AU5503400A - Diagnosis of, and vaccination against, a positive stranded RNA virus using an isolated, unprocessed polypeptide - Google Patents

Description

Document-31/08/00 -1-

AUSTRALIA

Patents Act 1990 COMPLETE SPECIFICATION FOR A DIVISIONAL PATENT

(ORIGINAL)

Regulation 3.2 o Name of Applicant: Actual Inventors: Address for Service: BioNova Corporation Jaw-Ching LIAO and Cheng-nan WANG DAVIES COLLISON CAVE, Patent Attorneys, Level 3, 303 Coronation Drive, Milton, Queensland, 4064, Australia Invention Title: "Diagnosis of, and vaccination against, a positive stranded RNA virus using an isolated, unprocessed polypeptide" Details of Parent Application No: 59575/96 The following statement is a full description of this invention, including the best method of performing it known to us: DIAGNOSIS OF. AND VACCINATION AGAINST. A POSITIVE STRANDED RNA VIRUS USING AN ISOLATED, UNPROCESSED

POLYPEPTIDE

Technical Field The present invention relates generally to methods and compositions for the highly specifi, highly sensitive diagnosis of a positive-stranded RNA virus. The methods and compositions are also suitable for the elicitation of an immune response in an animal, and for the vaccination of an animal, against a positive-stranded RNA virus.

Background of the Invention Acquired immune deficiency syndrome (AIDS) is caused by a group of retroviruses known as HIV (Barre-Sinoussi et al., Science 220:868-871, 1983; Gallo et al., Science 224:500-503, 1984; Coffin et al., Science 232:697, 1986). The first member "9 of the group has been designated HIV-1 and is responsible for a majority of cases of AIDS worldwide. It is distinguished from HIV-2, an isolate discovered from WAf (Clavel et al., Science 233:343-346, 1986). Although HIV-2, like HIV-1, produces symptoms of immune deficiency in man, it is also genetically distinct from HIV (Guyader et al., Nature 326:662-669, 1987).

The genomes of the HIV isolates, like those of other retroviruses, include three basic genes: gag, pol and env (Weiss et al., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1985). In addition, the genomes contain several other genes whose products play important roles in the regulation of viral gene expression (Dayton et al., Cell 44:941-947, 1986; Fisher et al., Nature 320:367-371, 1986; Sodroski et al., Nature 321:412-417, 1986).

HIV-1 is typically transmitted by sexual contact, by exposure to blood or certain blood products, or by an infected mother to her fetus or child (Piot et al., Science 239:573-579, 1988). The first examples of transfusion-associated HIV-2 infection have been disclosed (Courouce et al., AIDS 2:261-265, 1988). Therefore, the demand for sensitive and specific methods for detecting HIV in contaminated blood or blood products is significant.

EIAs, based on whole virus or viral lysate, have been developed for the detection of HV. However, it has been found that the EIAs have unacceptable, nonspecific reaction with specimens from individuals with non-HIV conditions such as autoimmune diseases, a history of multiple pregnancies, anti-HLA, EBV infections or hypergammaglobulinemia.

In order to avoid such non-specific reactions and in an attempt to detect anti-HIV-1 and/or anti-HIV-2 in samples, an ELISA has been developed and commercialized by Abbott Laboratories for serological diagnosis of HIV infection using the HIV-I core and HIV-1 envelope and HIV-2 envelope proteins. However, this ELISA has not provided the highly specific, highly sensitive detection needed for superior protection of the blood supply, or for early diagnosis of HIV in a patient.

10 Thus, in order to provide superior protection of the blood supply, and in order to provide superior diagnosis of HIV in a patient, there has gone unmet a need for products and methods capable of highly specific, highly sensitive detection of HIV.

There has also gone unmet a need for products and methods capable of eliciting an immune response to HIV, especially an immunoprotective immune response to HIV.

The present invention provides these and other related advantages.

In addition to the problems associated with HIV, other positive-stranded RNA viruses also pose significant health risks throughout the world. One example of such a positive-stranded RNA virus is the Hepatitis C virus (HCV). HCV is distinguishable from other forms of viral-associated liver diseases caused by known hepatitis viruses such as hepatitis A virus (HAV) and hepatitis B virus (HBV). Like HIV, HCV is often transferred via blood transfusion; post-transfusion hepatitis (PTH) occurs in approximately 10% of transfusion patients, and HCV Non-A, Non-B hepatitis (NANBH)) accounts for up to 90% of these cases. A major problem arising from this disease is the frequent progression to chronic liver damage (25-55%).

Therefore, the demand for sensitive, specific methods for detecting HCV in contaminated blood or blood products is significant.

The hepatitis C virus (HCV) was first identified by molecular cloning and characterization of its RNA genome by Choo et al. (Science 244:359-362, 1989). A specific assay using an HCV antigen designated C100-3 was then created, using recombinant DNA methods in yeast. The assay detects an antibody against HCV (Science 244:362-364). A detailed disclosure of the genome of HCV, and some cDNA sequences and polypeptides derived therefrom, as well as methodologies relating to such subject matter, is provided in EP 0 318 216 Al in the name of Chiron Corporation. In particular, this disclosure provides a synthesized polypeptide, C100-3, containing 363 virally-encoded amino acids that can be used for the detection of one type of HCV antibody. Presently, kits for detecting HCV antibodies on the basis of the C100-3 antigen have been commercialized by Abbott Laboratories.

As suggested in EP 0 318 216 Al, HCV may be a flavivirus or flavi-like virus. With respect to general morphology, a flavivirus contains a central nucleocapsid surrounded by a lipid bilayer. It is believed that hepatitis C virus protein is composed of structural proteins including a nucleocapsid (core) protein two glycosylated envelope proteins (El, E2) and several nonstructural proteins (NS1-5). It has been confirmed that C100-3 disclosed by Choo et al. is a protein encoded by part of nonstructural regions 3-4 of the HCV genome. It has been found that anti-C100-3 antibody is not detected in all post-transfusion NANBH cases. The failure to detect the anti-C100-3 antibody is possibly due to hypermutation of the nucleotide sequence in C100-3 region.

In addition to the work with the nonstructural C100-3 antigen, an enzyme-linked immunosorbent assay (ELISA) has been developed for serological diagnosis of hepatitis C virus (HCV) infection using the HCV core protein (p22). The core protein was synthesized by a recombinant baculovirus, as reported in Chiba et al.

(Proc. Natl. Acad Sci. USA 88:4641-4645, 1991). Thus, the assay of Chiba, et al. used a nonglycosylated 22-kDa nucleocapsid (core) protein, in an effort to establish an antibody-based, specific, sensitive method for diagnosing HCV infection. However, this core protein-based assay failed to detect a significant number of cases of HCV infection, even when relatively large sample volumes were available.

Thus, as with other positive-stranded RNA viruses, there has gone unmet a need for products and methods capable of highly specific, highly sensitive detection of :HCV. There has also gone unmet, as with other positive-stranded RNA viruses, a need for products and methods capable of eliciting an immune response to HCV, especially an immunoprotective immune response to HCV. The present invention provides these and other related advantages.

Summary of the Invention The present invention is directed toward the concept that unprocessed entire polypeptide(s) a polyprotein) or unprocessed partial polypeptide(s) in the structural region and proteins from the non-structural region of positive-stranded stranded) RNA viruses can provide a superior antigenicity and therefore an improved detection and diagnosis of a positive-stranded RNA virus in a sample. The present invention also provides improved immunoactivation, including an improved immunoprotective response from an animal.

