 in the
presence of different concentrations of NP-40. No viral RNA could be
detected in the precipitate (bound) and supernatant (unbound fraction)
at 0.025 and 0.0025% NP-40. ECL, electrochemiluminescence.
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As described before, Mc6 was shown to be specific for a linear
epitope. Therefore, the specificity of Mc6 could be proved
by
competition: HGV RNA-positive serum BM7822 was incubated with
Mc6 in
the presence of saturating amounts of peptides 277 and
281, which share
the sequence LTGGFYEPL, and two peptides with
nonrelated
sequences, 229 (biotin- spacer-MTRIRDTLHLVEC) and 273
(biotin-spacer-SEALGGAGLTGGF), followed by
immunoprecipitation
and RT-PCR (Fig.
2c). As expected, only peptides
277 and 281 were
able to compete with the binding of Mc6 to viral
E2 (Fig.
2c).
All immunoprecipitation experiments described in this work so far were
performed under nonstringent conditions to minimize
disruption of
viral particles. All steps were carried out at 4°C,
serum was thawed
only once, and denaturating reagents were avoided.
Taken together,
these experiments indicate the presence of viral
particles
displaying E2 on their surfaces. In another experiment,
immunoprecipitation of HGV RNA-positive serum BM7822 in combination
with anti-E2-positive serum SB9700575 was performed in the presence
of
different concentrations of lipid solvent. Nonidet P-40 (NP-40)
concentrations below the critical micelle concentration (CMC)
(CMC = 0.00025%) had no influence on the experiments, and HGV
RNA
could be isolated from the precipitate (bound fraction) and
supernatant
(unbound fraction) (Fig.
2d). However, NP-40 concentrations
close to
the CMC (0.0025%) or clearly above the CMC (0.025%) completely
abolished immunoprecipitation. Importantly, HGV RNA could no longer
be
detected in either the precipitate or the supernatant in contrast
to
all previous experiments (Fig.
2d). Obviously, the viral particles
were
solubilized and the RNA was degraded by RNases present in
the serum.
Conclusions.
Reports on HCV imply that detergents like NP-40
used at concentrations far above the CMC remove the viral envelope
while leaving the capsid structure intact (10, 21). In these
studies (10, 21), HCV RNA could be detected in
detergent-treated virus preparations. This is in contrast to our
observation that detection of HGV RNA in the supernatant (unbound
fraction) was impaired at NP-40 concentrations around or above the CMC.
Obviously, HGV RNA is not as well protected as HCV RNA, as already
discussed in another study (19). The lack of core-like
coding sequences in all HGV isolates analyzed so far might be related
to the reduced stability of the viral RNA after disruption of the
envelope.
Immunoprecipitation from human sera failed when
conformation-dependent E2-specific MAbs were used. Their
epitopes might be
masked on the virus surface for several
reasons, e.g., by complexation
with envelope protein E1 or E2
oligomerization. Studies of HCV
have shown that E1 and E2
glycoproteins interact to form a heterodimeric
complex, which has been
proposed to be a functional subunit of
the HCV virion (
4).
Such oligomerization processes might induce
conformational changes,
altering the epitopes recognized by our
MAbs generated against
recombinant E2. As described for HCV, viral
particles could also be
associated with either Ig (
8) or lipoproteins
(
27), making some epitopes inaccessible. Although the
conformation-dependent
MAbs were not able to precipitate viral
particles, they share
epitopes with antibodies present in sera of
HGV-infected individuals.
This was demonstrated by competition studies;
binding of MAbs
specific for the epitope cluster (groups II and III)
could be
competed by polyclonal antibodies present in sera of
HGV-infected
individuals, whereas the others (group I and Mc30) could
hardly
be competed by human antibodies. Future studies utilizing both
Mc6 and conformation-dependent E2-specific MAbs will hopefully
help to
address open questions on HGV, such as the site of viral
replication
and clinical manifestations of HGV.
| |
ACKNOWLEDGMENTS |
We thank the animal facilities of BM, Christa Huebner-Parajsz for
helping us to generate MAbs, Volker Schlueter for support on RT-PCR
methods, and Christoph Seidel for peptide synthesis.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Boehringer
Mannheim GmbH, R&D Infectious Diseases, Nonnenwaldstr. 2, D-82377
Penzberg, Germany. Phone: (49 8856) 60-3509. Fax: (49 8856)
60-3129. E-mail: ALFRED_ENGEL{at}BMG.BOEHRINGER-MANNHEIM.COM.
| |
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J Virol, May 1998, p. 4541-4545, Vol. 72, No. 5
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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