WO1994024565A1 - Hepatitis c virus immunodiagnostic antigens and antibodies - Google Patents

Hepatitis c virus immunodiagnostic antigens and antibodies Download PDF

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Publication number
WO1994024565A1
WO1994024565A1 PCT/US1994/004174 US9404174W WO9424565A1 WO 1994024565 A1 WO1994024565 A1 WO 1994024565A1 US 9404174 W US9404174 W US 9404174W WO 9424565 A1 WO9424565 A1 WO 9424565A1
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seq
antigen
val
ser
leu
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PCT/US1994/004174
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French (fr)
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Gregory R. Reyes
Jungsuh P. Kim
Randolph Moeckli
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Genelabs Technologies, Inc.
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Priority to AU67695/94A priority Critical patent/AU6769594A/en
Publication of WO1994024565A1 publication Critical patent/WO1994024565A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • C07K16/1081—Togaviridae, e.g. flavivirus, rubella virus, hog cholera virus
    • C07K16/109—Hepatitis C virus; Hepatitis G virus
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/576—Immunoassay; Biospecific binding assay; Materials therefor for hepatitis
    • G01N33/5767—Immunoassay; Biospecific binding assay; Materials therefor for hepatitis non-A, non-B hepatitis
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/24011—Flaviviridae
    • C12N2770/24211—Hepacivirus, e.g. hepatitis C virus, hepatitis G virus
    • C12N2770/24222—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • This invention relates to specific peptide viral antigens which are immunoreactive with sera from patients infected with parenterally trans ⁇ mitted non-A, non-B hepatitis (PT-NANBH) virus, to antibodies immunoreactive with these antigens, and to methods of using the antibodies for detecting PT-NANBH infection in human sera.
  • PT-NANBH non-A, non-B hepatitis
  • NANBH non-A, non-B hepatitis
  • HAV hepatitis E virus
  • the second NANB virus type known as parenterally transmitted NANBH, or PT-NANBH
  • PT-NANBH is transmitted by parenteral routes, typically by exposure to blood or blood products.
  • RNA isolated from infected chimpanzee sera has been used to construct cDNA libraries in an expression vector for immunoscreening with chronic-state human PT- NANBH serum. This procedure identified a PT-NANBH specific cDNA clone and the viral sequence was then used as a probe to identify fragments making up 7,300 contiguous basepairs of a PT-NANBH viral agent (Houghton, et al . , (A), EP Pub. No. 0318216; Houghton, et al . , (B) , EP Pub. No. 0388232).
  • the present invention provides a method for detecting the presence of hepatitis C virus (HCV) antigens in a sample.
  • the sample is contacted with at least one antibody which is reactive with an HCV antigen.
  • the antibody is usually attached to a solid support and is immunoreactive with a portion of the polypeptide presented as SEQ ID NO:6.
  • the support-bound antibody is then examined for the presence of bound HCV antigen. This examining typically involves reacting the solid support with an antigen-reporter complex, where the HCV antigen competes with binding of the antigen-reporter complex to the antibody.
  • the level of antigen- reporter complex, which is bound to the solid support is then determined.
  • the method can be used to analyze samples from a variety of sources including, but not limited to, the following: tissue culture medium, chimpanzee serum, and human serum.
  • a number of solid supports are useful in the method of the present invention, including microtiter plates.
  • two or more antibodies, with their corresponding antigen-reporter complexes can also be used.
  • Antigens useful in generating antibodies for the method of the present invention include the polypeptides selected from the group of sequences presented as SEQ ID NO:6, SEQ ID NO:17, SEQ ID NO:18, and SEQ ID NO:19, and derivatives thereof. These antigens are also useful in antibody-capture assays, where the antigen polypeptide is attached to a solid support and serum is screened for antibodies capable of binding to the antigens.
  • a number of reporters can be employed in the present method.
  • the antigen-reporter complexes can contain one or more of the following: enzymatic reporters, radioactive reporters, and fluorescent reporters.
  • An exemplary enzymatic reporter is horse radish peroxidase.
  • kits for use in the screening samples for the presence of hepatitis C virus (HCV) antigen, by the method of the present invention.
  • kits typically include (i) at least one antibody which is immunoreactive with a portion of the polypeptide presented as SEQ ID NO:6, and (ii) an antigen-reporter complex, where the HCV antigen competes with binding of the antigen-reporter complex to the antibody. Further, the antibody can be attached to a solid support.
  • the antigens and antibodies are as described above.
  • the present invention includes purified antibodies that are immunoreactive with a polypeptide consisting essentially of a sequence selected from the following: SEQ ID NO:6, SEQ ID NO:17, SEQ ID NO:18, and SEQ ID NO:19. These antibodies can be polyclonal or monoclonal antibodies.
  • Another aspect of the present invention includes polypeptides consisting essentially of the following sequences: SEQ ID NO:6, SEQ ID NO:17, SEQ ID NO:18, and SEQ ID NO:19. These polypeptides, and polypeptides containing other epitopes included in the clone 36 sequence, are useful in antibody-capture assays, the generation of antibodies for the diagnostic method of the present invention, and the generation of antibodies for use in immunoprophylaxis. Further, these polypeptides may be useful as polypeptide antigen vaccines effective against HCV.
  • Figure 1 illustrates the construction of a modified clone 36 insert lacking internal Ncol and BamHI sites.
  • Figure 2 schematically illustrates the basis design of an antibody based competition immunoassay.
  • PT-NANBH hepatitis viral agent
  • HCV HCV
  • HCV PT-NANBH viral agent whose polynucleotide sequence includes the sequence of the 7,300 basepair region of HCV (Houghton, et al . , (A), EP Pub. No. 0318216; Houghton, et al . , (B) , EP Pub. No. 0388232), and variations of the sequence, such as degenerate codons, or variations which may be present in different isolates or strains of HCV.
  • Two nucleic acid fragments are "homologous" if they are capable of hybridizing to one another under hybridization conditions described in Maniatis et al., op. cit.. pp. 320- 323, using the following wash conditions: 2 x SCC, 0.1% SDS, room temperature twice, 30 minutes each; then 2 x SCC, 0.1% SDS, 50°C once, 30 minutes; then 2 x SCC, room temperature twice, 10 minutes each, homologous sequences can be identified that contain at most about 25-30% basepair mismatches. More preferably, homologous nucleic acid strands contain 15-25% basepair mismatches, even more preferably 5-15% basepair mismatches. These degrees of homology can be selected by using more stringent wash or hybridization conditions for identification of clones from gene libraries (or other sources of genetic material) , as is well known in the art.
  • a DNA fragment is “derived from” HCV if it has substantially the same basepair sequence as a region of the HCV viral genome which was defined in (2) above.
  • a protein is "derived from” a PT-NANBH or HCV viral agent if it is encoded by an open reading frame of a cDNA or RNA fragment derived from a PT-NANBH or HCV viral agent, respectively.
  • the inserts were radioactively labelled and used as probes against Hindlll / EcoRI doubly-digested human peripheral lymphocyte (PBL) DNA (Example 3) . Approximately 46% (43/93) of the inserts hybridized with normal human PBL DNA and were therefore not pursued. Inserts from 11 PT-NANBH-immunopositive clones derived from chimpanzee #771 sera were characterized as exogenous to normal human PBL DNA (Example 3) . Of these 11 clones 2 PT-NANBH clones were identified having the following characteristics.
  • clone 40 was clearly exogenous by repeated hybridization tests against normal human PBL DNA, had a relatively small insert size (approximately 0.5 kilobases) , and was quite unreactive with negative control serum.
  • the second clone (clone 36) was shown to be reactive with multiple PT-NANBH antisera, had a relatively large insert size (approximately 1.5 kilobases) , and was exogenous by hybridization testing against normal human PBL DNA.
  • the immunoreactive characteristics of clones 36 and 40 are summarized in Table 1 (Example 3) .
  • Clone 36 was immunoreactive with chimpanzee #771 sera and two HCV-positive human sera, AG and BV.
  • the clone 36 antigen did not immunoreact with the negative control serum SKF.
  • Clone 40 was immunoreactive with chimpanzee #771 sera and was cleanly nonreactive when the negative control sera was used for screening.
  • the DNA sequences of clones 36 and 40 were determined and are presented as SEQ ID NO:5 and SEQ ID N0:l, respectively.
  • the clone 36 sequence corresponds to nucleotides 5010 to 6516 of the HCV sequence given in Houghton, et al . , (A), EP Pub. No. 0318216.
  • the clone 40 sequence is homologous to the HCV sequence (Houghton, et al . , (A), EP Pub. No. 0318216) in the region of approximately nucleotides 6515 to 7070.
  • the sequences for clones 36 and 40 are contiguous sequences, with the clone 36 sequences being located 5' of the clone 40 sequences as presented in Houghton, et al . , (A), EP Pub. No. 0318216, and Houghton, et al . , (B) , EP Pub. No. 0388232. Accordingly, these two clones represent isolation of a significant block of the HCV genome by the above-described immunoscreening methods.
  • the inserts of two other chimpanzee #771 clones, clones 44 and 45, were found to be homologous to clone 40 by hybridization and sequence analysis (Example 4) . Isolation and characterization of the four lambda gtll clones 36, 40, 44, and 45 were previously described in PCT International Application PCT/US91/02370, Publication No. WO 91/15516, Publication Date 17 October 1991.
  • Table 2 presents the data for preliminary immunoscreening of clone 36 and 45 antigens (the insert of clone 45 is essentially the same as the insert of clone 40) against well- documented PT-NANBH chronic sera which showed strong immunoreactivity to the 5-1-1 HCV peptide antigen (Kuo) .
  • the 5-1-1 HCV peptide antigen has previously been identified as immunoreactive against a high percentage of human PT-NANBH chronic sera.
  • the 5-1-1 antigen is encoded by the sequence between basepairs 3731 and 3857 in the HCV genome (Houghton, et al. , (A), EP Pub. No. 0318216; Houghton, et al .
  • the antigens produced by clones 36 and 40 yield HCV-specific immunopositive signals with selected samples. Additionally, subfragments of clone 36, when expressed in a lambda gtll expression system, were immunopositive with HCV- positive serum samples.
  • the data presented in Table 3 suggest that clone 36 identified all HCV-positive sera (within a selected sample) that were identified by any other single HCV antigen or combination of HCV antigens, including the C100 antigen which was employed in a commercial HCV diagnostic assay (the C-100 antigen corresponds essentially to clone 5-1-1, Table 3) .
  • the results show that the clone 36 antigen detected six unique positive sera samples.
  • the clone 36 antigen tested negative with all negative control sera.
  • the results presented in Table 2 and 3 support the use of the clone 36 antigen in immunodiagnostic assays directed to the detection of HCV.
  • the amplified subfragments were cloned into lambda gtll vectors (gtll-36-1, gtll-36-2, and gtll-36-3) for expression of the clone 36 polypeptide fragments encoded by each subfragment. These clones were used to test for expression of polypeptides capable of reacting with HCV-positive antisera in an immunoscreening assay.
  • the antigens were immunoscreened for reactivity with a number of human anti-sera including well- characterized HCV positive sera (Example 9) .
  • Each of the three sub-cloned clone 36 fragments tested immunopositive with at least one of the human anti-sera and tested negative with known HCV- negative control sera. These results indicate that each of the three portions contained at least one epitope immunoreactive with HCV-positive sera.
  • antigen containing portions of the clone 36 coding sequence can be identified by the methods described above.
  • the recombinant peptides of the present invention can be purified by standard protein purification procedures which may include differential precipitation, molecular sieve chromatography, ion-exchange chromatography, isoelectric focusing, gel electrophoresis and affinity chromatography.
  • a fused protein such as the beta-galactosidase fused proteins prepared as above
  • the fused protein can be isolated readily by affinity chromatography, by passing cell lysis material over a solid support having surface-bound anti-beta-galactosidase antibody.
  • affinity chromatography purification of a beta- galactosidase/fusion protein, derived from clone 36 coding sequences, by affinity chromatography is described in Examples 6.
  • a fused protein pontaining the clone 36 peptide fused with glutathione-S-transferase (Sj26) protein has also been expressed using the pGEX vector system in E. coli KM392 cells (Smith) .
  • This expression system has the advantage that the fused protein is generally soluble and therefore can be isolated under non-denaturing conditions.
  • the fused Sj26 protein can be isolated readily by glutathione substrate affinity chromatography
  • Example 7 This method of expressing this fusion protein is given in Example 7 and is applicable to any of the other antigen coding sequences described by the present invention.
  • Example 11 Yet another protein isolation method is described in Example 11 for the isolation of the pET-clone 36 antigen. Briefly, production of the pET-clone 36-encoded HCV antigen is induced in bacterial cells. The cells are lysed and the pelleted proteins are subjected to repeated rounds of suspension and re-pelleting. Then the protein pellet is suspended, filtered and subjected to size fractionation. Size fractionation can be achieved by any standard method including FPLC. Fractions resulting from the size fractionation are then assayed for the presence of the protein.
  • Fractions containing the antigenic protein of interest can be identified by a number of means including, immunoreactivity (e.g., ELISA assays) or separation on SDS polyacrylamide gels (identification by molecular weight) .
  • the polypeptide antigens of the present invention have a number of uses including, but not limited to, the following: antigens in immunoassays, such as ELISA (Example 10) ; vaccine compositions; and the generation of monoclonal and polyclonal antibodies.
  • the present invention includes antibodies specific against the recombinant antigens described above, including the clone 36 and clone 40 antigens, and further derivatives of either.
  • polyclonal antibodies typically, a host animal, such as a rabbit, is immunized with the purified antigen or fused protein antigen.
  • the host serum or plasma is collected following an appropriate time interval, and this serum is tested for antibodies specific against the antigen.
  • Example 7 describes the production of rabbit serum antibodies which are specific against the clone 36 antigens in the Sj26/clone 36 fusion protein. These techniques are equally applicable to the other antigens of the present invention.
  • the gamma globulin fraction or the IgG antibodies of immunized animals can be obtained, for example, by use of saturated ammonium sulfate or DEAE Sephadex, or other techniques known to those skilled in the art for producing polyclonal antibodies.
  • Hybridomas Alternatively, the purified antigen or fused antigen protein may be used for producing monoclonal antibodies.
  • the spleen or lymphocytes from an immunized animal are removed and immortalized or used to prepare hybridomas by methods known to those skilled in the art (Mishell) .
  • mice are immunized by intraperitoneal injection of the purified or partially purified antigen derived from clone 36 containing phage infected cells.
  • the antigen is mixed with an adjuvant, such as killed Bordatella pertussis organisms.
  • Hybridomas are formed by fusion of splenic B-lymphocytes and stable myeloma fusion partner cells.
  • Hybridomas are selected by growth in selective medium, then are tested for production of antibodies reactive with the specific im unogen, in one or more assays, such as a solid phase (ELISA) assay, plaque immunoscreening, or Western blot analysis (Ausubel, et al . ) .
  • assays such as a solid phase (ELISA) assay, plaque immunoscreening, or Western blot analysis (Ausubel, et al . ) .
  • Hybridomas found to produce reactive antibodies are further clone purified by limiting dilution.
  • Example 10 describes the generation of monoclonal antibodies directed against the pET36 antigen.
  • Hybrido a cells were formed by fusion of splenic B-lymphocytes from seropositive mice and stable myeloma fusion partner cells. Hybridomas were selected by growth in selective medium and tested for production of anti-pET-36 antibodies in a solid phase assay (ELISA) . Forty-nine positive clones were identified and isolated in one such selection. Hybridomas found to produce antibodies to pET 36 were further clone purified by limiting dilution. From the 49 positive clone, 5 were subcloned by limiting dilution.
  • Human hybridomas can also be produced by fusing a human lymphocyte with an appropriate immortalized fusion partner.
  • a donor known to be infected with an HCV virus may serve as a suitable lymphocyte donor.
  • Lymphocytes can be isolated from a peripheral blood sample or from spleen cells, if the donor is subject to splenectomy.
  • antibody- secreting B-lymphocytes are activated prior to selection using a transforming virus, such as Epstein-Barr virus, or can alternatively be achieved by exposure of the cells to other B-cell activators known in the art, such as pokeweed mitogen, or to the specific antigen recognized by the cells.
  • cells are grown in culture, then examined for production of specific anti-HCV antigen activity, using an appropriate antibody detection assay.
  • Cells showing activity in such an assay are selected for immortalization, by fusion with a heteromyeloma fusion partner.
  • Formation of a stable hybridoma that secretes a human anti-HCV antibody is achieved by fusing an activated B-lymphocyte with a heteromyeloma cell such as the K6H6-B5 cell line (Carroll, et al . ) or the H73C11 cell line (Perkins, et al.), originally produced by fusing activated human lymphocytes with a mouse myeloma fusion partner.
  • Such fusion can be achieved by a number of methods known in the art (Harlow, et al . ) including exposure of mixed cells to polyethylene glycol or exposure of cells to strong electric field (electrofusion) .
  • Hybridomas are selected by growth in selective medium, then are tested for HCV and antigen specificity in one or more i munoassays.
  • Primary in vitro immunization with peptide or protein antigens of hybridomas in culture can also be used in the generation of monoclonal antibodies.
  • Antibodies secreted by the immortalized cells are screened to determine the clones that secrete antibodies of the desired specificity.
  • FIG. 2 The basic design of the immunoassay is illustrated in Figure 2.
  • One example of the antigen detection assay is described in Example 12.
  • a rabbit anti-clone 36 antibody coated well and a clone 36 antigen-horse radish peroxidase (HRPO) conjugate are used in the assay.
  • Microwells were coated with the above- described monoclonal antibodies.
  • the antibody coated wells is then incubated with the test samples, such as, clone 36 polypeptide, HCV infected tissue culture media, human sera or chimpanzee sera. After incubation, clone 36-HRPO conjugate is added to each well.
  • Combination of the conjugate to the solid phase antibody coat was detected by the addition of the substrate-2 , 2 ' - azino-bis(3-ethylbenzthiazoline-6-sulfonic acid (ABTS) .
  • An alternative substrate which can be used is 5-aminosalicylic acid (5AS) .
  • the presence of a clone 36 HCV-associated viral antigen is identified by color diminution due to successful inhibition of binding clone 36-HRPO.
  • reporter labels other than HRPO can be used in the method of the present invention, including the following: enzymatic reporter systems, such as HRPO alkaline phosphatase, ⁇ -galactosidase, and glucose oxidase (Pierce, Rockford IL) ; fluorochrome reporters, such as fluorescein, R-phycoerythrin, rhodamine, rhodamine 600, and "TEXAS RED" (Pierce); biotin and avidin (Pierce) ; radioactive labelling, such as 125 I or synthesis of antigen polypeptides containing 3 H or 14 C; light emitting reporters, such as luciferase (de Wet, et al . ) ; and chromophors, such as heme (Sigma, St. Louis MO) . Reporter labels are conjugated to antigen peptides by appropriate standard methods in the art.
  • fluorochrome reporters such as fluorescein, R-phycoery
  • the antigen detection assay of the present invention can also be tested by addition of increasing quantities of free clone 36 polypeptide.
  • the addition of the clone 36 polypeptide over a linear range of concentrations results in a linear inhibition curve.
  • a method to confirm the specificity of the antigen detection reaction is described in Example 13. This confirmation assay is based on blocking the inhibition of clone 36-HRPO binding to its cognate antibody, which usually occurs by binding of a clone 36-based antigen to the same antibody.
  • the blocking of inhibition is accomplished by addition of anti-clone 36 positive sera to the antigen positive plasma before testing in the antibody coated plates.
  • the antigens obtained by the methods of the present invention are advantageous for use as diagnostic agents for anti-HCV antibodies present in HCV-infected sera; particularly, clone 36 antigen and related antigens 36-1, 36-2 and 36-3.
  • the clone 36 antigen provides an advantage over known HCV antigen reagents 5-1-1 and C-100 in that clone 36 is immunoreactive with a wider range of PT-NANBH infected sera.
  • test serum is reacted with a solid phase reagent having a surface-bound HCV antigen obtained by the methods of the present invention, e.g., the clone 36 antigen.
  • a solid phase reagent having a surface-bound HCV antigen obtained by the methods of the present invention e.g., the clone 36 antigen.
  • the reagent is reacted with reporter- labeled anti-human antibody to bind reporter to the reagent in proportion to the amount of bound anti-PT-NANBH antibody on the solid support.
  • the reagent is again washed to remove unbound labeled antibody, and the amount of reporter associated with the reagent is determined.
  • the reporter is an enzyme which is detected by incubating the solid phase in the presence of a suitable fluorometric or colorimetric substrate (Harlow, et al . ) .
  • a suitable fluorometric or colorimetric substrate Hard, et al .
  • the solid surface reagent in the above assay is prepared by known techniques for attaching protein material to solid support material, such as polymeric beads, dip sticks, 96-well plate or filter material. These attachment methods generally include non-specific adsorption of the protein to the support or covalent attachment of the protein, typically through a free amine group, to a chemically reactive group on the solid support, such as an activated carboxyl, hydroxyl, or aldehyde group.
  • homogeneous assay In a second diagnostic configuration, known as a homogeneous assay, antibody binding to a solid support produces some change in the reaction medium which can be directly detected in the medium.
  • Known general types of homogeneous assays proposed heretofore include (a) spin-labeled reporters, where antibody binding to the antigen is detected by a change in reported mobility (broadening of the spin splitting peaks) , (b) fluorescent reporters, where binding is detected by a change in fluorescence efficiency, (c) enzyme reporters, where antibody binding effects enzyme/substrate interactions, and (d) liposome- bound reporters, where binding leads to liposome lysis and release of encapsulated reporter.
  • the assay method involves reacting the serum from a test individual with the protein antigen and examining the antigen for the presence of bound antibody.
  • the examining may involve attaching a labeled anti-human antibody to the antibody being examined, either IgM (acute phase) or IgG (convalescent or chronic phase) , and measuring the amount of reporter bound to the solid support, as in the first method, or may involve observing the effect of antibody binding on a homogeneous assay reagent, as in the second method.