Accordingly, in a first aspect the present invention provides positive- 10 stranded RNA virus-derived compositions comprising an isolated, substantially complete, unprocessed polyprotein from a positive-stranded RNA virus. In alternative aspect, the present invention provides positive-stranded RNA virus-derived compositions comprising the following: a) an isolated polypeptide comprising a 5" positive-stranded RNA virus core-like antigen protein joined to an amino-terminal portion of an adjacent nucleic acid region of the positive-stranded RNA virus, wherein -the amino-terminal portion of the adjacent nucleic acid region is sized such that the .:Spolypeptide has an epitopic configuration specific to an unprocessed core-like-adjacent nucleic acid region of the positive-stranded RNA virus (this polypeptide is sometimes Sreferred to herein as a "core-like antigen-adjacent protein"); and b) an isolated 20 nonstructural protein of the positive-stranded RNA virus. As discussed further below, the full disclosure of this application relating to the core-like antigen-adjacent protein generally applies equally to a positive stranded RNA virus env protein, which env invention, the positive-stranded RNA virus is selected from the group consisting of Togaviridae, Coronaviridae, Retroviridae, Picornaviridae, Caliciviridae and Flaviviridae, further preferably from the group consisting of Hepatitis C virus, the Human Immunodeficiency virus (HIV) and the Human T-cell Leukemia virus (HTLV). Unless otherwise specifically stated, all preferred embodiments relate to each of the aspects of the present invention. Alternatively, the positive-stranded RNA virus is any positivestranded RNA virus other than HCV. In other preferred embodiments, the composition is produced by a suitable prokaryotic host cell, typically a bacterium, and preferably an coli BL21 (DE3). Alternatively, the isolated polypeptide is produced by a suitable eukaryotic host cell that is unable to process the isolated polypeptide.

In another aspect, the present invention provides a method of making a composition comprising an isolated, substantially complete, unprocessed polyprotein from a positive-stranded RNA virus. This aspect also provides a method of making multiple polypeptides obtained from a positive-stranded RNA virus, comprising the following steps: a) introducing into a first suitable host cell a first expression vector capable of expressing a nucleic acid molecule encoding an isolated polypeptide comprising a positive-stranded RNA virus core-like antigen protein joined to an aminoterminal portion of an adjacent nucleic acid region of the positive-stranded RNA virus, wherein the amino-terminal portion of the adjacent nucleic acid region is sized such that the polypeptide has an epitopic configuration specific to an unprocessed core-likeadjacent nucleic acid region of the positive-stranded RNA virus, b)incubating the first host cell under conditions suitable for the expression vector to produce the polypeptide, c) purifying the polypeptide to provide a purified polypeptide, and d) introducing into a second suitable host cell a second expression vector capable of expressing a nucleic acid molecule encoding an isolated nonstructural protein of the positive-stranded RNA virus, S*e) incubating the second host cell under conditions suitable for the nucleic acid molecule to produce the nonstructural protein, f) purifying the nonstructural protein to provide an purified nonstructural protein, and then g) combining the purified polypeptide and the purified nonstructural protein in the composition.

In a preferred embodiment, the method further comprises a) introducing into a suitable host cell an expression vector capable of expressing a first nucleic acid molecule encoding an isolated polypeptide comprising a positive-stranded RNA virus core-like antigen protein joined to an amino-terminal portion of an adjacent nucleic acid region of the positive-stranded RNA virus, wherein the amino-terminal portion of the adjacent nucleic acid region is sized such that the polypeptide has an epitopic configuration specific to an unprocessed core-like-adjacent nucleic acid region of the positive-stranded RNA virus, b) incubating the host cell under conditions suitable for the expression vector to produce the polypeptide and the nonstructural protein, and c) purifying the polypeptide and the nonstructural protein to provide a purified polypeptide and a purified nonstructural protein. In another preferred embodiment, the method further comprises binding the inventive polypeptide(s) to a solid substrate.

In a further aspect, the present invention provides a composition comprising the isolated, substantially complete, unprocessed polyprotein from a positive-stranded RNA virus wherein the polyprotein is bound to a solid substrate.

Alternatively, the composition comprises the core-like antigen-adjacent protein bound to a solid substrate, preferably further comprising a nonstructural protein of the positivestranded RNA virus bound to the solid substrate.

In another preferred embodiment, an assay for the detection of a positivestranded RNA virus in a sample, comprising: a) providing an isolated polypeptide comprising a positive-stranded RNA virus core-like antigen-adjacent protein, b) contacting the isolated polypeptide with the sample under conditions suitable and for a time sufficient for the polypeptide to bind to one or more antibodies specific for the positive-stranded RNA virus present in the sample, to provide an antibody-bound polypeptide, and c) detecting the antibody-bound polypeptide, and therefrom determining that the sample contains positive-stranded RNA virus. In an alternative embodiment, the method comprises, a) providing an isolated polypeptide comprising an isolated, substantially complete, unprocessed polyprotein from a positive-stranded

RNA

virus, b) contacting the isolated polypeptide with the sample under conditions suitable and for a time sufficient for the polypeptide to bind to one or more antibodies specific 10 for the positive-stranded RNA virus present in the sample, to provide an antibody-bound Spolypeptide, and c) detecting the antibody-bound polypeptide, and therefrom determining that the sample contains positive-stranded RNA virus.

In a preferred embodiment, the method further comprises a) in step a), providing a nonstructural protein of the positive-stranded RNA virus bound to the solid substrate, b) in step contacting the nonstructural protein with the sample under conditions suitable and for a time sufficient for the nonstructural protein to bind to one or more antibodies specific for the positive-stranded RNA virus present in the sample, to provide an antibody-bound positive-stranded RNA virus nonstructural protein, and c) in S. step detecting one or both of the antibody-bound polypeptide or the antibody-bound 20 nonstructural protein, and therefrom determining that the sample contains positivestranded RNA virus.

In another preferred embodiment, the assay further comprises the step of binding the isolated polypeptide, the nonstructural protein, or the polyprotein to a solid substrate. In another preferred embodiment, the sample is an unpurified sample, typically from an animal, and preferably from a human being. In yet other preferred embodiments, the assay is selected from the group consisting of a countercurrent immuno-electrophoresis (CIEP) assay, a radioimmunoassay, a radioimmunoprecipitation, an enzyme-linked immuno-sorbent assay (ELISA), a dot blot assay, an inhibition or competition assay, a sandwich assay, an immunostick (dip-stick) assays, a simultaneous assay, an immunochromatographic assay, an immunofiltration assay, a latex bead agglutination assay, an immunofluorescent assay, a biosensor assay, and a low-light detection assay. Still further, the assay is preferably not a western blot assay.

In still a further aspect, the present invention provides a method of producing an antibody, comprising the following steps: a) administering to an animal an isolated polypeptide comprising a positive-stranded RNA virus core-like antigen protein joined to an amino-terminal portion of an adjacent nucleic acid region of the positivestranded RNA virus, wherein the amino-terminal portion of the adjacent nucleic acid region is sized such that the polypeptide has an epitopic configuration specific to an unprocessed core-like-adjacent nucleic acid region of the positive-stranded RNA virus, and b) isolating the antibodies to the polypeptide. Alternatively, the invention provides a method of producing an antibody, comprising the following steps: a) administering to an animal an isolated polypeptide comprising an isolated, substantially complete, unprocessed polyprotein from a positive-stranded RNA virus, and b) isolating the antibodies to the polyprotein.

The present invention also provides the antibody produced according to either of the above methods, as well as an antibody to the other proteins disclosed herein (such as the nonstructural proteins). Preferably, the antibodies are bound to a solid substrate.

