Note: Descriptions are shown in the official language in which they were submitted.
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TITLE OF THE INVENTION
METHOD FOR INDUCING HEPATITIS C VIRUS (HCV) REPLICATION
IN VITRO, CELLS AND CELL LINES ENABLING ROBUST HCV
REPLICATION AND KIT THEREFOR
FIELD OF THE INVENTION
The present invention relates to hepatitis C virus
(HCV). More particularly, the invention relates to the development of a
tool suitable for the search and validation of novel HCV antiviral drugs
and therapies (e.g. vaccine). The invention further relates to methods
for inducing HCV replication in vitro, and more particularly to a simple in
vitro replication assay of HCV.
BACKGROUND OF THE INVENTION
The hepatitis C virus (HCV) is an enveloped RNA virus
of the Flaviviridae, which is classified within the Hepacivirus genus.
HCV is an important etiologic agent of chronic liver diseases. At this
time HCV infection is one of the primary causes of liver transplantation
in the US and other countries. Acute infections are usually subclinical or
associated with mild symptoms, but the virus persists in more than 80%
of infected individuals despite evidence of active, antiviral
immunological response (J. Viral Hepatitis 1997, 4:31-41; Hepatol 1998,
28:939-944; J. Viral Hepatitis 1999, 6:36-40). It is estimated than more
than 170,000,000 people are seropositive world-wide (Hepatology
1997, 26:62S-65S). The long-term outcome of HCV persistent infections
are varied, and they can range from an apparently healthy carrier state
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to chronic active hepatitis, liver cirrhosis, and eventually hepatocellular
carcinoma (N Engl J Med 1992, 327:1899-1905; Hepatology 1990,
12:671-675). The mechanism of such pervasive persistence is entirely
unknown. To date, there is no vaccine for HCV and the only available
therapy for chronic viral infections is treatment with interferon alpha
(IFN-a) either alone or in combination with the nucleoside analogue
ribavirin (J Hepatol 1999, 30:956-961; Mol Immunol 2001, 38:475-484).
Unfortunately, only ~40% of treated patients develop a sustained
response that is defined by absence of viral RNA for more than 6
months after cessation of therapy (J. Viral Hep. 1999, 6:35-47).
Moreover, during IFN-a treatment selection of viral variants resistant to
INF-a occurs frequently (Microbes & Infection 200, 2:1743-1756). In
addition, ribavirin can be used to treat patients. HCV resistance to
ribavirin is also common. The search for HCV drugs as well as the
development of an HCV vaccine is severely hampered by the lack of an
efficient tissue culture or simple animal system for the study of
replication and HCV pathogenicity. The only animal models currently
available for the study of this virus are the chimpanzee and a mouse
which possesses a chimeric human liver (Antiviral Research 2001, 52:1-
17; Nat Med 2001, 7:927-933). These facts cast HCV as an emerging
human pathogen of extreme medical significance (J Viral Hepat 1999,
6:35-47).
There thus remains a need to provide a simple assay
for HCV replication which would enable the study of HCV replication
and/or pathogenesis and enable the development of a treatment or
prophylaxy for HCV infections. There also remains a need to provide a
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HCV replication system which enables the screening of anti-HCV
compounds which can act in a larger number of stages of the HCV life
cycle such as infection, replication, translation and assembly. There
also remains a need to provide a system which enables the replication
of HCV from a patient so as to enable simpler and more efficient
genotyping thereof and/or phenotyping (e.g. to identify its
resistance/sensitivity characteristics toward anti-viral compounds).
While HCV infects a large number of individuals, no
efficient treatment or vaccine has been developed, despite a significant
effort by the pharmaceutical industry. Thus, most companies with
existing programs in the anti-infective area are focused towards the
discovery of agents that are active against this virus. Thus far the
human immunodeficiency virus (HIV) has provided a useful strategy for
HCV antiviral drug development (Drug Discov Today 1999, 4:518-529).
In fact the understanding of the function of anti-HIV drugs has outlined
the research platform of most of the companies screening for anti-HCV
drugs. Both viruses share interesting features. They lead to chronic
infection, are highly mutable, and they code for specific enzymes that
are not expected to be present in a normal non infected cell. Based on
the results of HIV therapy, it is possible that a combination therapy
involving at least two drugs directed against separate targets will be
more effective at reducing HCV load and preventing the emergence of
resistant strains than monotherapy. As the selected targets against HIV
have been the viral encoded protease and the viral reverse
transcriptase, it is not surprising to find that HCV protease and RNA
dependent RNA polymerase have often been mentioned as candidate
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antiviral targets. As judged by the lack of disclosures, the discovery of
anti-HCV agents has not been successful despite the functional
similarity of several HCV-enzymes with known targets from other
antiviral programs. Admittedly, part of this failure is because of the lack
of a tissue culture system, which in turn limits primary screens to isolate
viral protein targets. Interestingly, despite the fact that the enzyme
assays to test HCV protease are known, the discovery of a potential
drug candidate has met with little success. Taken together, it might be
concluded that putative chemotypes for inhibition of HCV-targets are
poorly represented in most industrial compound collections (Drug
Discov Today, 1999, 4:518-529).
Should a series of novel anti-HCV drugs be
developed, to advance these agents into the drug development
pipeline, several issues will need to be addressed, notably, their
mechanism of action. Unfortunately, tissue culture and in vivo control
experiments using whole virus are required to better determine the
mode of inhibition. As stated above, an efficient cell culture system for
the replication of HCV has not yet been provided (Drug Discov Today
1999, 4:518-529; Antiviral Res 2001, 52:1-17; J Mol Biol 2001, 313:451
464; Virus Res 2002, 82:35-32).
Attempts have been made, based on the use of
human cells of hepatocytic and lymphocytic origin, but low and variable
levels of replication and virus-induced cytotoxicity posed important
problems. Primary hepatocytes (derived from a human donor) can be
infected with HCV isolated from serum of viremic patients, and the virus
can be detected in the supernatant for several weeks after infection.
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HCV replication has been demonstrated by detection of minus-strand
RNA, an intermediate of virus replication, in primary hepatocytes
derived from a HCV-negative donor after infection with sera from HCV-
positive patients. However, the availability of primary hepatocytes is
limited, and their isolation is time-consuming and labor-intensive.
Consequently, such tissue culture systems are generally considered
unsuitable for intensive large-scale antiviral studies.
Another example of progress in this domain has been
the construction of subgenomic selective replicons cloned from a full-
length HCV consensus genome from an infected liver (Antiviral Res
2001, 52:1-17; J. Mol Biol 2001, 313:451-64; Virus Res 2002, 82:25-
32). Following transfection in human hepatoma cells, these RNAs were
found to replicate to high levels, allowing detailed molecular studies of
HCV and testing of antiviral drugs. One drawback of this system,
however, is that it only expresses the non-structural viral proteins
(Science 1999, 285:110-3). Therefore, studies aimed at assessing
target viral assembly and trafficking through the cytoplasm cannot be
carried out, with this reconstituted viral system. In other words, such
artificial system is of a more limited potential to identify antiviral agents.
As previously mentioned animal models currently exist
to study HCV replication. Although the chimpanzee model has
contributed significantly to the understanding of HCV infection, the high
cost and availability of these animals limit the extent to which antiviral-
drug or therapy studies can be carried out. Small laboratory animals,
including mice, are not susceptible to infection with HCV. An alternative
model such as a mouse model with a chimeric human liver has been
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generated (Nat Med 2001, 7:927-933). This system is considered
laborious and is known to require special expertise to isolate and
transplant human hepatocytes and maintain a colony of fragile
immunodeficient mice with an approximately 35% mortality in newborns
due to a defect in blood coagulation (Nat Med 2001, 7:927-933).
Nevertheless, when all the required conditions are met this mouse
model can provide an interesting system for testing antiviral agents.
There thus remains a need to provide a simple in vitro
system, which is suitable for the replication of HCV.
There also remains a need to provide an in vitro tissue
culture system for the replication of complete HCV.
There further remains a need to provide a tissue
culture system for HCV which enables the screening and development
of drugs and therapies for essentially all the different stages of virus
replication such as virus entry, cytoplasmic replication [viral (-) and (+)
strand synthesis], viral protein synthesis, virus assembly, virus
trafficking, and virus release.
The present invention seeks to meet these and other
needs.
The present description refers to a number of
documents, the content of which is herein incorporated by reference in
their entirety.
SUMMARY OF THE INVENTION
The invention relates to a simple in vitro culture
system, which is suitable for the replication of hepatis C virus (HCV).
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The invention further relates to an in vitro tissue
culture system which enables the replication of complete HCV.