  • an assay system or kit for carrying out the assay method just described is also forming part of the invention.
  • the kit generally includes a support with surface-bound recombinant HCV antigen (e.g., the clone 36 antigens), and a reporter labeled reporter-labeled anti-human antibody for detecting surface-bound anti-PT- NANBH-antigen antibody.
  • HCV antigen e.g., the clone 36 antigens
  • reporter labeled reporter-labeled anti-human antibody for detecting surface-bound anti-PT- NANBH-antigen antibody.
  • HCV antigens identified by the methods of the present invention can be formulated for use in a HCV vaccine.
  • the vaccine can be formulated by standard methods, for example, in a suitable diluent such as water, saline, buffered salines, complete or incomplete adjuvants, and the like.
  • the immunogen is administered using standard techniques for antibody induction, such as by subcutaneous administration of physiologically compatible, sterile solutions containing inactivated or attenuated virus particles or antigens.
  • An immune response producing amount of virus particles is typically administered per vaccinizing injection, typically in a volume of one milliliter or less.
  • a specific example of a vaccine composition includes, in a pharmacologically acceptable adjuvant, a recombinant clone 36 peptide.
  • the vaccine is administered at periodic intervals until a significant titer of anti-HCV antibody is detected in the serum.
  • test serum is reacted with a solid phase reagent having a surface-bound anti-HCV-antigen antibody, either polyclonal or monoclonal, obtained by the methods of the present invention: for example, an anti-clone 36 antigen antibody.
  • a solid phase reagent having a surface-bound anti-HCV-antigen antibody either polyclonal or monoclonal, obtained by the methods of the present invention: for example, an anti-clone 36 antigen antibody.
  • the solid phase is washed and then contacted with a reporter labeled antigen containing the epitope corresponding to the surface-bound anti-HCV-antigen antibody.
  • the level of reporter is then quantitated and the serum-antigen levels are determined based on the percent inhibition of antigen-reporter binding obtained in the presence of the antigen-containing serum: typically by comparison to a standard curve.
  • the reporter is an enzyme which is detected by incubating the solid phase in the presence of a suitable fluorometric or colorimetric substrate.
  • the solid surface reagent in the above assay is prepared by known techniques for attaching protein material to solid support material, such as polymeric beads, dip sticks, 96-well plate or filter material (such as nitrocellulose) (Harlow, et al . ) .
  • These attachment methods generally include non-specific adsorption of the protein to the support or covalent attachment of the protein, typically through a free amine group, to a chemically reactive group on the solid support, such as an activated carboxyl, hydroxyl, or aldehyde group.
  • the assay method involves reacting the serum from a test individual with a support bound anti-HCV antibody and examining the antibody for the presence of bound antigen.
  • kits for carrying out the assay method just described.
  • the kit generally includes a support with surface-bound anti-HCV antibody and a reporter-labeled cognate antigen (e.g. , clone 36-HRPO) for detecting antibody bound HCV-antigen.
  • a reporter-labeled cognate antigen e.g. , clone 36-HRPO
  • the more antigen, from a test sample, bound to the antibody the more inhibition of reporter-antigen binding: accordingly, lower levels of detectable reporter.
  • Polyclonal and monoclonal antibodies for use in the present invention, can be prepared as described above utilizing the peptides of the present invention.
  • the antibodies can be purified by standard methods to provide antibody preparations which are substantially free of serum proteins that may affect reactivity (e.g., affinity purification (Harlow et al . ) ) .
  • a number of the antigenic peptides (the 409- 1-1 series, clone 40, clone 36, and clone 36 subclones, e .g . , 36-1, 36-2, and 36-3) of the present invention can be used singly or in combination in the antigen-inhibition assay of the present invention.
  • Antibodies generated against these peptides can be combined with each other and/or anti-clone 36 antibodies for immunological detection of HCV infected sera.
  • the multiple antigen-reporter molecules can be similarly or differentially labeled.
  • microtiter plate wells i.e., multiwell plates (Corning)
  • the probe is then a mixture of, for example, 36-1-HRPO and 36-3-HRPO.
  • the results of this assay are then read as a single-specificity.
  • the two antigen-reporter molecules can be differentially labeled by, for example, labeling one antigen with HRPO and the second antigen with alkaline phosphatase.
  • differential labeling is the use of two fluorescent reporters having different emission wavelengths: for example, phycoerythrin (PE) and fluorescein isothiocyanate (FICT) .
  • PE phycoerythrin
  • FICT fluorescein isothiocyanate
  • Multi-well microtiter plates can then be scanned (Dynatech Corp. , Cambridge MA) and the relative levels of antigens determined based on the emission spectra.
  • the present invention also includes kits containing multiple antibodies and cognate antigen-reporter complexes, as well as antibodies generated against the antigens of the present invention.
  • the anti-HCV antibodies of the invention can be used as a means of enhancing an anti-HCV immune response since antibody-virus complexes are recognized by macrophages and other effector cells.
  • the antibodies can be administered in amounts similar to those used for other therapeutic administrations of antibody. For example, pooled gamma globulin is administered at 0.02-0.1 ml/lb body weight during the early incubation of other viral diseases such as rabies, measles and hepatitis B to interfere with viral entry into cells.
  • antibodies reactive with, for example, the 409-1-1(c-a) antigen can be passively administered alone in a "cocktail" with other anti-viral antibodies or in conjunction with another anti-viral agent to a host infected with an PT-NANBH virus to enhance the immune response and/or the effectiveness of an antiviral drug.
  • E. coli DNA polymerase I (Klenow fragment) was obtained from Boehringer Mannheim Biochemicals (Indianapolis, IN) .
  • T4 DNA ligase and T4 DNA polymerase were obtained from New England Biolabs (Beverly, MA) ; Nitrocellulose filters were obtained from Schleicher and Schuell (Keene, NH) .
  • Synthetic oligonucleotide linkers and primers were prepared using commercially available automated oligonucleotide synthesizers. Alternatively, custom designed synthetic oligo- nucleotides may be purchased, for example, from Synthetic Genetics (San Diego, CA) .
  • cDNA synthesis kit and random priming labeling kits were obtained from Boehringer-Mannheim Biochemical (BMB, Indianapolis, IN) . Standard manipulations of molecular biology have been carried out as previously described (Ausubel, et al . ; Sambrook, et al . ; Maniatis, et al . ) . Methods for antibody preparation and standard diagnostic applications are described in Harlow, et al .
  • PLC Polystyrene 96 well plates Immulon II
  • Antisera are typically diluted in 0.1 M PBS, pH 7.2. The desired dilution(s) of antisera (0.1 mL) are added to each well and the plate incubated 1 hours at 37°C. The plates are then washed 5 times with PBS 0.5% "TWEEN-20 M . A detection antibody is then used to detect the binding of antibodies present in the anti- serum, to the support bound antigen. For example, if rabbit anti-sera is used, horseradish peroxidase (HRP) conjugated goat anti-rabbit antiserum (Cappel) is diluted 1/5,000 in PBS. 0.1 L of this solution is added to each well. The plate is incubated 30 min at 37°C, then washed 5 times with PBS.
  • HRP horseradish peroxidase
  • Cappel horseradish peroxidase conjugated goat anti-rabbit antiserum
  • Sigma ABTS (substrate) is prepared just prior to addition to the plate.
  • the reagent consists of 50 mL 0.05 M citric acid, pH 4.2, 0.078 mL 30% hydrogen peroxide solution and 15 mg ABTS. 0.1 mL of the substrate is added to each well, then incubated for 30 min at room temperature. The reaction is stopped with the addition of 0.050 mL 5% SDS (w/v) . The relative absorbance for each sample well is determined at 410 nm.
  • RNA pellet was recovered by centrifugation in a microfuge at 12,000 x g for 15 minutes at 5°C. The pellet was washed in 70% ethanol and dried under vacuum.
  • SAM S-adenosyl-methionine
  • the reaction mixture was extracted with an equal volume of phenol/chloroform (1:1) and then with an equal volume of chloroform/isoamyl alcohol (24:1) .
  • the cDNA was precipitated with two volumes of ethanol. To maximize the number of blunt ends for the addition of linkers (Maniatis et al , 1982) the cDNA was then treated with the Klenow fragment of DNA polymerase I. The pelleted cDNA was resuspended in 11.5 ⁇ l of distilled water.
  • the reaction mixture was incubated at room temperature for 30 minutes.
  • the reaction mixture was extracted with phenol/chloroform and chloroform isoamyl alcohol as described above, and then precipitated with two volumes of ethanol.
  • the linkers used in the construction of the cDNAs contained an EcoRI site which allowed for direct insertion of the amplified cDNAs into lambda gtlO and gtll vectors (Promega, Madison WI) .
  • Lambda vectors were purchased from the manufacturer (Promega) which were already digested with EcoRI and treated with bacterial alkaline phosphatase, to remove the 5' phosphate and prevent self-ligation of the vector.
  • the _E.coi?J-linkered cDNA preparations were ligated into both lambda gtlO and gtll (Promega) .
  • the ligation reaction tubes were placed at 14°C overnight (12-18 hours).
  • the ligated cDNA was packaged the following morning by standard procedures using a lambda DNA packaging system (GIGAPAK, Stratagene, LaJolla, CA) , and then plated at various dilutions to determine the titer and recombinant frequency of the libraries.
  • a standard X-gal blue/white assay was used to screen the lambda gtll libraries (Miller; Maniatis et al . ) .
  • E. coli HG415 (from Howard Gersenfeld, Dept.of Pathology, Stanford School of Medicine) plating bacteria, which allows only plaque formation by recombinant clones, was used for plating the lambda gtlO libraries.
  • the standard strain, E. coli C600Hfl may be used as an alternative to E . coli HG415.
  • the five lambda gtll libraries generated in Example 1 were screened for specific HCV encoded viral antigens by immunoscreening.
  • the phage were plated for plaque formation using the Escherichia coli bacterial plating strain E. coli KM392 (Kevin Moore, DNax, Palo Alto, CA) .
  • E. coli Y1088 may be used.
  • the fusion proteins expressed by the lambda gtll clones were screened with serum antibodies (Young et al . ) from the following sources: chimpanzee #771 and various human PT-NANBH sera (including EGM, BV, WEH and AG) .
  • Human peripheral blood lymphocyte (PBL) DNA was restriction digested with Hindlll and EcoRI, loaded on a 0.7% agarose gel (as above, except 10 ⁇ g of DNA was loaded per lane) and the fragments separated electrophoretically.
  • the DNA fragments in the agarose gels were transferred to nitrocellulose filters (Southern) and the genomic DNA probed with the nick-translated lambda gtll inserts which were prepared above.
  • the filters were washed (Southern; Maniatis et al . ) and exposed to X-ray film. Forty-three of the 93 lambda clone inserts displayed a positive hybridization reaction with the human PBL DNA.
  • Clone 40 was clearly exogenous, i.e., not derived from normal human DNA, as evidenced by repeated hybridization tests against normal human PBL DNA, and a second clone, designated clone 36, was not only exogenous but also reactive with multiple PT- NANBH antisera.
  • Commercially available sequencing primers New England Biolabs
  • homologous to flanking lambda sequences at the 5' and 3' ends of the inserts were initially used for sequencing.
  • sequencing progressed primers were constructed to correspond to newly discovered sequences.
  • Synthetic oligonucleotide primers were prepared using commercially available automated oligonucleotide synthesizers. Alternatively, custom designed synthetic oligonucleotides may be purchased, for example, from Synthetic Genetics (San Diego, CA) .
  • DNA sequences were determined for the complete insert of clones 36 and 40 (SEQ ID NO:5 and SEQ ID N0:1, respectively).
  • the clone 36 sequence corresponds to nucleotides 5010 to 6515 of the HCV genome (Houghton, et al . , (A), EP Pub. No. 0318216; Houghton, et al . , (B) , EP Pub. No. 0388232) .
  • the clone 40 sequence corresponds to nucleotides 6516 to 7070 of the HCV genome.
  • the screening sera were as follows: GLI-1 sera was a human chronic PT-NANBH sera; BV, community acquired NANBH; SKF, PT-NANBH negative; and FEC, PT-NANBH positive.
  • the numbered sera samples correspond to human clinical serum samples which were PT-NANBH positive: these samples were obtained from Dr. Francoise Fabiani-Lunel, Hospital La Pitie Salpetriere, Paris, France.
  • the antigens produced by clones 36 and 40 yield HCV-specific immunopositive signals.
  • Clone 36 was also used for sera immunoscreening which employed an ELISA format instead of plaque screening.
  • the following Table presents a comparison of the abilities of 5 different antigens to identify HCV positive sera among a number of HCV-positive and HCV-negative sera samples.
  • HCV antigens correspond to portions of the following HCV proteins: 409-1-1, a portion of the NS3 protein; 33CU, a portion of the NS3 protein; NC450, a portion of the capsid protein; and 5-1-1, a portion of the NS4 protein.
  • the sera samples in the above table were as follows: samples 1-27 (human) and 28-30 (animal) , correspond to sera where the HCV-status of each serum was not previously characterized (NC) ; samples 31-35 and 39-40 were sera previously characterized as HCV-positive; and samples 36-38 were HCV-negative control sera. Empty cells in Table 3 indicate a negative ELISA result. Cells containing a "+" indicate a positive ELISA result.
  • EXAMPLE 6 Isolation of Clone 36 Fusion Protein Sepharose 4B beads conjugated with anti-beta galactosidase were purchased from Promega. The beads are packed in a 2 ml column and washed successively with phosphate-buffered saline with 0.02% sodium azide and 10 ml TX buffer (10 mM Tris buffer, pH 7.4, 1% aprotinin) .
  • BNN103 lysogens infected with gtll/clone 36 are used to inoculate 500 ml of NZYDT broth.
  • the culture is incubated at 32°C with aeration to an O.D. of about .2 to .4, then brought to 43°C quickly in a 43°C water bath for 15 minutes to induce gtll peptide synthesis, and incubated further at 37°C for 1 hour.
  • the cells are pelleted by centrifugation, suspended in 10 ml of lysis buffer (10 mM Tris, pH 7.4 containing 2% "TRITON X-100" and 1% aprotinin added just before use.
  • the resuspended cells are frozen in liquid nitrogen, then thawed, resulting in substantially complete cell lysis.
  • the lysate is treated with DNasel to digest bacterial and phage DNA, as evidenced by a gradual loss of viscosity in the lysate.
  • Non-solubilized material is removed by centrifugation.
  • the clarified lysate material is loaded on the Sepharose column, the ends of the column are closed, and the column is placed on a rotary shaker for 2 hrs. at room temperature and 16 hours at 4°C. After the column settles, it is washed with 10 ml of TX buffer.
  • the fused protein is eluted with 0.1 M carbonate/bicarbonate buffer, pHlO.
  • the eluate from the affinity column can be concentrated by filtration, for example, using "CENTRICON-30" cartridges (Amicon, Danvers, Mass.).
  • the final protein concentrate is resuspended in PBS buffer. Protein purity can be analyzed by SDS-PAGE.
  • the clone 36 digest fragments from lambda gtll were released by EcoRI digestion of the phage and the insert purified by gel electrophoresis. The purified fragment is introduced into the pGEX expression vector (Smith) . Expression of gluta- thione S-transferase fused protein (Sj26 fused protein) containing the clone 36 polypeptide antigen can be achieved in E. coli strain KM392 (above) . The fusion protein is isolated from lysed bacteria, and isolated by affinity chro ⁇ matography on a column packed with glutathione- conjugated beads, according to published methods (Smith) .
  • the purified Sj26/clone 36 fused protein is injected subcutaneously in Freund's adjuvant in a rabbit. Typically, approximately 1 mg of fused protein is injected at days 0 and 21, and rabbit serum collected on days 42 and 56.
  • a control rabbit is typically immunized with purified Sj26 protein obtained from control bacterial lysate. Minilysates from the bacterial cultures are prepared are screened for the present of clone 36 and Sj26 antigens using the sera from the immunized rabbits.
  • the clone 36-insert was cloned into a pET3d vector (Novagen, Madison, WI) , essentially according to conventional methods.
  • the clone 36 insert was modified so that it lacked internal Ncol and BamHI sites. This modification was achieved as follows.
  • Three polymerase chain reaction primer pairs were selected from the sequence of the clone 36 insert that defined three regions of the insert ( Figure 1) : 36R, SEQ ID NO:11; 36F, SEQ ID NO:12; 36 Nco Block F, SEQ ID NO:13; 36 Nco Block R, SEQ ID NO:14; 36 Bam Block R, SEQ ID NO:15; and 36 Bam Block F, SEQ ID NO:16.
  • the primers were designed to eliminate internal Ncol and BamHI sites by modifying third position nucleotides in the nucleic acid sequence, where such modification did not affect the protein coding sequence.
  • the primers were used in three amplification reactions (Mullis; Mullis, et al . ; supplies from Perkin Elmer/Cetus) .
  • the reactions were overlap polymerase chain reactions designed to regenerate the full length clone 36 insert without the internal WcoJ and BamHI sites.
  • the amplification reactions were run for 20 cycles each (1 minute denaturation, 1 minute annealing at 55°C, and 2 minutes of extension) .
  • the amplification products were examined on agarose gels. If insufficient amplification had occurred the amplification reactions were continued for 10 more cycles.
  • the original template DNA (used in reaction 1, below) was typically the original clone 36 gtll phage.
  • the primers were used in the three amplification reactions shown in Table 4 to generate template DNAs.
  • the resulting insert was cloned into the pET vector and designated pET-36.
  • the resulting amplification products were gel purified by standard procedures.
  • the clone 36 subfragments, 36-1 (614 bp) , 36-2 (661 bp) , and 36-3 (497 bp) respectively span the length of the clone 36 coding sequence 5' to 3'.
  • Each fragment contained _E.co.RJ sites at each of its 5' and 3' ends.
  • the amplified subfragments contain
  • the amplified subfragments were cloned into lambda gtll vectors (gtll-36-1, gtll-36-2, and gtll-36-3) for expression of the clone 36 polypeptide fragments encoded by each subfragment
  • amplified DNA regions 36-1, 36-2 and 36-3 were ligated into a lambda gtll vector using standard procedures.
  • KM392 bacterial cells were infected to yield titers of 7.3 x 10 4 pfu/ml for 36-1, 7.3 x 10 4 for 36-2, and 7.3 x 10 4 pfu/ml for 36-3.
  • These phage were plated and nitrocellulose filter lifts prepared (Schleicher and Schuell, (Keene, NH) ) .
  • the filters were immunoscreened (Ausubel, et al.) for reactivity with the following human anti-sera DPII-C1, DPII-clO, FEC, SKF and AG described above.
  • DPII stands for the panel II received from Sanofi Diagnostics Pasteur.
  • C2 and CIO are well characterized HCV samples (i.e., the sera tested positive by immunoscreening for the following HCV antigens: C100, 5-1-1, 409- 1-1, and clone 36) .
  • Sera were pre-adsorbed, prior to testing, with semi-confluent gtll phage lysates.
  • Three positive plaques selected from each plate were amplified in bacterial culture. DNA isolated from the each selected plaque was amplified using the overlapping fragment primer pairs described in Example 8 (36-1F/36-1R, 36- 2F/36-2R, and 36-3F/36-3R) . Insert sizes were confirmed by size fractionation on agarose gels.
  • EXAMPLE 10 Production of Monoclonal Antibodies A. Protein Isolation
  • a single bacterial colony containing pET-36 was inoculated into 500 ml LB + 100 ug/ml ampicillin and grown at 37°C, with vigorous aeration, until an O.D. of 0.7 at A600 was achieved. Production of the HCV antigen was induced by addition of 1.0 mM IPTG to the culture. After induction the culture was grown for approximately three hours.
  • the cells were pelleted by centrifugation at 4°C and held overnight at -21°C.
  • the frozen cells were resuspend cells in 25 ml PBS, 1 mM PMSF, 5 mM DTT, at room temperature. To this suspension,
  • the pellet was once again resuspended in 25 ml PBS, 1 mM PMSF, 1 mM DTT, and the suspension pelleted. The final supernatant was discarded and the pellet resuspended in 30 ml of 4M Urea, 20 Mm NaOAc, 5 mM DTT at pH 4.5 (Buffer A). This solution was spun-down in a Beckman JA20 rotor for 10 minutes at 10K rpm, 4°C. The resulting supernatant was filtered through a 0.45 ⁇ m "MILLEX" Filter (Millipore) .
  • Murine monoclonal antibodies were produced according to standard procedures (Mishell; Harlow, et al . ) . Mice were immunized .intraperitoneally two times, with a 21 day interval, using 200 ⁇ g pET clone 36 expression peptide (pET 36) . Blood was collected from the mice 1 week after the second injection. Sera prepared from the blood samples were tested at dilutions of 1/100, 1/1000, 1/2000 and 1/5000 in an ELISA assay for reactivity with purified pET-36 antigen.
  • the ELISA for detection of antibodies was performed essentially as follows. Polystyrene 96- 2311 plates (Falcon 3072, Becton Dickinson, Oxnard, CA) were coated with pET36 in 0.1 M bicarbonate, pH 9.0 (2 mg GSA/100 mL buffer) by adding 0.1 mL of this solution per well. Plates were incubated for 2 hrs, at 37°C, then washed 3 with PBS/0.05% "TWEEN-20". Plates were blocked by the addition of PBS/BSA (1%) for 1 hour at 37°C. Plates were washed 3x with PBS/0.05% "TWEEN-20", sealed and stored at 4°C. Antisera were diluted in 1% BSA in PBS.
  • the following dilutions of antisera were tested: 1/100, 1/1000, 1/2000 and 1/5000.
  • the desired dilution(s) of antisera (0.1 ml) were added to each well and the plate incubated 2 hours, at 37°C. The solution was then removed from the plate, and the wells washed 3 times with PBS/0.05% "TWEEN-20".
  • Alkaline phosphatase conjugated goat anti- mouse antiseru (affinity purified — Zymed, Berkeley, CA) was diluted 1/3000 in PBS/1% BSA. 0.1 mL of this solution was added to each well. This solution was left on the plate for 1 hour at room temperature. The plate was then washed twice with PBS/Tween-20 and three times with PBS, as above.