So a In yet another aspect, the present invention provides an assay for the detection of a positive-stranded RNA virus in a sample, comprising: a) contacting the sample with one or more of the antibodies described above under conditions suitable and for a time sufficient for the given antibody to bind its antigen protein, to provide a bound antibody, and b) detecting the bound antibody, and therefrom determining that the sample contains positive-stranded RNA virus.

In a preferred embodiment, the sample is an unpurified sample, typically from an animal, and preferably from a human being. In yet other preferred 0 embodiments, the assay is selected from the group consisting of a countercurrent immuno-electrophoresis (CIEP) assay, a radioimmunoassay, a radioimmunoprecipitation, an enzyme-linked immuno-sorbent assay (ELISA), a dot blot assay, an inhibition or competition assay, a sandwich assay, an immunostick (dip-stick) assays, a simultaneous assay, an immunochromatographic assay, an immunofiltration assay, a latex bead agglutination assay, an immunofluorescent assay, a biosensor assay, and a low-light detection assay. Still further, the assay is preferably not a western blot assay.

In yet a further aspect, the present invention provides a composition capable of eliciting an immune response in an animal comprising an isolated polypeptide comprising a positive-stranded RNA virus core-like antigen-adjacent protein, in combination with a pharmaceutically c acore er antign-adjacent ratein, in combination with a pharmaceutically acceptable carrier or diluent. Preferably, the composition further comprises a nonstructural protein from the positive-stranded

RNA

virus. In an alternative aspect, the composition capable of eliciting an immune response in an animal comprises an isolated, substantially complete, unprocessed polyprotein from a positive-stranded RNA virus, in combination with a pharmaceutically acceptable carrier or diluent.

Preferably, for each of the immune-active aspects (as well as the other aspects) of the invention, the animal is a human being.

In still yet a further aspect, the present invention provides a vaccine against a positive-stranded RNA virus comprising an isolated polypeptide comprising a positive-stranded. RNA virus core-like antigen-adjacent protein, in combination with a pharmaceutically acceptable carrier or diluent. Preferably, the composition fu~rther comprises a nonstructural protein from the positive-stranded RNA virus. In an alternative aspect, the vaccine against a positive-stranded RNA virus comprises an isolated, substantially complete, unprocessed polyprotein from a positive-stranded

RNA

virus, in combination with a pharmaceutically acceptable carrier or diluent.

The present invention also provides a method of inducing an immune response in an animal comprising administering to the animal an isolated polypeptide comprising a positive-stranded RNA virus core-like antigen-adjacent protein, in combination with a pharmaceutically acceptable carrier or diluent. Preferably, the method further comprises administering a nonstructural protein from the positivestranded RNA virus. In an alternative aspect, the method of inducing an immune p. *15 response in an animal comprises administering to the animal an isolated, substantially complete, unprocessed polyprotein from a positive-stranded RNA virus, in combination with a pharmaceutically acceptable carrier or diluent.

The present invention further provides a method of vaccinating an animal a a comprising administering to the animal an isolated polypeptide comprising a positive- 20 stranded RNA virus core-like antigen-adjacent protein, in combination with a pharmaceutically acceptable carrier or diluent. Preferably, the method firther comprises p administering a nonstructural protein from the positive-stranded RNA virus. In an a: 94 alternative aspect, the method of vaccinating an animal comprises administering to the animal an isolated, substantially complete, unprocessed polyprotein from a positivestranded RNA virus, in combination with a pharmaceutically acceptable carrier or diluent.

In yet another aspect, the present invention provides a kit for the detection of a positive-stranded RNA virus, the kit comprising a) an isolated polypeptide comprising a positive-stranded RNA virus core-like antigen protein joined to an amninoterminal portion of an adjacent nucleic acid region of the positive-stranded RNA virus, wherein the amino-terminal portion of the adjacent nucleic acid region is sized such that the polypeptide has an epitopic configuration specific to an unprocessed core-likeadjacent nucleic acid region of the positive-stranded RNA virus, bound to a solid substrate, and b) means for detecting the isolated polypeptide. Preferably, the kit comprises a nonstructural protein from the positive-stranded RNA virus and means for detecting the nonstructural protein. Alternatively, the kit for the detection of a positivestranded RNA virus comprises a) an isolated, substantially complete, unprocessed polyprotein from a positive-stranded RNA virus, bound to a solid substrate, and b) means for detecting the isolated polyprotein.

In an alternative aspect, the present invention provides a kit for the detection of a positive-stranded RNA virus comprising: a) one or more of the antibodies discussed above, and b) means for detecting the antibody(s).

The kits may also comprise a)the composition capable of eliciting an immune response, or the vaccine, and b) means for administering the composition or vaccine to the animal.

Turning to another aspect, the present invention provides a positive- 10 stranded RNA virus-derived composition comprising the following: a) an isolated polypeptide comprising a positive-stranded RNA virus core-like antigen protein joined to an adjacent nucleic acid region of the positive-stranded RNA virus, wherein the adjacent nucleic acid region is sized such that the polypeptide has an epitopic configuration specific to an unprocessed core-like-adjacent nucleic acid region of the positive-stranded RNA virus; and b) a second protein capable of cooperatively interacting with the positive-stranded RNA virus core-like antigen protein joined to the adjacent nucleic acid region of the positive-stranded RNA virus to increase the antigenicity of the positive-stranded RNA virus core-like antigen protein joined to the adjacent nucleic acid region of the positive-stranded RNA virus. The present invention 20 also provides a method of making such composition comprising multiple polypeptides, including one or both of the polypeptide described above; the proteins may be derived from the same or different positive-stranded RNA viruses.

The present invention also provides a composition comprising a first isolated protein from the positive-stranded RNA virus and a second isolated protein from the positive-stranded RNA virus (preferably from the same positive-stranded

RNA

virus), wherein the first and second proteins are selected, in accordance with methods set forth below for other embodiments of the claimed invention, such that the first and second proteins provide a synergistic effect for the detection of the positive-stranded RNA virus and/or immunoenhancement of an animal against the positive-stranded

RNA

virus.

The invention also provides the isolated polypeptide comprising a positive-stranded RNA virus core-like antigen protein joined to an adjacent nucleic acid region of the positive-stranded RNA virus, wherein the adjacent nucleic acid region is sized such that the polypeptide has an epitopic configuration specific to an unprocessed core-like-adjacent nucleic acid region of the positive-stranded RNA virus, bound to a solid substrate, either alone or in combination with a second protein capable of cooperatively interacting with the positive-stranded RNA virus core-like antigen protein joined to the adjacent nucleic acid region of the positive-stranded RNA virus to increase the antigenicity of the positive-stranded RNA virus core-like antigen protein joined to the adjacent nucleic acid region of the positive-stranded RNA virus of the positivestranded RNA virus bound to the solid substrate.