In addition, the invention relates to a tissue culture
system for HCV which enables the screening and development of drugs
and therapies for essentially all the different stages of virus replication
such as virus entry, cytoplasmic replication [viral (-) and (+) strand
synthesis], viral protein synthesis, virus assembly, virus trafficking, and
virus release.
The present invention also provides the means to
diagnose HCV. In addition, it enables an identification of the response
of a particular strain of HCV, from a particular patient, to a candidate
antiviral compound or to a known antiviral compound.
The present invention further relates to a method of
activating the replication of HCV in PBMCs comprising obtention of
same from a HCV-infection patient and activating the replication of HCV
by incubating the PBMCs with an activation-inducing amount of at least
one mitogen (e.g. activator).
The invention in addition relates to a co-culturing
system for replicating HCV in vitro which comprises co-culturing PBMCs
(or PBLCs) infected with HCV, wherein the PBMCs have been activated
and in which the HCV can replicate, together with a cell line, wherein
the co-culturing enables infection of the cell line and replication of the
HCV thereinto. In a particular embodiment of the present invention, the
cell line is an immortalized cell line.
It is believed that the Applicant is the first to provide an
in vitro cell system which enables replication of a native HCV.
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It is believed that prior to the present invention, while
HCV could infect PBMCs, it was unknown that it could actively replicate
in them. The present invention demonstrates HCV tropism for PBMCs
and more particularly for PBLCs. As known in the art, PBMCs are a
mixture of cells which also include macrophages and PBLCs (which can
be obtained from PBMCs) contain about 85% T cells and 5% B cells.
It is also believed that this is the first demonstration
that the HCV produced in an in vitro system is infectious and that
sustainable replication of HCV can be achieved.
Before the present invention, large-scale production of
HCV was unthinkable. The methods and in vitro system of the present
invention enables active replication of HCV in cells for at least 9 days
and opens the way to large scale production.
Prior to the present invention, no tissue culture
technology currently existed to replicate HCV. The only animal models
currently available for the study of this virus are the chimpanzee and
mice models (mice with chimeric human livers). These animal based-
system are laborious and require special expertise and facilities.
RNA interference can be used in accordance with the
present invention using, for example, the teachings of 6,506,559.
BRIEF DESCRIPTION OF THE DRAWINGS
Having thus generally described the invention,
reference will now be made to the accompanying drawings, showing by
way of illustration a preferred embodiment thereof, and in which:
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Figure 1 shows the hepatitis C virus (HCV) genome
organization;
Figure 2 shows the hypothetical model of the HCV
replication cycle;
Figure 3 shows an experimental protocol. All
experiments were performed with 1,000,000 cells/ml. T1 = anti-CD3 (1
ug/~I final), IL-2 (final = 200 U). T2 = PHA (3 ug/ul), IL-2. T3 = PHA, IL-
2, SAC (1/104). T4 = PHA, IL-2, SAC, IL-4 (final = 200 U);
Figure 4 shows PBMC and PBLC purification from
blood samples;
Figure 5 shows the detection of HCV NS3 and NS5
proteins in cell extracts from treated PBMC from a HCV (+) patient;
Figure 6 shows a validation that the antibody used is
decorating the NS3 translated (if positive) in the replicon system and
that of the present invention activated (A) or non-activated (NA);
Figure 7 shows the time course of HCV-NS3 detection:
PBMCs from patient MLL-001;
Figure 8 shows the time course of HCV-NS3 detection:
PBMCs from patient MLL-002;
Figure 9 shows the detection of HCV-NS3 protein in
treated (N3) PBMCs from HCV9+) donors;
Figure 10 shows the detection of virus like particles by
scanning electron microscopy;
Figure 11 shows the electron microscopy of activated
PBLCs and detection of virus like particles;
Figure 12 shows a virus partial purification;
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Figure 13 shows the detection of HCV core protein in
supernatant of treated PBMC from an HCV(+) patient;
Figure 14 shows RNA quantification I (virus copies/ng
total RNA);
Figure 15 shows an infection assay; co-culture;
Figure 16 shows infection of MT-4 cells RNA
quantification II (virus copies/ng total RNA);
Figure 17 shows co-culture of Huh-7 and HCV (-)
PBMCs;
Figure 18 shows co-culture of Huh-7 and HCV (+)
PBMCs (SB006);
Figure 19 shows PHA activation of PBMCs from
patient SB004 (HCV is not in T cells);
Figure 20 shows the detection of HCV (E2) on Daudi
cells upon co-cultivation with infected PBMCs (the control for Fig.). Of
note, Daudi cells are a B cell line;
Figure 21 shows a comparison of different activation
treatments (PBMCs from donor MLL-010). T1 = PHA + IL-2. T2 = SAC
+ IL-2. T3 = T1 + T2; and
Figure 22 shows viral RNA in cell supernatant (real
time RT-PCR). T1, T2, T3 are the same as for the preceding figure. Of
note, further addition of IL-4 to T3 further increased activation.
Fig. 23 shows that HCV (+) and (-) strand RNA is
produced de novo in activated PBLs. A) HCV-RNA was detected in
PBLs from an HCV positive donor by a one step reverse transcription-
polymerase-chain reaction (RT-PCR) followed by a nested PCR
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amplification using primers that targeted the highly conserved 5'
untranslated region (on-line material and methods). Total RNA, from
either activated (P) or non-activated (N) cells, were prepared at the
indicated times. RNA from Huh7 cells stably expressing the HCV
replicon (Huh-Rep) (47) was used as positive control. RNA extracted
from PBLs from an HCV negative donor and yeast tRNA were used as
negative controls. B) Kinetics of HCV-RNA synthesis. PBLs from two
positive donors, MLL-038 (0) and MLL-039 (O), were stimulated by
method P. RNA was extracted at the indicated time of culture and the
level of HCV (-) strand RNA was determined using the Roche
LightCycler system. RNA levels were normalized against GAPDH and
are reported as a fold variation relative to the amount of (-) strand RNA
in non-treated PBLs. C, D) Bromo-uridine incorporation into de novo
synthesized RNA was detected in by immunofluorescence using an
anti-bromodeoxyuridine antibody. C) HCV positive donor MLL-069. D)
HCV negative donor.
Fig. 24 shows that HCV proteins are produced in
activated PBLs. PBLs were stimulated using method P. Protein extracts
were prepared following five days of activation. A) Extracts from either
treated (P) or non-treated (N) PBLs, from donor SB-1 were run side by
side with extracts from Huh-7 cells expressing the HCV replicon (Huh-
Rep) (47). NS3 was detected using polyclonal antibody K135. B)
Extracts from PBLs, either treated (P) or non-treated (N), from a HCV
negative donor were run side by side with extracts from donor SB-6.
NS3 was detected using monoclonal antibody 1 G3D2. C) Extracts from
Huh-7 cells and Huh-Rep, were run side by side with extracts, either
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treated (P) or non-treated (N), from an HCV negative and positive
donor. NSSB was detected using monoclonal antibody 5B-3B1 (48) or
5B-10 (IFA). D) Extracts from either treated (P or A) or non-treated (N)
PBLs from different HCV positive donors were run side by side with
extracts from an HCV negative donor, Huh-7 or Huh-Rep cells. NS3
was detected using monoclonal antibody 1 G3D2. E) Kinetics of NS3
synthesis following PBLC stimulation by methods P, S and PS. Extracts
were prepared on the indicated days and NS3 was detected using
monoclonal antibody 1 G3D2. F, G, H) Kinetics of NS3 accumulation in
donors MLL-001, MLL-002 and MLL-010 after stimulation using method
P. Extracts were prepared on the indicated days. An extracts from non-
treated cells was prepared either on day 3 (F and G) or on day 2 (H).
NS3 was detected using anti-NS3 monoclonal antibody 1 G3D2 (F and
G) or with an NS3 rabbit antiserum-RB (H). Actin or a non-specific
band, LC, identified by antibody 1 G3D2, were used as loading controls.
I, J, K. ) siRNA silencing of HCV RNA. Core-siRNA or a non-specific
RNA sequence (nsRNA) were electroporated into PBLs three days after
stimulation. Proteins and RNA were extracted 48 hr later. I) NS3 and
NSSB were detected with NS3 rabbit antiserum-RB and 5B-3B1
monoclonal antibody (48), respectively. Actin was used as an internal
control. J) RNA levels were quantified by real-time PCR (method I,
materials and methods). Absolute copy number of the HCV (+) strand
transcripts (D) and the amount of GAPDH (O) RNA are shown. K) HCV
RNA amounts were normalized against GAPDH. The ratio of
HCV/GAPDH was determined for the nsRNA and assigned an arbitrary
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value of 100. The Core-siRNA HCV/GAPDH ratios are expressed
relative to the negative control.
