Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
WO92/07001 PCI`/US91/07571
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HEPATITIS C VIRUS ANTIBODY
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The present invention relates to antibodies
which selectively bind to antigens associated with
hepatitis C viral particles isolated from infected
patients and to processes for isolating hepatitis C virus
from plasma including a procedure by which HCV is
partially purified. , `,
BACKGROUND OF THE INVENTION
It has been established that hepatitis C virus
or HCV (HCV has historically been referred to as non-A
non-B hepatitis or NANBH) may be transmitted by
contaminated blood and blood products. In transfused
patients, as many as 10% will contract post-transfusion
hepatitis. Of those patients contracting the disease,
approximately 90% will be diagnosed as having HCV. The
prevention of the t~ansmission of this form of hepatitis
by blood and blood products requires reliable, sensitive
and specific diagnostic and prognostic assays which may
be used to identify HCV carriers as well as contaminated
blood and blood products.
The development of these needed diagnostic and
prognostic assays has been burdened by the unavailability
of HCV-specific immunoreactive reagents. Progress in .
developing needed reagents has been particularly hampered
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WV92/07001 PCT/US91/07571
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by difficulties in identifying and isolating HCV antigens
and antibodies. See, for example, Wands, et al., U.S.
Patent 4,870,076, Hellings, vox San~, 51, Supp. 1:63-66
(1986), Llnke, et al. ~need cite: Production of
Monoclonal Antibodies Specific for Non-A, Non B Hepatitis
Infected Liver] Wands, et al., Proc. Nat'l. Acad. Sci.,
83:6608-6612 (1986), Ohori, et al., J. Med. Virol.,
12:161-178 (1983), Bradley, et al., Proc. Nat'l. Acad.
Sci., 84:6277-6281, (1987), Akatsuka, T., et al., J. Med.
Virol, 20:43-56 ~1986), Seto, B., et al., U.S. Patent
Application Number 07/234,641 (available from U.S.
Department of Commerce National Technical Information
Ser~ice, Springfield, Virginia, No. 89138168), Takahashi,
K., et al., European Patent Application No. 0 293 274,
published No~ember 30, 1988, and Seelig, R., et al., in
PCT Application PCT/EP88/00123. The recent report of
isolated cDNA sequences encoding antigens which react
immunologically with antibodies present in infected
patients has provided one diagnostic assay which may use
recombinantly produced antigens. See, Houghton et al.,
European Patent Application publication number 0 318 216
and the related papers Kuo et al., Science, 244:359-361
(1989) and Choo et al., Science, 244:362-364 (1989).
The infectious agent itself has not been
successfully isolated and characterized. See, for
example, Fowler, et al., J. Med. Virol., 12:205-213
~1983) and Weiner, et al., J. Med. Virol., 21:~39-247
(19~7). Mori et al., Lancet, 318 (Aug. 9, lg80), Seto et
al., Lancet 941-943 (October 27, 1984), and Bradley et
al., J. Med. Virol, 3:253-269 (1979). BradLey et al.,
GastroenteroloqY~ 88:773 (1985) reported sizing of HCV
viral particles with polycarbonate filters. A variation
of this technique using a graded series of polycarbonate
filters to purify human rota~irus from seeded chimpanzee
wos2/o7ool PCT/US91/07S7J
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blood plasma is more fully described by Miller et al.,
Proceedings of the 46th Annual Meeting of the Electron
Microscopy Society oE America, pages 76-77 (August,1988)
and Miller et al., Proceedings of the 12th International
Congress for Electron Microscopy, pages 308-309 (August,
1990) . ' , ~, ,
Clearly, there exists a need to isolate and
fully characterize the infectious agent associated with
HCV. ~he successful isolation of the infectious agent
will provide for further development of additional
diagnostic and prognostic assays.
SUMMARY OF_THE INVENTION
The present invention relates to an antibody
capable of selectively binding with one or more HCV
antigen which are associated with the whole virus
particle and which produce an immune response in a
patient. Preferably, HCV antigens associated with the
virus particle are isolated from a mammal selected from
the grPUp consisting of chimpanzees and humans.
Both monoclonal and polyclonal antibodies are
provided by this invention. Both of these types of
antibodies may be prepared according to processes known
in the art for generating hybridoma cells from
immuno-challenged mammals. Specifically, these
antibodies may be isolated ~rom cell lines selected from
the group consisting of the hybridoma cell lines ~18 C27
and H18 C68 which were deposited on August 15, 1990 with
the American Type Culture Collection (ATCC), 12301
Parklawn Drive, Rockville, Maryland, 20852 under ATCC
Nos. HB 10529 and HB 10530, respectively.
