EP0836708A1

EP0836708A1 - Method for antibody to hepatitis c virus second envelope glycoprotein

Info

Publication number: EP0836708A1
Application number: EP96917969A
Authority: EP
Inventors: Gregory F. Okasinski; Verlyn G. Schaefer; Thomas S. Suhar; Richard R. Lesniewski; James W. Scheffel
Original assignee: Abbott Laboratories
Current assignee: Abbott Laboratories
Priority date: 1995-06-07
Filing date: 1996-06-04
Publication date: 1998-04-22
Also published as: CA2223277A1; WO1996041196A1; JPH11507129A

Abstract

A method for detecting antibody to HCV in a test sample. The method includes utilizing a recombinant protein that is the expression product of mammalian cells transformed by a heterologous expression vector comprising a DNA sequence encoding an E2 truncated protein. Test kits which include this recombinant protein also are provided.

Description

METHOD FOR DETECTION OF ANTIBODY TO HEPATITIS C VIRUS SECOND ENVELOPE GLYCOPROTEIN

FIELD OF THE INVENTION The present invention relates generally to a method for detecting antibodies to hepatitis C virus (HCV), and more particularly, relates to a method for detecting antibodies to an additional marker for HCV infection, the second envelope glycoprotein (E2) of HCV.

BACKGROUND OF THE INVENTION

The introduction of first generation hepatitis C virus enzyme immunoassays (HCV 1.0 EIAs) in 1989 and second generation HCV enzyme immunoassays (HCV 2.0 EIAs) in 1992 has dramatically reduced the incidence of post-transfusion HCV (PT-HCV) infection in countries routinely screening donated blood using these tests [Donahue et al., N. Engl. J. Med. 327:369-73 (1992); Kleinman et al., Transfusion 32: 805-13 (1992); Alter, in Viral Hepatitis and Liver Disease 551-53 (Nishioka et al. eds., 1994)]. In the second generation assay, antibodies to HCV (anti-HCV) are detected using recombinant proteins derived from the core, NS3 (viral protease) and NS4 (unknown function) genes of the virus. Third generation screening assays (HCV 3.0 EIAs), which include an additional antigen from the NS5 region (viral polymerase and a second unknown function), are now available and are being used in Europe [Lavanchy etal., J. Clin. Microbiol. 32:2272-75 (1994)].

Additional unlicensed immunoblot assays such as an immunodot assay or a strip immunoblot assay (SLA)^' can be used for the detection of antibodies to individual HCV proteins, including core, NS3, NS4 and NS5. These assays are intended for use as an additional, more particular test for those human serum or plasma test samples found repeatedly reactive using a licensed anti-HCV screening assay, e.g., HCV 2.0 EIA. These assays are used as a means to confirm reactivity in a HCV 2.0 screening assay. According to the conventional interpretation of results for these assays, reactivity to at least two HCV proteins corresponding to antigens encoded by different parts of the HCV genome is interpreted as positive. Reactivity to only a single HCV protein is interpreted as mdeterminate.

Any donor demonstrating antibodies specific for 2 or more HCV gene products should be regarded as having seroconverted to HCV subsequent to infection with this virus [Alter, 1994; Bresters et al., Transfusion 33:634-38 (1993); Sayers & Gretch, Transfusion 33:809-13 (1993)]. In addition, because of the high chronicity rate associated with HCV infection [Di Bisceglie et al., Hepatology 14:969-74 (1991); Alter et al., N. Engl. J. Med. 327:1899-905 (1992)], these donors should be considered potentially infectious. With respect to the mdeterminate test samples, blood banks have a significant concern with respect to how to handle mdeterminate cases and the added expense of follow- , up testing.

Leon et al. , using multiple supplemental HCV tests to evaluate RLBA 2.0 mdeterminate/PCR negative samples, demonstrated that true HCV antibodies were present in 71.6% and 57.1 % of core and NS3 mdeterminate specimens respectively [Vox Sanguinus 66:245-46 (1994)]. Previous studies using PCR have indicated as high as 75% of RLBA 2.0 mdeterminate specimens may be HCV RNA positive [Half on et al., Journal of Infectious Diseases 166:449 (1992); Buffet et al. , J. Med. Virol. 43:259-61 (1994)] . A positive

PCR result on indeterminate specimens is compelling evidence of HCV exposure. On the other hand, indeterminate samples which are PCR negative is not enough evidence to conclude false reactivity. While one previous study [Tobler et al., Transfusion 34:130-34 (1994)] suggests that RLBA 2.0 mdeteraiinate samples which are PCR negative are most likely to indicate false reactivity for HCV, negative PCR results on indeterminate samples need to be regarded as inconclusive due to factors such as low virus concentration [Ulrich et al., J. Clin. Investigation 86:1609-14 (1990)], intermittent viremia during HCV infection [Allain et al., J. Clin. Investigation 88:1672-79 (1991); Prince et al., J. Infectious Diseases 167:1296-301 (1993); Peters et al., J. Med. Virol. 42:420-27 (1994)], and variability in PCR technique [Zaaijer et al., Lancet 341:722-24 (1993)]. Furthermore, conversion to HCV 3.0 screening assays warrants careful scrutiny because of the discordant results obtained between HCV 2.0 and 3.0 version assays, particularly among specimens identified with mdeterminate immunoblot reactivities, to ensure that all potentially infectious units currently detected by HCV 2.0 tests are also detected by the new HCV 3.0 assays. It is important, therefore, to further investigate and understand the true disposition of HCV 2.0 positive specimens containing antibody reactivity to only a single gene product.