Fig 25 shows that HCV Core protein was detected by
indirect immunofluorescence in day 3 stimulated (P) PBLs from MLL
059, using the RR8 polyclonal antibody. Stimulated PBLs from an HCV
negative donor were used as a control.
Fig. 26 shows that HCV is released from activated
HCV positive PBLs. A, B) Supernatant from stimulated PBLs (method
P) was collected and sedimented through a 20% sucrose cushion. A)
Sedimented proteins were resolved by SDS 15%-PAGE, transferred to
a nitrocellulose membrane (overnight, 30V) and detected using
MAB255P monoclonal anti-core antibody (Maine Biotechnology
Services, Inc.). HCV (-) corresponds to the negative control. B) RNA
was analyzed by nested RT-PCR. RNA from Huh-Rep was used as a
positive control. RNA from yeast tRNA, Huh-7, and an HCV negative
donor were used as negative controls. C) PBLs from donor SB-5 were
stimulated using methods B, P, and PS. Five days following activation,
the supernatant was collected and sedimented through a 20% sucrose
cushion. The quantity of HCV RNA was determined by real-time RT-
PCR on the ABI Prism 7700 Sequence Detection System. D) Following
metabolic labeling (35S Met/Cys) of PBLs from donor MLL-035, the
supernatant was sedimented through a 20% sucrose cushion. The
sediment was resuspended and analyzed by a flotation gradient.
Collected fractions were resolved on a SDS-15% PAGE, transferred to
a nitrocellulose membrane and exposed to a Kodak Biomax MR film. E)
Fractions were concentrated and HCV E2 glycoprotein visualized by
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Western bolting using monoclonal anti-E2 1864 (450-470AA) antibody.
F) RNA was extracted from the gradient fractions of Fig 4E. and the
absolute quantity of HCV RNA was determined by real time RT-PCR.
G) Fractions 1-4 (L) and 5-11 (H) from the flotation gradient were
concentrated and pooled. Proteins were resolved on a SDS-15%
PAGE. HCV E2 glycoprotein was detected using monoclonal antibody
1864 (450-470AA). Core protein was visualized using monoclonal anti-
core 515S (20-40AA) antibody. H) Activated PBLs from donors MLL-
059 and MLL-064 were metabolic labeled for 12h with 35S-Met/Cys or
32P-orthophosphate. Supernatants were sedimented through a 20%
sucrose cushion. The sediments were resuspended and analyzed by a
flotation gradient. The amount of incorporated radioactivity in each
fraction of the gradients was determined in a Beckman LS 6500
scintillation counter.
Fig. 27 shows that virus released from activated HCV
positive PBLs is infectious. A) Schematic representation of the co-
culture chambers used in these experiments. B) MT-4 cells were co-
cultured with either treated (P) or non-treated (N) MT-4 cells, PBLs from
two HCV negative donors or PBLs from donors SB-2 or SB-7. Extracts
were prepared following six days of co-culture. NS3 was detected using
monoclonal anti-NS3 antibody 1 G3D2. LC indicates a non-specific band
used as a loading control.
Figure 28 shows Bromo-uridine incorporation into de
novo synthesized RNA and detected by immunofluorescence using an
anti-bromodeoxyuridine antibody in PBLs from donor MLL-065.
Figure 29 shows the HCV replication cycle.
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Figure 30 shows a protocol to detect HCV RNA in
PBLs.
Figure 31 shows the detection of HCV protein by
immunoprecipitation.
Figure 32 shows the detection of HCV protein by
Western Blot.
Figure 33 shows immunofluorescence of HCV (-)
Control Polyclonal-anti Core RR8 (M. Kohara).
Figure 34 shows immunofluorescence of MLL-059
Anti-Core RRB.
Figure 35 shows immunofluorescence of MLL-059
Anti-Core RRB.
Figure 36 shows immunofluorescence of MLL-059
Anti-Core RRB.
Figure 37 shows immuno-electronmicroscopy of HCV
protein using an anti NS3 antibody.
Figure 38 shows electron microscopy of cells showing
HCV viral particle assembly.
Figure 39 shows an embodiment of a scheme for virus
partial purification.
Figure 40 shows density determination of HCV viral
particles purified according to Fig. 39.
Figure 41 shows that PBMC generate two HCV
subpopulations that can be partially purified by density gradient.
Figure 42 shows an embodiment of a protocol to
assess infectivity of isolated HCV.
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Figure 43 shows EBV-transformed B-Cell lines express
HCV proteins when stimulated.
Figure 44 shows HCV(-) PBLs are infected with HCV
when co-cultured with stimulated HCV(+) B-cell lines.
Figure 45 shows mechanisms that could explain the
HCV-activation results.
Figure 46 shows Crosslinking to the HCV IRES.
Figure 47 shows PBMCs Activation and HCV IRES
Crosslinking pattern.
Figure 48 shows Crosslinking competition to the HCV
IRES.
Figure 49 shows Crosslinking competition to the HCV
IRES.
Figure 50 shows Crosslinking competition to the HCV
IRES.
Other objects, advantages and features of the present
invention will become more apparent upon reading of the following
non-restrictive description of preferred embodiments with reference to
the accompanying drawings which is exemplary and should not be
interpreted as limiting the scope of the present invention.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
The existence of extrahepatic reservoirs of hepatitis C
virus (HCV) replication remains controversial. Several groups have
described the presence of hepatitis C virus (HCV) genomic sequences
(plus-strand) and replicative intermediate (minus-strand) in peripheral
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blood mononuclear cells (PBMC). The association of HCV RNA with
peripheral blood leukocytes has been documented since 1992 (Proc
Natl Acad Sci USA, 1992, 89:5477; J Virol. 1993, 67:1953; Hepatology
1996, 23:205; J Virol, 19976, 70:3325-9; J Virol 1996, 70:7219-23;
Antiviral Research 2001, 52:1-17). However, the specificity of the
methods used in these studies has been questioned. More recent
reports, which used an optimized negative strand-specific reverse-
transcriptase polymerase chain reaction (RT-PCR) assay, detected
negative-strand HCV only in PBMC taken from post-transplant or
human immunodeficiency virus (HIV)-coinfected HCV patients, and not
in PBMC from typical patients with chronic HCV infection. Of note, a
number of studies have also reported that human B and T cell lines are
capable of supporting a productive infection. However, the data
supporting viral production was only based on RNA detection (Proc.
Natl Acad Sci USA, 1992, 89:5477; J Virol 1993, 67:1953; Hepatology,
1996, 23:205; J Virol, 1996, 70:3325-9; J Virol 1996, 70:7219-23;
Antiviral Research 2001, 52:1-17). The validity of these data have been
questioned (Laskus et al. 1998, see below). Moreover, PBMC obtained
from HCV negative donors were successfully infected using HCV-
positive sera, demonstrating that PBMCs are permissive for HCV
replication in vitro (J Gen Virol 1995, 76:2485-2491 ). However,
replication of the virus therein was really low. Of note, only RNA was
detected. Thus, prior to the present invention, it remained unclear
whether HCV could actively replicate to workable levels in PBMCs.
Using an immunodeficiency (SCID) mouse model that
allow long-term survival of human hematopoietic cells Bronowicki et al.
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(1998) presented strong evidence for persistence of HCV RNA in
PBMCs obtained from HCV positive donors (Hepatology 1998, 28:211-
218). The susceptibility of PBMC to HCV infection has been
corroborated by in situ hybridization techniques showing both positive
and negative polarity RNA strands in circulating and/or bone marrow
recruited mononuclear cells. Recent reports have established that HCV
is in fact associated to B cells. Based on the model of Epstein-Barr virus
another B-cell-tropic virus, that remains latent while the host cell is
quiescent but is reactivated and enters a lytic replication phase once the
host cell is activated (J Virol Methods, 1988, 21:223-227; Annu Rev
Microbiol 2000, 54:19-48). Boisvert et al (2001 ) examined the possibility
that HCV could replicate in peripheral B cells, but under altered
physiological conditions, such as immunosupression or cellular
activation. The authors could not detect HCV replication in enriched B
cells obtained from HCV positive donors upon cell stimulation with
CD40L.
Considering the observations of Laskus et al. (1998)
showing the presence of active HCV replication in lymphoid tissue in
patients coinfected with HIV (not in non-HIV infected patients),
suggesting that co-infection of HIV would be required in HCV cell-based
assay, and those of Boisvert et al (2001 ), it was hypothesized that HCV
replication in peripheral blood leukocytes (PBML) requires cell activation
(e.g. in the mixture of the T-and B-cell population).