WO92/07~01 PC~/U~gl/~7~71
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This inven~ion also provides a process for
isolating an infectious HCV particle in a plasma sample
taken from an infected patient which includes clarifying
the plasma sample by centrifugation; filtering the
clarified plasma through a series of filters having
sequentially smaller pore sizes, wherein the pore size of
the filters is 3.0, l.0, 0.8, 0.6, 0.4, and 0.2 microns, ;
respectively; pelleting the filtered plasma through a 10%
sucrose solution; and resuspending the pelleted material.
The present process provides resuspended
material having infectivity titers up to and about 1 o7
CID/ml even though this material contains less than about
1%, and preferably less than 0.1%, of the total
pelletable protein of the original plasma sample.
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The antibodies of the present invention are
useful in an immunological assay for detecting an antigen
specifically associated with HCV.
Other aspects and advantages of the present
invention will be apparent upon consideration of the
following detailed description which includes
illustrative examples of the practice of this invention.
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DE~AILED DESCRIPTION
The present invention provides a method to
isolate and visualize the agent(s) known to cause human
and chimpanzee HCV. Past efforts to immune aggregate a
putative virus, raise hyperimmune sera, or clone this
illusive agent have been thwarted by the lack of
availability of adequately purified virus from blood
plasma. I.ittle was known about the physi~al properties
of this infectious agent except that it is probably a
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WO92/07001 PCT/US91/07571
chloroform and B-propiolactone sensltive vlrus capable of
passing through about a lO0 nm filter. Attempts by
others to determine the bouyant density of the virus have
yielded ambiguous results and, until now, there has not
been a reliable means for purifying and, at the same
time, concentrating the virus. The present invention
describes a scheme for purifying and enriching whole,
infectious blood plasma and using the isolate to generate
antibodies.
Methods for purifying virus particles from
blood plasma or cell extracts have traditionally been
based on the bouyant density of the virus to be isolated.
Both differential centrifugation or density gradient
methods, such as isopycnic or rate zonal banding, collect
all classes of cellular debris having densities similar
to the vi~us in a given centrifugation run. Such debris
varies considerably in overall size and the presence of
the debris generally makes it difficult to detect or
identify virus particles using electron microscopy.
. Sizing of virus particles by ultrafiltration
has been used as an aid in virus classification. As
previously discussed, Bradley et al. report the use of
polycarbonate filters of defined pore size to gain some
information about the size of the HCV particle. This
invention provides an HCV purification scheme based on
the sequential passage of blood plasma through a sexies
of polycarbonate filters of decreasing pore size.
To isolate the HCV particles, polycarbonate
filters are preferred because, unlike nitrocellulose,
polyester or other types of filters which are made of a
relatively thick meshwork of interwoven fibers,
polycarbonate filters are thin and contain precisely
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sized, strai~ht-bore holes. These features limit the
loss of particles caused by non-specific absorption or
entrapment in the filter matrix associated with
nitrocellulose, polyester or other types of filters.
In initial experiments, the ability of a graded
series of filters to sequentially reduce the sizes of
particles present in whole blood plasma was examined.
Nuclepore filters 25 mm in diameter were mounted in
Swinlok holders which were, in turn, coupled to lO cc
hypodermic syringes filled with plasma. This set up,
however, allowed only a small amount of plasma to
successfully pass ~hrough a series of filters before the
filters plugged. One solution, dilution of the plasma
was not pursued because this would only serve to increase
the o~erall ~mount of handling of plasma and lead to
excessive ultracentrifugation steps to concentrate final
viral isolate. Ultimately, a small press was used to
facilitate the filtration steps.
Table l illustrates a preferred scheme to
isolate HCV particles. Whole blood plasma was first
clarified by low speed centrifugation and then
sequentially pressure filtered through 3.0, l.O, 0.8,
0.6, 0.4 and 0.2 micron Nuclepore filters. The 0.2
micron filtrate was then layered on top of buffered 10%
sucrose and pelleted for 4 hours at 105,000 x g.
Following pelleting thru lO~ sucrose, fine protein was
removed. Pellets were resuspended in phosphate buffered
saline to yield a lOOX stock concentrate and then further
diluted as desired. The portion of the final concentrate
to be used for infectivity studies was diluted in buffer
containing 2% BSA as a cryroprotectant.