In yet another aspect, the present invention provides an assay for the detection of a positive-stranded RNA virus in a sample, comprising: a) providing an isolated polypeptide comprising a positive-stranded RNA virus core-like antigen protein joined to adjacent nucleic acid region of the positive-stranded RNA virus, wherein the adjacent nucleic acid region is sized such that the polypeptide has an epitopic configuration specific to an unprocessed core-like-adjacent nucleic acid region of the positive-stranded RNA virus, b) contacting the isolated polypeptide with the sample under conditions suitable and for a time sufficient for the polypeptide to bind to one or S more antibodies specific for the positive-stranded RNA virus present in the sample, to provide an antibody-bound polypeptide, and c) detecting the antibody-bound 15 polypeptide, and therefrom determining that the sample contains positive-stranded

RNA

virus. The assay may also comprise, a) in step providing a a second protein capable of cooperatively interacting with the positive-stranded RNA virus core-like antigen protein joined to the adjacent nucleic acid region of the positive-stranded RNA virus to increase the antigenicity of the positive-stranded RNA virus core-like antigen protein 20 joined to the adjacent nucleic acid region of the positive-stranded RNA virus, bound to the solid substrate, b) in step contacting the second protein with the sample under conditions suitable and for a time sufficient for the second protein to cooperatively interact with the positive-stranded RNA virus core-like antigen protein joined to the adjacent nucleic acid region of the positive-stranded RNA virus, and c) in step c), detecting bound antibodies, and therefrom determining that the sample contains positivestranded RNA virus.

In a preferred embodiment, the assay further comprises the step of binding the isolated polypeptide or the second protein to a solid substrate. Further preferably, the assay is selected from the group consisting of a countercurrent immunoelectrophoresis (CIEP) assay, a radioimmunoassay, a radioimmunoprecipitation, an enzyme-linked immuno-sorbent assay (ELISA), a dot blot assay, an inhibition or competition assay, a sandwich assay, an immunostick (dip-stick) assays, a simultaneous assay, an immunochromatographic assay, an immunofiltration assay, a latex bead agglutination assay, an immunofluorescent assay, a biosensor assay, and a low-light detection assay, but is not a western blot assay.

The present invention also provides a method of producing an antibody, comprising a) administering to an animal an isolated polypeptide comprising a positive- 11 stranded RNA virus core-like antigen protein joined to an adjacent nucleic acid region of the positive-stranded RNA virus, wherein the adjacent nucleic acid region is sized such that the polypeptide has an epitopic configuration specific to an unprocessed core-likeadjacent nucleic acid region of the positive-stranded RNA virus, and b) isolating the antibodies to the polypeptide. The method may further comprise administering to the animal a second protein capable of cooperatively interacting with the positive-stranded RNA virus core-like antigen protein joined to the adjacent nucleic acid region of the positive-stranded RNA virus to increase the antigenicity of the positive-stranded

RNA

virus core-like antigen protein joined to the adjacent nucleic acid region of the positivestranded RNA virus. The present invention features an antibody produced as above, which antibodies may be bound to a solid substrate. The antibodies may also be used in assays, also as described above.

In yet another aspect, the present invention provides a composition capable of eliciting an immune response in an animal comprising an isolated polypeptide 15 comprising a positive-stranded RNA virus core-like antigen protein joined to an adjacent nucleic acid region of the positive-stranded RNA virus, wherein the adjacent nucleic acid region is sized such that the polypeptide has an epitopic configuration specific to an unprocessed core-like-adjacent nucleic acid region of the positive-stranded RNA virus, in combination with a pharmaceutically acceptable carrier or diluent. The composition S. 20 may further comprise a second protein capable of cooperatively interacting with the positive-stranded RNA virus core-like antigen protein joined to the adjacent nucleic acid region of the positive-stranded RNA virus to increase the antigenicity of the positivei stranded RNA virus core-like antigen protein joined to the adjacent nucleic acid region S* of the positive-stranded RNA virus. Preferably, the composition is a vaccine.

The present invention also provides a method of inducing an immune response in an animal comprising administering to the animal an isolated polypeptide comprising a positive-stranded RNA virus core-like antigen protein joined to an adjacent nucleic acid region of the positive-stranded RNA virus, wherein the adjacent nucleic acid region is sized such that the polypeptide has an epitopic configuration specific to an unprocessed core-like-adjacent nucleic acid region of the positive-stranded RNA virus, in combination with a pharmaceutically acceptable carrier or diluent. Preferably, the method further comprises administering a second protein capable of cooperatively interacting with the positive-stranded RNA virus core-like antigen protein joined to the adjacent nucleic acid region of the positive-stranded RNA virus to increase the antigenicity of the positive-stranded RNA virus core-like antigen protein joined to the adjacent nucleic acid region of the positive-stranded RNA virus. Further preferably, the method comprises a vaccination.

In another aspect, the present invention provides a kit for the detection of a positive-stranded RNA virus comprising: a) an isolated polypeptide comprising a positive-stranded RNA virus core-like antigen protein joined to an adjacent nucleic acid region of the positive-stranded RNA virus, wherein the adjacent nucleic acid region is sized such that the polypeptide has an epitopic configuration specific to an unprocessed core-like-adjacent nucleic acid region of the positive-stranded RNA virus, bound to a solid substrate, and b) means for detecting the isolated polypeptide. Preferably, the kit further comprises a second protein capable of cooperatively interacting with the positive-stranded RNA virus core-like antigen protein joined to the adjacent nucleic acid region of the positive-stranded RNA virus to increase the antigenicity of the positivestranded RNA virus core-like antigen protein joined to the adjacent nucleic acid region Sof the positive-stranded RNA virus and means for detecting the second protein.

Alternatively, the kit for the detection of a positive-stranded RNA virus may comprise: a) an antibody produced as described above, and b) means for detecting the antibody.

These and other aspects of the present invention will become evident upon reference to the following detailed description and attached drawings. In addition, as noted above, various references are set forth throughout the present specification that describe in more detail certain procedures or compositions plasmids, etc.); such 20 references are incorporated by reference in their entirety.

Brief Description of the Drawings Fig. 1A depicts the nucleotide sequence of a nucleic acid molecule encoding a polypeptide comprising an HCV core antigen protein joined to an aminoterminal portion of an HCV envelope region.

Fig. 1B depicts the amino acid sequence encoded by the nucleotide sequence depicted in Fig. 1A.

Fig. 2 shows the structure of the expression vector pEN-2, which was constructed by inserting a cDNA encoding an HCV core antigen protein joined to an amino-terminal portion of an HCV envelope region into a plasmid. The figure also shows a restriction map illustrating certain significant features of the vector pEN-2.

Fig. 3A depicts the nucleotide sequence of a nucleic acid molecule encoding a polypeptide comprising an NS5 nonstructural region.

Fig. 3B depicts the amino acid sequence encoded by the nucleotide sequence depicted in Fig. 3A.

13 Fig. 4 shows the structure of the expression vector pEN-1, which was constructed by inserting a cDNA encoding an NS5 nonstructural region into a plasmid.

The figure also shows a restriction map illustrating certain significant features of the vector pEN- 1.

Detailed Description of the Invention The present invention is based on the discovery that the unprocessed polyprotein initially translated from the genome of a positive-stranded RNA virus contains epitopic configurations that are not retained in the processed proteins. In particular, the core protein region (or other protein encoded by the viral genome that serves the equivalent purpose as the "core" protein) loses an epitopic configuration upon processing at the cleavage site between the genomic region gene) encoding the core protein and the genomic region encoding the protein adjacent the amino-terminal end of the core protein, such as the envelope protein in HCV. As discussed below in the 15 Examples portion of the present disclosure, the unprocessed epitopic configuration of the core region provides a surprisingly improved ability to detect the presence of the 9"positive-stranded RNA virus, or antibodies to the positive-stranded RNA virus, in a 9999 sample, including an unpurified sample or a sample of very small volume (which can be particularly helpful when testing a sample from an infant or other person having very little blood (or other suitable material) available for testing).

Even more surprising, combining the unprocessed core region with a non-structural protein (such as an NS5 protein or an unprocessed NS3-NS4 fusion protein from HCV) results in a synergistic effect that greatly enhances the already .e improved sensitivity and specificity provided by the unprocessed core region.