Until now, all studies of HCV replication have
concentrated on documenting the presence of the replicative
intermediate (minus-strand) RNA. However, the validity of these reports
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has been criticized because the presence of viral proteins was not
demonstrated. It stands to reason that in order for replication to occur,
protein expression is required. Therefore, in order to sustain the
observations relating to activated PBMCs, non-structural (NS) HCV
proteins were chosen as an indicator of viral replication. The studies
presented hereinbelow clearly demonstrated that PBMCs obtained from
HCV seropositive donors are able to support at least one cycle of viral
replication upon activation. For this a simple method that actively
induces virus replication within the infected cell was developed.
Most circulating leukocytes are in a resting state, but
remain responsive to mitogenic sigal that can induce cell activation.
Lymphocyte activation in response to extrinsic signals results in either
progression through the cell cycle, or activation of proapoptotic
pathways) (Cell 1991, 65:921-923; Science 1996, 274:1664-1672).
Lymphocyte activation correlates with a strong increase in translation
rates and expression of translation initiation factors (J Immunol. 1998,
160: 3269-3273). As shown therein, the change in the cellular
environment associated with immune activation could induce HCV
protein synthesis and initiate a cascade of events leading to an
impaired cell cycle and an enhanced viral replication.
The activation of PBMCs (or PBLCs) is achieved using
at least one mitogenic (or activating agent). In one particular
embodiment, the activating agent is a mixture of antigen-nonspecific T
and/or B cell activators (Anti-CD3 antibody, phytohemagglutinin (PHA),
CD40L, Staphylococcus aureus crown I (SAC), IL2 and IL4). Of course,
it will be realized that other T and B cell activating agents exist and are
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well-known in the art. Such agents could be used in the methods and
culture systems of the present invention. In one particular embodiment,
Ag-specific T and/or B cell activating agents could also be used. It will
also be understood that the present invention provides assays which
can be used to identify further activating agents, mixtures thereof or
other nutrients which can further activate the HCV-producing cells of the
present invention and/or promote a longer survival thereof in culture.
HCV non-structural proteins (NS3 and NS5) were
detected by Western blot analysis (data not shown). Virus-like particles
could be detected within the infected cells by electron microscopy
demonstrating that viral proteins are assembling (data not shown). Viral
particles could be isolated from the PBMCs supernatant. The presence
of virus was evidenced from Western blot (anti-Core) analysis and
genomic RNA detection by real time RT-PCR, this observation shows
that upon assembly viral particles were actively being liberated to the
supernatant.
Moreover, using a co-culture method it was
demonstrated that the HCV particles produced in PBMC could infect
other cells. Non-limiting examples thereof include Huh-7 (liver), Daudi
(B-cell) and MT4 (T-cell) cell lines. Thus, not only can HCV replicate,
and assemble in the tissue culture system of the present invention, it
can also infect other cells. Infection was monitored by detection of viral
RNA (real time RT-PCR). The results generated by these experiments
will have a significant impact on the testing of anti-HCV agents. Of
course, it also serves as a proof of principle that PBMC are able to
sustain HCV infection and generate infective HCV. Moreover these data
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strongly suggest that both the serum and PBMCs obtained from HCV
positive donors can be used as a source of infectious virus to infect
naive cells such as monocyte and/or monocyte-derived dendritic cells
(DCs). Therefore, the instant invention which enables the infection of
cells with HCV is by itself a significant achievement.
A novel tool for developing a HCV vaccine
Adoptive transfer of donor-derived virus-specific T
cells generated in cultures with antigen-bearing autologous monocyte-
derived dendritic cells (DCs) has attracted considerable attention as a
promising tool to generate a strong immune response (Int. J. Cancer.
2009, 94:459-73; Exp. Hematol. 2007, 29:7247-55; Trends Mol. Med.
2007, 7 :388-94). This technique has not only proved useful as an
alternative anti-cancer strategy but also as a novel anti-virus therapy.
For example, when DCs were pulsed with human cytomegalovirus virus
(HCMV) antigen and cocultured with autologous peripheral blood
lymphocytes from HCMV-seropositive individuals, there was an
increase in the numbers of cytolytic T cells. This technique was used to
enhance immunity in HCMV-seropositive transplant patients (Blood.
2000, 97: 994-7000).
Now having developed a technology to infect cells with
HCV, it becomes possible to adapt the dendritic cells (DCs) technology
mentioned above, to generate T-cell responses to HCV. Advantages for
using DCs for this purpose include: i) they are considered the most
potent of the antigen-presenting cells (APCs) (Blood. 7997, 90:3245-
3287; Nature. 7998, 392:245-252); ii) their role in resistance against
experimental malignancies and infections is well documented (J.
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Immunol. 1998, 161:2094-2098; J. Virol. 1998, 72:3812-3818); iii) DCs
can be easily generated from bone marrow, cord blood, and peripheral
blood; iv) DCs have the unique ability to process exogenously supplied
antigen efficiently and present peptides on both class 1 and class 2
HLA molecules along with an array of costimulatory molecules (Nature.
1998, 392:245-252; Nature. 1999, 398:77-80). The presentation of both
helper and CTL-defined epitopes suggests that both CD4+ and CD8+
HCV-specific T cells will be generated. This will allow both the
generation of cytolytic effector function and the potential for re-
establishment of longer-term immune memory, which may be important
in preventing subsequent viral reactivation; vi) The lack of an absolute
knowledge of the presented peptides means that this technique can be
used for patients of any HLA type and will trigger T-cell reactivity to
undefined immunogenic determinants, thereby allowing a greater
potential for augmentation of a broader T-cell response. It is thus
expected that this will reduce the possibility that selective pressure will
be applied to HCV in vivo. Based on the foregoing, it is predicted that
the approach described herein (together with possible adaptations by a
person of ordinary skill using the knowledge in the art) will contribute
significantly to the design of a vaccine therapy towards HCV infection.
The present invention is illustrated in further detail by the
following non-limiting examples.
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CYA1111~1 C A
Robust Hepatitis C virus replication in peripheral blood
lymphocytes from infected donors
There is considerable evidence that hepatitis C virus (HCV)
resides in an extrahepatic reservoir. Although peripheral blood
lymphocytes (PBLs) have been suspected of harboring HCV, virus
production was not achieved in these cells despite many attempts.
Here, we show that PBLs from HCV positive, injection drug users,
harbor the virus and support viral replication. HCV replication was
activated by ex vivo cell stimulation, with the use of a mixture of T and B
cell activators. The presence of viral positive and negative RNA strands
and HCV proteins is documented. Virus particles were isolated from cell
supernatant and analyzed by density gradients centrifugation. Virus
structural proteins and viral RNA could be readily detected in the
supernatant of activated PBLs by Western blotting and real time RT-
PCR, respectively. Virus particles contain de novo synthesized genomic
RNA and structural proteins as shown by metabolic labeling with 32P-
orthophosphate and 35S-labeled aminoacids. Finally, HCV particles,
released from cells, are infectious as demonstrated by co-culturing.
Studies using this novel HCV replication system should contribute to the
understanding of the virus life cycle, host-virus relationship,
pathogenesis and importantly to the discovery and validation of new
anti-HCV agents.
Hepatitis C virus (HCV) is a significant etiologic agent of
chronic liver disease (1 ). It is estimated that more than 170 million
people world-wide are seropositive. About 85% of primary infections
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become chronic, and ~20% of patients with chronic HCV develop
serious complications, such as liver cirrhosis, end-stage liver disease,
hepatocellular carcinoma, and death due to liver failure (2). To date,
there is no vaccine against HCV and the most effective therapy is
treatment with peginterferon in combination with ribavirin (3, 4). The
search and validation of novel HCV drugs is severely hampered by the
lack of a robust cellular system that supports virus replication. These
facts cast HCV as a human pathogen of extreme medical significance.
HCV is an enveloped RNA virus of the Flaviviridae family,
classified within the Hepacivirus genus. It contains a 5'uncapped
positive strand RNA genome of 9.4 kb, that possesses two overlapping
open reading frames: one is translated into a single polyprotein of 3010
aminoacids, while the other yields a 17 kDa protein (5-7). The viral
polyprotein is processed to generate at least 10 different structural and
nonstructural proteins (5, 6). The genome of HCV is highly
heterogeneous and the virus circulates as quasispecies in a single
infected individual (8). HCV is primarily hepatotropic, but it has also
been implicated in lymphoproliferative diseases such as mixed
cryoglobulinaemia, B-cell non-Hodgkin's lymphoma, and Sjogren's
syndrome (9). The case for HCV replication in PBLs is suggested by the
following observations: a) PBLs from HCV positive donors are capable
of transmitting viral infection when inoculated into chimpanzees (10),
and b) HCV minus-strand RNA can be detected in PBLs from HCV
carriers upon injection into SCID mice (11). However, despite the
growing evidence that supports HCV entry into PBLs, viral RNA
synthesis is still a matter of debate and virus replication in PBLs has not
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been demonstrated (9, 12). Detection of HCV genomic sequences
(plus-strand) and replicative intermediates (minus-strand) in PBLs from
chronically infected donors (13-16) or infected chimpanzees has been
reported (17, 18). But, the presence of viral proteins or virus particles
has never been documented. To examine HCV extrahepatic replication,
we used PBLs from seventy-eight HCV positive, HIV-negative, injection
drug users (IDUs; all obtained with written consent; table S1 detailing
the available information on the participants is included in the on-line
supplement). PBLs from the IDUs were treated with a mixture of T and
B cell activators to show replication of HCV and infectivity of the de
novo produced virus. The rationale behind the selection of IDUs as a
source of PBLs is addressed below.