WO92/07001 PCT/US91/07571
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TA LE 1
Step #l Whole Undiluted Plasma
(Clarify at 2000 rpm, 20 min)
Step ~2 3.0 MicrTn Filter
Step #3 T
Step #4 0-8 MicrTn Filter
Step #5 T
Step #6 0 4 MicrTn Filter
Step ~7 0-2 MicrTn Filter
Step #8 10 % Sucrose Pellet
(105,000 x g for 4 hrs)
Step #9 Resuspend to 100 x
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The partially purified plasma passed easily
through the filters and at ~ach step of the purification
process lO0 microliter aliquots of plasma were prepared
for electron microscopic viewing using a method developed
by Miller (Miller, M.F., Electron MicroscopY in B}oloqy,
Vol.2, J.D. Griffith, ed., John Wiley and Sons, Inc., New
York, N.Y., 1982, pp. 305-339) which employs the
commercially available Beckman Airfuge ultracentrifuge
and Em-90 electron microscopy particle counting rotor.
Briefly, lO0 microliters of starting plasma and plasma
derived from various stages of the polycarbonate filter
purification process is sedimented at lO0,000 x g in the -~
EM 90 rotor onto 5mm2 Millipore filter supports. Supports
with sedimented particulates are fixed with 3~ -
glutaraldehyde followed by osmium tetroxide, dehydrated
and embedded in epoxy resin. Embedded filter supports
are then thin-sectioned and stained with uranyl acetate
followed by lead citrate.
At low magnification, the appearance of whole,
unfiltered chimpanzee blood plasma prepared for EM
viewin~ showed large quantities of fine proteinaceous
material, clumps of fibrin and a variety of sizes of
particulates. At higher magnification, the appearance of
a 0.2 micron filtrate of chimp plasma showed that all
particulates larger than 0.2 micron were removed by
filtration and that the fine protein was removed
following the 10% sucrose pelleting step. These results
clearly demonstrated the efficacy of the purification
scheme for selectively eliminating larger particulates
and removing ~Lne protein.
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To evaluate virus recovery using this
filtration procedure, whole chimp plasma was seeded with
approximately lO'~ particles per ml of highly purified
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human rotavirus. Rotavirus, although non-blood born, is
about 65-70 nm in diameter and is relatively easy to
recognize and count in thin sections of impure samples.
Seeded plasma samples derived from various stages of
purification ware processed for EM viewing as described
above and the virus particles were counted. Samples were
also assayed for total pelletable protein using a
s-nsitive spectrophotometric protein assay. It was found
that only a fraction of a log of virus was lost during
purification but there was an overall 250-fold reduction
of total pelletable protein.
The filtration procedure may be used to provide
concentrated plasma needed to isolate the infectious
agent associated with HCV. One method to isolate ~CV,
immune aggregation electron microscopy, may be performed
by mixing appropriate concentrations of the plasma
concentrate and a suitable antibody and visualizing
resultant immune aggregates using a transmission electron
microscope. The antibody is generally pre-centri~uged at
about 40r000 RPM for l0 minutes to remove undesirable
backgro~nd particulates found in most sera. When working
with unknown antibody titers it may be necessary to
experimentally determine optimum dilutions using a
systematic trial and error approach. Too dilute or
concentrated an antibody solution will result in
incomplete or no aggregation.
The preferred aggregation procedure to study a
variety of hepatitis viruses includes: a) mixing 20
microliters of well dispersed plasma concentrate with 50
microliters of PBS, pH 7.2, and 20 microliters of
antibody, b) incubating the mixture for one hour at room
temperature and then overnight at 4C, c) pelleting the
immune aggregates for 30 minutes at 20 psi in a Beckman
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2~3919 10
Airfuge Ultracentrifuge, d) discarding the superna~ant
and resupsending the pellet in 20 microliters of water,
e) applying 5 microliter droplets of suspension to
Formvar coated EM specimen grids, f) blotting away the
droplet with absorbent paper, and g) negatively staining
adherent particulates with 2~ phosphotungstic acid.
Specimen grids are then carefully surveyed for immune
aggregates with a transmission electron microscope at
magnifications ranging from lO to 45,000 x.
The monoclonal antibody or other monoclonal
antibodies derived using the described isolated HCV
preparations may be used in diagnostic tests for antigens
of HCV separately, in combination with each other or in
combination with human, chimpanzee, rabbit or other
mammalian polyclonal antibodies.
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Se~eral alternative assay configurations may be
used with the antibodies of the invention. For example,
the antibody may be bound to a suitable solid support,
contacted with a sample or aliquot containing HCV, and
then contacted with a second labeled antibody which may
be developed to provide a detectable signal. An assay
which uses antibody of the present invention which has
been labeled with a signal generating moiety is an
al~ernative procedure. In addition, the labeled antibody
may be used in a competitive antibody assay. In this type
of assay, the labeled antibody is incubated with the
sample or aliquot in the presence of a solid support
which is coated with an antigen containing the
immunodominant epitope o the antigen contained in the
sample. Antibodie9 to HCV present in the sample will
compete for binding with the labeled antibody and with
the bound antibody. Therefore, a sample containing an
antibody to HCV will produce a detectably lower signal
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compared to the signal produced in an assay which has no
added labeled antibody.