These significant advantages in antigenicity and epitopic configuration also provide surprisingly enhanced compositions and methods for the induction of immune responses in an animal, as well as enhanced vaccination of such an animal.

Accordingly, the present invention features compositions and methods utilizing an isolated, substantially complete, unprocessed polyprotein from a positivestranded RNA virus.

The present invention also features compositions and methods utilizing an isolated polypeptide comprising the positive-stranded RNA virus core antigen protein joined to an amino-terminal portion of the adjacent protein of the positive-stranded RNA virus, wherein the amino-terminal portion of the positive-stranded RNA virus envelope region is sized such that the polypeptide has an epitopic configuration specific to an unprocessed core-adjacent protein region of the positive-stranded RNA virus. The present invention additionally features the combination of such an unprocessed coreadjacent protein region in a composition or method with a nonstructural protein, thereby providing surprisingly sensitive and specific interactions with the given positive-stranded The present invention provides the first discovery that the full polyprotein does have unique configurations, and that such configurations result in antigenically important differences. The present invention also provides the first discovery that a lost epitopic configuration occurs in the core protein-adjacent protein region.

An "isolated, substantially complete, unprocessed polyprotein" from a positive-stranded RNA virus is the polyprotein that is initially translated from the genome of the positive-stranded RNA virus. Such polyprotein has not been subjected to processing, and thus the processing sites between the proteins of the polyprotein are not leaved. The polyprotein is also isolated, which means that the polyprotein has been separated from its encoding genome. The polyprotein is substantially complete when it 15 retains all of the functional elements necessary to provide the immune-active features of the present invention, particularly epitopic configuration(s) that are present only in the .polyprotein and not in the processed proteins or subunits that are obtained from the polyprotein. However, with respect to this and other proteins of the present invention, it is within the skill of the art to make conservative amino acid substitutions, or insignificant amino acid additions, modifications or deletions, that may change the amino acid sequence of the protein but do not significantly alter the functioning of the protein the unprocessed epitopic configuration is retained). However, such modifications may, when desired, delete the processing signals and/or sites of the protein. These modifications are discussed further below. The completeness of the polyprotein ma be determined, for example, by SDS-PAGE followed by amino acid sequencing. The completeness may also be determined by utilizing the polyprotein in question in one or more of the assays discussed below, and detecting effects of epitopic configurations specific to the unprocessed state.

A "core-like" protein is a structural protein that provides the same type of functions as the core protein of HCV. Examples of "core-like" proteins from other viruses include the Japanese encephalitis virus core protein and the HIV gag protein.

A

"core-like antigen protein" is a structural "core-like" protein that includes the portion of the core-like protein that displays the antigenicity of the core-like protein. Although alteration ofepitopic configuration upon processing was not known in the art, core-like proteins generally, and regions of the core-like proteins that can be important to antigenicity, are well known in the art (see, Okamoto, et al. J Virol. 188:331, 1992; Wang, U.S. Patent No. 5,106,726). A core-like antigen protein may be determined for a desired positive-stranded RNA virus, for example, by ELISA or western blotting, or both, for traditional core-type antigenic reactivity, as is well known in the art. The core-like antigen protein may also be determined by SDS-PAGE followed by amino acid sequencing.

Typically, the core-like antigen protein is joined to an amino-terminal portion of the adjacent protein or peptide region of the positive-stranded RNA virus to provide the unprocessed "core-like antigen-adjacent protein" of the invention.

However, in some embodiments, particularly where the core-like protein is not the first protein region of the polyprotein, the core-like protein is joined to a carboxyi-terminal portion of the adjacent protein of the positive-stranded RNA virus in unprocessed form to provide the inventive unprocessed core-like antigen-adjacent protein of the invention.

In unprocessed form means that the core-like region and the adjacent region are typically, and preferably, maintained precisely as they are joined encoded) in a native positive-stranded RNA virus. As with the polyprotein and other proteins herein, 15 the core-like antigen protein may be insignificantly modified without changing the inventive functioning of the core-like antigen protein.

The portion of the "adjacent protein" that is adjacent the core-like antigen protein is sized such that the fusion protein has an epitopic configuration specific to an unprocessed core-like-adjacent protein of the positive-stranded RNA virus. Thus, *20 typically, the amino-terminal portion of the adjacent protein region must be of sufficient length to permit the fusion protein to display the transient epitopic configuration specific to the unprocessed core-like region.

In addition to traditional core-like proteins, the env protein of a positive- S: stranded RNA virus can also provide the surprisingly enhanced antigenic conformation and interactions shown by the core-like antigen-adjacent proteins described herein. This is particularly true when the env protein is used in combination with a second protein, also as described herein. Preferably, the env protein includes an unprocessed connection to an adjacent protein (which may itself be an adjacent env protein, such as gp120 and gp41 in HIV), similar to that found with the core-like antigen-adjacent protein disclosed herein. Additionally, the second protein may be a core-like protein, such as the gag protein of HIV. Because the env region provides similar enhanced detection and immune-induction shown by the core-like antigen-adjacent protein of the present invention, unless stated otherwise or otherwise clear from the context, reference herein to the core-like antigen-adjacent protein applies equally to env and\or env-adjacent proteins. Determination of whether a given env or env-adjacent protein displays such enhanced detection and immune-induction can be effected by assaying as with a corelike antigen-adjacent protein, as discussed below.

Determination of whether a given polypeptide displays the epitopic configuration of the inventive core-like antigen-adjacent protein can be performed as follows. A core-like antigen-adjacent protein in question can be included in a panel of core-like antigen-adjacent proteins comprising an established core-like antigen-adjacent protein, such as EN-80-2. The panel is placed in a series of wells on a microtiter plate.

The panel can also include other core-like antigen-adjacent proteins having different lengths of adjacent protein. In a separate well is placed an established nonstructural, or other, protein capable of synergistic cooperation with the core-like antigen-adjacent protein, such as EN-80-1. An antiserum is selected for the established core-like antigenadjacent protein that reacts weakly with the established core-like antigen-adjacent *protein and that also is nonreactive with the established nonstructural protein. The basis for selection is that the antiserum will react with the separated proteins as expected, but the antiserum will react much more strongly when both a suitable core-like antigenadjacent protein and the established nonstructural protein are present in the sample.

15 Many examples of such an antiserum, such as G614 (diluted 8-fold), G614 (diluted 16fold), G615 (diluted 8-fold), G615 (diluted 16-fold), and 8-5, are set forth below in the Examples. The antiserum is introduced to the sample proteins under conditions suitable for elicitation and detection of a reaction between the antiserum and the given protein, and detect and measure such response. The established nonstructural protein is then combined with a firther sample of each member of the core-like antigen-adjacent Sprotein panel. Next, the antiserum is introduced to the combined proteins under conditions suitable for elicitation and detection of a reaction between the antiserum and the proteins, and such response is detected and measured. Those core-like antigenadjacent proteins that provide a cooperative effect are suitable for use in the present invention. Preferably, the antiserum will react at least about 1.25 or 1.5 times as strongly with the combined proteins when compared to the additive reaction of the antiserum with each protein, alone. Further preferably, the antiserum will react at least about twice as strongly. Each of the above-recited steps is routine in the art, in light of the present specification.

The core-like antigen-adjacent protein is preferably isolated, which means that the core-like antigen-adjacent protein is .separated from the remainder of the polyprotein originally translated from the genome of the positive-stranded RNA virus.