Difficulties in the expression and purification of the putative viral envelope proteins (El, E2) have prevented detailed research and possible incorporation of these proteins as targets in blood screening assays. The 5¹ end of the HCV genome encodes two putative envelope proteins, El and E2. Both proteins contain multiple glycosy lation sites [Takeuchi et al., J. Gen. Virol. 71:3027-33 (1990)] and share some nucleotide and amino acid sequence homologies with pestivirus envelope proteins [Miller & Purcell, Proc. Natl. Acad. Sci. USA 87:2057-61 (1990); Takeuchi et al. , supra, 1990; Choo et al. , Proc. Natl. Acad. Sci. USA 88:2451-55 (1991)]. In addition, the E2 protein has been shown to contain a hypervariable region with mutation characteristics similar to that observed in the hypervariable V3 loop of gpl20 of the human immunodeficiency virus [Kato et al. , Molecular and Biological Medicine 7:495-501 (1990); Houghton et al., Hepatology 14:381-388 (1991); Weiner et al., Proc. Natl. Aca. Sci. USA 89:3468-72 (1992)]. As envelope proteins, El and E2 are likely to be located partially or entirely at the surface of the virion particle. This association with the structural surface of the virus would seem to make these proteins prime targets for humoral immune responses to infection with HCV. As evidence of this, the hypervariable region of HCV E2 appears to mutate in response to humoral immune selective pressure [Inchauspe et al. , Proc. Natl. Acad. Sci. USA 88:10292-96 (1991); Ogata et al., Proc. Natl. Acad. Sci. USA 88:3392-96 (1991); Lesniewski et al., J. Med. Virol. 40:150-56 (1993)].

However, prior to the present invention, some investigators reported low reactivity between HCV E2 recombinant proteins and the antibodies of individuals infected with HCV. Mita et al. [Biochemical and Biophysical Research Communications 183:925-30 (1992)] and Hsu et al. lΗepatologv 17:763-71 (1993)] reported that antibodies to E2 were detected in 17% and 10%, respectively, of patients with chronic type C hepatitis liver disease. The conformation and degree of glycosylation of the recombinant E2 protein may affect its immunoreactivity. Chien et al. reported that antibodies to E2 were detected in 97% of chronic non-A, non-B hepatitis (NANBH) patients using a carboxyl-truncated E2 protein (amino acids [aa] 384-661) produced in CHO cells [Lancet 342:933 (1993)]. Thus, there is a need for the development of additional assay reagents and methods to identify acute infection and viremia which may be present, but not currently detectable by commercially available screening and confirmatory assays. These assay reagents and methods are needed in order to help distinguish between those individuals with acute and persistent, on-going and/or chronic infection and those individuals whose HCV infections are likely to be resolved, and to defme the prognostic course of NANB hepatitis infection in order to develop preventive or therapeutic strategies. The present invention provides a heretofore unrealized confirmatory marker for deteπnimng true HCV infection.

SUMMARY OF THE INVENTION

The present invention provides an improved method for detecting the presence of antibodies to HCV antigen which may be present in a test sample comprising contacting said sample with HCV antigen and deteπnining whether antibodies are bound to said HCV antigen, wherein the improvement comprises employing as said HCV antigen at least one recombinant HCV protein comprising a recombinant polypeptide that is the expression product of mammalian cells transformed by a heterologous expression vector comprising a DNA sequence encoding an E2 truncated protein, a DNA sequence encoding a rabbit heavy chain signal sequence and a DNA sequence encoding an amino- terminal sequence of human pro-urokinase, wherein said HCV antigen DNA sequence is located downstream to said other DNA sequences.

The present invention further provides a test kit for detecting the presence of antibodies to HCV antigen which may be present in a test sample, comprising a container containing a recombinant polypeptide that is the expression product of mammalian cells transformed by a heterologous expression vector comprising a DNA sequence encoding an E2 truncated protein, a DNA sequence encoding a rabbit heavy chain signal sequence and a DNA sequence encoding an ammo-terminal sequence of human pro-urokinase, wherein said HCV antigen DNA sequence is located downstream to said other DNA sequences.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1 shows the immunofluorescent staining pattern of HCV E2 antigen in transfected CHO cells using (A) rabbit anti-peptide serum (amino acids 509-551); and (B) HCV positive human serum.

Fig. 2 shows the results of a RIP A analysis of HCV E2 antigen produced in CHO cells. ³⁵S-labeled E2 antigen was i munoprecipitated with: pre-immune rabbit sera (Lane 2), hyperimmune serum from a rabbit immunized with synthetic peptide aa 509-551 (Lane 3), normal human plasma (Lane 4), and three different HCV antibody positive human plasma (Lanes 5-7). Lane 1 contains radioactive molecular weight markers.

Fig. 3 is a SDS-PAGE gel of purified HCV E2 antigen produced in CHO cells. Lane 1: Molecular weight markers. Lanes 2 and 3: Purified E2 antigen. DETAILED DESCRIPTION OF THE INVENTION

The present invention provides an improved method for detecting the presence of antibodies to HCV antigen which may be present in a test sample comprising contacting said sample with HCV antigen and determining whether antibodies are bound to said HCV antigen, wherein the improvement comprises employing as said HCV antigen at least one recombinant HCV protein comprising a recombinant polypeptide that is the expression product of mammalian cells transformed by a heterologous expression vector comprising a DNA sequence encoding an E2 truncated protein, a DNA sequence encoding a rabbit heavy chain signal sequence and a DNA sequence encoding an amino- terminal sequence of human pro-urokinase, wherein said HCV antigen DNA sequence is located downstream to said other DNA sequences.

Recombinant polypeptides produced as described herein in mammalian expression systems provide antigens for diagnostic assays which can be used to determine the viremia of a patient based on a strong correlation between the presence of E2 antigen and patients found to be viremic using reverse transcriptase polymerase chain reaction (RT-PCR) amplification. The antigens also are useful as an early marker of seroconversion, and provide another means for determining true HCV exposure in indeterminate test samples tested by commercially available confirmatory tests. The antigens also provide a means for resolving discrepant results between commercially ^■: available second generation and third generation HCV screening assays.

The present invention confers several technical advantages over the prior art. For example, the presence of antibodies to HCV E2 in specimens already positive for HCV antibody provides additional, and therefore, more compelling evidence of true HCV infection. Furthermore, the presence of antibodies to HCV E2, along with reactivity in a licensed HCV 2.0 screening assay as well as reactivity to the HCV core or NS3 protein in an immunoblot assay, point to a consistent interpretation that individuals with these serologic profiles have had previous, or have ongoing, HCV infection. The recombinant polypeptides produced can be provided in the form of a kit with one or more containers such as vials or bottles, with each container containing a separate reagent such as a recombinant polypeptide, packaged as test kits for the convenience of performing assays. Other aspects of the present invention include a recombinant polypeptide comprising an HCV E2 epitope attached to a solid phase.