  • Color reagent ⁇ substrate was either BCIP (5- bromo-4-chloro-3-indolyl phosphate P-toluidine salt, 50 mg/ml in 100% dimethyl formamide) or NBT (Nitro blue tetrazoliu Grad. Ill, 50 mg/ml in 100% dimethyl formamide) , both reagents are available from Sigma ⁇ was prepared just prior to addition to the plate. 0.1 mL of the color reagent was added to each well, then incubated for 30 min at room temperature prior to determination of the absorbance, at 405 n , relative to the blank well, containing no mouse sera. Spleens were removed from mice exhibiting seroreactivity of at least 0.5 O.D.
  • Hybridoma cells were formed by fusion of splenic B-lymphocytes from seropositive mice and stable myeloma fusion partner cells, SP2/0 cells (SP2/0-Agl4; ATCC CRL 1581, American Type Culture Collection, Rockville, MD) , mixed at a ratio of about 1:1 (splenic cells:myeloma cells). Fusion of the cells was promoted by addition to the cells of polyethylene glycol (PEG-1500; Sigma Chemical Co., St. Louis, MO; final concentration: 50%).
  • PEG-1500 polyethylene glycol
  • Hybridomas were selected by growth in selective (HAT) medium, then were tested for production of anti-pET-36 antibodies in a solid phase assay (ELISA) as described above. Hybridomas found to produce antibodies to pET 36 were further clone purified by limiting dilution.
  • Example 8 The following clones were plated as described in Example 8: subfragment clones gtll-36-1, gtll- 36-2, and gtll-36-3; and lambda gtll. Each plate of phage-infected bacteria was overlaid with a nitrocellulose sheet. The expression products from the plaques transferred to filter paper. The plate and filter were indexed for matching corresponding plate and filter positions. The filter was removed after 6-12 hours, washed three times in TBS buffer (10 mM Tris, pH 8.0, 150 mM NaCl) , blocked with AIB (TBS buffer with 1% gelatin) , washed again in TBS, and incubated overnight with hybridoma supernatants from the 49 positive clones identified in Example 9. Each filter was washed twice in TBS, then incubated with alkaline-phosphatase-conjugated anti-human IgG to attach the labeled antibody at filter sites containing antigen recognized by the antiserum.
  • the filter was developed in a substrate medium containing 33 ⁇ l NBT (50 mg/ml stock solution maintained at 4°C) mixed with 16 ⁇ l BCIP (50 mg/ml stock solution maintained at 4°C) in 5 ml of alkaline phosphatase buffer (100 mM Tris, 9.5, 100 mM NaCl, 5 mM MgC12) . Reacted substrate precipitated at points of antigen production, as recognized by the antiserum.
  • NBT 50 mg/ml stock solution maintained at 4°C
  • BCIP 50 mg/ml stock solution maintained at 4°C
  • alkaline phosphatase buffer 100 mM Tris, 9.5, 100 mM NaCl, 5 mM MgC12
  • Results from immunoscreening experiments using selected hybridoma supernatants are shown in Tables 6 and 7.
  • the hybridoma supernatants used in Table 6 were strongly positive in the original screening against the entire clone 36 coding region (Example 10) .
  • the hybridoma supernatants used in Table 7 were less strongly positive than those used in Table 6.
  • the first column of each table presents the clone number used to produce the hybridoma supernatant. The presence of plaques showing antigen-positive reaction is indicated by "+" in these tables.
  • the results of overall immunoreactivity of each of the clone 36 coded sub-polypeptides, and the negative control (lambda gtll) are presented in the tables.
  • This example describes the use of an HCV non- structural protein antigen to design an antigen- based detection system for HCV.
  • sera containing polyclonal antibodies are initially fractionated by addition of ammonium sulfate.
  • the supernatant is then passed over a gel filtration column (Pierce) and the IgG containing fraction identified by absorbance (A 280 ) • IgG molecules can be isolated by a number of standard procedures (Garvey, et al . ) , including affinity chromatography, or by the use of commercially available kits (Pierce) .
  • microtiter plates (Dynatech) are coated with the antibody as follows (Harlow, et al . ) . Approximately 50 ⁇ l of purified antibody (approximately 20 ⁇ g/ml) , in phosphate buffered saline (PBS) (Maniatis, et al . ) is added to each well of polyvinylchloride plates. The plates are sealed and incubated for either 4 hours at room temperature or overnight at 4°C. Alternatively, polystyrene 96 well plates
  • IMMULON II PPC
  • suitable plastic plates Corning Biotechnology, Corning NY
  • 5 ug/ L (100 ⁇ L per well) antibodies in 0.1 M carb/bicarbonate buffer, pH 9.5, the plates sealed with parafilm and stored at 4°C overnight.
  • the wells can also be coated with an anti-mouse IgG antibody, followed by addition of the mouse monoclonal antibodies. After incubation the wells are washed twice with binding buffer (PBS or carb/bicarbonate buffer) .
  • binding buffer PBS or carb/bicarbonate buffer
  • BSA bovine serum albumin
  • the plates were then incubated for approximately 2 hours at room temperature and the liquid removed.
  • the antibody coated wells were then incubated with the test samples, such as, the clone 36- encoded polypeptide, HCV infected tissue culture media, human sera, or chimpanzee sera, for 1 hr. After incubation, clone 36 polypeptide-HRPO (horse radish peroxidase) conjugate was added to each well. Polypeptide-HRPO conjugates were formed using commercially available activated HRPO (Pierce) .
  • HRPO is coupled to peptides using one of the following techniques: (i) the glutaraldehyde technique that links through epsilon-amino acid groups on lysine residues, or other free amino groups (Pierce) ; or (ii) a two-step procedure using m-maleimidobenzoyl sulfosuccinimide ester (sulfo-MBS) to link through free disulfide linkages (Pierce) .
  • sulfo-MBS m-maleimidobenzoyl sulfosuccinimide ester
  • a cysteine residue or a lysine residue can be added to the N-terminus of the peptide.
  • Combination of the conjugate to the solid phase antibody coat was detected by the addition of the substrate-2,2'-azino-bis(3- ethylbenzthiazoline-6-sulfonic acid (ABTS) (Pierce) .
  • ABTS substrate-2,2'-azino-bis(3- ethylbenzthiazoline-6-sulfonic acid
  • the presence of a HCV-NS5 associated viral antigen was identified by color diminution due to successful inhibition of binding clone 36- HRPO.
  • the above protocol is first tested by addition of increasing quantities of free clone 36 polypeptide.
  • a linear inhibition curve is generally established, for example, over the range of 10 ⁇ g/ml to O.ooi ⁇ g/ml.
  • EXAMPLE 13 Antigen Confirmatory Assay A confirmatory assay was designed and tested to confirm positive antigen reactive plasma.
  • the basic design is a blocking of the clone 36-HRPO inhibition assay (Blocking of Inhibition) by addition of anti-clone 36 positive sera to antigen positive plasma before testing in the antibody coated plates.
  • the reagents for the confirmatory assay were evaluated by titration. HCV antibody positive sera are tested for their ability to block the inhibition reaction.
  • NAME Desjardins, Cathleen M.
  • ORGANISM Hepatitis C Virus
  • MOLECULE TYPE protein
  • ORGANISM Hepatitis HCV Virus
  • ORGANISM Hepatitis C Virus
  • GTA ACC CAC ATC AAC TCC GTG TGG AAA GAC CTT CTG GAA GAC AAT GTA 1296 Val Thr His He Asn Ser Val Trp Lys Asp Leu Leu Glu Asp Asn Val 420 425 430
  • ORGANISM Hepatitis C Virus
  • ORGANISM Hepatitis C Virus
  • MOLECULE TYPE protein
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE protein
  • HYPOTHETICAL NO
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)

Abstract

Antigen and antibody compositions immunoreactive with hepatitis C virus infected sera are disclosed. The antigen composition include a peptide corresponding to the predicted NS5 protein of HCV. The antibody composition contains monoclonal antibodies immunoreactive with several epitopes of this antigen. The antigens are useful in generating antibodies for use in antigen-inhibition immunodiagnostic methods for detecting the presence of HCV antigens in test samples.

Description

HEPATITIS C VIRUS IMMUNODIAGNOSTIC ANTIGENS AND ANTIBODIES
Field of Invention This invention relates to specific peptide viral antigens which are immunoreactive with sera from patients infected with parenterally trans¬ mitted non-A, non-B hepatitis (PT-NANBH) virus, to antibodies immunoreactive with these antigens, and to methods of using the antibodies for detecting PT-NANBH infection in human sera.
References
Ausubel, F. M. , et al . , Current Protocols in Molecular Biology. John Wiley and Sons, Inc., Media PA.
Bradley, D.W. , et al . , J. Infec. Dis., 148:2 (1983) .
Bradley, D.W. , et al . , J Gen. Virol., 69:1 (1988).
Bradley, D.W. , et al . , Proc. Nat. Acad. Sci., USA, 84.:6277 (1987) .
Carroll, W.P., et al . , J. Immunol. Methods 9:61 (1986). Chomczyns i, P., et al . , Anal Biochem, 162:156 (1987).
Choo, Q.-L., et al , Science, 244:359 (1989).
Current Protocols in Molecular Biology. Wiley Interscience, Chapter 10. de Wet, J.R. , et al . , Mol. Cell. Biol. 2:725 (1987).
Dienstag, J.L. , et al , Sem Liver Disease,
Figure imgf000003_0001
Feramisco, J. R. , et al . , J. Biol. Chem. 257.(18) :11024 (1982).
Garvey, J.S., et al . , Methods in Immunology. Third Edition, Benjamin Cummings, publisher (1977) .
Gubler, U. , et al , Gene, 2.5:263 (1983). Harlow, E. , et al . , Antibodies: A Laboratory Manual. Cold Spring Harbor Press, Cold Spring Harbor, NY (1988) .
Houghton, M. , et al., EPO Patent Application No. 88/310922.5, Publication No. 0 318 216 Al, published 31 May 1989.
Houghton, M. , et al . , EPO Patent Application No. 90/302866.0, Publication No. 0 388 232 Al, published 19 Sept. 1990. Hunyh, T.V. , et al, in DNA Cloning
Techniques: A Practical Approach (D. Glover, ed.) IRL Press (1985) .
Kuo, G. , et al . , Science, 244:362 (1989). Maniatis, T. , et al. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory (1982) .
Miller, J.H. , Experiments in Molecular Genetics.. Cold Spring Harbor Laboratories, Cold Spring Harbor, NY (1972) . Mullis, K.B., U.S. Patent No. 4,683,202, issued 28 July 1987.
Mullis, K.B., et al., U.S. Patent No. 4,683,195, issued 28 July 1987.
Perkins, S., et al . , in Borrebaeck, C.A.K., Hagen, I. (eds) Electromanipulation in Hvbridoma Technology, A Laboratory Manualf Stockton Press, New York (1989) .
Perkins, S., et al . Hum . Antibod. Hybridomas 2:155-159 (1991). Reyes, G. , et al , Science, 247:1335 (1990).
Sa brook, J. , et al . , In Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Vol. 2 (1989) .
Scharf, S.J., et al . , Science 233:1076 (1986). Mishell, B.D. , et al . , eds., Selected Method in Cellular Immunology. W.H. Freeman and Co. , pp416-440 (1980) .
Smith. D.B., et al , Gene, €>7.:31 (1988). Southern, E. , Methods in Enzymology 69:152 (1980) .
Woo, S.L.C., Methods in Enzymology 68:389 (1979) .
Young, R.A. and R.W. Davis, Proc. Natl . Acad . Sci . USA 80:1194-1198 (1983).
Background of the Invention
Viral hepatitis resulting from a virus other than hepatitis A virus (HAV) and hepatitis B virus (HBV) has been referred to as non-A, non-B hepatitis (NANBH) . More recently, it has become clear that NANBH encompasses at least two, and perhaps more, quite distinct viruses. One of these, known as enterically transmitted NANBH or ET-NANBH, is contracted predominantly in poor- sanitation areas where food and drinking water have been contaminated by fecal matter. The molecular cloning of a portion of this virus, referred to as the hepatitis E virus (HEV) , has recently been described (Reyes, et al . ) . The second NANB virus type, known as parenterally transmitted NANBH, or PT-NANBH, is transmitted by parenteral routes, typically by exposure to blood or blood products. Approximately 10% of transfusions cause PT-NANBH infection, and about half of these go on to a chronic disease state (Dienstag) .
Human sera documented as having produced post-transfusion NANBH in human recipients has been used successfully to produce PT-NANBH infection in chimpanzees (Bradley) . RNA isolated from infected chimpanzee sera has been used to construct cDNA libraries in an expression vector for immunoscreening with chronic-state human PT- NANBH serum. This procedure identified a PT-NANBH specific cDNA clone and the viral sequence was then used as a probe to identify fragments making up 7,300 contiguous basepairs of a PT-NANBH viral agent (Houghton, et al . , (A), EP Pub. No. 0318216; Houghton, et al . , (B) , EP Pub. No. 0388232).
Summary of the Invention
The present invention provides a method for detecting the presence of hepatitis C virus (HCV) antigens in a sample. In the method of the present invention the sample is contacted with at least one antibody which is reactive with an HCV antigen. The antibody is usually attached to a solid support and is immunoreactive with a portion of the polypeptide presented as SEQ ID NO:6. The support-bound antibody is then examined for the presence of bound HCV antigen. This examining typically involves reacting the solid support with an antigen-reporter complex, where the HCV antigen competes with binding of the antigen-reporter complex to the antibody. The level of antigen- reporter complex, which is bound to the solid support, is then determined.
The method can be used to analyze samples from a variety of sources including, but not limited to, the following: tissue culture medium, chimpanzee serum, and human serum.
A number of solid supports are useful in the method of the present invention, including microtiter plates. In the method of the present invention two or more antibodies, with their corresponding antigen-reporter complexes, can also be used. Antigens useful in generating antibodies for the method of the present invention include the polypeptides selected from the group of sequences presented as SEQ ID NO:6, SEQ ID NO:17, SEQ ID NO:18, and SEQ ID NO:19, and derivatives thereof. These antigens are also useful in antibody-capture assays, where the antigen polypeptide is attached to a solid support and serum is screened for antibodies capable of binding to the antigens. A number of reporters can be employed in the present method. The antigen-reporter complexes can contain one or more of the following: enzymatic reporters, radioactive reporters, and fluorescent reporters. An exemplary enzymatic reporter is horse radish peroxidase.
The present invention also includes a diagnostic kit for use in the screening samples for the presence of hepatitis C virus (HCV) antigen, by the method of the present invention. Such kits typically include (i) at least one antibody which is immunoreactive with a portion of the polypeptide presented as SEQ ID NO:6, and (ii) an antigen-reporter complex, where the HCV antigen competes with binding of the antigen-reporter complex to the antibody. Further, the antibody can be attached to a solid support. The antigens and antibodies are as described above.
Further, the present invention includes purified antibodies that are immunoreactive with a polypeptide consisting essentially of a sequence selected from the following: SEQ ID NO:6, SEQ ID NO:17, SEQ ID NO:18, and SEQ ID NO:19. These antibodies can be polyclonal or monoclonal antibodies. Another aspect of the present invention includes polypeptides consisting essentially of the following sequences: SEQ ID NO:6, SEQ ID NO:17, SEQ ID NO:18, and SEQ ID NO:19. These polypeptides, and polypeptides containing other epitopes included in the clone 36 sequence, are useful in antibody-capture assays, the generation of antibodies for the diagnostic method of the present invention, and the generation of antibodies for use in immunoprophylaxis. Further, these polypeptides may be useful as polypeptide antigen vaccines effective against HCV.
Brief Description of the Drawings
Figure 1 illustrates the construction of a modified clone 36 insert lacking internal Ncol and BamHI sites. Figure 2 schematically illustrates the basis design of an antibody based competition immunoassay.
Detailed Description of the Invention I- Definitions
The terms defined below have the following meaning herein:
A. "Parenterally transmitted non-A, non-B hepatitis viral agent (PT-NANBH)" means a virus, virus type, or virus class which (i) causes parenterally transmitted infectious hepatitis, (ii) is transmissible in chimpanzees, (iii) is serologically distinct from hepatitis A virus (HAV) , hepatitis B virus (HBV) , and hepatitis E virus (HEV) .
B. "HCV (HCV)" means a PT-NANBH viral agent whose polynucleotide sequence includes the sequence of the 7,300 basepair region of HCV (Houghton, et al . , (A), EP Pub. No. 0318216; Houghton, et al . , (B) , EP Pub. No. 0388232), and variations of the sequence, such as degenerate codons, or variations which may be present in different isolates or strains of HCV.
C. Two nucleic acid fragments are "homologous" if they are capable of hybridizing to one another under hybridization conditions described in Maniatis et al., op. cit.. pp. 320- 323, using the following wash conditions: 2 x SCC, 0.1% SDS, room temperature twice, 30 minutes each; then 2 x SCC, 0.1% SDS, 50°C once, 30 minutes; then 2 x SCC, room temperature twice, 10 minutes each, homologous sequences can be identified that contain at most about 25-30% basepair mismatches. More preferably, homologous nucleic acid strands contain 15-25% basepair mismatches, even more preferably 5-15% basepair mismatches. These degrees of homology can be selected by using more stringent wash or hybridization conditions for identification of clones from gene libraries (or other sources of genetic material) , as is well known in the art.
D. A DNA fragment is "derived from" HCV if it has substantially the same basepair sequence as a region of the HCV viral genome which was defined in (2) above. E. A protein is "derived from" a PT-NANBH or HCV viral agent if it is encoded by an open reading frame of a cDNA or RNA fragment derived from a PT-NANBH or HCV viral agent, respectively.
II. Molecular Clone Selection by Immunoscreening In the screening procedure reported in Examples 1-3, individual cDNA libraries were prepared from the serum of one PT-NANBH infected chimpanzee (#771) and four PT-NANBH infected humans (designated EGM, BV, WEH, and AG) . These five libraries were immunoscreened using PT-NANBH positive human or chimpanzee sera (Example 2) : 111 lambda gtll clones were identified which were immunoreactive with at least one of the sera. Of these 111 clones, 93 were examined for insert hybridization with normal DNA. The inserts were radioactively labelled and used as probes against Hindlll / EcoRI doubly-digested human peripheral lymphocyte (PBL) DNA (Example 3) . Approximately 46% (43/93) of the inserts hybridized with normal human PBL DNA and were therefore not pursued. Inserts from 11 PT-NANBH-immunopositive clones derived from chimpanzee #771 sera were characterized as exogenous to normal human PBL DNA (Example 3) . Of these 11 clones 2 PT-NANBH clones were identified having the following characteristics.
One clone (clone 40) was clearly exogenous by repeated hybridization tests against normal human PBL DNA, had a relatively small insert size (approximately 0.5 kilobases) , and was quite unreactive with negative control serum.
The second clone (clone 36) was shown to be reactive with multiple PT-NANBH antisera, had a relatively large insert size (approximately 1.5 kilobases) , and was exogenous by hybridization testing against normal human PBL DNA. The immunoreactive characteristics of clones 36 and 40 are summarized in Table 1 (Example 3) . Clone 36 was immunoreactive with chimpanzee #771 sera and two HCV-positive human sera, AG and BV. The clone 36 antigen did not immunoreact with the negative control serum SKF. Clone 40 was immunoreactive with chimpanzee #771 sera and was cleanly nonreactive when the negative control sera was used for screening. The DNA sequences of clones 36 and 40 were determined and are presented as SEQ ID NO:5 and SEQ ID N0:l, respectively. The clone 36 sequence corresponds to nucleotides 5010 to 6516 of the HCV sequence given in Houghton, et al . , (A), EP Pub. No. 0318216. The clone 40 sequence is homologous to the HCV sequence (Houghton, et al . , (A), EP Pub. No. 0318216) in the region of approximately nucleotides 6515 to 7070. The sequences for clones 36 and 40 are contiguous sequences, with the clone 36 sequences being located 5' of the clone 40 sequences as presented in Houghton, et al . , (A), EP Pub. No. 0318216, and Houghton, et al . , (B) , EP Pub. No. 0388232. Accordingly, these two clones represent isolation of a significant block of the HCV genome by the above-described immunoscreening methods. The inserts of two other chimpanzee #771 clones, clones 44 and 45, were found to be homologous to clone 40 by hybridization and sequence analysis (Example 4) . Isolation and characterization of the four lambda gtll clones 36, 40, 44, and 45 were previously described in PCT International Application PCT/US91/02370, Publication No. WO 91/15516, Publication Date 17 October 1991.
III. Immunoreactive of The Clone 36 and 40 Encoded Antigens
A. Immunoreactivity Screening
Table 2 (Example 5) presents the data for preliminary immunoscreening of clone 36 and 45 antigens (the insert of clone 45 is essentially the same as the insert of clone 40) against well- documented PT-NANBH chronic sera which showed strong immunoreactivity to the 5-1-1 HCV peptide antigen (Kuo) . The 5-1-1 HCV peptide antigen has previously been identified as immunoreactive against a high percentage of human PT-NANBH chronic sera. The 5-1-1 antigen is encoded by the sequence between basepairs 3731 and 3857 in the HCV genome (Houghton, et al. , (A), EP Pub. No. 0318216; Houghton, et al . , (B) , EP Pub. No. 0388232) and is itself contained in a larger peptide antigen C-100 encoded by the sequence between basepairs 3531 and 4442. The latter peptide is employed in a commercial diagnostic kit for detection of human HCV infection (Ortho/Chiron) . The kit is reported to react positively with about 80% of human chronic PT- NANBH samples, and about 15% of human acute PT- NANBH sera, as noted above.
The 409-1-1 (c-a) phage (immunoscreening and plaque purified) and a related clone designated 409-1-1(abc) were previously identified in PCT International Application PCT/US91/02370,
Publication No. WO 91/15516, Publication Date 17 October 1991. The sequences of the 409-1-1 antigens are presented in the Sequence Listing.