The purified virus prepared by the process of
this invention may also be used to characterize unknown
HCV antigens. The purified virus used to generate an
antibody may be labeled with radioactive iodine using
both Chloramine T and Bolton Hunter reaction conditions
according to established procedures before ~nd after
solubilization of the virus with detergents. The
antibodies may be used in an immunoprecipitation
experiment where the antibody is allowed to react in
solu~ion with the solubilized labeled ~irus. After this
incubation period, several micrograms of fixed
Sta~halococcus aureus (Staph A) cells are incubated with
the antigen-antibody mixture. Immunoglobulin receptors
on these cells will bind the antigen-antibody complexes
as well as free immunoglubulins. The bound cells are
pelleted by centrifugation and washed repeatedly to
remove unadsorbed labeled antigen. The Staph A cells
with the adsorbed complexes are then dissociated by
heatin~ to 100C in a solution containing 2-
mercaptoethanol, detergents and buffer and then
electrophoresed on polyacrylamide slab gels. After
electrophoresis the gels are fixed, treated with
chemicals known to enhance detection of radioactivity,
dried and exposed to x-ray film at -70C for an extended
period of days, weeks or even months. Upon development
of the x-ray film bands will appear on the film at
positions where the radiolabeled antigens migrated in the
gel ~ased on their molecular size~ By comparing the
positions of these bands to those of radioactive
standards of known molecular weight, the molecular size
of the precipitated viral antigen can be determined. The
information may be valuable in the determination of the
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WO92/07~1 PCT/~S91/~7S71
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size of the actual gene products of HCV which are unknown
at this time.
variations of this technique may also be
utilized to identify labeled viral antigens produced in
cell lines capable of supporting viral replication. In
essence, this techni~ue can be used to screen various
cell lines for the presence of viral replication and
therefore is useful as a tool for establishing a tissue
culture system for hepatitis C diagnosis.
It is expected that monoclonal antibodies
according to the in~ention will be useful in assays for
detecting HCV infection and infectious agents in bodily
fluids, tissues, experimental reagents and fluids.
The monoclonal antibodies of the invention may
also be used to isolate antigens specifically associated
with HCV by affinity chromatography. Such immunopurified
antigen may be used to immunize non-primate species such
as goats and rabbits, in order to obtain antibodies
having multiple epitope specificities. Alternatively,
the isolated antigen may be used to immunize mice or rats
allowing for preparation of hybridoma cell lines capable
of producing additional monoclonal antibodies.
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Antibodies generated according to the present
inventions will allow the characterization o~ antigens
specifically associated with HCV infection and inectious
agents. In this regard, standard methods such as
analytical and preparative gel electrophoresis may be
employed to determine the molecular weight of antigens
reactive with the antibodies of this invention. Reactive
isolates so obtained may be subjected to further
characterization through use of various selective agents
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2v~33,9l9
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including reducing agents, proteases, lipases,
glycosidases, and the like. Antibodies which react with
HCV viral antigens may be utilized in a variety of
immunoassays including competitive and non-competitive
immunoassays, radioimmunoassays, ELISA, EIA and the like.
.
The following examples illustrate the practice
of this invention and are included only for the
illustrative purposes. These examples are not intended
to limi~ the scope of the invention or the scope of the
appended claims.
Example l describes a process to purify HCV
from an infected chimpanzee,/ Example 2 describes the
production of a monoclonal antibody, and Example 3
describes a diagnostic test.
EXAMPLE 1
Purification of Hepatitis C Virus
Usinq Polycarbonate Filters
Chimpanzee blood plasma having an infectivity
titer of 105 CID/ml (chimp infectious doses/milliliterj
of HCV was purified as follows. ~aw plasma was first
clarified by low speed centrifugation for 20 minutes at
lOOOXg in a tabletop centrifuge to remove large celluiar
debris and then sequentially pressure filtered through
polycarbonate membrane filters having pore sizes of 3.0,
l.0, 0.8, 0.6, 0.4 and 0.2 micron diameter. Particulates
remaining in the 0.2 um filtrate were then pelleted by
ultracentrifugation through phosphate buf~ered 10%
sucrose for 4 hours at 105,000 X g. This step removed
fine proteins and served to concentrate the virus.