The core-like antigen-adjacent protein is also preferably separated from its encoding nucleic acid molecule.

In a preferred embodiment, the core-like antigen-adjacent protein of the present invention is used in combination with a second protein. The second protein is preferably from a positive-stranded RNA virus, is further preferably from the same positive-stranded RNA virus as the core-like antigen-adjacent protein, and is most preferably a nonstructural protein from a positive-stranded RNA virus (preferably from the same positive-stranded RNA virus as the core-like antigen-adjacent protein).

In one preferred embodiment, the second protein is a nonstructural protein. In positive stranded RNA viruses other than HCV, the nonstructural proteins may be referred to by other names, as is well known in the art. For the purposes of the present specification, all such nonstructural-like proteins shall be referred to herein as "nonstructural proteins." As noted above, the nonstructural coding regions of positivestranded RNA viruses are well known in the art.

The determination of an appropriate second protein that is suitable for use with the core-like antigen-adjacent protein, which second protein may include portions of nonstructural coding regions comprising more than one nonstructural protein .(or less than all of one nonstructural protein), can be performed as follows.

A second protein in question can be included in a panel of second proteins comprising an established second protein, such as EN-80-2. The panel is placed in a series of wells on a microtiter plate. The panel can also include other second proteins having different lengths of adjacent protein. In a separate well is placed an established core-like antigen-adjacent protein capable of synergistic cooperation with the second protein, such as EN-80-1. An antiserum is selected for the established second 20 protein that reacts weakly with the established second protein and that also is nonreactive with the established core-like antigen-adjacent protein. The basis for selection is that the antiserum will react with the separated proteins as expected, but the antiserum will react much more strongly when both a suitable second protein and the established core-like antigen-adjacent protein are present in the sample. Many examples of such an antiserum are set forth below in the Examples. The antiserum is introduced to the sample proteins under conditions suitable for elicitation and detection of a reaction between the antiserum and the given protein, and such response is detected and measured. The established core-like antigen-adjacent protein is combined with each member of the second protein panel. Next, the antiserum is introduced to the combined proteins under conditions suitable for elicitation and detection of a reaction between the antiserum and the proteins, and such response is detected and measured. Those second proteins that provide a cooperative effect are suitable for use in the present invention.

Each of the above-recited steps is routine in the art, in light of the present specification.

The present invention also provides antibodies, preferably monoclonal antibodies, to the substantially complete polyprotein, the core-like antigen-adjacent protein, and/or nonstructural protein of the present invention, as well as other proteins of the present invention. The antibodies are preferably used in combination to provide particularly sensitive and specific detection of the positive-stranded RNA virus in a sample.

Still further, the present invention provides compositions and methods for the elicitation on an immune response in an animal (either humoral, cellular, or both).

Even further, the compositions and methods can vaccinate an animal against the positive-stranded RNA virus.

Preferably, the methods and compositions of the present invention, including those for detection, immune response elicitation and vaccination, are applied to a human being.

One example of the present invention is the Hepatitis C virus (HCV).

The following discussion focuses generally on HCV, and even further on the HCV core antigen protein joined to an amino-terminal portion of an HCV envelope region. The discussion also focuses on such core antigen-envelope region in combination with an HCV nonstructural protein (particularly the HCV NS5 and NS3-NS4 nonstructural proteins), or in combination with a second protein from another positive-stranded RNA virus (particularly the HIV envelope protein and the HTLV-I envelope protein). As S, noted above, this discussion is predictive of the results to be obtained with the core-like antigen-adjacent proteins of positive-stranded RNA viruses generally. The discussion is also predictive of the results to be obtained with the substantially complete polyprotein 20 of positive-stranded RNA viruses generally, and the substantially complete polyprotein of HCV in particular.

Nucleic Acid Molecules Encoding The Unprocessed Polvpeptides, And Other Polvpeptides. Of The Invention As noted above, the present invention includes a nucleic acid molecule encoding a polypeptide comprising a substantially complete positive-stranded RNA virus polyprotein. The present invention also provides a nucleic acid molecule encoding a polypeptide comprising a core-like antigen-adjacent protein, such as the HCV core antigen protein joined to an amino-terminal portion of the HCV envelope region. The present invention further provides a nucleic acid molecule encoding a polypeptide comprising a nonstructural protein of such positive-stranded RNA virus. In a preferred embodiment, the nucleic acid molecule is DNA.

In a preferred embodiment, the nucleic acid molecule is a DNA molecule encoding an unprocessed core antigen-envelope protein that was isolated from nucleic acid sequences present in the plasma of an HCV infected patient. As discussed further below, the isolation of the molecule included the steps of isolating viral particles from the patient's plasma, extracting and purifying the viral nucleic acid sequences, and then cloning the desired DNA molecule via a Polymerase Chain Reaction (PCR) technique.

The primers used for cloning were as follows: 5'-GGATCCATGAGCACAAATCCTAAACCT-3' (SEQ ID No. 1) and (fi) 5'-GAATTCGGTGTGCATGATCATGTCCGC-3, (SEQ ID No. 2).

The cloned DNA molecule was sequenced in order to confirm its identity. The molecule thus obtained was designated EN-80-2. The DNA sequence of the molecule EN-80-2 is given in Fig. IA (SEQ ID No. and has 669 bp. The amino acid sequence of the molecule EN-80-2 is given in Fig. 1B (SEQ ID No. and has 223 residues. The molecule EN-80-2, in E. coli strain BL21(DE3), was deposited with the American Type Culture Collection (ATCC) Rockville Maryland 20852, on July 14, 1993, and has been aaccorded ATCC Designation 55451. The culture has been deposited the conditions of 15 the Budapest Treaty.

In another preferred embodiment, the nucleic acid molecule is a DNA molecule encoding an HCV NS5 nonstructural protein that was isolated from nucleic acid sequences present in the plasma of an HCV infected patient. As with the isolation of the unprocessed core antigen-envelope protein discussed above (although with a 20 different patient), the isolation included the steps of isolating viral particles from the "-.patient's plasma, extracting and purifying the viral nucleic acid sequences, and then cloning the desired DNA molecule via a Polymerase Chain Reaction (PCR) technique.

o The primers used in the PCR were as follows: a a a 5'-GGATCCCGGTGGAGGATGAGAGGGAAATATCCG-3' (SEQ ID No. 3) and (ii) 53GAATTCCCGGACGTCCTTCGCCCCGTAGCCAAATTT-3' (SEQ ID No. 4) The isolated DNA molecule was subjected to sequence analysis in order to confirm its identity. The molecule thus obtained was designated EN-80-1. The DNA.

sequence of the molecule EN-80-1 is given in Fig. 3A (SEQ ID No. 9) and has 803 bp.

The amino acid sequence of the molecule EN-80-1 is given in Fig. 3B (SEQ ID No. and has 267 residues. The molecule EN-80-1, in E. coli strain BL21(DE3), was deposited with the American Type Culture Collection (ATCC) Rockville Maryland 20852, on July 14, 1993, and has been accorded ATCC Designation 55450. The culture has been deposited under the conditions of the Budapest Treaty.

Figure 2 depicts an expression plasmid, pEN-2, that contains the DNA molecule encoding the unprocessed core antigen-envelope protein isolated using the primers SEQ ID Nos. I and 2, discussed above. Figure 4 depicts an expression plasmid, pEN-1, that contains the DNA molecule encoding the NS5 nonstructural protein isolated using the primers SEQ ID Nos. 1 and 2, discussed above.