As can be seen from the results presented in Table 2, the antigens produced by clones 36 and 40 yield HCV-specific immunopositive signals with selected samples. Additionally, subfragments of clone 36, when expressed in a lambda gtll expression system, were immunopositive with HCV- positive serum samples.
Further, the data presented in Table 3 suggest that clone 36 identified all HCV-positive sera (within a selected sample) that were identified by any other single HCV antigen or combination of HCV antigens, including the C100 antigen which was employed in a commercial HCV diagnostic assay (the C-100 antigen corresponds essentially to clone 5-1-1, Table 3) . In addition, the results show that the clone 36 antigen detected six unique positive sera samples. The clone 36 antigen tested negative with all negative control sera. The results presented in Table 2 and 3 support the use of the clone 36 antigen in immunodiagnostic assays directed to the detection of HCV.
B. Clone 36 and Clone 36 Subfraqments In order to further investigate the nature of the clone 36 antigen epitopes, sub-clones of the clone 36 antigen were produced (Example 8) . The clone 36-insert was cloned into a pET3d vector and modified such that it lacked internal WcoJ and BamHI sites (Example 8) . Using sequence specific primers in a series of polymerase chain reactions, three subfragment clones of clone 36 were generated.
The amplified subfragments were cloned into lambda gtll vectors (gtll-36-1, gtll-36-2, and gtll-36-3) for expression of the clone 36 polypeptide fragments encoded by each subfragment. These clones were used to test for expression of polypeptides capable of reacting with HCV-positive antisera in an immunoscreening assay. The antigens were immunoscreened for reactivity with a number of human anti-sera including well- characterized HCV positive sera (Example 9) . Each of the three sub-cloned clone 36 fragments tested immunopositive with at least one of the human anti-sera and tested negative with known HCV- negative control sera. These results indicate that each of the three portions contained at least one epitope immunoreactive with HCV-positive sera.
Other antigen containing portions of the clone 36 coding sequence can be identified by the methods described above.
C. Antigen Polypeptide Purification The recombinant peptides of the present invention can be purified by standard protein purification procedures which may include differential precipitation, molecular sieve chromatography, ion-exchange chromatography, isoelectric focusing, gel electrophoresis and affinity chromatography. In the case of a fused protein, such as the beta-galactosidase fused proteins prepared as above, the fused protein can be isolated readily by affinity chromatography, by passing cell lysis material over a solid support having surface-bound anti-beta-galactosidase antibody. For example, purification of a beta- galactosidase/fusion protein, derived from clone 36 coding sequences, by affinity chromatography is described in Examples 6.
A fused protein pontaining the clone 36 peptide fused with glutathione-S-transferase (Sj26) protein has also been expressed using the pGEX vector system in E. coli KM392 cells (Smith) . This expression system has the advantage that the fused protein is generally soluble and therefore can be isolated under non-denaturing conditions. The fused Sj26 protein can be isolated readily by glutathione substrate affinity chromatography
(Smith) . This method of expressing this fusion protein is given in Example 7 and is applicable to any of the other antigen coding sequences described by the present invention. Yet another protein isolation method is described in Example 11 for the isolation of the pET-clone 36 antigen. Briefly, production of the pET-clone 36-encoded HCV antigen is induced in bacterial cells. The cells are lysed and the pelleted proteins are subjected to repeated rounds of suspension and re-pelleting. Then the protein pellet is suspended, filtered and subjected to size fractionation. Size fractionation can be achieved by any standard method including FPLC. Fractions resulting from the size fractionation are then assayed for the presence of the protein. Fractions containing the antigenic protein of interest can be identified by a number of means including, immunoreactivity (e.g., ELISA assays) or separation on SDS polyacrylamide gels (identification by molecular weight) . The polypeptide antigens of the present invention have a number of uses including, but not limited to, the following: antigens in immunoassays, such as ELISA (Example 10) ; vaccine compositions; and the generation of monoclonal and polyclonal antibodies.
IV. Anti-HCV Antigen Antibodies
The present invention includes antibodies specific against the recombinant antigens described above, including the clone 36 and clone 40 antigens, and further derivatives of either.
A. Polyclonal Antibodies
Typically, to prepare polyclonal antibodies, a host animal, such as a rabbit, is immunized with the purified antigen or fused protein antigen. The host serum or plasma is collected following an appropriate time interval, and this serum is tested for antibodies specific against the antigen. Example 7 describes the production of rabbit serum antibodies which are specific against the clone 36 antigens in the Sj26/clone 36 fusion protein. These techniques are equally applicable to the other antigens of the present invention. The gamma globulin fraction or the IgG antibodies of immunized animals can be obtained, for example, by use of saturated ammonium sulfate or DEAE Sephadex, or other techniques known to those skilled in the art for producing polyclonal antibodies.
B. Monoclonal Antibodies
1. Hybridomas. Alternatively, the purified antigen or fused antigen protein may be used for producing monoclonal antibodies. Here the spleen or lymphocytes from an immunized animal are removed and immortalized or used to prepare hybridomas by methods known to those skilled in the art (Mishell) . In one exemplary method, described in Example 10, mice are immunized by intraperitoneal injection of the purified or partially purified antigen derived from clone 36 containing phage infected cells. The antigen is mixed with an adjuvant, such as killed Bordatella pertussis organisms. Hybridomas are formed by fusion of splenic B-lymphocytes and stable myeloma fusion partner cells. Such fusion can be achieved by a number of methods known in the art (Harlow; Mishell) including exposure of mixed cells to polyethylene glycol and exposure of cells to strong electric field (electrofusion) . Hybridomas are selected by growth in selective medium, then are tested for production of antibodies reactive with the specific im unogen, in one or more assays, such as a solid phase (ELISA) assay, plaque immunoscreening, or Western blot analysis (Ausubel, et al . ) .
Hybridomas found to produce reactive antibodies are further clone purified by limiting dilution.
Example 10 describes the generation of monoclonal antibodies directed against the pET36 antigen. Hybrido a cells were formed by fusion of splenic B-lymphocytes from seropositive mice and stable myeloma fusion partner cells. Hybridomas were selected by growth in selective medium and tested for production of anti-pET-36 antibodies in a solid phase assay (ELISA) . Forty-nine positive clones were identified and isolated in one such selection. Hybridomas found to produce antibodies to pET 36 were further clone purified by limiting dilution. From the 49 positive clone, 5 were subcloned by limiting dilution.
The subclones of clone 36 (Example 8) were screened to evaluate their immunoreactivity relative to the hybridoma supernatants (Example 11) . Supernatants from the 49 hybridomas identified above were screened against the clone 36 subclones. The results of these screening assays are presented in Tables 6 and 7 (Example
11) . These data demonstrate the identification of at least two independent epitopes in the clone 36 polypeptide.
2. Human Hybridomas. Human hybridomas can also be produced by fusing a human lymphocyte with an appropriate immortalized fusion partner. A donor known to be infected with an HCV virus (where infection has been shown for example by the presence of anti-virus antibodies in the blood) may serve as a suitable lymphocyte donor. Lymphocytes can be isolated from a peripheral blood sample or from spleen cells, if the donor is subject to splenectomy. Generally, antibody- secreting B-lymphocytes are activated prior to selection using a transforming virus, such as Epstein-Barr virus, or can alternatively be achieved by exposure of the cells to other B-cell activators known in the art, such as pokeweed mitogen, or to the specific antigen recognized by the cells. Following activation, cells are grown in culture, then examined for production of specific anti-HCV antigen activity, using an appropriate antibody detection assay. Cells showing activity in such an assay are selected for immortalization, by fusion with a heteromyeloma fusion partner. Formation of a stable hybridoma that secretes a human anti-HCV antibody is achieved by fusing an activated B-lymphocyte with a heteromyeloma cell such as the K6H6-B5 cell line (Carroll, et al . ) or the H73C11 cell line (Perkins, et al.), originally produced by fusing activated human lymphocytes with a mouse myeloma fusion partner. Such fusion can be achieved by a number of methods known in the art (Harlow, et al . ) including exposure of mixed cells to polyethylene glycol or exposure of cells to strong electric field (electrofusion) . Hybridomas are selected by growth in selective medium, then are tested for HCV and antigen specificity in one or more i munoassays.
Primary in vitro immunization with peptide or protein antigens of hybridomas in culture can also be used in the generation of monoclonal antibodies. Antibodies secreted by the immortalized cells are screened to determine the clones that secrete antibodies of the desired specificity.
V. Development of an Immunoassav for Detection of HCV NS5 Antigen
The basic design of the immunoassay is illustrated in Figure 2. One example of the antigen detection assay is described in Example 12. In this example, a rabbit anti-clone 36 antibody coated well and a clone 36 antigen-horse radish peroxidase (HRPO) conjugate are used in the assay. Microwells were coated with the above- described monoclonal antibodies. The antibody coated wells is then incubated with the test samples, such as, clone 36 polypeptide, HCV infected tissue culture media, human sera or chimpanzee sera. After incubation, clone 36-HRPO conjugate is added to each well. Combination of the conjugate to the solid phase antibody coat was detected by the addition of the substrate-2 , 2 ' - azino-bis(3-ethylbenzthiazoline-6-sulfonic acid (ABTS) . An alternative substrate which can be used is 5-aminosalicylic acid (5AS) . The presence of a clone 36 HCV-associated viral antigen is identified by color diminution due to successful inhibition of binding clone 36-HRPO.
A number of reporter labels, other than HRPO can be used in the method of the present invention, including the following: enzymatic reporter systems, such as HRPO alkaline phosphatase, β-galactosidase, and glucose oxidase (Pierce, Rockford IL) ; fluorochrome reporters, such as fluorescein, R-phycoerythrin, rhodamine, rhodamine 600, and "TEXAS RED" (Pierce); biotin and avidin (Pierce) ; radioactive labelling, such as 125I or synthesis of antigen polypeptides containing 3H or 14C; light emitting reporters, such as luciferase (de Wet, et al . ) ; and chromophors, such as heme (Sigma, St. Louis MO) . Reporter labels are conjugated to antigen peptides by appropriate standard methods in the art.
The antigen detection assay of the present invention can also be tested by addition of increasing quantities of free clone 36 polypeptide. The addition of the clone 36 polypeptide over a linear range of concentrations results in a linear inhibition curve. A method to confirm the specificity of the antigen detection reaction is described in Example 13. This confirmation assay is based on blocking the inhibition of clone 36-HRPO binding to its cognate antibody, which usually occurs by binding of a clone 36-based antigen to the same antibody. The blocking of inhibition is accomplished by addition of anti-clone 36 positive sera to the antigen positive plasma before testing in the antibody coated plates.
VI. Utility A. Antibody Capture: Diagnostic Method and Kit The antigens obtained by the methods of the present invention are advantageous for use as diagnostic agents for anti-HCV antibodies present in HCV-infected sera; particularly, clone 36 antigen and related antigens 36-1, 36-2 and 36-3. As noted above, the clone 36 antigen provides an advantage over known HCV antigen reagents 5-1-1 and C-100 in that clone 36 is immunoreactive with a wider range of PT-NANBH infected sera.
In one preferred diagnostic configuration, test serum is reacted with a solid phase reagent having a surface-bound HCV antigen obtained by the methods of the present invention, e.g., the clone 36 antigen. After binding anti-HCV antibody to the reagent and removing unbound serum components by washing, the reagent is reacted with reporter- labeled anti-human antibody to bind reporter to the reagent in proportion to the amount of bound anti-PT-NANBH antibody on the solid support. The reagent is again washed to remove unbound labeled antibody, and the amount of reporter associated with the reagent is determined. Typically, the reporter is an enzyme which is detected by incubating the solid phase in the presence of a suitable fluorometric or colorimetric substrate (Harlow, et al . ) . The solid surface reagent in the above assay is prepared by known techniques for attaching protein material to solid support material, such as polymeric beads, dip sticks, 96-well plate or filter material. These attachment methods generally include non-specific adsorption of the protein to the support or covalent attachment of the protein, typically through a free amine group, to a chemically reactive group on the solid support, such as an activated carboxyl, hydroxyl, or aldehyde group.
In a second diagnostic configuration, known as a homogeneous assay, antibody binding to a solid support produces some change in the reaction medium which can be directly detected in the medium. Known general types of homogeneous assays proposed heretofore include (a) spin-labeled reporters, where antibody binding to the antigen is detected by a change in reported mobility (broadening of the spin splitting peaks) , (b) fluorescent reporters, where binding is detected by a change in fluorescence efficiency, (c) enzyme reporters, where antibody binding effects enzyme/substrate interactions, and (d) liposome- bound reporters, where binding leads to liposome lysis and release of encapsulated reporter. The adaptation of these methods to the protein antigen of the present invention follows conventional methods for preparing homogeneous assay reagents. In each of the assays described above, the assay method involves reacting the serum from a test individual with the protein antigen and examining the antigen for the presence of bound antibody. The examining may involve attaching a labeled anti-human antibody to the antibody being examined, either IgM (acute phase) or IgG (convalescent or chronic phase) , and measuring the amount of reporter bound to the solid support, as in the first method, or may involve observing the effect of antibody binding on a homogeneous assay reagent, as in the second method. Also forming part of the invention is an assay system or kit for carrying out the assay method just described. The kit generally includes a support with surface-bound recombinant HCV antigen (e.g., the clone 36 antigens), and a reporter labeled reporter-labeled anti-human antibody for detecting surface-bound anti-PT- NANBH-antigen antibody.
B. Peptide-Based Vaccine The HCV antigens identified by the methods of the present invention, e.g. clone 36, can be formulated for use in a HCV vaccine. The vaccine can be formulated by standard methods, for example, in a suitable diluent such as water, saline, buffered salines, complete or incomplete adjuvants, and the like. The immunogen is administered using standard techniques for antibody induction, such as by subcutaneous administration of physiologically compatible, sterile solutions containing inactivated or attenuated virus particles or antigens. An immune response producing amount of virus particles is typically administered per vaccinizing injection, typically in a volume of one milliliter or less. A specific example of a vaccine composition includes, in a pharmacologically acceptable adjuvant, a recombinant clone 36 peptide. The vaccine is administered at periodic intervals until a significant titer of anti-HCV antibody is detected in the serum.
C. Antigen Capture: Diagnostic Method and Kit In one preferred diagnostic configuration, test serum is reacted with a solid phase reagent having a surface-bound anti-HCV-antigen antibody, either polyclonal or monoclonal, obtained by the methods of the present invention: for example, an anti-clone 36 antigen antibody. After exposure to the test serum, the solid phase is washed and then contacted with a reporter labeled antigen containing the epitope corresponding to the surface-bound anti-HCV-antigen antibody. The level of reporter is then quantitated and the serum-antigen levels are determined based on the percent inhibition of antigen-reporter binding obtained in the presence of the antigen-containing serum: typically by comparison to a standard curve.
Generally, the reporter is an enzyme which is detected by incubating the solid phase in the presence of a suitable fluorometric or colorimetric substrate. A number of alternative- reporter systems have been described above.
The solid surface reagent in the above assay is prepared by known techniques for attaching protein material to solid support material, such as polymeric beads, dip sticks, 96-well plate or filter material (such as nitrocellulose) (Harlow, et al . ) . These attachment methods generally include non-specific adsorption of the protein to the support or covalent attachment of the protein, typically through a free amine group, to a chemically reactive group on the solid support, such as an activated carboxyl, hydroxyl, or aldehyde group.
In each of the assays described above, the assay method involves reacting the serum from a test individual with a support bound anti-HCV antibody and examining the antibody for the presence of bound antigen.
Also forming part of the invention is an assay system or kit for carrying out the assay method just described. The kit generally includes a support with surface-bound anti-HCV antibody and a reporter-labeled cognate antigen (e.g. , clone 36-HRPO) for detecting antibody bound HCV-antigen. The more antigen, from a test sample, bound to the antibody the more inhibition of reporter-antigen binding: accordingly, lower levels of detectable reporter.
Polyclonal and monoclonal antibodies, for use in the present invention, can be prepared as described above utilizing the peptides of the present invention. The antibodies can be purified by standard methods to provide antibody preparations which are substantially free of serum proteins that may affect reactivity (e.g., affinity purification (Harlow et al . ) ) .
D. Combined Antigen-Inhibition Immunoassays A number of the antigenic peptides (the 409- 1-1 series, clone 40, clone 36, and clone 36 subclones, e .g . , 36-1, 36-2, and 36-3) of the present invention can be used singly or in combination in the antigen-inhibition assay of the present invention. Antibodies generated against these peptides can be combined with each other and/or anti-clone 36 antibodies for immunological detection of HCV infected sera.
When multiple anti-HCV antibodies are used the multiple antigen-reporter molecules can be similarly or differentially labeled. For example, microtiter plate wells (i.e., multiwell plates (Corning) ) can be coated with a mixture of antibodies containing equal quantities of anti-36- 1-antigen and anti-36-3-antigen antibodies. The probe is then a mixture of, for example, 36-1-HRPO and 36-3-HRPO. The results of this assay are then read as a single-specificity. Alternatively, the two antigen-reporter molecules can be differentially labeled by, for example, labeling one antigen with HRPO and the second antigen with alkaline phosphatase. Another embodiment of differential labeling is the use of two fluorescent reporters having different emission wavelengths: for example, phycoerythrin (PE) and fluorescein isothiocyanate (FICT) . Multi-well microtiter plates can then be scanned (Dynatech Corp. , Cambridge MA) and the relative levels of antigens determined based on the emission spectra. The present invention also includes kits containing multiple antibodies and cognate antigen-reporter complexes, as well as antibodies generated against the antigens of the present invention.
E. Passive Immunoprophylaxis The anti-HCV antibodies of the invention can be used as a means of enhancing an anti-HCV immune response since antibody-virus complexes are recognized by macrophages and other effector cells. The antibodies can be administered in amounts similar to those used for other therapeutic administrations of antibody. For example, pooled gamma globulin is administered at 0.02-0.1 ml/lb body weight during the early incubation of other viral diseases such as rabies, measles and hepatitis B to interfere with viral entry into cells. Thus, antibodies reactive with, for example, the 409-1-1(c-a) antigen can be passively administered alone in a "cocktail" with other anti-viral antibodies or in conjunction with another anti-viral agent to a host infected with an PT-NANBH virus to enhance the immune response and/or the effectiveness of an antiviral drug.
The following examples illustrate various aspects of the invention, but are in no way intended to limit the scope thereof.
Materials E. coli DNA polymerase I (Klenow fragment) was obtained from Boehringer Mannheim Biochemicals (Indianapolis, IN) . T4 DNA ligase and T4 DNA polymerase were obtained from New England Biolabs (Beverly, MA) ; Nitrocellulose filters were obtained from Schleicher and Schuell (Keene, NH) . Synthetic oligonucleotide linkers and primers were prepared using commercially available automated oligonucleotide synthesizers. Alternatively, custom designed synthetic oligo- nucleotides may be purchased, for example, from Synthetic Genetics (San Diego, CA) . cDNA synthesis kit and random priming labeling kits were obtained from Boehringer-Mannheim Biochemical (BMB, Indianapolis, IN) . Standard manipulations of molecular biology have been carried out as previously described (Ausubel, et al . ; Sambrook, et al . ; Maniatis, et al . ) . Methods for antibody preparation and standard diagnostic applications are described in Harlow, et al .
General ELISA Protocol for Detection of Antibodies.
Polystyrene 96 well plates Immulon II (PGC) are coated with 5 ug/mL (100 μL per well) peptide in 0.1 M carb/bicarbonate buffer, pH 9.5. Plates are sealed with parafilm and stored at 4°C overnight. Plates are aspirated and blocked with 300 uL 10% NGS and incubated at 37°C for 1 hr.
Plates are washed 5 times with PBS 0.5% "TWEEN-20". Antisera are typically diluted in 0.1 M PBS, pH 7.2. The desired dilution(s) of antisera (0.1 mL) are added to each well and the plate incubated 1 hours at 37°C. The plates are then washed 5 times with PBS 0.5% "TWEEN-20M. A detection antibody is then used to detect the binding of antibodies present in the anti- serum, to the support bound antigen. For example, if rabbit anti-sera is used, horseradish peroxidase (HRP) conjugated goat anti-rabbit antiserum (Cappel) is diluted 1/5,000 in PBS. 0.1 L of this solution is added to each well. The plate is incubated 30 min at 37°C, then washed 5 times with PBS.
Sigma ABTS (substrate) is prepared just prior to addition to the plate.
The reagent consists of 50 mL 0.05 M citric acid, pH 4.2, 0.078 mL 30% hydrogen peroxide solution and 15 mg ABTS. 0.1 mL of the substrate is added to each well, then incubated for 30 min at room temperature. The reaction is stopped with the addition of 0.050 mL 5% SDS (w/v) . The relative absorbance for each sample well is determined at 410 nm.
EXAMPLE 1
Construction of NANB-containing cDNA Libraries A. Infection of a Chimpanzee with HCV A chimpanzee (#771) was inoculated with a Factor VIII preparation which was known to cause parenterally transmitted non-A non-B hepatitis (PT-NANBH) in human patients treated with the Factor VIII concentrate (Bradley) . Post-infection ultrastructural changes in liver tissue were observed by electron microscopy and ALT (alanine amino transferase) elevation was observed in the infected chimpanzee. These observations are consistent with PT-NANBH infection.