The present invention provides assays which utilize the recombinant proteins produced as described herein in various formats, any of which may employ a signal generating compound which generates a measurable signal in the assay. All of the assays described generally detect antibody, and include contacting a test sample with at least one HCV antigen provided herein to form at least one antigen-antibody complex and detecting the presence of the complex so formed. These assays are described in detail herein.

The term "test sample" refers to any component of an individual's body which can be a source of the antibodies of interest. These components are well known in the art. These test samples include biological samples which can be tested by the methods of the present invention described herein and include human and animal body fluids such as whole blood, serum, plasma, cerebrospinal fluid, urine, lymph fluids, and various external sections of the respiratory, intestinal and genitourinary tracts, tears, saliva, milk, white blood cells, myelomas and the like, biological fluids such as cell culture supematants, fixed tissue specimens and fixed cell specimens.

"Solid phases" ("solid supports") are known to those in the art but not critical and include the walls of wells of a reaction tray, test tubes, polystyrene beads, magnetic or non-magnetic beads, nitrocellulose strips, membranes, microparticles such as latex particles, plastic tubes, glass or silicon chips and sheep red blood cells are all suitable examples and others. Suitable methods for immobilizing peptides on solid phases include ionic, hydrophobic, covalent interactions and the like. A "solid phase", as used herein, refers to any material which is insoluble, or can be made insoluble by a subsequent reaction. The solid phase can be chosen for its intrinsic ability to attract and immobilize the capture reagent. Alternatively, the solid phase can retain an additional receptor which has the ability to attract and immobilize the capture reagent. The additional receptor can include a charged substance that is oppositely charged with respect to the capture reagent itself or to a charged substance conjugated to the capture reagent. As yet another alternative, the receptor molecule can be any specific binding member which is attached to the solid phase and which has the ability to immobilize the capture reagent through a specific binding reaction. The receptor molecule enables the indirect binding of the capture reagent to a solid phase material before the performance of the assay or during the performance of the assay.

It is contemplated and within the scope of the invention that the solid phase also can comprise any suitable porous material with sufficient porosity to allow access by detection antibodies and a suitable surface affinity to bind antigens. Microporous structures are generally preferred, but materials with gel structure in the hydrated state may be used as well. Such useful solid supports include: natural polymeric carbohydrates and their synthetically modified, cross-linked or substituted derivatives, such as agar, agarose, cross¬ linked alginic acid, substituted and cross-linked guar gums, cellulose esters, especially with nitric acid and carboxylic acids, mixed cellulose esters, and cellulose ethers; natural polymers containing nitrogen, such as proteins and derivatives, including cross-linked or modified gelatins; natural hydrocarbon polymers, such as latex and rubber; synthetic polymers which may be prepared with suitably porous structures, such as vinyl polymers, including polyethylene, polypropylene, polystyrene, polyvmylchloride, polyvinylacetate and its partially hydrolyzed derivatives, polyacrylamides, polymethacrylates, copolymers and terpolymers of the above polycondensates, such as polyesters, polyamides, and other polymers, such as polyurethanes or polyepoxides; porous inorganic materials such as sulfates or carbonates of alkaline earth metals and magnesium, including barium sulfate, calcium sulfate, calcium carbonate, silicates of alkali and alkaline earth metals, aluminum and magnesium; and aluminum or silicon oxides or hydrates, such as clays, alumna, talc, kaolin, zeolite, silica gel, or glass (these materials may be used as filters with the above polymeric materials); and mixtures or copolymers of the above classes, such as graft copolymers obtained by initializing polymerization of synthetic polymers on a pre-existing natural polymer. All of these materials may be used in suitable shapes, such as films, sheets, or plates, or they may be coated onto or bonded or laminated to appropriate inert carriers, such as paper, glass, plastic films, or fabrics.

The porous structure of nitrocellulose has excellent absorption and adsorption qualities for a wide variety of reagents. Nylon also possesses similar characteristics and also is suitable. It is contemplated that such porous solid supports described herein above are preferably in the form of sheets of thickness from about 0.01 to about 0.5 mm, preferably about 0.1 mm. The pore size may vary within wide limits, and is preferably from about 0.025 to about 15 microns, especially from about 0.15 to about 15 microns. The surfaces of such supports may be activated by chemical processes which cause covalent linkage of the antigen to the support. The irreversible binding of the antigen is obtained, however, in general, by adsorption on the porous material by poorly understood hydrophobic forces. Suitable solid supports also are described in U.S. Patent Application Serial No. 227,272. The "indicator reagent" comprises a "signal generating compound"

(label) which generates a measurable signal detectable by external means conjugated to a specific binding member for HCV. "Specific binding member" as used herein means a member of a specific binding pair, that is, two different molecules where each of the molecules through chemical or physical means specifically binds to the other molecule. An immunoreactive specific binding member can be an antibody, an antigen, or an antibody /antigen complex that is capable of binding either to HCV as in a sandwich assay, to the capture reagent as in a competitive assay, or to the ancillary specific binding member as in an indirect assay. In addition to being an antibody member of a specific binding pair for HCV, the indicator reagent also can be a member of other specific binding pairs, including hapten-anti-hapten systems such as biotin or anti-biotin and avidin or biotin, a carbohydrate or a lectin, a complementary nucleotide sequence, an effector or a receptor molecule, an enzyme cofactor or an enzyme, an enzyme inhibitor or an enzyme, and the like.