HCV (+) and (-) strand RNA and viral proteins are produced de
novo in activated PBLs.
Viral RNA was detected in non-stimulated and stimulated
PBLs from a HCV positive donor by nested RT-PCR (Fig. 23A). Viral
RNA was not detected in HCV negative donors or in negative controls
(Fig. 23A; Note that nested RT-PCR is neither strand specific nor
quantitative). These results confirm early evidence showing that PBLs
harbor HCV RNA (12-16). To obtain quantitative results, total RNA
extracted from activated cells was subjected to a strand specific real
time RT-PCR analysis to demonstrate the presence of HCV (-) RNA
strand (Fig. 23B). The kinetics of HCV RNA induction was similar in
activated PBLs from two carriers, MLL-038 and MLL-039 (Figs. 23B).
The amount of (-) strand RNA increases slightly, but significantly, early
(1 day) upon cell activation then decreases at later times (1-3 days), but
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increases again afterwards (5-7 days) (Fig. 23B). Although these
kinetics are not readily explained, the presence of HCV (-) RNA strand
supports the notion of virus replication in PBLs. HCV life cycle is
cytoplasmic (5), therefore, to show that RNA synthesis occurs in the
cytoplasm, bromo-substituted uridine (BrU) together with actinomycin D
(ActD) was added to stimulated PBLs (19). Incorporated BrU was
detected by immunofluorescence using antibodies to 5'-
bromodeoxyuridine (19). Cytoplasmic RNA synthesis was detected in
activated HCV positive PBLs from two HCV positive donors (Fig. 23C
and 28). In contrast, no incorporation of BrU was detected in ActD
treated PBLs from a HCV negative donor (Fig. 23D). In the absence of
ActD, strong incorporation of BrU in newly synthesized RNA was
detected in the nucleus (Figs. 23C and D). Taken together, our data
clearly show that HCV RNA synthesis occurs in activated PBLs from
IDUs.
Next, we wished to document HCV-directed translation in
PBLs. Upon mitogen stimulation of HCV positive PBLs, NS3 and NSSB
proteins were readily detected by Western blotting using several
different antibodies (Figs. 24A-C). The quantity and kinetics of NS3
appearance was dependent on the particular procedure of stimulation
(Figs. 24D and E) and the HCV carrier (Figs. 24F-H). This suggests that
the kinetics of HCV protein production in stimulated PBLs is modulated
by host factors. To show that the appearance of the proteins, which
interact with the NS3 and NSSB antibodies, is dependent on HCV
replication, we used siRNA against the core protein coding sequence
(Figs. 241-K). NS3 and NSSB levels decreased drastically following
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electroporation of the Core-siRNA in a dose-dependent manner when
compared a to a non-specific unrelated RNA (inverted 4E-T-siRNA; see
Materials and Methods, below) (Fig. 241). siRNA silencing resulted from
a decrease of HCV RNA, as compared a to a non-specific RNA, as
demonstrated by real-time PCR quantification (Figs. 24J, K).
The presence of core protein in the cytoplasm of activated
HCV positive PBLs was further confirmed by indirect
immunofluorescence (Fig. 25). Based on surveying 10 fields, we
estimate that 1 to 3 % of the cells expressed high levels of HCV core
protein. Taken together, the data demonstrate that translation of the
HCV (+) strand RNA (Figs. 24 and 25 and transcription of the (-) strand
RNA (Fig. 23) occur in activated PBLs.
To examine whether HCV particles are produced and
released into the culture medium, the supernatant from PBLs was
harvested and sedimented by centrifugation through a 20% sucrose
cushion. The presence of HCV particles was demonstrated by Western
blotting with an anti-core monoclonal antibody, MAB225P (Fig. 26A).
Similar results were obtained when other anti-core antibodies
(monoclonal 515S (20) and polyclonal RR8) were used (data not
shown). Viral RNA co-sedimented with the HCV core protein as
demonstrated by nested RT-PCR (Fig. 26B). PBLs were stimulated by
methods B, P and PS (detailed in Materials and Methods) and genomic
RNA isolated from the cell supernatant was quantified by real time RT-
PCR (Fig. 26C). Consistent with the protein data shown above, the
amount of viral RNA in the cell supernatant varied among the different
stimulation procedures (Fig. 26C). To further support the evidence for
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virus production, particles were examined following metabolic labeling
with 35S-methionine/cysteine (Figs. 26D-G). Particles were sedimented
through a 20% sucrose cushion, resuspended and floated on OptiprepT""
density gradients (21 ) (Fig. 26D). The sedimentation range of the
labeled particles (1.13-1.215 g/ml) was similar to that reported by others
(22-28). HCV-E2 protein was present in the particles as determined by
Western blotting using monoclonal anti-E2 1864 (Fig 26E). The
absolute quantity of HCV (+) strand RNA present in each faction was
determined by real-time RT-PCR (Fig. 26F). The HCV genomic RNA
and E2 co-sedimented through the density gradient (Fig. 26F).
Interestingly, Western blotting revealed that the HCV core protein
sedimented throughout the gradient (data not shown). To further
examine this behavior fractions 1-4 and 5-11 from the gradient were
pooled and the presence of HCV E2 and core proteins was determined.
The high (H) density complexes (1.111 to 1.215 g/ml) contained E2 and
core protein and are likely to represent viral particles, while the low (L)
density complexes (1.006 to 1.1 g/ml) contained only core (Fig. 26G).
The biological significance of this observation is not immediately clear.
However, it was suggested earlier that different types of particles are
found in serum from chronically infected individuals (23, 29), and in the
supernatant of cells expressing the full length HCV RNA (21 ). RNA and
proteins were isolated following metabolic labeling with 35S-
methionine/cysteine or 32P-orthophosphate (the latter in the presence of
ActD) to determine whether the viral proteins and genomic RNA isolated
from the different fractions was synthesized de novo. Supernatant was
collected after labeling (Fig. 26H). Significantly, labeled RNA and
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proteins co-sedimented through the density gradient (Fig. 26H). Thus,
the results show that virus particles containing de novo synthesized
proteins and genomic RNA were released to the supernatant.
HCV particles released from HCV positive PBLs are infectious.
It was highly pertinent to examine whether the HCV particles
released from stimulated PBLs are infectious. As it is impossible to
estimate the real ratio of infectious to non-infectious virus particles
produced by activated PBLs, a co-culture strategy, in which two
different cell types in two chambers are separated by a 0.45 pm
polyethylene terephthalate track-etched membrane, was used (Fig.
27A). The HTLV-1 transformed T cell line, MT-4 was chosen as the
target cell of infection (30-33). Total RNA was extracted from infected
cells and the quantity of HCV RNA was determined. Strikingly, viral
RNA (average of 1600 copies/~,g of total RNA; as determined by real-
time RT-PCR, data not shown) and NS3 protein were detected in MT-4,
upon co-culture with activated PBLs (Fig. 27B), demonstrating that the
released viral particles are infectious and that cell-to-cell contact is not
required for infection. No viral proteins were detected in MT-4 cells
when co-cultured with PBLs from two HCV negative donors (Fig. 27B).
In conclusion, we demonstrated that robust HCV replication
occurs in PBLs. Without being limited to a particular theory, our success
in showing replication, while earlier studies failed, can be attributed to
two important factors: activation of the PBLs and the use of IDU donors.
IDUs were selected because they experience a long-term altered
immune response (34-36) and HCV replication in PBLs has been
associated with induced immunodeficiencies (37-39). Drugs have a
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variety of effects on the immune system including suppressed cell-
mediated immunity (34-36). This is reflected in a depressed level of T-
dependent antibody production by B lymphocytes and in an alteration of
T lymphocyte function. The clinical consequences of this suppression
include an increase in the incidence of viral infections such as HIV and
HCV (40-42). Thus, our observations support the notion that
immunosuppression in combination with cell activation act as
"cofactors" in HCV pathogenesis. Studies including HCV infected
individuals who are not IDUs and non-IDU immuno-suppressed
individuals are required to support this hypothesis.