This general procedure has also been used to isolate a representative nucleic acid molecule from the NS3-NS4 nonstructural region of HCV. See also Simmonds, Lancet 336: 1469-1472, 1990. The primers used for the cloning were as follows: (i 5'-CACCCAGACAGTCGATTTCAG-3' (SEQ ID No. 5) and (ii 5'-GTATTTGGTGACTGGGTGCGTC-3' (SEQ ID No. 6) The molecule thus obtained was designated EN-80-4. The polypeptide encoded by the isolated molecule has a molecular weight of about 20,000 Daltons as measure by electrophoresis through SDS-PAGE.

Additional examples of polypeptides useful as the second protein include 20 the HIV envelope protein (molecular wieght about 18,000 daltons) and the HTLV envelope protein (molecular wieght about 18,000 daltons).

The present invention provides for the manipulation and expression of the above described nucleic acid molecules by culturing host cells containing a construct capable of expressing the above-described genes.

Numerous vector constructs suitable for use with the nucleic acid molecules of the present invention can be prepared as a matter of convenience. Within the context of the present invention, a vector construct is understood to typically refer to a DNA molecule, or a clone of such a molecule (either single-stranded or doublestranded), that has been modified through human intervention to contain segments of DNA combined and juxtaposed in a manner that as a whole would not otherwise exist in nature. Vector constructs of the present invention comprise a first DNA segment encoding one or more of an unprocessed core-like antigen-adjacent protein and a nonstructural protein of a positive stranded RNA virus operably linked to additional DNA segments required for the expression of the first DNA segment. Within the context of the present invention, additional DNA segments will include a promoter and will generally include transcription terminators, and may further include enhancers and other elements. See WO 94/25597 and WO/25598.

Mutations in nucleotide sequences constructed for expression of the inventive proteins preferably preserve the reading frame of the encoding sequences.

Furthermore, the mutations will preferably not create complementary regions that could hybridize to produce secondary mRNA structures, such as loops or hairpins, that would adversely affect translation of the mRNA. Although a mutation site may be predetermined, it is not necessary that the nature of the mutation per se be predetermined. For example, in order to select for optimum characteristics of mutants at a given site, random mutagenesis may be conducted at the target codon and the expressed mutants screened for indicative biological activity.

Mutations may be introduced at particular loci by synthesizing oligonucleotides containing a mutant sequence, flanked by restriction sites enabling ligation to fragments of the native sequence. Following ligation, the resulting reconstructed sequence encodes a derivative having the desired amino acid insertion, substitution or deletion.

Alternatively, oligonucleotide-directed, site-specific mutagenesis procedures may be employed to provide an altered gene having particular codons altered according to the substitution, deletion, or insertion required. Exemplary methods of making the alterations set forth above are disclosed by Walder et al. (Gene 42:133, 1986); Bauer et al. (Gene 37:73, 1985); Craik (BioTechniques, January 1985, 12-19); 20 Smith et al. (Genetic Engineering: Principles and Methods, Plenum Press, 1981); and Sambrook et al. (supra).

The primary amino acid structure of the above described proteins may also be modified by forming covalent or aggregative conjugates with other chemical moieties, such as glycosyl groups, lipids, phosphate, acetyl groups, or with other proteins or polypeptides, provided that such modifications should not interfere with the antigenicity of the proteins. (See U.S. Patent No. 4,851,341; see also Hopp et al., Bio/Technology 6:1204, 1988). For example, such modifications should not interfere with the epitopic configuration (including access to the epitope and other antigenic considerations) specific to the unprocessed core-like antigen-adjacent protein.

A preferred type of vector construct is known as an expression vector.

As noted above, the plasmids pEN-1 and pEN-2 are examples of such an expression vector, and contain nucleic acid molecules encoding an HCV NS5 nonstructural region and an unprocessed HCV core antigen-envelope protein, respectively.

For expression, a nucleic acid molecule, typically DNA, as described above is inserted into a suitable vector construct, which in turn is used to transform or transfect appropriate host cells for expression. Preferably, the host cell for use in expressing the gene sequences of the present invention is a prokaryotic host cell, further 22 preferably a bacterium such as E. coli. Other suitable host cells include Salmonella, Bacillus, Shigella, Pseudomonas, Streptomyces and other genera known in the art. In a further preferred embodiment, the host cell is an E. coli containing a DE3 lysogen or T7 RNA polymerase, such as BL21(DE3), JM109(DE3) or BL21(DE3) pLysS.

Vectors used for expressing cloned DNA sequences in bacterial hosts will generally contain a selectable marker, such as a gene for antibiotic resistance, and a promoter that functions in the host cell. Appropriate promoters include the trp (Nichols and Yanofsky, Meth. Enzymol. 101:155-164, 1983), lac (Casadaban et al., J. Bacteriol.

143:971-980, 1980), and phage X (Queen, J. Mol. Appl. Genet. 2:1-10, 1983) promoter systems. The expression units may also include a transcriptional terminator. Plasmids :**useful for transforming bacteria include the pUC plasmids (Messing, Meth. Enzymol.

101:20-78, 1983; Vieira and Messing, Gene 19:259-268, 1982), pBR322 (Bolivar et al., Gene 2:95-113, 1977), pCQV2 (Queen, ibid.), and derivatives thereof. Plasmids may contain both viral and bacterial elements.

15 In another embodiment, the host cell may be a eukaryotic cell, provided that either the host cell has been modified such that the host cell cannot process, for example, the unprocessed core-like antigen-adjacent protein or unprocessed nonstructural region (such as the NS3-NS4 nonstructural protein), or the processing signals and/or processing sites in the unprocessed polypeptide have been modified such 20 that the protein is no longer susceptible to processing (such modifications should not affect the antigenicity of the unprocessed protein). Eukaryotic host cells suitable for use in practicing the present invention include mammalian, avian, plant, insect and fungal cells. Preferred eukaryotic cells include cultured mammalian cell lines rodent or human cell lines), insect cell lines Sf-9) and fungal cells, including species of yeast Saccharomyces spp., particularly S. cerevisiae, Schizosaccharomyces spp., or Kluyveromyces spp.) or filamentous fungi Aspergillus spp., Neurospora spp.).

Techniques for transforming these host cells, and methods of expressing foreign DNA sequences cloned therein, are well known in the art (see, Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, 1982; Sambrook et al., supra; "Gene Expression Technology," Methods in Enzymology, Vol. 185, Goeddel Academic Press, San Diego, Calif, 1990; "Guide to Yeast Genetics and Molecular Biology," Methods in Enzymology, Guthrie and Fink (eds.), Academic Press, San Diego, Calif., 1991; Hitzeman et al., J. Biol. Chem. 255:12073- 12080, 1980; Alber and Kawasaki, J Mol. Appl. Genet. 1:419-434, 1982; Young et al., in Genetic Engineering of Microorganisms for Chemicals, Hollaender et al. p- 355, Plenum, New York, 1982; Ammerer, Meth. Enzymol. 101:192-201, 1983; McKnight et al., U.S. Patent No. 4,935,349).

In general, a host cell will be selected on the basis of its ability to produce the protein of interest at a high level. In this way, the number of cloned DNA sequences that must be introduced into the host cell can be minimized and overall yield of biologically active protein can be maximized. Given the teachings provided herein, promoters, terminators and methods for introducing such expression vectors encoding the proteins of the present invention into desired host cells would be evident to those of skill in the art.