The various "signal generating compounds" (labels) contemplated include chromogens, catalysts such as enzymes, luminescent compounds such as fluorescein and rhodamine, chemiluminescent compounds such as acridinium, phenanthridinium and dioxetane compounds including those described in co¬ pending U.S. Patent Application Serial No. 0_/921,979 corresponding to EP Publication No. 0 273,115, which enjoys common ownership and which is incorporated herein by reference, radioactive elements, and direct visual labels. Examples of enzymes include alkaline phosphatase, horseradish peroxidase, B- galactosidase, and the like. The selection of a particular label is not critical, but it will be capable of producing a signal either by itself or in conjunction with one or more additional substances. Other embodiments which utilize various other solid phases also are contemplated and are within the scope of this invention. For example, ion capture procedures for separating an immobilizable reaction complex with a negatively charged polymer, described in co-pending U.S. Patent Application Serial No. 150,278 corresponding to EP Publication No. 0 326,100, and U.S. Patent Application Serial No. 375,029 corresponding to EP Publication No. 0 406,473, both of which enjoy common ownership and are incorporated herein by reference, can be employed according to the present invention to effect a fast solution-phase immunochemical reaction. An immobilizable immune complex is separated from the rest of the reaction mixture by ionic interactions between the negatively charged poly-anion/immune complex and the previously treated, positively charged porous matrix and detected by using various signal generating systems previously described.

Also, the methods of the present invention can be adapted for use in systems which utilize microparticle technology including in automated and semi-automated systems wherein the solid phase comprises a microparticle.

Such systems include those described in pending U.S. Patent Application Nos. 425,651 and 425,643, which correspond to published EP Publication Nos. 0 425,633 and 0424,634, respectively, both of which enjoy common ownership and are incorporated herein by reference.

After preparing the recombinant proteins as described herein, these recombinant proteins can be used to develop unique assays as described by the present invention to detect the presence of anti-HCV in test samples. For example, a test sample is contacted with a solid phase to which at least one recombinant HCV protein comprising E2 antigen is attached. The test sample and solid phase are incubated for a time and under conditions sufficient to form antigen-antibody complexes. Following incubation, the antigen-antibody complexes are detected. Indicator reagents may be used to facilitate detection, depending upon the assay system chosen.

In another assay format, a test sample is contacted with a solid phase to which at least one recombinant HCV protein comprising E2 antigen produced as described herein is attached and also is contacted with a monoclonal or polyclonal antibody specific for the HCV protein(s), which preferably has been labeled with an indicator reagent. After incubation for a time and under conditions sufficient to form antigen-antibody complexes, the solid phase is separated from the free phase, and the label is detected in either the solid or free phase as an indication of the presence of anti-HCV.

Other assay formats utilizing the proteins of the present invention are contemplated. These include contacting a test sample with a solid phase to which at least one recombinant HCV protein comprising E2 antigen produced as described herein is attached, incubating the solid phase and test sample for a time and under conditions sufficient to form antigen-antibody complexes, and then contacting the solid phase with a labeled recombinant antigen to form antigen-antibody-antigen sandwiches. Assays such as this and others are described in U.S. Patent No. 5,254,458 which enjoys common ownership and is incorporated herein by reference. While the present invention discloses the preference for the use of solid phases, it is contemplated that the proteins of the present invention can be utilized in non-solid phase assay systems. These assay systems are known to those skilled in the art, and are considered to be within the scope of the present invention.

The present invention will now be described by way of examples, which are meant to illustrate, but not to limit, the spirit and scope of the invention.

MATERIALS AND METHODS

E2 Cloning and Expression The viral source for the envelope gene was second passage

HCV-H strain plasma [Ogata et al., supra, 1991] from a chimpanzee which represents a Type la genotype of HCV [Mishiro & Bradley, in Viral Hepatitis and Liver Disease 283-85 (Nishioka et al. eds., 1994)]. The envelope gene cDNA was isolated by RT-PCR amplification. A truncated E2 sequence was obtained using PCR amplification of the region coding for amino acids 388-664 of the large open reading frame of HCV. E2 complementary DNA (cDNA) was inserted into a plasmid vector downstream of both a rabbit heavy chain signal sequence and a human pro-urokinase amino terminal sequence to enhance signal protease processing, efficient secretion and final product stability in cell culture fluids. The expression system is described more fully in co-pending

U.S. patent application serial no. 08/ , [Atty. Docket No. 5763.US.01], filed concurrently herewith and which is incorporated herein by reference. Briefly, plasmid 577 containing a 2.3 kb fragment of pBR322 including the bacterial gene beta-lactamase and the origin of DNA replication, a 1.8 kb cassette directing expression of a neomycin resistance gene under the control of herpes simplex virus-1 (HSV-1) thymidine kinase promoter and poly-A addition signals, and a 1.9 kb cassette directing expression of a mouse dihydrofolate reductase gene under the control of simian virus 40 (SV40) T-antigen promoter and transcription enhancer and poly-A addition signals (for selection and amplification) was modified to include a 3.5 kb cassette directing expression of a truncated E2 gene under the control of SV40 T-antigen promoter and enhancer, hepatitis B virus surface antigen enhancer I and a fragment of HSV-1 genome containing poly-A addition signals.

The 3.5 kb E2 expression cassette contained a duplex synthetic oligonucleotide that had been digested with Spel and Xbal inserted at the Xbal cloning site of the cassette by sticky end ligation. The synthetic oligonucleotide sequence contained a sequence derived from a rabbit immunoglobulin gamma heavy chain signal peptide and other sequences to create restriction sites for cloning purposes, inserted downstream of a promoter element and transcription start site. The E2 expression cassette also contained the PCR-derived E2 fragment inserted as an Xbal fragment downstream of the rabbit sequence. Immediately after the Xbal site, the sequence encoding the amino terminal sequence of human pro-urokinase~serine, asparagine, glutamic acid and leucine (SNEL)~followed. The pro-urokinase sequence promoted signal protease processing, efficient secretion and product stability in culture fluids. Immediately following the SNEL sequence, the sequence encoding the amino acid sequence of the HCV putative envelope gene from aa 338 to aa 664, a duplicate stop codon and a Xbal site for cloning purposes.