It is most probable that HCV enters lymphocytes during the
primary infection and remains latent in resting cells. Viral latency is well
documented for Epstein-Barr virus (EBV), which remains dormant in
quiescent host B-cells, but enters a lytic replication phase once the cell
is activated (43, 44). Interestingly, EBV can also infect T cells (45, 46).
Therefore, a number of intriguing parallels can be drawn between the
HCV and EBV life cycles. It is conceivable that like in EBV infection, T
cell immunity plays a critical role in limiting the number of HCV infected
PBLs and that during a sustained immunodeficiency state, such as that
manifested in IDUs, clonal proliferation of virus infected cells will be
favored. Most importantly, in this report we describe a simple cell-based
system that supports robust HCV replication. The implications of these
findings are paramount for several reasons. First, they clearly implicate
PBLs in HCV pathogenesis. Second, they provide a model that should
be useful in the quest to gain understanding of the HCV life cycle, host-
virus relationship, viral infectivity and in the discovery and validation of
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novel anti-HCV agents. For the latter purpose we have established
EBV-transformed B-cell lines from HCV-infected donors which should
facilitate the discovery of anti-HCV drugs (see below).
EXAMPLE 2
Materials and Methods
Antibodies. NS3 rabbit anti-serum-RB was provided by Dr. R.
Bartenschlager, Department of Molecular Virology, Institute of Hygiene,
University of Heidelberg, Germany. Monoclonal anti-NSSB, 5B-3B1 was
from Dr. D. Moradpour, Department of Medicine II, University of
Freiburg, Germany. Monoclonal anti-NS3 antibody, 1 G3D2 and
polyclonal anti-NS3, K135 were from Dr. D. Lamarre (Boehringer
Ingelheim Canada Ltd). Monoclonal anti-E2 1864 (450-470AA),
monoclonal anti-5B 10 (IFA), monoclonal anti-Core 515S (20-40AA),
and Core rabbit anti-serum RR8 were developed in The Tokyo
Metropolitan Institute of Medical Science. Monoclonal anti-Core
(Cat.No.: MAB255P; Lot:hcv-core-2-4) was purchased from Maine
Biotechnology services, Inc. Monoclonal anti-human F-Actin (ab205)
was purchased from Abcam Limited. Monoclonal anti-human (i-Actin
(clone AC-15) was purchased from Sigma-Aldrich CO. Anti-
Bromodeoxyuridine monoclonal antibody-Alexa fluor 488 conjugated,
and goat anti-rabbit Alexa fluor 594 conjugated were purchased from
Molecular Probes, Inc..
Blood Donors and lymphocyte purification. Participants were
recruited through the drug addiction unit of the Saint-Luc Hospital of the
Centre Hospitalier de I'Universite de Montreal (CHUM) and the Saint
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Luc Cohort study. Donors provided a written informed consent
approved by the CHUM Review Board before having their blood drawn.
Individuals from both sexes (87% males) were enrolled in this study
between 2001 and 2003. Their mean age was 42.1 years (sd ~ 8.8) and
the average time since their first injection was 16.5 years (sd ~ 9.6).
80% of the donors reported injecting drugs during the 6 month period
before blood was withdrawn for this study. Cocaine and opiates were
the most frequently used drugs, with 77% and 34.6% use, respectively.
All HCV positive donors tested positive in a serological screen for HCV
antibodies performed in the laboratory of microbiology at Saint-Luc
Hospital of the CHUM using two Enzyme Linked Immunosorbent
Assays (ELISA, AxSym and Cobas). Presence of HCV was confirmed
by HCV-RNA detection when ELISA data were discordant. All
participants recruited for this study were HIV-1 and HIV-2 negative.
Serological screening for HIV antibodies was performed in the
microbiology laboratory at Saint-Luc Hospital, CHUM, with an enzyme-
linked immunosorbent assay (ELISA). Similar procedures were used to
verify the HCV negative donors. HCV negative donors (six) were
recruited from the different participating laboratories as well as from the
support staff responsible for the St. Luc Cohort. Peripheral blood (20
ml) was collected from HCV positive IDU or HCV negative donors into
EDTA-containing Vacutainer tubes (Becton Dickinson).
Polymorphonuclear leukocytes and red blood cells were separated by
centrifugation over a density gradient (Lymphocyte separation medium,
cellgro~). Monocytes were then removed by plastic adherence under
serum free conditions as described in The Current protocols of
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Immunology. When required, cells were frozen in 10% DMSO
containing FCS and stored at -80°C prior to monocyte separation. Total
PBLs were cultured in 24-well plates at 1x106 cells per ml in RPMI 1640
supplemented with 10% heat-inactivated FCS and antibiotics.
PBLs stimulation. Mitogens were added to the media (RPMI 1640,
10% FBS, and antibiotics) upon starting the culture and maintained
throughout the experiment. The protocols used for PBCLs stimulation
were as follows: Method A, PBLs were grown in the presence of
irradiated L4.5 cells (murine fibroblasts expressing the CD40 ligand,
CD154) as described (49). Method B, 1 p,g/ml of anti-CD3 and 200
U/ml of IL-2 (Sigma-Aldrich CO) were added. Method P, 3 pg/ml
phytohemagglutinin (PHA, Sigma-Aldrich CO), and 200 U/ml IL-2 were
used. Method PS, 1:104 vol/vol of Staphylococcus aureus Cowan fixed
cells (SAC, Calbiochem) in combination with phytohemagglutinin and
200 U/ml IL-2 were added to the media. Method S, 1:104 vol/vol of SAC
and 200 U/ml of IL-4 (Sigma-Aldrich CO) were used. Cell activation was
verified by flow cytometry. Cells were rinsed twice with 1 ml cold
phosphate buffered saline (PBS: 137 mM NaCI, 2.7 mM KCI, 4.3 mM
Na2HP04, 1.4 mM KH2P04, pH 7.4) and fixed in 80% ethanol/PBS for
min at 4 °C. PBS (2 volumes) was added and cells were pelleted by
centrifugation. Cells were rinsed twice with 2 ml PBS and then
resuspended in 0.5 mL PBS containing 0.2 ~g/ml RNase A and
incubated for 40 min at 37°C. Propidium iodide was added to a final
25 concentration of 1.2 ~,g/ml and samples were analyzed by flow
cytometry using a single laser FACS instrument (Becton-Dickinson)
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combined with the CeIIQuestT"" software.
RNA purification. Total RNA was extracted from cells using TrizoIT""
(Invitrogen) according to the manufacturer's protocol. Yeast tRNA (1
mg/ml) was added as a carrier. RNA was resuspended in nuclease-free
water (Sigma-Aldrich CO). Total RNA was quantified by
PhosphoimagerT"' (STORM system, Molecular Dynamics) using the
RiboGreenT"~ RNA Quantification Kit (Molecular Probes, Inc).
Nested RT-PCR. HCV-RNA was detected in cells by a reverse
transcription-polymerase-chain reaction (one step RT-PCR reaction, 45
cycles, Qiagen) against the highly conserved 5' untranslated region
(sense primer from nucleotide 13 to 38 and the anti-sense primer from
nucleotide 383 to 359) of the HCV genome (strain H77 pCV-H77C,
EMBL:AF011751, MEDLINE: 97385173) followed by a second round of
amplification, nested PCR (45 cycles, sense primer from nucleotide 59
to 82 and the anti-sense primer from nucleotide 307 to 285, strain H77
pCV-H77C) using Taq DNA polymerase (MBI Fermentas). ~3-Actin was
amplified (30 cycles) using the sense primer 5'-
GTGGGGCGCCCCAGGCACCA-3' and antisense primer 5'-
GTCCTTAATGTCACGCACGATTTC-3'.