B. Isolation of RNA from Sera Serum was collected from the above described infected chimpanzee (#771) and four human PT-NANBH clinical sources (EGM, BV, CC and WEH) . Ten illiliters of each undiluted serum was pelleted by centrifugation at 3OK, for 3 hours in an SW40 rotor, at 4°C. RNA was extracted from each resulting serum pellet using the following modifications of the hot phenol method of
Feramisco et al . Briefly, for each individual serum sample, the pellet was resuspended in 0.5 ml of 50 M NaOAc, pH=4.8, containing 1% SDS. An equal volume of 60°C phenol was added and incubated for 15 minutes at 60°C with occasional vortexing. This mixture was transferred to a 1.5 ml microfuge tube and spun for two minutes at room temperature in a table top microfuge. The aqueous phase was transferred to a new microfuge tube. To the aqueous phase, 50 μl of 3 M NaOAc, pH=5.2, and two volumes of 100% ethanol were added. This solution was held at -70°C for approximately 10 minutes and then spun in a microfuge at 4°C for 10 minutes. The resulting pellet was resuspended in 100 μl of sterile glass distilled water. To this solution 10 μl of NaOAc, pH=5.2, and two volumes of 100% ethanol were added. The solution was held at -70°C for at least 10 minutes. The RNA pellet was recovered by centrifugation in a microfuge at 12,000 x g for 15 minutes at 5°C. The pellet was washed in 70% ethanol and dried under vacuum. C. Synthesis of cDNA
1. First Strand Synthesis. The synthesis of cDNA molecules was accomplished as follows. The above described RNA preparations were each resuspended in 26 μl of sterile glass distilled water (treated with diethyl pyrocarbonate, Maniatis et al.), 5 μl of 10 x reaction buffer (0.5 M Tris HCl, pH=8.5; 0.4 M KCl; 0.1 M MgCl2; 4 mM DTT) , 10 μl of a nucleotide solution (dGTP, dATP, dTTP, and dCTP, each at a concentration of 5 mM) , 5 μl random primer, 0.25 μl of 32P-dCTP, 2 μl AMV reverse transcriptase, and 2 μl of RNASIN (Promega) , in a total reaction volume of 50 μl. This mixture was incubated for one hour at 42°C.
2. Second Strand cDNA Synthesis. To the first strand synthesis reaction mixture the following components were added: 55 μl of 2 x second strand synthesis buffer (50 mM Tris HCl, pH=7.0; 60 mM KCl); 2 μl RNase H; 5 μl DNA polymerase I, and 2 μl of the above described nucleotide solution. The reaction was incubated for one hour at 12°C, followed by a one hour incubation at room temperature. The reaction mix- ture was extracted with an equal volume of 1:1 phenol/chloroform, followed by an extraction using 24:1 chloroform/isoa yl alcohol. To each reaction mixture l μl of 10 mg/ml tRNA was added as car¬ rier. The cDNA was precipitated by the addition of two volumes of 100% ethanol and chilling at - 70°C for 15 minutes. The cDNA was collected by centrifugation, the pellet washed with 70% ethanol and dried under vacuum.
3. Preparation of the Double Stranded cDNA for Cloning. To provide vector compatible ends each of the double stranded cDNA preparations was tailed with EcoRI linkers in the following manner.
The cDNA was treated with EcoRI methylase under the following conditions: The cDNA pellet was resuspended in 20 μl lx methylase buffer (50 mM Tris HCl, pH=7.5; 1 mM EDTA; 5 mM DTT) , 2 μl 0.1 mM S-adenosyl-methionine (SAM) and 2 μl EcoRI methylase (New England Biolabs) . The reaction was incubated for 30 minutes at 37°C. TE buffer (10 mM Tris-HCl, pH=7.5; 1 mM EDTA, pH=8.0) was added to achieve a final volume of 80 μl. The reaction mixture was extracted with an equal volume of phenol/chloroform (1:1) and then with an equal volume of chloroform/isoamyl alcohol (24:1) . The cDNA was precipitated with two volumes of ethanol. To maximize the number of blunt ends for the addition of linkers (Maniatis et al , 1982) the cDNA was then treated with the Klenow fragment of DNA polymerase I. The pelleted cDNA was resuspended in 11.5 μl of distilled water. The following components were added to the resuspended cDNA: 4 μl of 5 x NTB (10 x NTB stock solution: 0.5 M Tris.Cl pH=7.2; 0.1 M MgS04; 1 mM dithiothreitol (DTT) ; 500 μg/ml bovine serum albumin (BSA) ) ; 3 μl 0.1 M MgCl2, 1.5 μl 10GATC (a solution containing 10 mM of each nucleotide G, A, T, and C) , and 1 μl Klenow (Boehringer Mannheim Biochemicals) . The reaction mixture was incubated at room temperature for 30 minutes. The reaction mixture was extracted with phenol/chloroform and chloroform isoamyl alcohol as described above, and then precipitated with two volumes of ethanol.
The cDNA pellet was resuspended in 12 μl distilled water. To the resuspended linkers the following components were added: 5 μl EcoRI phosphorylated linkers (New England Biolabs) , 2 μl lOx ligation buffer (0.66 M Tris.Cl pH=7.6, 50 mM MgCl2, 50 mM DTT, 10 mM ATP) and 1 μl T4 DNA ligase. The reaction was incubated at 14°C overnight. The following morning the reaction was incubated at 67°C for three minutes to inactivate the ligase, then momentarily chilled. To the ligation reaction mixture 2.5 μl of 10 x high salt restriction digest buffer (Maniatis et al . ) and 2.5 μl of EcoRI enzyme were added and the mixture incubated at 37°C for at least 6 hours to overnight. To remove excess linkers the digestion mixture was loaded onto a 1.2% agarose gel and the reaction components size fractionated by electrophoresis. Size fractions of the 0.3-1.3 Kb and 1.3-7 Kb ranges were electroeluted onto NA45 paper (Schleicher and Schuell) . The NA45 paper, with the eluted cDNA bound to it, was placed in a 1.5 ml microfuge tube containing 0.5 ml of elution solution (50 mM arginine, 1 M NaCl, pH=9.0). The tube was then placed at 67°C for approximately one hour to allow the cDNA to be eluted from the paper into the solution. The solution was then phenol/- chloroform, chloroform/isoamyl alcohol extracted and precipitated with two volumes of ethanol. The resulting cDNA pellets were resuspended in 20 μl TE (pH=7.5).
4. Cloning of the cDNA into Lambda Vectors. The linkers used in the construction of the cDNAs contained an EcoRI site which allowed for direct insertion of the amplified cDNAs into lambda gtlO and gtll vectors (Promega, Madison WI) . Lambda vectors were purchased from the manufacturer (Promega) which were already digested with EcoRI and treated with bacterial alkaline phosphatase, to remove the 5' phosphate and prevent self-ligation of the vector. The _E.coi?J-linkered cDNA preparations were ligated into both lambda gtlO and gtll (Promega) . The conditions of the ligation reactions were as follows: 1 μl vector DNA (Promega, 0.5 mg/ml) ; 0.5 or 3 μl of insert cDNA; 0.5 μl 10 x ligation buffer (0.5 M Tris-HCl, pH=7.8; 0.1 M MgCl2; 0.2 M DTT; 10 mM ATP; 0.5 g/ml BSA) , 0.5 μl T4 DNA ligase (New England Biolabs) and distilled water to a final reaction volume of 5 μl. The ligation reaction tubes were placed at 14°C overnight (12-18 hours). The ligated cDNA was packaged the following morning by standard procedures using a lambda DNA packaging system (GIGAPAK, Stratagene, LaJolla, CA) , and then plated at various dilutions to determine the titer and recombinant frequency of the libraries. A standard X-gal blue/white assay was used to screen the lambda gtll libraries (Miller; Maniatis et al . ) . E. coli HG415 (from Howard Gersenfeld, Dept.of Pathology, Stanford School of Medicine) plating bacteria, which allows only plaque formation by recombinant clones, was used for plating the lambda gtlO libraries. The standard strain, E. coli C600Hfl, may be used as an alternative to E . coli HG415.
EXAMPLE 2
Screening the cDNA Library for Production of PT-NANBH Antigens The five lambda gtll libraries generated in Example 1 were screened for specific HCV encoded viral antigens by immunoscreening. The phage were plated for plaque formation using the Escherichia coli bacterial plating strain E. coli KM392 (Kevin Moore, DNax, Palo Alto, CA) . Alternatively, E. coli Y1088 may be used.. The fusion proteins expressed by the lambda gtll clones were screened with serum antibodies (Young et al . ) from the following sources: chimpanzee #771 and various human PT-NANBH sera (including EGM, BV, WEH and AG) .
From the lambda gtll libraries (Example 1) approximately 111 independent clones gave a positive immunological reaction with at least one of the chimp or human PT-NANBH sera. These phage clones were plaque purified and the recombinant phage grown for DNA purification (Maniatis et al . ) .
EXAMPLE 3
Genomic Hybridization Screening of Immunopositive Clones Out of the 111 plaque purified recombinant phage, obtained as in Example 2, 93 were isolated
(Maniatis et al . ) and digested with EcoRI as per the manufacturer's instructions (Bethesda Research
Laboratories, Gaithersburg, MD) . Approximately 1.0 microgram of each digested phage DNA sample was loaded into sample wells of 1.0% agarose gels prepared using TAE (0.04 m Tris Acetate, 0.001 M EDTA) . The DNA samples were then electrophoretically separated. DNA bands were visualized by ethidium bromide staining (Maniatis et al.) . Inserts were clearly identified for each of the 93 clones, purified by electroelution using NA45, and then radioactively labelled by nick translation (Maniatis et al . ) . Human peripheral blood lymphocyte (PBL) DNA was restriction digested with Hindlll and EcoRI, loaded on a 0.7% agarose gel (as above, except 10 μg of DNA was loaded per lane) and the fragments separated electrophoretically. The DNA fragments in the agarose gels were transferred to nitrocellulose filters (Southern) and the genomic DNA probed with the nick-translated lambda gtll inserts which were prepared above. The filters were washed (Southern; Maniatis et al . ) and exposed to X-ray film. Forty-three of the 93 lambda clone inserts displayed a positive hybridization reaction with the human PBL DNA. Among the remaining inserts which clearly did not hybridize with the PBL DNA, were 11 inserts derived from chimp #771 clones which were also clearly immunopositive from Example 2. Of these 11 clones, two of the clones had the immunoreactive characteristics summarized in Table 1. Chimpanzee #771 and humans AG, BV and WEH were chronic PT-NANBH sera samples and SKF was a normal human serum sample.
TABLE 1
Clone Designation
Sera
36 40
#771 + +
AG + -
BV + -
WEH - -
SKF - -
Clone 40 was clearly exogenous, i.e., not derived from normal human DNA, as evidenced by repeated hybridization tests against normal human PBL DNA, and a second clone, designated clone 36, was not only exogenous but also reactive with multiple PT- NANBH antisera.
EXAMPLE 4 Seguencing of Clones DNA sequencing was performed on clones 36 and 40. Commercially available sequencing primers (New England Biolabs) homologous to flanking lambda sequences at the 5' and 3' ends of the inserts were initially used for sequencing. As sequencing progressed primers were constructed to correspond to newly discovered sequences. Synthetic oligonucleotide primers were prepared using commercially available automated oligonucleotide synthesizers. Alternatively, custom designed synthetic oligonucleotides may be purchased, for example, from Synthetic Genetics (San Diego, CA) .
DNA sequences were determined for the complete insert of clones 36 and 40 (SEQ ID NO:5 and SEQ ID N0:1, respectively). The clone 36 sequence corresponds to nucleotides 5010 to 6515 of the HCV genome (Houghton, et al . , (A), EP Pub. No. 0318216; Houghton, et al . , (B) , EP Pub. No. 0388232) . The clone 40 sequence corresponds to nucleotides 6516 to 7070 of the HCV genome.
Subsequently, the inserts present in clones 44 and 45 (2 other clones of the 11 clones identified in Example 3) were found to cross- hybridize to the clone 40 insert. Partial sequencing of clones 44 and 45 showed that the sequences obtained from these two clones matched the sequence of clone 40.
EXAMPLE 5 HCV Sera Paneling of Selected Clones A. Paneling of Clones 36 and 45 Table 2 shows the results of the immunoscreening of clone 36 and 45 (corresponds to clone 40) encoded epitopes which were identified above.
TABLE 2 PANEL I: SEROCONVERSION SPECIMENS
Figure imgf000036_0001
Figure imgf000037_0001
Figure imgf000038_0001
The screening sera were as follows: GLI-1 sera was a human chronic PT-NANBH sera; BV, community acquired NANBH; SKF, PT-NANBH negative; and FEC, PT-NANBH positive. The numbered sera samples correspond to human clinical serum samples which were PT-NANBH positive: these samples were obtained from Dr. Francoise Fabiani-Lunel, Hospital La Pitie Salpetriere, Paris, France. As can be seen from the results presented in Table 2, the antigens produced by clones 36 and 40 yield HCV-specific immunopositive signals.
B. The Use of the Clone 36 Antigen as an HCV Diagnostic
Clone 36 was also used for sera immunoscreening which employed an ELISA format instead of plaque screening. The following Table presents a comparison of the abilities of 5 different antigens to identify HCV positive sera among a number of HCV-positive and HCV-negative sera samples.
TABLE 3
Sample HCV? Antigens Number
409- 33CU NC45D 5- Clone 1-1 1-1 36
1 NC + + + +
2 NC Sample HCV? Antigens Number
409- 33CU NC 5D 5- Clone 1-1 1-1 36
3 NC +
4 NC + +
5 NC
6 NC
7 NC + + + + +
8 NC + +
9 NC
10 NC
11 NC
12 NC
13 NC
14 NC
15 NC
16 NC +
17 NC
18 NC
19 NC
20 NC
21 NC +
22 NC +
23 NC
24 NC +
25 NC
26 NC +
27 NC + + + + +
28 NC
29 NC
30 NC
31 + + + + +
32 + + + + + +
33 + + + +
34 + + + + + +
35 + + + + + +
36 - Sample HCV? Antigens Number
409- 33CO NC45D 5- Clone 1-1 1-1 36
37 _
38 —
39 + + + + + +
40 + + + + + +
In Table 3 the HCV antigens correspond to portions of the following HCV proteins: 409-1-1, a portion of the NS3 protein; 33CU, a portion of the NS3 protein; NC450, a portion of the capsid protein; and 5-1-1, a portion of the NS4 protein. The sera samples in the above table were as follows: samples 1-27 (human) and 28-30 (animal) , correspond to sera where the HCV-status of each serum was not previously characterized (NC) ; samples 31-35 and 39-40 were sera previously characterized as HCV-positive; and samples 36-38 were HCV-negative control sera. Empty cells in Table 3 indicate a negative ELISA result. Cells containing a "+" indicate a positive ELISA result. The data presented in Table 3 show that clone 36 identified all HCV-positive sera in this selected sample that were identified by any other single HCV antigen or combination of HCV antigens. Clone 36 antigen also detected six unique positive sera samples. The clone 36 antigen tested negative with all negative control sera.
EXAMPLE 6 Isolation of Clone 36 Fusion Protein Sepharose 4B beads conjugated with anti-beta galactosidase were purchased from Promega. The beads are packed in a 2 ml column and washed successively with phosphate-buffered saline with 0.02% sodium azide and 10 ml TX buffer (10 mM Tris buffer, pH 7.4, 1% aprotinin) .
BNN103 lysogens infected with gtll/clone 36 are used to inoculate 500 ml of NZYDT broth. The culture is incubated at 32°C with aeration to an O.D. of about .2 to .4, then brought to 43°C quickly in a 43°C water bath for 15 minutes to induce gtll peptide synthesis, and incubated further at 37°C for 1 hour. The cells are pelleted by centrifugation, suspended in 10 ml of lysis buffer (10 mM Tris, pH 7.4 containing 2% "TRITON X-100" and 1% aprotinin added just before use. The resuspended cells are frozen in liquid nitrogen, then thawed, resulting in substantially complete cell lysis. The lysate is treated with DNasel to digest bacterial and phage DNA, as evidenced by a gradual loss of viscosity in the lysate. Non-solubilized material is removed by centrifugation. The clarified lysate material is loaded on the Sepharose column, the ends of the column are closed, and the column is placed on a rotary shaker for 2 hrs. at room temperature and 16 hours at 4°C. After the column settles, it is washed with 10 ml of TX buffer. The fused protein is eluted with 0.1 M carbonate/bicarbonate buffer, pHlO. The eluate from the affinity column can be concentrated by filtration, for example, using "CENTRICON-30" cartridges (Amicon, Danvers, Mass.). The final protein concentrate is resuspended in PBS buffer. Protein purity can be analyzed by SDS-PAGE.
EXAMPLE 7 Preparation of Anti-Clone 36 Antibodies
The clone 36 digest fragments from lambda gtll were released by EcoRI digestion of the phage and the insert purified by gel electrophoresis. The purified fragment is introduced into the pGEX expression vector (Smith) . Expression of gluta- thione S-transferase fused protein (Sj26 fused protein) containing the clone 36 polypeptide antigen can be achieved in E. coli strain KM392 (above) . The fusion protein is isolated from lysed bacteria, and isolated by affinity chro¬ matography on a column packed with glutathione- conjugated beads, according to published methods (Smith) .
The purified Sj26/clone 36 fused protein is injected subcutaneously in Freund's adjuvant in a rabbit. Typically, approximately 1 mg of fused protein is injected at days 0 and 21, and rabbit serum collected on days 42 and 56.
A control rabbit is typically immunized with purified Sj26 protein obtained from control bacterial lysate. Minilysates from the bacterial cultures are prepared are screened for the present of clone 36 and Sj26 antigens using the sera from the immunized rabbits.
EXAMPLE 8
Amplification of Clone 36 and Clone 36 Subfragments
The clone 36-insert was cloned into a pET3d vector (Novagen, Madison, WI) , essentially according to conventional methods. The clone 36 insert was modified so that it lacked internal Ncol and BamHI sites. This modification was achieved as follows. Three polymerase chain reaction primer pairs were selected from the sequence of the clone 36 insert that defined three regions of the insert (Figure 1) : 36R, SEQ ID NO:11; 36F, SEQ ID NO:12; 36 Nco Block F, SEQ ID NO:13; 36 Nco Block R, SEQ ID NO:14; 36 Bam Block R, SEQ ID NO:15; and 36 Bam Block F, SEQ ID NO:16. The primers were designed to eliminate internal Ncol and BamHI sites by modifying third position nucleotides in the nucleic acid sequence, where such modification did not affect the protein coding sequence.
The primers were used in three amplification reactions (Mullis; Mullis, et al . ; supplies from Perkin Elmer/Cetus) . The reactions were overlap polymerase chain reactions designed to regenerate the full length clone 36 insert without the internal WcoJ and BamHI sites. The amplification reactions were run for 20 cycles each (1 minute denaturation, 1 minute annealing at 55°C, and 2 minutes of extension) . The amplification products were examined on agarose gels. If insufficient amplification had occurred the amplification reactions were continued for 10 more cycles. The original template DNA (used in reaction 1, below) was typically the original clone 36 gtll phage. The primers were used in the three amplification reactions shown in Table 4 to generate template DNAs.
TABLE 4
Reaction Primers Template No. Product
1 36F (which contains a Ncol A site) and 36 Nco Block R
2 36 Nco Block F and 36 Bam B Block R
3 36 Bam Block F and 36R C
These templates were then used in the following sequential amplification reactions (Table 5) to generate a full length insert corresponding to clone 36, with the exception of the internal Ncol and BamHI sites.
TABLE 5
Reaction Primers Template No.
4 36F and 36 Bam Block R A and B
5 36F and 36R Reaction 4 and C
The resulting insert was cloned into the pET vector and designated pET-36.
Three overlapping subfragments of clone 36 were generated by polymerase chain reaction using clone 36 gtll DNA or pET-36. The primer pairs for the subfragments were as follows: 36-1F/36-1R
(SEQ ID NO:20, SEQ ID NO:21), .36-2F/36-2R (SEQ ID NO:22, SEQ ID NO:23), and 36-3F/36-3R (SEQ ID NO:24, SEQ ID NO:25) .
The resulting amplification products were gel purified by standard procedures. The clone 36 subfragments, 36-1 (614 bp) , 36-2 (661 bp) , and 36-3 (497 bp) , respectively span the length of the clone 36 coding sequence 5' to 3'. Each fragment contained _E.co.RJ sites at each of its 5' and 3' ends. Further, the amplified subfragments contain
Nco and BamHI internal to the EcoRI sites.
The amplified subfragments were cloned into lambda gtll vectors (gtll-36-1, gtll-36-2, and gtll-36-3) for expression of the clone 36 polypeptide fragments encoded by each subfragment
(protein sequences: SEQ ID NO:17, SEQ ID NO:18, and SEQ ID NO: 19, respectively; DNA sequences: SEQ ID NO:26, SEQ ID NO:27, and SEQ ID NO:28, respectively) . Other selected regions of the clone 36 coding sequence can be similarly cloned.
EXAMPLE 9 Antigenicitv of Clone 36-derived Polypeptides Amplified DNA fragments corresponding to the sub-fragments of the clone 36 expression protein were gel purified, digested with EcoRI, according to standard procedures (Maniatis, et al . ,
Sambrook, et al . ) , and individually ligated into a lambda gtll phage vector (Promega) Recombinant phage were then tested for expression of polypeptides capable of reacting with HCV-positive antisera in an immunoscreening assay (Young, et al . )
Briefly, amplified DNA regions 36-1, 36-2 and 36-3 were ligated into a lambda gtll vector using standard procedures. KM392 bacterial cells were infected to yield titers of 7.3 x 104 pfu/ml for 36-1, 7.3 x 104 for 36-2, and 7.3 x 104 pfu/ml for 36-3. These phage were plated and nitrocellulose filter lifts prepared (Schleicher and Schuell, (Keene, NH) ) . The filters were immunoscreened (Ausubel, et al.) for reactivity with the following human anti-sera DPII-C1, DPII-clO, FEC, SKF and AG described above. DPII stands for the panel II received from Sanofi Diagnostics Pasteur. C2 and CIO are well characterized HCV samples (i.e., the sera tested positive by immunoscreening for the following HCV antigens: C100, 5-1-1, 409- 1-1, and clone 36) . Sera were pre-adsorbed, prior to testing, with semi-confluent gtll phage lysates. Three positive plaques selected from each plate were amplified in bacterial culture. DNA isolated from the each selected plaque was amplified using the overlapping fragment primer pairs described in Example 8 (36-1F/36-1R, 36- 2F/36-2R, and 36-3F/36-3R) . Insert sizes were confirmed by size fractionation on agarose gels. EXAMPLE 10 Production of Monoclonal Antibodies A. Protein Isolation
A single bacterial colony containing pET-36 was inoculated into 500 ml LB + 100 ug/ml ampicillin and grown at 37°C, with vigorous aeration, until an O.D. of 0.7 at A600 was achieved. Production of the HCV antigen was induced by addition of 1.0 mM IPTG to the culture. After induction the culture was grown for approximately three hours.