Chinese hamster ovary (CHO) cells lacking dhfr (dhfr-) (Dxb- 111) were transfected with the HCV E2 plasmid and stable cell lines were obtained after several rounds of methotrexate selection. Uriacio, et al., Proc.. Acad. Sci. 77:4451-4466 (1980). These cells are available from the American Type Culture Collection (A.T.C.C), 12301 Parklawn Drive, Rockville, MD 20852, under Accession number CRL 9096. Cell cultures were grown on Ham's F12 custom minus formulation (without glycine, hypoxanthine or thymidine) supplemented with methotrexate hydrate, 5000 nm, G418 at an actual concentration of 300 ug/ml, dialyzed 10% fetal calf serum and HEPES buffer at a concentration of 8 ml per 500 ml of media (for non-CO2 incubation). Ham's F12 Custom Minus Medium was overlayed onto just confluent monolayers for 12-24 hours at 37°C in 5% CO2. Then the growth medium was removed and the cells were rinsed three times with phosphate buffered saline (PBS) (with calcium and magnesium) available from Gibco- BRL, to remove the remaining media/serum which might be present. Cells then were incubated with VAS Custom Medium (VAS Custom formulation with l-glutamine with HEPES without phenol red, available from JRH Bioscience, product number 52-08678P), for 1 hour at 37°C in 50% CO2. As a final wash, the VAS then was discarded. These procedures are detailed in co-pending U.S.

Serial No. (Attorney docket number 5763. US.01), incorporated herein by reference. Media containing secreted E2 was collected off confluent cell monolayers, pooled and stored frozen until purification. The cells typically were harvested at six to seven day intervals. An E2 antigen inhibition assay was used to screen the cells. A value of 100% was given to a HCV positive standard which was diluted to achieve an absorbance value of 1.0 in the assay. A standard curve was generated by spiking into the HCV positive standard known amounts of E2 antigen in the range of 0.4 ug/ml to 50 ug/ml. In the assay, 20 ul of the test sample (i.e., spiked standard or cell supernatant) were incubated with the standard positive control for approximately one hour at 40° C and then incubated with an E2 peptide-coated bead (HCV synthetic peptide comprising amino acids 509-551) for approximately one hour at 40° C. The amount of E2 antigen produced was measured by adding a gamma globulin specific goat anti- human-HRPO conjugate, incubating, adding OPD substrate and quenching the reaction with IN H₂SO₄. The unknown cell culture supernatant was read off the standard curve to determine E2 concentration. It was determined that 6-10 milligrams of CHO-E2 were produced per liter of culture fluid using this method. Expression of E2 was detected by immunofluorescent staining using polyclonal rabbit sera from animals immunized with an HCV synthetic peptide comprising amino acids 509-551 (anti-peptide 509-551 serum). Figure 1 shows the immunofluorescent staining pattern of HCV E2 antigen in transfected CHO cells using (A) rabbit anti-peptide serum (amino acids 509-551); and (B) HCV positive human serum. E2 expression also was detected by radioimmunoprecipitation analysis (RLPA) of lysed cell extracts from ³⁵S-labeled CHO cell cultures. Figure 2 shows the results of a RLPA analysis of HCV E2 antigen produced in CHO cells. Both rabbit and human antisera specific for HCV E2 antigen precipitated a heterogeneous E2 protein. E2 Purification

Purification of E2 was achieved by first concentrating the cell supernatants 50 fold followed by ion exchange and lectin chromatography. The ion exchange chromatography consisted of two columns, S-Sepharose and DEAE-Sepharose. The harvests were clarified at 1500 x g for thirty minutes and the supematants were concentrated to 50x with an Amicon stirred-cell concentrator and employing an Amicon YM10 membrane (available from Amicon, Beverly, MA). The 50x concentrate was 0.2 u final filtered and then extensively dialyzed against S-Sepharose running buffer comprising 0.02M sodium phosphate buffer (no salt), pH 6.5, conductivity approximately 2.0 mS. After dialysis, the supernatant was loaded onto an equilibrated 200 ml S- Sepharose column at a flow rate of 5ml per minute. The unbound flow was collected, concentrated to original volume and extensively dialyzed against DEAE-Sepharose running buffer comprising 0.2M Tris buffer, 0.1M NaCl, pH 8.5, conductivity approximately 12 mS. After dialysis, the supernatant was loaded onto a 200 ml DEAE-Sepharose column at a flow rate of 5 ml per minute. The unbound flow was collected, concentrated to original volume and extensively dialyzed against wheat germ agglutinin (WGA)-Sepharose 6MB running buffer comprising 0.01M sodium phosphate buffer, 0.13M NaCl, pH 7.0. After dialysis, the supernatant was loaded onto a 10 ml WGA-Sepharose 6MB column at a flow rate of 0.5 ml per minute. The unbound flow was collected and recirculated. The column flow was reversed and the purified CHO-E2 antigen was eluted using 10 mM N,N'-diacetylchitobiose in WGA- Sepharose running buffer. Purified antigen was dialyzed against phosphate buffered saline and stored at -70° C. Final purity was assessed using sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE, a procedure well-known to those of ordinary skill.. Figure 3 shows a SDS-PAGE gel of purified HCV E2 antigen produced in CHO cells. The purified E2 antigen ran as a heterogeneous band of approximately 62-72 kDa on SDS-PAGE. A similar heterogeneous band was confirmed to be E2 by RLPA analysis (Figure 2). The final purity was estimated to be greater than 90% using scanning densitometry of SDS-PAGE gels stained with Coomassie blue. During the harvest phase of growth, the CHO cells were grown in protein free media which greatly enhanced the efficiency of purification of this glycoprotein.