Real Time RT-PCR. Two methods were used to detect and quantify
HCV RNA. Method I: Reverse transcription was carried out at 50°C
for
20 minutes in a one-tube two-step RT-PCR reaction with
ThermoscriptT"" reverse transcriptase (Invitrogen), 10 ~M of HCV-
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tagged strand-specific RT primer and 100 ~.M of anti-sense GAPDH
primer (Table S2). The reverse transcriptase was inactivated by heating
for 5 minutes at 95°C and PCR (22 cycles) with Platinum Taq DNA
polymerase was performed in a Trio-thermocyclerT"" (Biometra): at 94°C
for 45 s, 60°C for 60 s, 68°C for 2 min. The first round PCR
products
were then amplified for 40 cycles in the Roche LightCycIerT"'
instrument: denaturation at 95°C for 60 s, and amplification and
quantification at 95°C for 15 s, 60°C for 10 s with a single
fluorescence
measurement, 72°C for 15 s. Real-time quantification of RNA copy
numbers for HCV and the human GAPDH gene was based on a set of
eight logio external standards covering 108 to 10' plasmid copies of a
pCRI I vector containing the 5' HCV leader (genotype 1 a) and the
GAPDH normalization PCR amplicons which were run in parallel with
the test samples. RNA extracted from PBLs of a HCV negative donor
was used as control. As a reaction control for the strand-specific signal,
the RT step of the RT-PCR was carried out without a HCV-tagged
primer. The presence of HCV non-structural proteins in the cell
samples used for RNA preparation was confirmed by Western blotting
(data not shown). Method II: Real-time RT-PCR was performed on the
ABI Prism 7700 Sequence Detection System using the TaqMan EZ RT-
PCR Kit (Applied Biosystems). RNA sample (5 p,l), combined with 45 NI
of Reagent Mix, was used for the Real-Time RT-PCR reaction. In vitro
transcribed replicon RNA was used as a standard to determine HCV
copy numbers (1 pg of replicon RNA equals 2.15x10" HCV copies). The
RNA copy number was normalized (RiboGreen RNA quantification,
Molecular Probes Inc.) and expressed as genome equivalents per ml of
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total supernatant.
Bromo-uridine labeling: Bromo-uridine (BrU, 5-Bromouridine 5'-
Triphosphate, Sigma-Aldrich CO) was incorporated into PBLs using a
modified version of the procedure of Haukenes et al. (50). BrU (10 mM)
was incubated with an equal volume of LipofectamineT"" 2000
transfection reagent (Invitrogen) for 30 min at room temperature and
added to 250 ~I of cells resuspended in optimem medium (Invitrogen) in
a 1:1 (vol/vol) ratio. The BrU/LipofectamineT"" 2000 mixture was added
to cells 6 h after activation. Cells were incubated for 5h, washed and
resuspended in mitogen (method P) containing culture medium. Cells
were collected after a 12 h incubation period at 37 °C in a 5% C02
environment. When actinomycin D (ActD, Sigma-Aldrich CO) was used,
cells were incubated with the drug (5 ~g/ml) starting 30 min prior to the
addition of BrU. ActD was maintained throughout the experiment.
Immunofluorescence. Immunofluorescence was performed on 5x104
cells. Following cytospin for 7 min at 1100 rpm in a Cytosin 2
(Shandon), cells were dried for 30 min at room temperature and fixed
for 30 min at -20°C in a mixture of acetone and methanol (1:1 vol/vol).
Cells were blocked for 30 min at room temperature in 10 mM Tris-HCI
pH 8.0 containing 1 % BSA. Slides were washed 3 times with PBS and
incubated at room temperature for 2 h with the polyclonal anti-core RR8
antibody (1/50) or overnight at 4°C with the anti-bromo-deoxyuridine
Alexa Fluor 488 conjugate antibody (2 ~g/ml) in a humidified box. Slides
were washed 3 times with PBS. For Core detection, slides were
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incubated 1 h at room temperature with an Alexa-594 conjugated
antibody (dilution 1/250). DAPI staining was performed for 7 min at
room temperature (1 ~g/ml final concentration). Mounted slides
(Permount mounting medium, Fisher Scientific) were stored overnight at
4°C prior to analysis. Conventional epifluorescence micrographs were
obtained on a Zeiss Cell Observer system equipped with an Axiovert
200 M microscope using the 100X oil lens. Images were digitally
deconvoluted with the AxioVision 3.1 software using the Nearest
Neighbor deconvolution method that uses the Agard's formula.
Western Blots. Proteins extracts were prepared by sonification in RIPA
buffer (150 mM NaCI, 1 % NP-40, 0.5% DOC, 0.1 % SDS, 50 mM Tris-
HCI pH 7.5) and quantified (BSA assay, BioRad). Proteins (10 p,g of
extracts from PBLs or 5 ~g of extract from Huh7 cells stably expressing
the HCV replicon (47)) were resolved on SDS-10% polyacrylamide gels
(PAGE) and transferred to 0.2 ~m Protran nitrocellulose membrane
(Schleider and Schuell) for 1 h at 100V. The membrane was blocked
with PBS containing 0. 5% Tween-20 (PBS-T) and 5% nonfat dry milk.
Blots were then incubated with the primary antibody for 2 h at room
temperature, washed 3 times with PBS-T and incubated for 1 h with a
horse radish peroxidase (HRP) conjugated secondary antibody. Blots
were visualized using an enhanced luminol reagent (ECL; PerkinElmer
Life Sciences Inc).
Radio labeling and gradient purification of virus particles. A total of
1 x106 activated PBLs were first preincubated in methionine- or
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phosphate-free RPMI for 30 min, and then incubated for 12 h in the
same media supplemented with [35S] protein labeling mix (1175
Ci/mmol) or carrier-free inorganic 32P (500 pCi/ml, H3P04, ICN
Biomedicals, INC), the latter in presence of ActD (5 ~,g/ml). Supernatant
was collected, cells and cellular debris was removed by low-speed
centrifugation at 1600 x g for 15 min at 4°C, followed by filtration
with
0.45 p.m pore size filter (Fisherbrand, Fisher scientific). Particles were
partially purified by ultracentrifugation through a 20% sucrose cushion
for a minimum of 6 h at 4°C (in Beckman L8-55 ultracentrifuge) at
35,000 rpm in a SW-41 rotor. Sediments were resuspended in serum
free RPMI and lodixanol (OptiprepT"", Invitrogen) was added to a final
concentration of 40% w/v (p=1.216). The sample was laid over a 60%
wt/vol OptiprepT"" solution (p=1.320 g/ml) and then overlaid with a linear
iodixanol gradient (p=1.038 to 1.205 g/ml) prepared in RPMI and spun
for 20 h at 4°C in Beckman L8-55 ultracentrifuge at 30,000 rpm using a
SW-41 rotor. Fractions were collected from the top of the tube and RNA
was prepared as described above. Half of the final RNA volume was
mixed with liquid scintillation cocktail (EcoLiteT"", ICN Biomedicals) and
s2P radioactivity was counted in a Beckman LS 6500 scintillation
counter. Proteins were extracted by directly adding 10X RIPA buffer to
a final concentration of 1X RIPA. 1/100th of the protein extract was
mixed with liquid scintillation cocktail and 35S radioactivity was
determined using a Beckman LS 6500 scintillation counter. 1/10 of the
protein extract was directly mixed with concentrated Laemmli sample
buffer, resolved on a SDS 15%-PAGE, and transferred to 0.2 ~m
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Protran nitrocellulose membrane over night at 30V. The membrane was
dried and exposed against Kodak BiomaxT"" MR film. The remaining
protein extract was concentrated by TCA precipitation (15% final).
Proteins were washed twice with ether, dried and dissolved in a solution
containing 3 M urea, 26 mM EDTA (pH 8), and 0.5 pg/ml of RNase A.
Samples were mixed with concentrated Laemmli sample buffer,
resolved on a SDS 10% PAGE and transferred to 0.2 pm Protran
nitrocellulose membrane for 1 h at 100V. Proteins were detected by
Western blotting as described above.
siRNA. The target sequence for the siRNA was chosen using the
Ambion web-based criteria. The selected RNA oligonucleotides, Core
(from nucleotide 371 to nucleotide 391, strain H77 pCV-H77C,
EMBL:AF011751, MEDLINE: 97385173) and the unrelated non-specific
RNA (inverted sequence for 4E-T from nucleotide 986 to nucleotide
1008; DDBJ/EMBL/GenBank database, accession No. AF240775),
were synthesized by Dharmacon Research (Lafayette, CO) and
handled according to the manufacturer's instructions. Varying amounts
(3 ~I or 5 ~I of a 20 ~M solution) of RNA duplexes were electroporated
using a Gene pulser~ II electroporator (BioRad), into 1x106 PBLs in
0.5 ml of serum free RPMI. Cells were treated with a pulse of 975 pF
and 300 V. Then 0.5 ml of RPMI containing 20% FCS was added and
the cells were seeded in a 24-well cell culture dish. Protein and RNA
extracts were harvested 48 h after electroporation. Immunoblots were
performed as described above using an NS3 rabbit antiserum-RB and
monoclonal anti-NSSB, 5B-3B1. HCV RNA levels were quantified by
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real-time RT-PCR using method I.