The cells were pelleted by centrifugation at 4°C and held overnight at -21°C. The frozen cells were resuspend cells in 25 ml PBS, 1 mM PMSF, 5 mM DTT, at room temperature. To this suspension,
5,000 units of DNasel was added, with continuous mixing until solution viscosity was reduced. The suspension was subjected to centrifugation and the resulting pellet resuspended, using a Dounce Ho ogenizer, in 25 ml PBS, 1 mM DTT, 1 mM PMSF. Once again the suspension was spun-down the supernatant discarded.
The pellet was once again resuspended in 25 ml PBS, 1 mM PMSF, 1 mM DTT, and the suspension pelleted. The final supernatant was discarded and the pellet resuspended in 30 ml of 4M Urea, 20 Mm NaOAc, 5 mM DTT at pH 4.5 (Buffer A). This solution was spun-down in a Beckman JA20 rotor for 10 minutes at 10K rpm, 4°C. The resulting supernatant was filtered through a 0.45 μm "MILLEX" Filter (Millipore) .
Ten milliliters of the filtered supernatant was subjected to FPLC separation (Pharmacia) . The supernatant was loaded at 1 ml/minute, onto a 1.6 cm x 12 cm S-Sepharose "FAST FLOW" column (Pharmacia, Piscataway NJ) , which was pre- equilibrated with Buffer A. The loaded column was washed with Buffer A for 30 minutes at 1 ml/minute.
A 0.0 M to 1.0 M NaCl gradient, in Buffer A, was then passed through the column at 1 ml/minute and fractions collected for 2 minute intervals. The fractions were then assayed for the presence of the fusion protein using SDS polyacrylamide gel electrophoresis. After fractions containing the antigenic protein have been identified, those fractions are then pooled. The pooled fractions may be concentrated by filtration.
B. Generation of Monoclonal Antibodies Murine monoclonal antibodies were produced according to standard procedures (Mishell; Harlow, et al . ) . Mice were immunized .intraperitoneally two times, with a 21 day interval, using 200 μg pET clone 36 expression peptide (pET 36) . Blood was collected from the mice 1 week after the second injection. Sera prepared from the blood samples were tested at dilutions of 1/100, 1/1000, 1/2000 and 1/5000 in an ELISA assay for reactivity with purified pET-36 antigen.
The ELISA for detection of antibodies was performed essentially as follows. Polystyrene 96- 2311 plates (Falcon 3072, Becton Dickinson, Oxnard, CA) were coated with pET36 in 0.1 M bicarbonate, pH 9.0 (2 mg GSA/100 mL buffer) by adding 0.1 mL of this solution per well. Plates were incubated for 2 hrs, at 37°C, then washed 3 with PBS/0.05% "TWEEN-20". Plates were blocked by the addition of PBS/BSA (1%) for 1 hour at 37°C. Plates were washed 3x with PBS/0.05% "TWEEN-20", sealed and stored at 4°C. Antisera were diluted in 1% BSA in PBS. The following dilutions of antisera were tested: 1/100, 1/1000, 1/2000 and 1/5000. The desired dilution(s) of antisera (0.1 ml) were added to each well and the plate incubated 2 hours, at 37°C. The solution was then removed from the plate, and the wells washed 3 times with PBS/0.05% "TWEEN-20".
Alkaline phosphatase conjugated goat anti- mouse antiseru (affinity purified — Zymed, Berkeley, CA) was diluted 1/3000 in PBS/1% BSA. 0.1 mL of this solution was added to each well. This solution was left on the plate for 1 hour at room temperature. The plate was then washed twice with PBS/Tween-20 and three times with PBS, as above.
Color reagent {substrate was either BCIP (5- bromo-4-chloro-3-indolyl phosphate P-toluidine salt, 50 mg/ml in 100% dimethyl formamide) or NBT (Nitro blue tetrazoliu Grad. Ill, 50 mg/ml in 100% dimethyl formamide) , both reagents are available from Sigma} was prepared just prior to addition to the plate. 0.1 mL of the color reagent was added to each well, then incubated for 30 min at room temperature prior to determination of the absorbance, at 405 n , relative to the blank well, containing no mouse sera. Spleens were removed from mice exhibiting seroreactivity of at least 0.5 O.D. units at a serum dilution of 1/5000, and dissociated spleen cells were employed for subsequent hybridoma generation. Hybridoma cells were formed by fusion of splenic B-lymphocytes from seropositive mice and stable myeloma fusion partner cells, SP2/0 cells (SP2/0-Agl4; ATCC CRL 1581, American Type Culture Collection, Rockville, MD) , mixed at a ratio of about 1:1 (splenic cells:myeloma cells). Fusion of the cells was promoted by addition to the cells of polyethylene glycol (PEG-1500; Sigma Chemical Co., St. Louis, MO; final concentration: 50%). Cells were incubated with PEG at 37°C for 1 minute, prior to removal of PEG by dilution with culture medium (RPMI 1640) and centrifugation. Cells were plated at a density of about 106 cells/ml in 96-well culture plates. Hybridomas were selected by growth in selective (HAT) medium, then were tested for production of anti-pET-36 antibodies in a solid phase assay (ELISA) as described above. Hybridomas found to produce antibodies to pET 36 were further clone purified by limiting dilution.
From one such fusion, 49 positive clones were identified and isolated. Subclones were then generated from five of the clones.
EXAMPLE 11 Immunoreactivity of pET 36 Subfragments The subclones of clone 36 (Example 8) were screened to evaluate their immunoreactivity relative to the hybridoma supernatants.
The following clones were plated as described in Example 8: subfragment clones gtll-36-1, gtll- 36-2, and gtll-36-3; and lambda gtll. Each plate of phage-infected bacteria was overlaid with a nitrocellulose sheet. The expression products from the plaques transferred to filter paper. The plate and filter were indexed for matching corresponding plate and filter positions. The filter was removed after 6-12 hours, washed three times in TBS buffer (10 mM Tris, pH 8.0, 150 mM NaCl) , blocked with AIB (TBS buffer with 1% gelatin) , washed again in TBS, and incubated overnight with hybridoma supernatants from the 49 positive clones identified in Example 9. Each filter was washed twice in TBS, then incubated with alkaline-phosphatase-conjugated anti-human IgG to attach the labeled antibody at filter sites containing antigen recognized by the antiserum.
After a final washing, the filter was developed in a substrate medium containing 33 μl NBT (50 mg/ml stock solution maintained at 4°C) mixed with 16 μl BCIP (50 mg/ml stock solution maintained at 4°C) in 5 ml of alkaline phosphatase buffer (100 mM Tris, 9.5, 100 mM NaCl, 5 mM MgC12) . Reacted substrate precipitated at points of antigen production, as recognized by the antiserum.
Results from immunoscreening experiments using selected hybridoma supernatants (Example 10) are shown in Tables 6 and 7. The hybridoma supernatants used in Table 6 were strongly positive in the original screening against the entire clone 36 coding region (Example 10) . The hybridoma supernatants used in Table 7 were less strongly positive than those used in Table 6. The first column of each table presents the clone number used to produce the hybridoma supernatant. The presence of plaques showing antigen-positive reaction is indicated by "+" in these tables. The results of overall immunoreactivity of each of the clone 36 coded sub-polypeptides, and the negative control (lambda gtll) are presented in the tables.
TABLE 6 MAPPING OF ANTI-36 HYBRIDOMAS
Clone gtll gtn- gtll- gtll-
36-1 36-2 36-3
5D7 - - - -
6E7 - + - +
7E2 - - - +
4B11 - - - + Clone gtll gtn- gti - gtn-
36-1 36-2 36-3
4C2 — — — +
4F7 - - + +
3B5 - - - +
3B9 - - - +
3C4 - -_. - +
3D10 - - - +
3E11 - - - +
3F8 - - - +
7D4 - - - +
7B7 - - - +
7F5 - - - +
4E11 - - - +
3F2 - - - +
4D11 - — - -
5C9 - + - +
6E3 - - - +
3D8* 7 7 7 7
4G7 - - - +
TOTAL 0/21 2/21 1/21 19/21
= high background nd = not determined
TABLE 7 IMMUNOSCREENING OF 36 ANTIBODIES
Sample gtll- gtn- gtll- gtll 36-1 36-2 36-3
2B3 _ _ _ _
2G3 - - — -
1F4 - - — -
6-E8 - - — -
7-E4 - - - - Sample gtn- gtil- gtll- gtll
36-1 36-2 36-3
7F11 — — — -
2G11 — — — -
6G7 — — — -
7B8 — — — -
7D8 - - - -
7B4 - - — -
6G8 — — —
3D6 + — —
3C5 _ _ — —
4F9 - — — -
5F10 + — - -
7D6 _ — — —
3D5 - - - -
3B3 - - - -
1F4 — - — —
5B6 — — — -
5D4 — — — -
7-E9 - - — -
3D7 — - - -
3F7 - - - -
7F2 + - - -
7-E5 - - - -
F511 - - - -
TOTAL 3/28 0/28 0/28 0/28
The results suggest that there is no single epitope in any of the clone 36 subcloned fragments that reacts with all of the hybridoma supernatants which are reactive with pET-36. Accordingly, the clone 36 coding region, when presented in its entirety, may contain an epitope not present in any individual fragment. EXAMPLE 12
Development of an Immunoassay for Detection of HCV NS5 Antigen
This example describes the use of an HCV non- structural protein antigen to design an antigen- based detection system for HCV.
Typically, sera containing polyclonal antibodies are initially fractionated by addition of ammonium sulfate. The supernatant is then passed over a gel filtration column (Pierce) and the IgG containing fraction identified by absorbance (A280) • IgG molecules can be isolated by a number of standard procedures (Garvey, et al . ) , including affinity chromatography, or by the use of commercially available kits (Pierce) .
The basic design of the immunoassay is illustrated in Figure 2. Microwells were coated with IgG derived by gel filtration from high titer rabbit anti-clone 36 sera or with the monoclonal antibodies described above. The following example employs monoclonal antibodies.
The wells of microtiter plates (Dynatech) are coated with the antibody as follows (Harlow, et al . ) . Approximately 50 μl of purified antibody (approximately 20 μg/ml) , in phosphate buffered saline (PBS) (Maniatis, et al . ) is added to each well of polyvinylchloride plates. The plates are sealed and incubated for either 4 hours at room temperature or overnight at 4°C. Alternatively, polystyrene 96 well plates
"IMMULON II" (PGC) (or suitable plastic plates, Corning Biotechnology, Corning NY) were coated with 5 ug/ L (100 μL per well) antibodies in 0.1 M carb/bicarbonate buffer, pH 9.5, the plates sealed with parafilm and stored at 4°C overnight. The wells can also be coated with an anti-mouse IgG antibody, followed by addition of the mouse monoclonal antibodies. After incubation the wells are washed twice with binding buffer (PBS or carb/bicarbonate buffer) . To each well is added approximately 200 μl of 3% bovine serum albumin (BSA) in PBS, containing 0.02% sodium azide. The plates were then incubated for approximately 2 hours at room temperature and the liquid removed.
The antibody coated wells were then incubated with the test samples, such as, the clone 36- encoded polypeptide, HCV infected tissue culture media, human sera, or chimpanzee sera, for 1 hr. After incubation, clone 36 polypeptide-HRPO (horse radish peroxidase) conjugate was added to each well. Polypeptide-HRPO conjugates were formed using commercially available activated HRPO (Pierce) . Alternatively, HRPO is coupled to peptides using one of the following techniques: (i) the glutaraldehyde technique that links through epsilon-amino acid groups on lysine residues, or other free amino groups (Pierce) ; or (ii) a two-step procedure using m-maleimidobenzoyl sulfosuccinimide ester (sulfo-MBS) to link through free disulfide linkages (Pierce) . For polypeptides that lacked any free epsilon-amino groups or sulfhydryl groups, a cysteine residue or a lysine residue can be added to the N-terminus of the peptide.
Combination of the conjugate to the solid phase antibody coat was detected by the addition of the substrate-2,2'-azino-bis(3- ethylbenzthiazoline-6-sulfonic acid (ABTS) (Pierce) . The presence of a HCV-NS5 associated viral antigen was identified by color diminution due to successful inhibition of binding clone 36- HRPO. The above protocol is first tested by addition of increasing quantities of free clone 36 polypeptide. A linear inhibition curve is generally established, for example, over the range of 10 μg/ml to O.ooi μg/ml.
EXAMPLE 13 Antigen Confirmatory Assay A confirmatory assay was designed and tested to confirm positive antigen reactive plasma. The basic design is a blocking of the clone 36-HRPO inhibition assay (Blocking of Inhibition) by addition of anti-clone 36 positive sera to antigen positive plasma before testing in the antibody coated plates.
The reagents for the confirmatory assay were evaluated by titration. HCV antibody positive sera are tested for their ability to block the inhibition reaction.
Although the invention has been described with reference to particular embodiments, methods, construction and use, it will be apparent to those skilled in the art that various changes and modifications can be made without departing from the invention.
SEQUENCE LISTING
) GENERAL INFORMATION :
( i ) APPLICANT :
(A) NAME: Genelabs Technologies, Inc.
(B) STREET: 505 Penobscot Drive
(C) CITY: Redwood City
(D) STATE: CA
(E) COUNTRY: USA
(F) POSTAL CODE: 94063
(ii) TITLE OF INVENTION: HCV Immunodiagnostic Antigens and Antibodie
(iii) NUMBER OF SEQUENCES: 28
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Genelabs Technologies, Inc.
(B) STREET: 505 Penobscot Drive
(C) CITY: Redwood City
(D) STATE: CA
(E) COUNTRY: USA
(F) ZIP: 94063
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: Patentln Release #1.24
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE:
(C) CLASSIFICATION:
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: US 08/052,542
(B) FILING DATE: 22-APR-1993
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Desjardins, Cathleen M.
(B) REGISTRATION NUMBER: 35,856
(C) REFERENCE/DOCKET NUMBER: G20P3PCT/4600-0107 (ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: 415-369-9500
(B) TELEFAX: 415-368-0709
(2) INFORMATION FOR SEQ ID NO:l:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 561 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA to mRNA
(iii) HYPOTHETICAL: N
(iv) ANTI-SENSE: N
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Hepatitis C Virus
(B) STRAIN: CDC
(vii) IMMEDIATE SOURCE:
(B) CLONE: 304-12-1, clone 40
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..561 (D) OTHER INFORMATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:
GAA TTC CTC GTG CAA GCG TGG AAG TCC AAG AAA ACC CCA ATG GGG TTC 48 Glu Phe Leu Val Gin Ala Trp Lys Ser Lys Lys Thr Pro Met Gly Phe 1 5 10 15
TCG TAT GAT ACC CGC TGC TTT GAC TCC ACA GTC ACT GAG AGC GAC ATC 96 Ser Tyr Asp Thr Arg Cys Phe Asp Ser Thr Val Thr Glu Ser Asp lie 20 25 30
CGT ACG GAG GAG GCA ATC TAC CAA TGT TGT GAC CTC GAC CCC CAA GCC 144 Arg Thr Glu Glu Ala lie Tyr Gin Cys Cys Asp Leu Asp Pro Gin Ala 35 40 45 CGC GTG GCC ATC AAG TCC CTC ACC GAG AGG CTT TAT GTT GGG GGC CCT 192 Arg Val Ala lie Lys Ser Leu Thr Glu Arg Leu Tyr Val Gly Gly Pro 50 55 60
CTT ACC AAT TCA AGG GGG GAG AAC TGC GGC TAT CGC AGG TGC CGC GCG 240 Leu Thr Asn Ser Arg Gly Glu Asn Cys Gly Tyr Arg Arg Cys Arg Ala 65 70 75 80
AGC GGC GTA CTG ACA ACT AGC TGT GGT AAC ACC CTC ACT TGC TAC ATC 288 Ser Gly Val Leu Thr Thr Ser Cys Gly Asn Thr Leu Thr Cys Tyr lie 85 90 95
AAG GCC CGG GCA GCC TGT CGA GCC GCA GGG CTC CAG GAC TGC ACC ATG 336 Lys Ala Arg Ala Ala Cys Arg Ala Ala Gly Leu Gin Asp Cys Thr Met 100 105 110
CTC GTG TGT GGC GAC GAC TTA GTC GTT ATC TGT GAA AGC GCG GGG GTC 384 Leu Val Cys Gly Asp Asp Leu Val Val lie Cys Glu Ser Ala Gly Val 115 120 125
CAG GAG GAC GCG GCG AGC CTG AGA GCC TTC ACG GAG GCT ATG ACC AGG 432 Gin Glu Asp Ala Ala Ser Leu Arg Ala Phe Thr Glu Ala Met Thr Arg 130 135 140
TAC TCC GCC CCC CCC GGG GAC CCC CCA CAA CCA GAA TAC GAC TTG GAG 480 Tyr Ser Ala Pro Pro Gly Asp Pro Pro Gin Pro Glu Tyr Asp Leu Glu 145 150 155 160
CTC ATA ACA TCA TGC TCC TCC AAC GTG TCA GTC GCC CAC GAC GGC GCT 528 Leu lie Thr Ser Cys Ser Ser Asn Val Ser Val Ala His Asp Gly Ala 165 170 175
GGA AAG AGG GTC TAC TAC CTC ACC CGG GAA TTC 561
Gly Lys Arg Val Tyr Tyr Leu Thr Arg Glu Phe 180 185
(2) INFORMATION FOR SEQ ID NO:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 187 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
Glu Phe Leu Val Gin Ala Trp Lys Ser Lys Lys Thr Pro Met Gly Phe 1 5 10 15
Ser Tyr Asp Thr Arg Cys Phe Asp Ser Thr Val Thr Glu Ser Asp He 20 25 30
Arg Thr Glu Glu Ala He Tyr Gin Cys Cys Asp Leu Asp Pro Gin Ala 35 40 45
Arg Val Ala He Lys Ser Leu Thr Glu Arg Leu Tyr Val Gly Gly Pro 50 55 60
Leu Thr Asn Ser Arg Gly Glu Asn Cys Gly Tyr Arg Arg Cys Arg Ala 65 70 75 80
Ser Gly Val Leu Thr Thr Ser Cys Gly Asn Thr Leu Thr Cys Tyr He 85 90 95
Lys Ala Arg Ala Ala Cys Arg Ala Ala Gly Leu Gin Asp Cys Thr Met 100 105 110
Leu Val Cys Gly Asp Asp Leu Val Val He Cys Glu Ser Ala Gly Val 115 120 125
Gin Glu Asp Ala Ala Ser Leu Arg Ala Phe Thr Glu Ala Met Thr Arg 130 135 140
Tyr Ser Ala Pro Pro Gly Asp Pro Pro Gin Pro Glu Tyr Asp Leu Glu 145 150 155 160