Preliminary experiments with endoglycosidase H treatment demonstrated that the E2 protein appeared to be glycosylated as evidenced by the reduction in size from a 62-72 kDa heterogeneous band to a distinct 32 kDa protein band after treatment with endoglycosidase H. Solid Phase Immunoassays

Screening for HCV antibody was carried out using commercially available 2.0 and 3.0 HCV EIAs from Abbott Laboratories (North Chicago, Illinois) and Ortho Diagnostics Inc. (Raritan, New Jersey). All testing was carried out according to the manufacturer's instructions. Supplemental, commercially available, immunoblot assays (RLBA HCV 2.0, Chiron

Corporation, Emeryville, California; MATRIX HCV 1.0 and MATRIX HCV 2.0, Abbott Laboratories, North Chicago, Illinois) were used to establish the specific antibody reactivity patterns. RLBA HCV 2.0 and MATRIX HCV 1.0 assays were performed according to the manufacturer's instructions. MATRIX HCV 2.0 is a second generation MATRIX immunoblot assay which contains an NS5 antigen in addition to core, NS3 and NS4. The assay procedure is the same as that for MATRIX HCV 1.0, as previously described [Vallari et al., Clin. Microbiol. 30: 552-56 (1992)]. Specimens Chronic and acute NANBH specimens were obtained from multiple U.S. sites. Serially collected specimens from individuals seroconverting to HCV antigens were obtained from commercial plasma vendors. Archived samples which were HCV RNA positive (N=495) were obtained from a large virology reference laboratory in the U.S. without linkage to patients or donors. The RNA extraction and PCR amplification procedures have been described [Gretch et al. , J. Clin. Microbiol. 30:2145-49 (1992)] . Additional HCV RNA positive specimens were collected from Japan (N=59) and The Netherlands (N=33) [Zaaijer et al., J. Med. Virol. 44:395-97 (1994)]. Specimens (N=304) from blood donors at risk for HCV infection with ALT values greater than 100 IU/L were provided by Dr. Alfred Prince, New York Blood Center. Samples indeterminately reactive on MATRIX 1.0 for core (N=139) and NS3 (N=149) were obtained from the Abbott Virology Reference Laboratory, North Chicago, Illinois and represented a mix of HCV 2.0 reactive blood donors and patients. A commercially available anti-HCV panel comprised of varying titer of anti-HCV (PHV203), well characterized with regard to HCV serological markers, was obtained from Boston Biomedica, Incorporated (BBI), West Bridgewater, Massachusetts. HCV 2.0 reactive plasma specimens were obtained from North American Biologicals Incorporated (NABI) of which only samples concordantly reactive in both HCV 2.0 EIA's (Abbott and Ortho) were analyzed further.

EXAMPLES

Example 1

Anti-E2 Antigen EIA

Purified HCV E2 antigen, prepared as described above, was coated onto polystyrene beads at a concentration of 1.0-2.0 ug/ml in a 0.1M borate, 0.15M NaCl buffer, pH 9.0. The antigen coating buffer composition was adjusted with respect to pH and ionic strength to provide optimum sensitivity and specificity in the assay.

Briefly, the assay procedure used was as follows. Specimens were diluted with specimen diluent and mixed. All specimens were tested at a 1:41 dilution in the assay. The specimen diluent comprised a phosphate and TRIS- EDTA buffered saline solution containing bovine serum albumin, fetal bovine serum and goat serum with 0.002% Triton X-100^® detergent. The HCV E2 antigen-coated beads were added, one to each well of the tray, and incubated at 37° C for approximately 60 minutes in the rotation mode. The unbound materials were removed by washing the beads with water. Anti-E2 remaining bound to the bead was detected by adding to each well containing a bead 200 ul of conjugate comprising goat anti-human IgG labeled with horseradish peroxidase as the label [Dawson et al., J. Clin. Microbiol. 29:1479-86 (1991)]. The beads were incubated at 37° C for approximately 30 minutes in the rotation mode. The unbound materials were removed by washing the beads with water. Color development was obtained by adding o-Phenylenediamine (OPD) solution containing hydrogen peroxide to the beads, and, after incubation for approximately 30 minutes, a yellow-orange color developed in proportion to the amount of anti-E2 which was bound to the bead. The enzyme reaction was stopped by adding 1 ml of IN H₂SO₄. The intensity of the color was measured using a spectrophotometer at a wavelength of 492 nm. Example 2 Specificity

The specificity of the anti-E2 EIA described in Example 1 was established by testing specimens from several populations of volunteer blood donors totaling 750 serum and plasma specimens. A cutoff value for the E2 antibody assay was established at a signal to negative control absorbance (S/N) ratio of 4.0. This cutoff represented a minimum of six (range of from six to ten) standard deviations from the mean of the absorbance distribution for any of these populations. Example 3 Correlation between Anti-E2 EIA and Prior Art HCV Assays

One hundred fifty-nine patients diagnosed with chronic NANBH were tested using the Abbott HCV 2.0 test and the anti-E2 EIA of Example 1. Of those patients, 147 (92.5%) patients were positive with HCV 2.0, while 141 (88.6%) patients also had antibody to E2. Overall, there was 96.2% agreement between the HCV 2.0 and anti-E2 assays. A high correlation (94%) between HCV core and E2 antibodies also was observed in this population.

A similar high concordance was seen between the HCV 2.0 and anti-E2 assays in acute NANBH patients. One hundred thirteen specimens were tested using the Abbott HCV 2.0 test and the anti-E2 EIA of Example 1. Ninety-nine (87.6%) specimens gave concordant results (51 positive and 48 negative), while 10 specimens reacted exclusively with HCV 2.0 and 4 specimens were positive only in the E2 antibody assay. The overall reactive rates in acute patients for HCV 2.0 and anti-E2 EIA were 54% and 49%, respectively. Example 4 Seroconversion Samples

Serially collected specimens from five individual plasma donors who seroconverted to multiple HCV antigens were tested using the anti-E2 EIA of Example 1. In three of the five patients, anti-E2 was the first antibody detectable during seroconversion. Anti-E2 eventually appeared in all five cases. Table I. Seroconversion to HCV Proteins in Plasma Donors

HCV Antibody Specificity

Core NS3 NS4 NS5 E2

Donor No. (Days)^* (Days)" (Days)" (Days)^* (Days)^*

1 4 4 20 41 0

2 17 17 37 NS 7

3 0 29 71 0 29

4 14 7 12 14 5

5 5 12 NS NS 19

Expressed as days to seroconversion after the first ALT elevation (ALT =50 IU L or greater)

"O" Indicates seroconversion to that marker occurred by the time the ALT value reached 50 IU/L