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Table Sl. Characteristics of the IDU donors, enrolled between March 2001 and
April
2003:
Participant Age Sex IDU Under IDU Opioids excl Cocaine
(years) duration Methadon (past 6 methadone (past 6
(years) a months) (past 6 months)
treatment months)
SB-1 41 male 22 yes no no no
SB-2 42 female 20 yes yes yes yes
SB-4 35 male 11 yes yes yes yes
SB-5 21 female 3 yes yes yes no
SB-6 32 male 1 yes yes yes yes
SB-7 45 male 18 yes no no no
MLL 001 48 male 31 no yes yes yes
MLL 002 39 male 3 no yes no yes
MLL 003 38 male 10 no yes yes yes
MLL 004 47 male 32 no yes yes yes
MLL 005 38 male 21 no yes no no
MLL 006 49 male 37 yes yes yes no
MLL 007 61 male 36 no yes no no
MLL 008 39 male 13 no no no yes
MLL 009 23 male 5 no yes no no
MLL 010 40 male 21 no no no yes
MLL 011 45 male 6 no yes no yes
MLL 012 48 male 14 no yes yes yes
MLL 013 49 male 24 no no yes no
MLL 014 41 male 18 no yes no yes
MLL 015 38 male 6 no yes yes yes
MLL 016 34 male 11 no no no no
MLL 018 42 male 13 no yes no yes
MLL 019 51 male 10 no yes no yes
MLL 020 38 male 13 no yes no yes
MLL 021 35 female 5 no no no no
MLL 022 43 male 29 no yes no yes
MLL 023 52 male 20 no yes no yes
MLL 024 37 male 13 no yes no yes
MLL 025 36 male 18 yes yes yes yes
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Participant Age Sex IDU Under IDU Opioids Cocaine
excl
(years) durationMethadon(past methadone (past
6 6
(years)a months)(past 6 months)
treatment months)
MLL 026 29 female 13 yes yes yes yes
MLL 027 52 male 11 no yes yes yes
MLL 028 45 male 6 no yes yes yes
MLL 029 42 male 6 no yes yes yes
MLL 030 43 male 10 no yes no yes
MLL 031 36 male 19 yes yes no yes
MLL 032 22 male 11 yes yes yes no
MLL 033 24 male 7 yes yes yes yes
MLL 034 52 male 26 no yes no yes
MLL 035 61 male 36 no yes no no
MLL 036 49 male 31 no yes no yes
MLL 037 57 male 36 no no no no
MLL 038 27 male 11 no yes yes yes
MLL 039 42 female 17 yes yes yes no
MLL 040 53 male 40 no yes yes yes
MLL 041 34 male 11 no no no yes
MLL 042 47 male 7 no yes no yes
MLL 043 42 female 23 no no no no
MLL 044 30 male 11 no no no yes
MLL 045 41 male 22 no yes no yes .
MLL 046 43 male 21 no yes yes yes
MLL 047 41 male 18 no yes no yes
MLL 048 47 male 22 no yes no yes
MLL 049 52 male 11 no no no yes
MLL 050 33 male 10 no yes no yes
MLL 051 45 male 30 yes yes no yes
MLL 052 33 male 8 no yes no yes
MLL 053 43 female 12 no yes no yes
MLL 054 46 male 22 no yes no yes
MLL 055 36 female 21 yes yes no yes
MLL 056 40 male 14 no no no yes
MLL 057 37 male 9 no yes yes yes
MLL 058 45 male 30 yes yes no yes
MLL 059 50 male 30 no yes yes yes
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ParticipantAge Sex IDU Under IDU Opioids Cocaine
excl
(years) durationMethadon(past methadone (past
6 6
(years)a months)(past 6 months)
treatment months)
MLL 060 35 male 12 yes yes yes no
MLL 061 46 male 7 no no no yes
MLL 062 48 male 11 yes yes yes yes
MLL 063 66 female 35 no yes no yes
MLL 064 38 male 3 no yes no yes
MLL 065 33 male 10 no yes no yes
MLL 066 48 male 11 yes no no no
MLL 067 46 male 11 no yes no yes
MLL 068 42 male 6 no yes no yes
MLL 069 42 male 23 no yes yes yes
MLL 070 44 male 11 no yes no yes
MLL 071 47 female 22 no yes no yes
MLL 072 61 male 16 no yes no yes
MLL 073 37 male 9 yes no no no
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Table S2. Probes and primers used in Real Time RT-PCR method 1.
Name
Orientation Used in: Target Nucleotide position in target
H250 sense 5' external HCV 64-84
HC110 antisense 3' external RT and HCV 456-475
1St round PCR
G.24 sense 5' external GAPDH 15-32
6.589antisense 3' externalGAPDH 581-597
H190 sense 5' internal HCV 142-161
C40 antisense 3' internalreal-time PCR HCV 385-405
6.174sense 5' internal GAPDH 166-182
G.S11antisense 3' internalGAPDH 502-520
297.P1sense FL1 probe HCV 274-297
300.P2sense FL2 probe Hybridization HCV 300-324
Probes
G.P1 antisense FL1 probeGAPDH 187-212
G.P2 antisense FL2 probeGAPDH 214-238
EXAMPLE 3
Established EBV-Transformed gRobust Hepatitis
Cell Line enablin
C virus replicationperipheral blood lymphocytes from infected
in
donors
It is show n herein that HCV can
naturally infect blood
cell
and can replicate
therein (Figs.
29-41 ). In order
to assess whether
the
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produced HCV was infectious the protocol of Fig. 42 was followed. We
show that HCV replicating in naturally infected PBLs was indeed
infectious. We further went on to generate an HCV exprssing cell line. In
an embodiment, we developed an EBV-cell line that is able to replicate
HCV.
B-cells from infected donors were identified as the cells
that harbored HCV virus. These cells were immortalized by EBV
infection. Interestingly, when grown under normal conditions, the EBV-
immortalized B cells from infected donors, do not produce detectable
amounts of HCV proteins. However, when stimulated (independent from
the stimulation procedure P, S or PS) virus proteins (NS3 and NS5)
become detectable (Fig 43).
Peripheral blood lymphocytes (PBLs) obtained from an
HCV negative donor can be infected by co-culturing with stimulated
EBV-transformed B-cells from an HCV positive donor (Fig 44). This
implies: a) PBLs are infectable, thus HCV has tropism for these cells, b)
HCV produced by the EBV-transformed B-cells from an HCV positive
donor is infectious.
Advantages of this system include:
a) EBV-transformed B-cells grow in culture. Therefore, a cell based
replication system for HCV has been developed. b) EBV-transformed B-
cells proliferate under normal culture conditions (RPMI 1640, Antibiotics
and 10% serum), but produce the virus only when stimulated. c) the
released virus is infectious. Therefore, this system can be used for HCV
receptor identification. d) This system should prove useful in the
discovery and validation of new anti-HCV agents at all levels of the virus
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life cycle (entry, protein synthesis, RNA replication, assembly and
release).
CONCLUSIONS
The present invention relates among other things to
the fact that: (1 ) HCV has PBMC tropism; (2) HCV can naturally infect
blood cells; (3) HCV can replicate in PBMCs and PBMLs; (4) HCV
replicating in naturally infected PBMCs is infectious; (5) HCV can
replicate in extrahepatic tissue; and (6) HCV has a latent phase during
PBMC infection, which can be ended by activation.
It is interesting to note that HCV replication is activated
upon immune response. Thus, a person of ordinary skill in the art will be
able to provide other methods of activation than those disclosed herein
(or complementary thereto) to activate HCV replication in PBMCs or
PBLCs, without undue experimentation.
The present invention provides the tools to study
hepatitis C virus replication in a simple cell culture based system. This
simple culturing tool is suitable for the search and validation of novel
HCV antiviral drugs and therapies (vaccine). The assays and methods
of the present invention enable the performance of screening assays to
identify antiviral agents. Of course, the assays can be highthroughput.
Compound libraries can now be used to identify candidate anti-HCV
agents. These assays can thus be used to generate lead compounds
for pharmaceutical anti-HCV formulations.
The novel replication system of the present invention,
in one embodiment, based on PBMCs (or PBMLs) is simple, does not
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require facilities other than those normally used for HIV research, and
allows experiments with the complete HCV. Thus, novel drugs and
therapies can be screened to target all the different stages of virus
replication such as virus entry, cytoplasmic replication (viral (-) and (+)
strand synthesis), viral protein synthesis, virus assembly, virus
trafficking, and virus release.
Although the present invention has been described
hereinabove by way of preferred embodiments thereof, it can be
modified without departing from the spirit and nature of the subject
invention as defined in the appended claims.
The present invention thus shows that: 1 ) HCV can
replicate in extrahepatic tissue; 2) HCV has PBL tropism; 3) Virus
produced by PBLs is infectious; 4) HCV has a latent phase during PBL
infection; 5) EBV-transformed HCV producing B cells were generated
(and thus demonstrate one means of generating established cell lines
which can produce HCV); and 6) HCV latency is most probably due to
the presence of an inhibitor of viral translation initiation.
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