Leu He Thr Ser Cys Ser Ser Asn Val Ser Val Ala His Asp Gly Ala 165 170 175
Gly Lys Arg Val Tyr Tyr Leu Thr Arg Glu Phe 180 185
(2) INFORMATION FOR SEQ ID NO:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 252 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double ( D ) TOPOLOGY : linear
( ii ) MOLECULE TYPE : cDNA to mRNA
( iii ) HYPOTHETICAL : N
( iv ) ANTI -SENSE : N
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Hepatitis HCV Virus
(B) STRAIN: CDC
(vii) IMMEDIATE SOURCE:
(B) CLONE: 303-1-4, clone 36 carboxy terminus
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..252 (D) OTHER INFORMATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
AAC TCC GTG TGG AAA GAC CTT CTG GAA GAC AAT GTA ACA CCA ATA GAC 48 Asn Ser Val Trp Lys Asp Leu Leu Glu Asp Asn Val Thr Pro He Asp 1 5 10 15
ACT ACC ATC ATG GCT AAG AAC GAG GTT TTC TGC GTT CAG CCT GAG AAG 96 Thr Thr He Met Ala Lys Asn Glu Val Phe Cys Val Gin Pro Glu Lys 20 25 30
GGG GGT CGT AAG CCA GCT CGT CTC ATC GTG TTC CCC GAT CTG GGC GTG 144 Gly Gly Arg Lys Pro Ala Arg Leu He Val Phe Pro Asp Leu Gly Val 35 40 45
CGC GTG TGC GAA AAG ATG GCT TTG TAC GAC GTG GTT ACC AAG CTC CCC 192 Arg Val Cys Glu Lys Met Ala Leu Tyr Asp Val Val Thr Lys Leu Pro 50 ' 55 60
TTG GCC GTG ATG GGA AGC TCC TAC GGA TTC CAA TAC TCA CCA GGA CAG 24 Leu Ala Val Met Gly Ser Ser Tyr Gly Phe Gin Tyr Ser Pro Gly Gin 65 70 75 80
CGG GTT GAA TTC 25
Arg Val Glu Phe (2) INFORMATION FOR SEQ ID NO:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 84 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:
Asn Ser Val Trp Lys Asp Leu Leu Glu Asp Asn Val Thr Pro He Asp 1 5 10 15
Thr Thr He Met Ala Lys Asn Glu Val Phe Cys Val Gin Pro Glu Lys 20 25 30
Gly Gly Arg Lys Pro Ala Arg Leu He Val Phe Pro Asp Leu Gly Val 35 40 45
Arg Val Cys Glu Lys Met Ala Leu Tyr Asp Val Val Thr Lys Leu Pro 50 55 60
Leu Ala Val Met Gly Ser Ser Tyr Gly Phe Gin Tyr Ser Pro Gly Gin 65 70 75 80
Arg Val Glu Phe
(2) INFORMATION FOR SEQ ID NO:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1512 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA to mRNA
(iii) HYPOTHETICAL: N
(iv) ANTI-SENSE: N (vi) ORIGINAL SOURCE:
(A) ORGANISM: Hepatitis C Virus
(vii) IMMEDIATE SOURCE:
(B) CLONE: 303-1-4, clone 36
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..1512 (D) OTHER INFORMATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:
GAA TTC TTC ACA GAA TTG GAC GGG GTG CGC CTA CAT AGG TTT GCG CCC 48 Glu Phe Phe Thr Glu Leu Asp Gly Val Arg Leu His Arg Phe Ala Pro 1 5 10 15
CCC TGC AAG CCC TTG CTG CGG GAG GAG GTA TCA TTC AGA GTA GGA CTC 96 Pro Cys Lys Pro Leu Leu Arg Glu Glu Val Ser Phe Arg Val Gly Leu 20 25 30
CAC GAA TAC CCG GTA GGG TCG CAA TTA CCT TGC GAG CCC GAA CCG GAT 144 His Glu Tyr Pro Val Gly Ser Gin Leu Pro Cys Glu Pro Glu Pro Asp 35 40 45
GTG GCC GTG TTG ACG TCC ATG CTC ACT GAT CCC TCC CAT ATA ACA GCA 192 Val Ala Val Leu Thr Ser Met Leu Thr Asp Pro Ser His He Thr Ala 50 55 60
GAG GCG GCC GGG CGA AGG TTG GCG AGG GGA TCA CCC CCC TCT GTG GCC 240 Glu Ala Ala Gly Arg Arg Leu Ala Arg Gly Ser Pro Pro Ser Val Ala 65 70 75 80
AGC TCC TCG GCT AGC CAG CTA TCC GCT CCA TCT CTC AAG GCA ACT TGC 288 Ser Ser Ser Ala Ser Gin Leu Ser Ala Pro Ser Leu Lys Ala Thr Cys 85 90 95
ACC GCT AAC CAT GAC TCC CCT GAT GCT GAG CTC ATA GAG GCC AAC CTC 336 Thr Ala Asn His Asp Ser Pro Asp Ala Glu Leu He Glu Ala Asn Leu 100 105 110 CTA TGG AGG CAG GAG ATG GGC GGC AAC ATC ACC AGG GTT GAG TCA GAA 384 Leu Trp Arg Gin Glu Met Gly Gly Asn He Thr Arg Val Glu Ser Glu 115 120 125
AAC AAA GTG GTG ATT CTG GAC TCC TTC GAT CCG CTT GTG GCG GAG GAG 432 Asn Lys Val Val He Leu Asp Ser Phe Asp Pro Leu Val Ala Glu Glu 130 135 140
GAC GAG CGG GAG ATC TCC GTA CCC GCA GAA ATC CTG CGG AAG TCT CGG 480 Asp Glu Arg Glu He Ser Val Pro Ala Glu He Leu Arg Lys Ser Arg 145 150 155 160
AGA TTC GCC CAG GCC CTG CCC GTT TGG GCG CGG CCG GAC TAT AAC CCC 528 Arg Phe Ala Gin Ala Leu Pro Val Trp Ala Arg Pro Asp Tyr Asn Pro 165 170 175
CCG CTA GTG GAG ACG TGG AAA AAG CCC GAC TAC GAA CCA CCT GTG GTC 576 Pro Leu Val Glu Thr Trp Lys Lys Pro Asp Tyr Glu Pro Pro Val Val 180 185 190
CAT GGC TGT CCG CTT CCA CCT CCA AAG TCC CCT CCT GTG CCT CCG CCT 624 His Gly Cys Pro Leu Pro Pro Pro Lys Ser Pro Pro Val Pro Pro Pro 195 200 205
CGG AAG AAG CGG ACG GTG GTC CTC ACT GAA TCA ACC CTA TCT ACT GCC 672 Arg Lys Lys Arg Thr Val Val Leu Thr Glu Ser Thr Leu Ser Thr Ala 210 215 220
TTG GCC GAG CTC GCC ACC AGA AGC TTT GGC AGC TCC TCA ACT TCC GGC 720 Leu Ala Glu Leu Ala Thr Arg Ser Phe Gly Ser Ser Ser Thr Ser Gly 225 230 235 240
ATT ACG GGC GAC AAT ACG ACA ACA TCC TCT GAG CCC GCC CCT TCT GGC 768 He Thr Gly Asp Asn Thr Thr Thr Ser Ser Glu Pro Ala Pro Ser Gly 245 250 255
TGC CCC CCC GAC TCC GAC GCT GAG TCC TAT TCC TCC ATG CCC CCC CTG 816 Cys Pro Pro Asp Ser Asp Ala Glu Ser Tyr Ser Ser Met Pro Pro Leu 260 265 270
GAG GGG GAG CCT GGG GAT CCG GAT CTT AGC GAC GGG TCA TGG TCA ACG 864 Glu Gly Glu Pro Gly Asp Pro Asp Leu Ser Asp Gly Ser Trp Ser Thr 275 280 285 GTC AGT AGT GAG GCC AAC GCG GAG GAT GTC GTG TGC TGC TCA ATG TCT 912 Val Ser Ser Glu Ala Asn Ala Glu Asp Val Val Cys Cys Ser Met Ser 290 295 300
TAC TCT TGG ACA GGC GCA CTC GTC ACC CCG TGC GCC GCG GAA GAA CAG 960 Tyr Ser Trp Thr Gly Ala Leu Val Thr Pro Cys Ala Ala Glu Glu Gin 305 310 315 320
AAA CTG CCC ATC AAT GCA CTA AGC AAC TCG TTG CTA CGT CAC CAC AAT 1008 Lys Leu Pro He Asn Ala Leu Ser Asn Ser Leu Leu Arg His His Asn 325 330 335
TTG GTG TAT TCC ACC ACC TCA CGC AGT GCT TGC CAA AGG CAG AAG AAA 1056 Leu Val Tyr Ser Thr Thr Ser Arg Ser Ala Cys Gin Arg Gin Lys Lys 340 345 350
GTC ACA TTT GAC AGA CTG CAA GTT CTG GAC AGC CAT TAC CAG GAC GTA 1104 Val Thr Phe Asp Arg Leu Gin Val Leu Asp Ser His Tyr Gin Asp Val 355 360 365
CTC AAG GAG GTT AAA GCA GCG GCG TCA AAA GTG AAG GCT AAC TTG CTA 1152 Leu Lys Glu Val Lys Ala Ala Ala Ser Lys Val Lys Ala Asn Leu Leu 370 375 380
TCC GTA GAG GAA GCT TGC AGC CTG ACG CCC CCA CAC TCA GCC AAA TCC 1200 Ser Val Glu Glu Ala Cys Ser Leu Thr Pro Pro His Ser Ala Lys Ser 385 390 395 400
AAG TTT GGT TAT GGG GCA AAA GAC GTC CGT TGC CAT GCC AGA AAG GCC 1248 Lys Phe Gly Tyr Gly Ala Lys Asp Val Arg Cys His Ala Arg Lys Ala 405 410 415
GTA ACC CAC ATC AAC TCC GTG TGG AAA GAC CTT CTG GAA GAC AAT GTA 1296 Val Thr His He Asn Ser Val Trp Lys Asp Leu Leu Glu Asp Asn Val 420 425 430
ACA CCA ATA GAC ACT ACC ATC ATG GCT AAG AAC GAG GTT TTC TGC GTT 1344 Thr Pro He Asp Thr Thr He Met Ala Lys Asn Glu Val Phe Cys Val 435 440 445
CAG CCT GAG AAG GGG GGT CGT AAG CCA GCT CGT CTC ATC GTG TTC CCC 1392 Gin Pro Glu Lys Gly Gly Arg Lys Pro Ala Arg Leu He Val Phe Pro 450 455 460 GAT CTG GGC GTG CGC GTG TGC GAA AAG ATG GCT TTG TAC GAC GTG GTT 1440 Asp Leu Gly Val Arg Val Cys Glu Lys Met Ala Leu Tyr Asp Val Val 465 470 475 480
ACC AAG CTC CCC TTG GCC GTG ATG GGA AGC TCC TAC GGA TTC CAA TAC 1488 Thr Lys Leu Pro Leu Ala Val Met Gly Ser Ser Tyr Gly Phe Gin Tyr 485 490 495
TCA CCA GGA CAG CGG GTT GAA TTC 1512
Ser Pro Gly Gin Arg Val Glu Phe 500
(2) INFORMATION FOR SEQ ID NO:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 504 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:
Glu Phe Phe Thr Glu Leu Asp Gly Val Arg Leu His Arg Phe Ala Pro 1 5 10 15
Pro Cys Lys Pro Leu Leu Arg Glu Glu Val Ser Phe Arg Val Gly Leu 20 25 30
His Glu Tyr Pro Val Gly Ser Gin Leu Pro Cys Glu Pro Glu Pro Asp 35 40 45
Val Ala Val Leu Thr Ser Met Leu Thr Asp Pro Ser His He Thr Ala 50 55 60
Glu Ala Ala Gly Arg Arg Leu Ala Arg Gly Ser Pro Pro Ser Val Ala 65 70 75 80
Ser Ser Ser Ala Ser Gin Leu Ser Ala Pro Ser Leu Lys Ala Thr Cys 85 90 95
Thr Ala Asn His Asp Ser Pro Asp Ala Glu Leu He Glu Ala Asn Leu 100 105 110 Leu Trp Arg Gin Glu Met Gly Gly Asn He Thr Arg Val Glu Ser Glu 115 120 125
Asn Lys Val Val He Leu Asp Ser Phe Asp Pro Leu Val Ala Glu Glu 130 135 140
Asp Glu Arg Glu He Ser Val Pro Ala Glu He Leu Arg Lys Ser Arg 145 150 155 160
Arg Phe Ala Gin Ala Leu Pro Val Trp Ala Arg Pro Asp Tyr Asn Pro 165 170 175
Pro Leu Val Glu Thr Trp Lys Lys Pro Asp Tyr Glu Pro Pro Val Val 180 185 190
His Gly Cys Pro Leu Pro Pro Pro Lys Ser Pro Pro Val Pro Pro Pro 195 200 205
Arg Lys Lys Arg Thr Val Val Leu Thr Glu Ser Thr Leu Ser Thr Ala 210 215 220
Leu Ala Glu Leu Ala Thr Arg Ser Phe Gly Ser Ser Ser Thr Ser Gly 225 230 235 240
He Thr Gly Asp Asn Thr Thr Thr Ser Ser Glu Pro Ala Pro Ser Gly
245 250 255
Cys Pro Pro Asp Ser Asp Ala Glu Ser Tyr Ser Ser Met Pro Pro Leu
260 265 270
Glu Gly Glu Pro Gly Asp Pro Asp Leu Ser Asp Gly Ser Trp Ser Thr 275 280 285
Val Ser Ser Glu Ala Asn Ala Glu Asp Val Val Cys Cys Ser Met Ser 290 295 300
Tyr Ser Trp Thr Gly Ala Leu Val Thr Pro Cys Ala Ala Glu Glu Gin 305 310 315 320
Lys Leu Pro He Asn Ala Leu Ser Asn Ser Leu Leu Arg His His Asn 325 330 335
Leu Val Tyr Ser Thr Thr Ser Arg Ser Ala Cys Gin Arg Gin Lys Lys 340 345 350
Val Thr Phe Asp Arg Leu Gin Val Leu Asp Ser His Tyr Gin Asp Val 355 360 365 Leu Lys Glu Val Lys Ala Ala Ala Ser Lys Val Lys Ala Asn Leu Leu 370 375 380
Ser Val Glu Glu Ala Cys Ser Leu Thr Pro Pro His Ser Ala Lys Ser 385 390 395 400
Lys Phe Gly Tyr Gly Ala Lys Asp Val Arg Cys His Ala Arg Lys Ala 405 410 415
Val Thr His He Asn Ser Val Trp Lys Asp Leu Leu Glu Asp Asn Val 420 425 430
Thr Pro He Asp Thr Thr He Met Ala Lys Asn Glu Val Phe Cys Val 435 440 445
Gin Pro Glu Lys Gly Gly Arg Lys Pro Ala Arg Leu He Val Phe Pro 450 455 460
Asp Leu Gly Val Arg Val Cys Glu Lys Met Ala Leu Tyr Asp Val Val 465 470 475 480
Thr Lys Leu Pro Leu Ala Val Met Gly Ser Ser Tyr Gly Phe Gin Tyr 485 490 495
Ser Pro Gly Gin Arg Val Glu Phe 500
(2) INFORMATION FOR SEQ ID NO:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 477 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA to mRNA
(iii) HYPOTHETICAL: N
(iv) ANTI-SENSE: N
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Hepatitis C Virus
(B) STRAIN: CDC (C) INDIVIDUAL ISOLATE: Rodney
(vii) IMMEDIATE SOURCE:
(B) CLONE: 409-1-1 (c-a)
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..477
(D) OTHER INFORMATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:
GAA TTC CGC ACG CCC GCC GAG ACT ACA GTT AGG CTA CGG GCG TAC ATG 48 Glu Phe Arg Thr Pro Ala Glu Thr Thr Val Arg Leu Arg Ala Tyr Met 1 5 10 15
AAC ACT CCG GGG CTT CCC GTG TGC CAG GAC GGA ATT CCG TCC CCG TCC 96 Asn Thr Pro Gly Leu Pro Val Cys Gin Asp Gly He Pro Ser Pro Ser 20 25 30
ACC ACC GGA GAG ATC CCT TTT TAC GGC AAG GCT ATC CCC CTC GAA GTA 144 Thr Thr Gly Glu He Pro Phe Tyr Gly Lys Ala He Pro Leu Glu Val 35 40 45
ATC AAG GGG GGG AGA CAT CTC ATC TTC TGT CAT TCA AAG AAG AAG TGC 192 He Lys Gly Gly Arg His Leu He Phe Cys His Ser Lys Lys Lys Cys 50 55 60
GAC GAA CTC GCC GCA AAG CTG GTC GCA TTG GGC ATC AAT GCC GTG GCC 240 Asp Glu Leu Ala Ala Lys Leu Val Ala Leu Gly He Asn Ala Val Ala 65 70 75 80
TAC TAC CGC GGT CTT GAC GTG TCC GTC ATC CCG ACC AGC GGC GAT GTT 288 Tyr Tyr Arg Gly Leu Asp Val Ser Val He Pro Thr Ser Gly Asp Val 85 90 95
GTC GTC GTG GCA ACC GAT GCC CTC ATG ACC GGC TAT ACC GGC GAC TTC 336 Val Val Val Ala Thr Asp Ala Leu Met Thr Gly Tyr Thr Gly Asp Phe 100 105 110
GAC TCG GTG ATA GAC TGC AAT ACG TGT GTC ACC CAG ACA GTC GAT TTC 384 Asp Ser Val He Asp Cys Asn Thr Cys Val Thr Gin Thr Val Asp Phe 115 120 125 AGC CTT GAC CCT ACC TTC ACC ATT GAG ACA ATC ACG CTC CCC CAG GAT 432 Ser Leu Asp Pro Thr Phe Thr He Glu Thr He Thr Leu Pro Gin Asp 130 135 140
GCT GTC TCC CGC ACT CAA CGT CGG GGC AGG ACT GGC ACG GAA TTC 477
Ala Val Ser Arg Thr Gin Arg Arg Gly Arg Thr Gly Thr Glu Phe 145 150 155
(2) INFORMATION FOR SEQ ID NO:8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 159 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:
Glu Phe Arg Thr Pro Ala Glu Thr Thr Val Arg Leu Arg Ala Tyr Met 1 5 10 15
Asn Thr Pro Gly Leu Pro Val Cys Gin Asp Gly He Pro Ser Pro Ser 20 25 30
Thr Thr Gly Glu He Pro Phe Tyr Gly Lys Ala He Pro Leu Glu Val 35 40 45
He Lys Gly Gly Arg His Leu He Phe Cys His Ser Lys Lys Lys Cys 50 55 60
Asp Glu Leu Ala Ala Lys Leu Val Ala Leu Gly He Asn Ala Val Ala 65 70 75 80
Tyr Tyr Arg Gly Leu Asp Val Ser Val He Pro Thr Ser Gly Asp Val 85 90 95
Val Val Val Ala Thr Asp Ala Leu Met Thr Gly Tyr Thr Gly Asp Phe 100 105 110
Asp Ser Val He Asp Cys Asn Thr Cys Val Thr Gin Thr Val Asp Phe 115 120 125 Ser Leu Asp Pro Thr Phe Thr He Glu Thr He Thr Leu Pro Gin Asp 130 135 140
Ala Val Ser Arg Thr Gin Arg Arg Gly Arg Thr Gly Thr Glu Phe 145 150 155
(2) INFORMATION FOR SEQ ID NO:9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 558 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA to mRNA
(iii) HYPOTHETICAL: N
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Hepatitis C Virus
(B) STRAIN: CDC
(vii) IMMEDIATE SOURCE:
(B) CLONE: 409-1-1 (abc)
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..558 (D) OTHER INFORMATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:
TCC ACC ACC GGA GAG ATC CCT TTT TAC GGC AAG GCT ATC CCC CTC GAA 48 Ser Thr Thr Gly Glu He Pro Phe Tyr Gly Lys Ala He Pro Leu Glu 1 5 10 15
GTA ATC AAG GGG GGG AGA CAT CTC ATC TTC TGT CAT TCA AAG AAG AAG 96 Val He Lys Gly Gly Arg His Leu He Phe Cys His Ser Lys Lys Lys 20 25 30
TGC GAC GAA CTC GCC GCA AAG CTG GTC GCA TTG GGC ATC AAT GCC GTG 144 Cys Asp Glu Leu Ala Ala Lys Leu Val Ala Leu Gly He Asn Ala Val 35 40 45 GCC TAC TAC CGC GGT CTT GAC GTG TCC GTC ATC CCG ACC AGC GGC GAT 192
Ala Tyr Tyr Arg Gly Leu Asp Val Ser Val He Pro Thr Ser Gly Asp 50 55 60
GTT GTC GTC GTG GCA ACC GAT GCC CTC ATG ACC GGC TAT ACC GGC GAC 240
Val Val Val Val Ala Thr Asp Ala Leu Met Thr Gly Tyr Thr Gly Asp 65 70 75 80
TTC GAC TCG GTG ATA GAC TGC AAT ACG TGT GTC ACC CAG ACA GTC GAT 288
Phe Asp Ser Val He Asp Cys Asn Thr Cys Val Thr Gin Thr Val Asp 85 90 95
TTC AGC CTT GAC CCT ACC TTC ACC ATT GAG ACA ATC ACG CTC CCC CAG 336
Phe Ser Leu Asp Pro Thr Phe Thr He Glu Thr He Thr Leu Pro Gin
100 105 110
GAT GCT GTC TCC CGC ACT CAA CGT CGG GGC AGG ACT GGC AGG GGG AAG 384
Asp Ala Val Ser Arg Thr Gin Arg Arg Gly Arg Thr Gly Arg Gly Lys 115 120 125
CCA GGC ATC TAC AGA TTT GTG GCA CCG GGG GAG CGC CCC TCC GGC ATG 432
Pro Gly He Tyr Arg Phe Val Ala Pro Gly Glu Arg Pro Ser Gly Met 130 135 140
TTC GAC TCG TCC GTC CTC TGT GAG TGC TAT GAC GCA GGC TGT GCT TGG 480
Phe Asp Ser Ser Val Leu Cys Glu Cys Tyr Asp Ala Gly Cys Ala Trp 145 150 155 160
TAT GAG CTC ACG CCC GCC GAG ACT ACA GTT AGG CTA CGA GCG TAC ATG 528
Tyr Glu Leu Thr Pro Ala Glu Thr Thr Val Arg Leu Arg Ala Tyr Met 165 170 175
AAC ACC CCG GGG CTT CCC GTG TGC CAG GAC 558
Asn Thr Pro Gly Leu Pro Val Cys Gin Asp
180 185
(2) INFORMATION FOR SEQ ID NO:10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 186 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:
Ser Thr Thr Gly Glu He Pro Phe Tyr Gly Lys Ala He Pro Leu Glu 1 5 10 15
Val He Lys Gly Gly Arg His Leu He Phe Cys His Ser Lys Lys Lys 20 25 30
Cys Asp Glu Leu Ala Ala Lys Leu Val Ala Leu Gly He Asn Ala Val 35 40 45
Ala Tyr Tyr Arg Gly Leu Asp Val Ser Val He Pro Thr Ser Gly Asp 50 55 60
Val Val Val Val Ala Thr Asp Ala Leu Met Thr Gly Tyr Thr Gly Asp 65 70 75 80
Phe Asp Ser Val He Asp Cys Asn Thr Cys Val Thr Gin Thr Val Asp 85 90 95
Phe Ser Leu Asp Pro Thr Phe Thr He Glu Thr He Thr Leu Pro Gin 100 105 110
Asp Ala Val Ser Arg Thr Gin Arg Arg Gly Arg Thr Gly Arg Gly Lys 115 120 125
Pro Gly He Tyr Arg Phe Val Ala Pro Gly Glu Arg Pro Ser Gly Met 130 135 140
Phe Asp Ser Ser Val Leu Cys Glu Cys Tyr Asp Ala Gly Cys Ala Trp 145 150 155 160
Tyr Glu Leu Thr Pro Ala Glu Thr Thr Val Arg Leu Arg Ala Tyr Met 165 170 175
Asn Thr Pro Gly Leu Pro Val Cys Gin Asp 180 185
(2) INFORMATION FOR SEQ ID NO:11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: 36R, primer
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:
CTAGGATCCT TAGAATTCAA CCCGCTGTCC 30
(2) INFORMATION FOR SEQ ID NO:12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: 36F, primer
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:
GGCCCCATGG AATTTTTCAC AGAATTGGAC 30
(2) INFORMATION FOR SEQ ID NO:13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO (vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: #36 Nco Block F, primer
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:
TGGTCCACGG CTGTCCGCTT 20
(2) INFORMATION FOR SEQ ID NO:14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: #36 Nco Block R, primer
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:
AAGCGGACAG CCGTGGACCA 20
(2) INFORMATION FOR SEQ ID NO:15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: 36 Bam Block R, primer (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:
CTAAGATCCG GGTCCCCAGG 20
(2) INFORMATION FOR SEQ ID NO:16: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: 36 Bam Block F, primer
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:
CCTGGGGACC CGGATCTTAG 20
(2) INFORMATION FOR SEQ ID NO:17:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 195 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: NO
(v) FRAGMENT TYPE: N-terminal