Pelleted virus was resuspended in phosphate buffered
W092/~7U01 PCT/US91/07~71
~U93 9 19 14
saline to yield a final l00X concentrate The portion of
the concentrate to be used for infectivity studies
was diluted with buffer containing 0.2~ Bovine Serum
Albumin as a cryoprotectant.
Electron micrographs showing sedimented and
thin-sectioned particulates derived from the purified
chimpanzee plasma were compared to the similarly prepared
raw starting plasma. Using a sensitive
spectrophotometric assay it was found that total
pelletable protein was decreased more than 250 fold. When
purified virus product was inoculated into chimpanzees it
was found to be infectious (100% of titer recovered as
evidanced by the characteristic elevation of serum
enzymes and appearane o~ hepatocyte ultrastructural
alterations)~ ~he purified product was used to generate
monoclonal antibodies.
EXAMPLE 2
Production of a Monoclonal Antibody
Six female Balb c/J mice were selected on the
basis of low reactivity of their sera by
immunofluorescent microscopy on liver sections from
hepatitis C infected chimpanzees. These selected mice
were used for inoculation with purified HCV.
Pre-inoculation hleeds were collected from all mice prior
to ihoculation with virus. Purified virus obtained by
the procedure described in Example 1 was used as the
inoculum. Two mice each were inoculated according to the
following regimen: intraperitoneally (~P) with 0.l ml o~
the purified ~irus stock, 0.2 ml o a l/10 dilution of
the ~rirus stock in normal saline, or 0.2 ml of a 1~l00
dilution of the virus stock in normal saline. Blood
samples were collected by eye bleeds at day 21. On day
w~s2/o7~l PCT/US91/0757~
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35, all mice were boosted (IP) with the same inocula and
dose as their primary immunization regimen. Blood
samples were again collected by eye bleeds on day 49. On
day 129, all mice were again boosted, (IP) as before.
Blood samples were collected by eye bleeds on day 141.
On day 199, one mouse from each dosage category was
inoculated intravenously (IV) with 0.1 ml of a 1/10
dilution of the purified virus stock. Three days later,
the IV boos~ed mice were sacrificed and their spleen
lymphocyte cells were isolated and fused to SP 2/0 mouse
myeloma cells following the procedure of Kohler and
Milstein (Kohler G., and Milstein C., Nature,
256:495-497, 1975). On day 241 the remaining 3 mice were
boosted IV as previously described, and sacrificed three
days later and their spleen lymphocytes were isolated and
fused to SP 2/0 cells.
Supernates from hybridoma cells resulting from
the fusions of mouse lymphocytes with SP 2/0 cells were
screened by an enzyme immunoassay (EIA) using synthetic
peptides representing sequences from the Chiron C-100
clone (described in European Patent Application
publication number 0 318 216 published May, 31, 1989) as
the antigen coated onto a solid polystyrene bead. A
hybridoma cell line producing a positive signal in the
peptide EIA was cloned by limiting dilution. Monoclonal
cell lines producing Ig~ class antibodies specific for an
epitope contained within the Chiron C-100 sequence were
isolated. Two of these cell lines, H18 C27 and H18 C68,
were expanded in cell culture and frozen under liquid
nitrogen and are now on deposlt with the ATCC under
accession nos. HB 10529 and HB 10530, respectively.
WO92/07001 PCT~US91/07571
3~ ~9 16
EXAMPLE 3
A Diaqnostic Test
The monoclonal antibody prepared according to
the procedure of example 2 is used as an antigen capture
reagent bound to a solid phase polystyrene bead support.
An aliquot of bodily fluid such as serum, plasma or
cerebral spinal fluid from a patient is incubated with
the antibody bound to the bead. During the incubation,
an antigen or whole virus binds to the solid phase.
After incubation, unbound antigens are washed away from
the solid support. In the second step of the assay, the
same antibody, or another monocLonal antibody binding to
a different epitope of an HCV antigen, or a polyclonal
antibody, coupled to horseradish peroxidase is incubated
with the bead. This labeled antibody will bind to
antigen trapped on the solid phase forming an
antibody-antigen-antibody sandwich. Excess peroxidase
labeled antibody is washed away and the bead is exposed
to a suitable enzyme substrate such as OPD
(orthophenylene diamine). The resulting colored product
is detected spectrophotometrically in amounts directly
proportional to the amount of antigen which was present
in the original aliquot.
While the present invention has been
illustrated in terms of specific methods and
compositions, it is understood that variations and
modifications will occur to those skilled in the art upon
conslderstion oE the present invention.
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