NS: No seroconversion detected The results of Table I show that antibodies to E2 as detected by the anti-E2 EIA are a good index of HCV exposure by demonstrating that all five individuals who seroconverted to other HCV proteins also seroconverted to E2. E2 antibodies appeared as the first serological marker of HCV infection in three of the five cases indicating that E2 antibodies are produced early after HCV infection in some individuals. Example 5 RNA Positive Specimens

In order to establish how frequently E2 antibodies appear during active HCV infection, 587 HCV RNA positive plasma specimens, identified in the section "Specimens" above, were tested using the anti-E2 EIA of Example 1 as well using the MATRIX HCV 2.0 assay to test for other individual HCV antibodies. As shown in Table π, 571 (97.3%) of these RNA positive specimens were shown to contain antibodies to E2 (Table LL), including 56 (94.9%) of the 59 specimens collected in Japan. All E2 positive samples contained other HCV antibodies as detected by MATRIX but no single antibody occurred with greater frequency than E2 antibody in this population.

Table LL. Correlation of HCV E2 Reactivity with Presence of HCV RNA

No Specimens No E2 Antibody %

Panel Testes No. PCR Positive Positive

1 33 33 29 88

2 59 59 56 95

3 195 195 193 99

4 300 300 293 98

Total 587 587 571 97.3

E2 antibodies were found in 97.3% of these patients demonstrating that the presence of E2 antibody and HCV RNA, as detected by PCR, are very closely and positively correlated. Since 94.9% of 59 HCV patients from Japan (where Genotype lb predominates) were reactive for E2 antibodies, it appears that there must be conserved E2 epitopes among Type la and Type lb viruses. The close correlation between HCV RNA and E2 antibody suggests that the presence of E2 antibody alone is not sufficient for virus clearance and calls into question the role these antibodies may play in virus neutralization. Example 6 HCV Populations Several HCV populations, identified in the section "Specimens" above, were tested in the anti-E2 EIA of Example 1. Among the cohort of New York Blood Center blood donors with ALT values greater than 100 IU/L, 48 (15.8%) were positive for E2 antibodies. Samples reactive with E2 antigen were verified to determine whether the samples reacted with additional HCV serological markers. Forty six (95.8%) of these 48 donors also were reactive in the Abbott HCV 2.0 EIA and were confirmed positive (except for one sample which tested core antigen-reactive only) in the MATRIX HCV 2.0 assay. Thus, the data indicate that a positive E2 antibody test has a high positive predictive value for HCV exposure. Among the specimens interpreted as indeterminate by MATRLX

HCV 1.0, 59 (42.4%) of 139 core reactive only specimens were found to contain E2 antibodies and 23 (15.4%) of 149 NS3 reactive only specimens were found to contain E2 antibodies. The data show that the combined high specificity and high predictive value of the anti-E2 EIA of Example 1 is useful in evaluating indeterminate samples. Example 7 Samples with Discordant Results between HCV 2.0 and HCV 3.0 Assays

BBI Panel PHV203 specimen members were evaluated with the Abbott HCV 3.0 EIA and anti-E2 EIA of Example 1. Testing results from the commercially available assays Abbott HCV 2.0 EIA, Ortho HCV 2.0 EIA, Ortho HCV 3.0 EIA, MATRLX HCV 1.0 (Abbott) and RLBA HCV 2.0 (Chiron) were provided by BBI. Two panel members reported by BBI as HCV negative were also E2 antibody negative.

Eighteen (78.3%) of 23 HCV 2.0 EIA positive (by both Abbott and Ortho assays) specimens tested anti-E2 positive. Among the 23 HCV 2.0 concordantly positive specimens, six samples (26.1%) tested negative in the Ortho HCV 3.0 EIA but remained reactive in the Abbott HCV 3.0 EIA. Table LU sets forth the serological profile for each of these samples. As shown in Table HI, three (50%) of the six specimens (panel members -01, -02 and -10) contained antibodies to multiple HCV proteins, of which two samples (panel members -02 and -10) had antibodies to E2. The two anti-E2 positive samples also were reactive to core in both the RLBA and MATRIX assays.

Thus, the data show that two (33.3%) of six BBI panel members, which were concordantly positive in both manufacturer's HCV 2.0 EIAs but negative in the Ortho 3.0 EIA, contained both core and E2 antibodies.

The anti-E2 EIA of Example 1 was used to help evaluate NABI plasma donor specimens with discordant reactivity between licensed HCV 2.0 EIA tests and HCV 3.0 EIA tests currently sold only outside the United States. In order to remove any pre-selection bias, only those donor specimens •■' (N= 104) repeatedly reactive by both the Abbott and Ortho 2.0 EIAs were tested using HCV 3.0 blood screening tests from each of these companies.

Thirteen (12.5%) of 104 specimens, representing 12 unique donors, were non-reactive in either one or both HCV 3.0 EIAs. A complete analysis of the HCV antibody patterns present in these 13 specimens was done using RLBA 2.0, MATRLX 2.0 and the anti-E2 EIA of Example 1. Table TV sets forth the serological profile for each specimen. Specimen numbers 5 and 8 represent sequential donations from an individual donor.

Table HI. Serological Profile for HCV 2.0/3.0 EIA Discordant BBI Panel Members

HCV Screening EIA's SuoDlemeπtal Testine

Abbott 2.0 Ortho 2.0 Abbott 3.0 Ortho 3.0 Antibodies Detected E2

Panel Member s/co s/co s/co s/co RIBA 2.0 MATRIX 1.0 S/N

PHV203-01 1.4 1.9 1.6 0.6 Core, NS4 Core, NS3, 2.4 NS4

PHV203-02 1.7 2.5 2.7 0.8 Core Core 13. 2

PHV203-03 2.2 1.9 2.6 0.3 NR NS4 1.6

PHV203-10 1.3 2.3 2.7 0.8 Core Core, NS3 21. 6

PHV203-24 1.4 1.6 6.0 0.2 Core Core 3.2

PHV203-25 2.3 >4.4 2.9 0.2 NS4 NS4 1.6

S/CO: Sample to cutoff value, considered positive if = 1.0 or greater

S/N: Sample to negative control absorbance ratio, considered positive if = 4.00 or greater