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: Clone 36-1 protein coding sequence
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:
Met Glu Phe Phe Thr Glu Leu Asp Gly Val Arg Leu His Arg Phe Ala 1 5 10 15 Pro Pro Cys Lys Pro Leu Leu Arg Glu Glu Val Ser Phe Arg Val Gly 20 25 30
Leu His Glu Tyr Pro Val Gly Ser Gin Leu Pro Cys Glu Pro Glu Pro 35 40 45
Asp Val Ala Val Leu Thr Ser Met Leu Thr Asp Pro Ser His He Thr 50 55 60
Ala Glu Ala Ala Gly Arg Arg Leu Ala Arg Gly Ser Pro Pro Ser Val 65 70 75 80
Ala Ser Ser Ser Ala Ser Gin Leu Ser Ala Pro Ser Leu Lys Ala Thr 85 90 95
Cys Thr Ala Asn His Asp Ser Pro Asp Ala Glu Leu He Glu Ala Asn 100 105 110
Leu Leu Trp Arg Gin Glu Met Gly Gly Asn He Thr Arg Val Glu Ser 115 120 125
Glu Asn Lys Val Val He Leu Asp Ser Phe Asp Pro Leu Val Ala Glu 130 135 140
Glu Asp Glu Arg Glu He Ser Val Pro Ala Glu He Leu Arg Lys Ser 145 150 155 160
Arg Arg Phe Ala Gin Ala Leu Pro Val Trp Ala Arg Pro Asp Tyr Asn 165 170 175
Pro Pro Leu Val Glu Thr Trp Lys Lys Pro Asp Tyr Glu Pro Pro Val 180 185 190
Val His Gly 195
(2) INFORMATION FOR SEQ ID NO:18:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 211 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: NO
(v) FRAGMENT TYPE: internal
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: Clone 36-2 protein sequence
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:
Met Val Trp Ala Arg Pro Asp Tyr Asn Pro Pro Leu Val Glu Thr Trp 1 5 10 15
Lys Lys Pro Asp Tyr Glu Pro Pro Val Val His Gly Cys Pro Leu Pro 20 25 30
Pro Pro Lys Ser Pro Pro Val Pro Pro Pro Arg Lys Lys Arg Thr Val 35 40 45
Val Leu Thr Glu Ser Thr Leu Ser Thr Ala Leu Ala Glu Leu Ala Thr 50 55 60
Arg Ser Phe Gly Ser Ser Ser Thr Ser Gly He Thr Gly Asp Asn Thr 65 70 75 80
Thr Thr Ser Ser Glu Pro Ala Pro Ser Gly Cys Pro Pro Asp Ser Asp 85 90 95
Ala Glu Ser Tyr Ser Ser Met Pro Pro Leu Glu Gly Glu Pro Gly Asp 100 105 110
Pro Asp Leu Ser Asp Gly Ser Trp Ser Thr Val Ser Ser Glu Ala Asn 115 120 125
Ala Glu Asp Val Val Cys Cys Ser Met Ser Tyr Ser Trp Thr Gly Ala 130 135 140
Leu Val Thr Pro Cys Ala Ala Glu Glu Gin Lys Leu Pro He Asn Ala 145 150 155 160
Leu Ser Asn Ser Leu Leu Arg His His Asn Leu Val Tyr Ser Thr Thr 165 170 175
Ser Arg Ser Ala Cys Gin Arg Gin Lys Lys Val Thr Phe Asp Arg Leu 180 185 190 Gin Val Leu Asp Ser His Tyr Gin Asp Val Leu Lys Glu Val Lys Ala 195 200 205
Ala Ala Ser 210
(2) INFORMATION FOR SEQ ID NO:19:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 156 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: NO
(v) FRAGMENT TYPE: C-terminal
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: Clone 36-3 protein coding sequence
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
Met Glu Arg Gin Lys Lys Val Thr Phe Asp Arg Leu Gin Val Leu Asp 1 5 10 15
Ser His Tyr Gin Asp Val Leu Lys Glu Val Lys Ala Ala Ala Ser Lys 20 25 30
Val Lys Ala Asn Leu Leu Ser Val Glu Glu Ala Cys Ser Leu Thr Pro 35 40 45
Pro His Ser Ala Lys Ser Lys Phe Gly Tyr Gly Ala Lys Asp Val Arg 50 55 60
Cys His Ala Arg Lys Ala Val Thr His He Asn Ser Val Trp Lys Asp 65 ' 70 75 80
Leu Leu Glu Asp Asn Val Thr Pro He Asp Thr Thr He Met Ala Lys 85 90 95
Asn Glu Val Phe Cys Val Gin Pro Glu Lys Gly Gly Arg Lys Pro Ala 100 105 110 Arg Leu He Val Phe Pro Asp Leu Gly Val Arg Val Cys Glu Lys Met 115 120 125
Ala Leu Tyr Asp Val Val Thr Lys Leu Pro Leu Ala Val Met Gly Ser 130 135 140
Ser Tyr Gly Phe Gin Tyr Ser Pro Gly Gin Arg Val 145 150 155
(2) INFORMATION FOR SEQ ID NO:20:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 35 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: primer 36-1F
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:
CCCGAATTCA CCATGGAATT TTTCACAGAA TTGGA 35
(2) INFORMATION FOR SEQ ID NO:21:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 39 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: primer 36-1R (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:
CCCGAATTCG GATCCCTATT AGCCGTGGAC CACAGGTGG 39
(2) INFORMATION FOR SEQ ID NO:22:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 33 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: Primer 36-2
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:
CCCGAATTCA CCATGGTTTG GGCGCGGCCG GAC 33
(2) INFORMATION FOR SEQ ID NO:23:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 39 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: Primer 36-2R
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:
CCCGAATTCG GATCCCTATT ATGACGCCGC TGCTTTAAC 39 (2) INFORMATION FOR SEQ ID NO:24:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 36 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: Primer 36-3F
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:
CCCGAATTCA CCATGGAAAG GCAGAAGAAA GTCACA 36
(2) INFORMATION FOR SEQ ID NO:25:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 39 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: Primer 36-3R
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:
CCCGAATTCG GATCCCTATT AAACCCGCTG TCCTGGTGA 39
(2) INFORMATION FOR SEQ ID NO:26:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 599 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: Clone 36-1 nucleic acid coding sequence
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 3..587
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:
CC ATG GAA TTT TTC ACA GAA TTG GAC GGG GTG CGC CTA CAT AGG TTT 47
Met Glu Phe Phe Thr Glu Leu Asp Gly Val Arg Leu His Arg Phe 1 5 10 15
GCG CCC CCC TGC AAG CCC TTG CTG CGG GAG GAG GTA TCA TTC AGA GTA 95 Ala Pro Pro Cys Lys Pro Leu Leu Arg Glu Glu Val Ser Phe Arg Val 20 25 30
GGA CTC CAC GAA TAC CCG GTA GGG TCG CAA TTA CCT TGC GAG CCC GAA 143 Gly Leu His Glu Tyr Pro Val Gly Ser Gin Leu Pro Cys Glu Pro Glu 35 40 45
CCG GAC GTG GCC GTG TTG ACG TCC ATG CTC ACT GAT CCC TCC CAT ATA 191 Pro Asp Val Ala Val Leu Thr Ser Met Leu Thr Asp Pro Ser His He 50 55 60
ACA GCA GAG GCG GCC GGG CGA AGG TTG GCG AGG GGA TCA CCC CCC TCT 239 Thr Ala Glu Ala Ala Gly Arg Arg Leu Ala Arg Gly Ser Pro Pro Ser 65 70 75
GTG GCC AGC TCC TCG GCT AGC CAG CTA TCC GCT CCA TCT CTC AAG GCA 287 Val Ala Ser Ser Ser Ala Ser Gin Leu Ser Ala Pro Ser Leu Lys Ala 80 85 90 95 ACT TGC ACC GCT AAC CAT GAC TCC CCT GAT GCT GAG CTC ATA GAG GCC 335 Thr Cys Thr Ala Asn His Asp Ser Pro Asp Ala Glu Leu He Glu Ala ' 100 105 110
AAC CTC CTA TGG AGG CAG GAG ATG GGC GGC AAC ATC ACC AGG GTT GAG 383 Asn Leu Leu Trp Arg Gin Glu Met Gly Gly Asn He Thr Arg Val Glu 115 120 125
TCA GAA AAC AAA GTG GTG ATT CTG GAC TCC TTC GAT CCG CTT GTG GCG 431 Ser Glu Asn Lys Val Val He Leu Asp Ser Phe Asp Pro Leu Val Ala 130 135 140
GAG GAG GAC GAG CGG GAG ATC TCC GTA CCC GCA GAA ATC CTG CGG AAG 479 Glu Glu Asp Glu Arg Glu He Ser Val Pro Ala Glu He Leu Arg Lys 145 150 155
TCT CGG AGA TTC GCC CAG GCC CTG CCC GTT TGG GCG CGG CCG GAC TAT 527 Ser Arg Arg Phe Ala Gin Ala Leu Pro Val Trp Ala Arg Pro Asp Tyr 160 165 170 175
AAC CCC CCG CTA GTG GAG ACG TGG AAA AAG CCC GAC TAC GAA CCA CCT 575 Asn Pro Pro Leu Val Glu Thr Trp Lys Lys Pro Asp Tyr Glu Pro Pro 180 185 190
GTG GTC CAC GGC TAATAGGGAT CC 599
Val Val His Gly 195
(2) INFORMATION FOR SEQ ID NO:27:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 647 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: Clone 36-2 nucleic acid coding sequence ( ix ) FEATURE :
(A) NAME/KEY: CDS
(B) LOCATION: 3..635
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:
CC ATG GTT TGG GCG CGG CCG GAC TAT AAC CCC CCG CTA GTG GAG ACG 4
Met Val Trp Ala Arg Pro Asp Tyr Asn Pro Pro Leu Val Glu Thr 1 5 10 15
TGG AAA AAG CCC GAC TAC GAA CCA CCT GTG GTC CAC GGC TGT CCG CTT 9 Trp Lys Lys Pro Asp Tyr Glu Pro Pro Val Val His Gly Cys Pro Leu 20 25 30
CCA CCT CCA AAG TCC CCT CCT GTG CCT CCG CCT CGG AAG AAG CGG ACG 14 Pro Pro Pro Lys Ser Pro Pro Val Pro Pro Pro Arg Lys Lys Arg Thr 35 40 45
GTG GTC CTC ACT GAA TCA ACC CTA TCT ACT GCC TTG GCC GAG CTC GCC 19 Val Val Leu Thr Glu Ser Thr Leu Ser Thr Ala Leu Ala Glu Leu Ala 50 55 60
ACC AGA AGC TTT GGC AGC TCC TCA ACT TCC GGC ATT ACG GGC GAC AAT 23 Thr Arg Ser Phe Gly Ser Ser Ser Thr Ser Gly He Thr Gly Asp Asn 65 70 75
ACG ACA ACA TCC TCT GAG CCC GCC CCT TCT GGC TGC CCC CCC GAC TCC 28 Thr Thr Thr Ser Ser Glu Pro Ala Pro Ser Gly Cys Pro Pro Asp Ser 80 85 90 95
GAC GCT GAG TCC TAT TCC TCC ATG CCC CCC CTG GAG GGG GAG CCT GGG 33 Asp Ala Glu Ser Tyr Ser Ser Met Pro Pro Leu Glu Gly Glu Pro Gly 100 105 110
GAT CCG GAT CTT AGC GAC GGG TCA TGG TCA ACG GTC AGT AGT GAG GCC 38 Asp Pro Asp Leu Ser Asp Gly Ser Trp Ser Thr Val Ser Ser Glu Ala 115 120 125
AAC GCG GAG GAT GTC GTG TGC TGC TCA ATG TCT TAC TCT TGG ACA GGC 43 Asn Ala Glu Asp Val Val Cys Cys Ser Met Ser Tyr Ser Trp Thr Gly 130 135 140 GCA CTC GTC ACC CCG TGC GCC GCG GAA GAA CAG AAA CTG CCC ATC AAT 479 Ala Leu Val Thr Pro Cys Ala Ala Glu Glu Gin Lys Leu Pro He Asn 145 150 155
GCA CTA AGC AAC TCG TTG CTA CGT CAC CAC AAT TTG GTG TAT TCC ACC 527 Ala Leu Ser Asn Ser Leu Leu Arg His His Asn Leu Val Tyr Ser Thr 160 ' 165 170 175
ACC TCA CGC AGT GCT TGC CAA AGG CAG AAG AAA GTC ACA TTT GAC AGA 575 Thr Ser Arg Ser Ala Cys Gin Arg Gin Lys Lys Val Thr Phe Asp Arg 180 185 190
CTG CAA GTT CTG GAC AGC CAT TAC CAG GAC GTA CTC AAG GAG GTT AAA 623 Leu Gin Val Leu Asp Ser His Tyr Gin Asp Val Leu Lys Glu Val Lys 195 200 205
GCA GCG GCG TCA TAATAGGGAT CC 647
Ala Ala Ala Ser
210 ,
(2) INFORMATION FOR SEQ ID NO:28:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 482 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: Clone 36-3 nucleic acid coding sequence
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 3..470 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28: CC ATG GAA AGG CAG AAG AAA GTC ACA TTT GAC AGA CTG CAA GTT CTG 4
Met Glu Arg Gin Lys Lys Val Thr Phe Asp Arg Leu Gin Val Leu 1 5 10 15
GAC AGC CAT TAC CAG GAC GTA CTC AAG GAG GTT AAA GCA GCG GCG TCA 9 Asp Ser His Tyr Gin Asp Val Leu Lys Glu Val Lys Ala Ala Ala Ser 20 25 30
AAA GTG AAG GCT AAC TTG CTA TCC GTA GAG GAA GCT TGC AGC CTG ACG 14 Lys Val Lys Ala Asn Leu Leu Ser Val Glu Glu Ala Cys Ser Leu Thr 35 40 45
CCC CCA CAC TCA GCC AAA TCC AAG TTT GGT TAT GGG GCA AAA GAC GTC 19 Pro Pro His Ser Ala Lys Ser Lys Phe Gly Tyr Gly Ala Lys Asp Val 50 55 60
CGT TGC CAT GCC AGA AAG GCC GTA ACC CAC ATC AAC TCC GTG TGG AAA 23 Arg Cys His Ala Arg Lys Ala Val Thr His He Asn Ser Val Trp Lys 65 70 75
GAC CTT CTG GAA GAC AAT GTA ACA CCA ATA GAC ACT ACC ATC ATG GCT 28 Asp Leu Leu Glu Asp Asn Val Thr Pro He Asp Thr Thr He Met Ala 80 85 90 95
AAG AAC GAG GTT TTC TGC GTT CAG CCT GAG AAG GGG GGT CGT AAG CCA 33 Lys Asn Glu Val Phe Cys Val Gin Pro Glu Lys Gly Gly Arg Lys Pro 100 105 110
GCT CGT CTC ATC GTG TTC CCC GAT CTG GGC GTG CGC GTG TGC GAA AAG 38 Ala Arg Leu He Val Phe Pro Asp Leu Gly Val Arg Val Cys Glu Lys 115 120 125
ATG GCT TTG TAC GAC GTG GTT ACA AAG CTC CCC TTG GCC GTG ATG GGA 43 Met Ala Leu Tyr Asp Val Val Thr Lys Leu Pro Leu Ala Val Met Gly 130 135 140
AGC TCC TAC GGA TTC CAA TAC TCA CCA GGA CAG CGG GTT TAATAGGGAT 48 Ser Ser Tyr Gly Phe Gin Tyr Ser Pro Gly Gin Arg Val 145 150 155
CC 48

Claims

IT IS CLAIMED:
1. A method for detecting the presence of hepatitis C virus (HCV) antigens in a sample, comprising contacting the sample with at least one antibody which is reactive with an HCV antigen, where the antibody is attached to a solid support and where the antibody is immunoreactive with a portion of the polypeptide presented as SEQ ID NO:6, examining the antibody for the presence of bound HCV antigen, where said examining involves reacting the solid support with an antigen- reporter complex, where the HCV antigen competes with binding of the antigen-reporter complex to the antibody, and detecting antigen-reporter complex which is bound to the solid support.
2. The method of claim 1, where said detecting includes WP of the level of reporter that remains bound to the solid support.
3. The method of claim 1, where the sample is selected from the group consisting of tissue culture medium, chimpanzee serum, and human serum.
4. The method of claim 1, where the solid support is the well of a microtiter plate.
5. The method of claim 1, where the antigen used to generate the antibody comprises a polypeptide antigen selected from the group of sequences presented as SEQ ID NO:6, SEQ ID NO:17, SEQ ID NO:18, and SEQ ID NO:19.
6. The method of claim 1, where the antigen of the antigen-reporter complex comprises a polypeptide antigen selected from the group consisting of SEQ ID NO:6, SEQ ID NO:17, SEQ ID NO:18, and SEQ ID NO:19.
7. The method of claim 1, where two antibodies and two antigen-reporter complexes are used.
8. The method of claim 1, where the reporter of the antigen-reporter complex is selected from the group consisting of enzymatic reporters, radioactive reporters, and fluorescent reporters.
9. The method of claim 8, where the reporter is the enzymatic reporter horse radish peroxidase.
10. A diagnostic kit for use in screening samples for the presence of hepatitis C virus
(HCV) antigen comprising at least one antibody which is immunoreactive with a portion of the polypeptide presented as SEQ ID NO:6, and an antigen-reporter complex, where the HCV antigen competes with binding of the antigen- reporter complex to the antibody.
11. The kit of claim 10, where said antibody is attached to a solid support.
12. The kit of claim 10, where the antigen of the antigen-reporter complex comprises a polypeptide antigen selected from the group consisting of SEQ ID NO:6, SEQ ID NO:17, SEQ ID NO:18, and SEQ ID NO:19..
13. The kit of claim 10, where the reporter of the antigen-reporter complex is selected from the group consisting of enzymatic reporters, radioactive reporters, and fluorescent reporters.
14. Purified antibodies that are immunoreactive with a polypeptide consisting essentially of a sequence selected from the group consisting of SEQ ID NO:6, SEQ ID NO:17, SEQ ID NO:18, and SEQ ID NO:19.
15. The antibodies of claim 14, which are polyclonal antibodies.
16. The antibodies of claim 14, which are monoclonal antibodies.
17. Monoclonal antibodies that are immunoreactive with a polypeptide consisting essentially of a sequence selected from the group consisting of SEQ ID NO:6, SEQ ID NO:17, SEQ ID NO:18, and SEQ ID NO:19.
18. A polypeptide consisting essentially of a sequence selected from the group consisting of
SEQ ID NO:6, SEQ ID NO:17, SEQ ID NO:18, and SEQ ID NO:19.
PCT/US1994/004174 1993-04-22 1994-04-15 Hepatitis c virus immunodiagnostic antigens and antibodies WO1994024565A1 (en)

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Cited By (2)

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GB2294690A (en) * 1994-11-01 1996-05-08 United Biomedical Inc Polypeptides for the diagnosis and therapy of hepatitis C virus(HCV)
FR2738827A1 (en) * 1995-09-18 1997-03-21 Bio Merieux DETECTION OF ENTEROBACTERIA

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EP0318216A1 (en) * 1987-11-18 1989-05-31 Chiron Corporation NANBV diagnostics and vaccines
EP0388232A1 (en) * 1989-03-17 1990-09-19 Chiron Corporation NANBV diagnostics and vaccines
WO1991015516A2 (en) * 1990-04-06 1991-10-17 Genelabs Incorporated Hepatitis c virus epitopes
WO1993006488A1 (en) * 1991-09-16 1993-04-01 Genelabs Technologies, Inc. Peptide based hepatitis c virus immunoassays

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Publication number Priority date Publication date Assignee Title
EP0318216A1 (en) * 1987-11-18 1989-05-31 Chiron Corporation NANBV diagnostics and vaccines
EP0388232A1 (en) * 1989-03-17 1990-09-19 Chiron Corporation NANBV diagnostics and vaccines
WO1991015516A2 (en) * 1990-04-06 1991-10-17 Genelabs Incorporated Hepatitis c virus epitopes
WO1993006488A1 (en) * 1991-09-16 1993-04-01 Genelabs Technologies, Inc. Peptide based hepatitis c virus immunoassays

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Title
G.KUO, Q-.L. CHOO ET AL.: "An Assay for Circulating Antibodies to a Major Etiologic Virus of Human Non-A, Non-B Hepatitis", SCIENCE, vol. 244, no. 4902, 21 April 1989 (1989-04-21), LANCASTER,PA, US, pages 362 - 364 *
Q.-L. CHOO ET AL.: "Genetic organization and diversity of the hepatitis C virus", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF USA, vol. 88, no. 6, 15 March 1991 (1991-03-15), WASHINGTON US, pages 2451 - 2455 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB2294690A (en) * 1994-11-01 1996-05-08 United Biomedical Inc Polypeptides for the diagnosis and therapy of hepatitis C virus(HCV)
GB2294690B (en) * 1994-11-01 1998-10-28 United Biomedical Inc Peptides effective for diagnosis and detection of hepatitis C infection
FR2738827A1 (en) * 1995-09-18 1997-03-21 Bio Merieux DETECTION OF ENTEROBACTERIA
US5786147A (en) * 1995-09-18 1998-07-28 Bio Merieux Detection of enterobacteria

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