NR: Nonreactive Table IV. Serological Profiles for Thirteen HCV 2.0 Positive/HCV 3.0 Negative Plasma Donors

Screening Assay

Reactivity

HCV 2.0 HCV 3.0 HCV 3.0 SuϋDlemental Testing

Specimen Abbott/ Abbott Ortho Antibodies Detected E2 EIA

Number Ortho S/CO S/CO RIBA 2.0 MATRIX 2.0 S/N

1 +/+ 2.90 0.70 CORE CORE 5.92

2 +/+ 1.59 0.31 CORE CORE 2.80

3 +/+ 1.96 0.32 CORE CORE 2.40

4 +/+ 1.99 0.79 CORE CORE, NS4Y^* 10.12

5 +/+ 2.72 0.52 CORE CORE 4.92

6 +/+ 2.24 0.68 CORE CORE, NS3 2.40

7 +/+ 3.55 0.08 CORE NS4Y^* 0.80

8 +/+ 3.01 0.47 CORE CORE 5.52

9 +/+ 1.97 0.96 CORE CORE, NS5 0.60

10 +/+ 2.09 0.04 NS4 NS4Y' 1.00

11 +/+ 1.51 0.15 NS4 NR 1.20

12 +/+ 0.65 0.05 NS4 NS4, NS5 1.40

13 +/+ 0.27 0.05 NS4 NS4Y^* 1.20

S/CO: Sample to cutoff value, considered positive if = 1.00 or greater " * Denotes reactivity to the clOO (NS4) antigen expressed in yeast. Reactivity to both the yeast and

E.coli clOO antigens must occur in order to be considered reactive to NS4 on MATRIX 2.0 NR: Nonreactive S/N: Sample to negative control absorbance ratio, considered positive if = 4.00 or greater

As shown in Table IV, specimen numbers 5 and 8 which represent sequential donations from an individual donor are core and E2 antibody positive demonstrating the reproducibility and specificity of these assays. Four specimens which reacted to core antigen on both RLBA 2.0 and MATRLX 2.0 also contained antibody to E2. These four specimens were reactive in the Abbott HCV 3.0 assay (S/CO values 1.99-3.01) but were negative in the Ortho HCV 3.0 assay (S/CO values 0.47-0.79).

Three other specimens had antibodies to two distinct HCV proteins other than E2 on MATRIX (core/NS3, core/NS5 and NS4/NS5). Of these specimens, the two core reactive specimens were positive in the Abbott HCV 3.0 EIA (S/CO values 2.24 and 1.97) and negative or borderline negative in the Ortho 3.0 EIA (S/CO values 0.68 and 0.96). The other specimen which contained antibodies to NS4 and NS5 was negative in both the Abbott and Ortho HCV 3.0 EIA. The data show that four (30.7%) of 13 samples which were concordantly positive in both manufacturer's HCV 2.0 ELAs but negative in the Ortho 3.0 EIA, contained antibodies to both core and E2.

Claims

WHAT IS CLAIMED IS:

1. In a method for detecting the presence of antibodies to HCV antigen which may be present in a test sample comprising contacting said sample with HCV antigen and determining whether antibodies are bound to said HCV

^" antigen, wherein the improvement comprises employing as said HCV antigen at least one recombinant HCV protein comprising a recombinant polypeptide that is the expression product of mammalian cells transformed by a heterologous expression vector comprising a DNA sequence encoding an E2 truncated protein, a DNA sequence encoding a rabbit heavy chain signal sequence and a DNA sequence encoding an ammo-terminal sequence of human pro-urokinase, wherein said HCV antigen DNA sequence is located downstream to said other DNA sequences.

2. The method of claim 1, wherein said DNA sequence encoding an E2 truncated protein comprises amino acids 388-664 of the large open reading frame of HCV.

3. The method of claim 1, wherein said HCV antigen comprises an E2 truncated protein and at least one other HCV antigen selected from the group consisting of core, NS3, NS4 and NS5.

4. The method of claim 1, wherein said contacting step comprises incubating said test sample with a solid phase to which at least one recombinant HCV protein comprising E2 antigen is attached for a time and under conditions sufficient to form antigen-antibody complexes.

5. The method of claim 4, wherein said determining step comprises contacting the solid phase with a member of a specific binding pair.

6. The method of claim 5, wherein said specific binding member is an antibody.

7. The method of claim 6, wherein said antibody is anti-human IgG.

8. The method of claim 5, wherein said specific binding member is a recombinant antigen.

9. The method of claim 5, wherein said specific binding member is conjugated to a signal generating compound.

10. The method of claim 9, wherein said signal generating compound is selected from the group consisting of chromogens, enzymes, luminescent compounds, chemiluminescent compounds, radioactive elements and direct visual labels.

11. The method of claim 1, wherein said contacting step comprises incubating said test sample with a solid phase to which at least one recombinant HCV protein comprising E2 antigen is attached and an antibody specific for said at least one recombinant protein for a time and under conditions sufficient to form antigen- antibody complexes.

12. The method of claim 11, wherein said deterniining step comprises contacting the solid phase or free phase with a member of a specific binding pair.

13. The method of claim 12, wherein the specific binding member is an antibody.

14. The method of claim 13, wherein the specific binding member is anti-human IgG.

15. The method of claim 12, wherein said specific binding member is conjugated to a signal generating compound.

16. The method of claim 15, wherein said signal generating compound is selected from the group consisting of chromogens, enzymes, luminescent compounds, chemiluminescent compounds, radioactive elements and direct visual labels.

17. A test kit for detecting the presence of antibodies to HCV antigen which may be present in a test sample comprising a container containing a recombinant polypeptide that is the expression product of mammalian cells transformed by a heterologous expression vector comprising a DNA sequence encoding an E2 truncated protein, a DNA sequence encoding a rabbit heavy chain signal sequence and a DNA sequence encoding an ammo-teπninal sequence of human pro-urokinase, wherein said HCV antigen DNA sequence is located downstream to said other DNA sequences.