Host cells containing vector constructs of the present invention are then cultured to express a DNA molecule as described above. The cells are cultured according to standard methods in a culture medium containing nutrients required for growth of the chosen host cells. A variety of suitable media are known in the art and generally include a carbon source, a nitrogen source, essential amino acids, vitamins and minerals, as well as other components, growth factors or serum, that may be required by the particular host cells. The growth medium will generally select for cells S. 15 containing the DNA construct(s) by, for example, drug selection or deficiency in an essential nutrient which is complemented by the selectable marker on the DNA construct or co-transfected with the DNA construct.

PolvDpetides Comprising The Unprocessed Polvpeptides Of The Invention 20 As noted above, the invention provides a polypeptide comprising an unprocessed, substantially complete polyprotein from a positive-stranded RNA virus.

The invention also provides a polypeptide comprising a core-like antigen protein, such as the HCV core protein, joined to an amino-terminal portion of an adjacent protein, such as the HCV envelope region. The present invention also provides certain nonstructural proteins. In one preferred embodiment, the amino acid sequence of the core-like antigen protein is that depicted in Fig. 1B (Seq. ID No. In such a preferred embodiment, the polypeptide has a molecular weight of about 25,000 daltons as measured by electrophoresis through a sodium dodecyl sulfate-polyacrylamide gel and has been deduced to have about 223 amino acids.

The unprocessed polypeptide from the positive-stranded RNA virus is capable of binding antibodies specific to the positive-stranded RNA virus. In the case of HCV, this has been confirmed by Western Blotting and by enzyme-linked immunosorbent assay (ELISA). The unprocessed core antigen-envelope protein has been found to be specifically reactive with the sera of patients with HCV, and therefore is not reactive with the sera of persons without HCV. The unprocessed polypeptide from the positive-stranded RNA virus is also capable of detecting the presence of antibodies in samples specific to the positive-stranded RNA virus, and therefore is useful for detection and diagnosis of the positive-stranded RNA virus in a patient, particularly a human being.

The present invention also provides a polypeptide comprising a nonstructural protein from the positive-stranded RNA virus. In a preferred embodiment, the polypeptide has the amino acid sequence of the polypeptide given in Fig. 3B (SEQ ID No. 10). The polypeptide of Figure 3B (SEQ ID No. 10) has a molecular weight of about 29,000 daltons as measured by electrophoresis through a sodium dodecyl sulfatepolyacrylamide gel (SDS-PAGE) and has been deduced to have about 267 amino acids.

The nonstructural protein of the present invention is capable of binding antibodies specific to the positive-stranded RNA virus, which in the case of HCV has been confirmed by Western Blotting and (ELISA) for both the NS5 and the NS3-NS4 nonstructural proteins disclosed herein. The nonstructural protein of the present invention is specifically reactive with the sera of patients infected with the positivestranded RNA virus, and therefore is not reactive with the sera of persons without the 15 positive-stranded RNA virus. The nonstructural protein is also capable of detecting the presence of antibodies specific to the positive-stranded RNA virus the conditions of the .Budapest Treaty, and in samples, and therefore is useful for diagnosis of the positivestranded RNA virus in a patient, particularly a human being.

Where the protein of the present invention is encoded by a portion of a 20 native gene, a derivative of a native gene, or has been otherwise modified, the protein maintains substantially the same biological activity of the native protein. For example, the structure of proteins corresponding to the unprocessed, substantially complete S- polyprotein from a positive-stranded RNA virus, the core-like antigen-adjacent protein, or the nonstructural protein can be predicted from the primary translation products thereof using the hydrophobicity plot function of, for example, P/C Gene or Intelligenetics Suite (Intelligenetics, Mountain View, Calif), or according to the methods described by Kyte and Doolittle Mol. Biol. 157:105-132, 1982).

In a preferred embodiment, the present invention provides isolated proteins. Proteins can be isolated by, among other methods, culturing suitable host and vector systems to produce the recombinant translation products of the present invention.

Supernatants from such cell lines, or protein inclusions or whole cells where the protein is not excreted or secreted into the supernatant, can then be treated by a variety of purification procedures in order to isolate the desired proteins. For example, the supernatant may be first concentrated using commercially available protein concentration filters, such as an Amicon or Millipore Pellicon ultrafiltration unit.

Following concentration, the concentrate may be applied to a suitable purification matrix such as, for example, an anti-protein antibody bound to a suitable support.

Alternatively, anion or cation exchange resins may be employed in order to purify the protein. As a further alternative, one or more reverse-phase high performance liquid chromatography (RP-HPLC) steps may be employed to further purify the protein.

Other methods of isolating the proteins of the present invention are well known in the skill of the art. See WO 94/25597 and WO/25598.

A protein is deemed to be "isolated" within the context of the present invention if no other (undesired) protein is detected pursuant to SDS-PAGE analysis followed by coomassie blue staining. Within other embodiments, the desired protein can be isolated such that no other (undesired) protein, and preferably no lipopolysaccharide (LPS), is detected pursuant to SDS-PAGE analysis followed by silver staining. Within still other embodiments, the protein is isolated if no other protein having significant antigenic activity that significantly interferes with detection assays or immunological events is included with the protein.

15 Bindine Partners To The Unprocessed Polvpentides Of The Invention The present invention also provides monoclonal and polyclonal antibodies directed against the unprocessed positive-stranded RNA virus polyprotein, ~the core-like antigen-adjacent protein of a positive-stranded RNA virus, the nonstructural protein of such positive-stranded RNA virus or other proteins of the 20 invention. The antibodies are produced by using the polypeptide of the invention as an immunogen through standard procedures for preparing a hybridoma, and/or other methods. The resulting antibodies are particularly useful for detecting the positive- S:.i stranded RNA virus in a sample, preferably a sample from a human being. See WO 94/25597 and WO/25598.

Polyclonal antibodies can be readily generated by one of ordinary skill in the art from a variety of warm-blooded animals such as horses, cows, goats, sheep, dogs, chickens, turkeys, rabbits, mice, or rats. Briefly, the desired protein or peptide is utilized to immunize the animal, typically through intraperitoneal, intramuscular; intraocular, or subcutaneous injections. The immunogenicity of the protein or peptide of interest may be increased through the use of an adjuvant such as Freund's complete or incomplete adjuvant. Following several booster immunizations, small samples of serum are collected and tested for reactivity to the desired protein or peptide.

Once the titer of the animal has reached a plateau in terms of its reactivity to the protein, larger quantities of polyclonal antisera may be readily obtained either by weekly bleedings, or by exsanguinating the animal.

Monoclonal antibodies can also be readily generated using well-known techniques (see U.S. Patent Nos. RE 32,011, 4,902,614, 4,543,439, and 4,411,993; see also Monoclonal Antibodies, Hybridomas A New Dimension in Biological Analyses, Plenum Press, Kennett, McKearn, and Bechtol 1980, and Antiboies.: 1 aboratory Manual, supra). Briefly, in one embodiment, a subject animal such as a rat or mouse is injected with a desired rotein or eptide If desired, arious techniques may be utilized in order to increase the resultant immune resp r ne v e ted bu the protein, in order to develop greater antibody reactivity. For examle the desire protein or peptide may be coupled to another rotein such as valbumin or keyhole limpet hemocyanin (KLH), or through the use f adjuvants such as Freund's complete or icomplete adjuvants. The initial elicitation f an immune response may be through intraperitoneal, intramuscular, intraocular, or subcutaneous routes.

may Between one and three weeks after the initial immunization, the animal may be reimmunized with booster immunization. The animal may then be test bled and he serum tested for bind to the unprocessed olypeptide uing assays as described S pabove. Additional immunizations may also be accomplished until the animal has reached a plateau in its reacivity to the desired protein or peptide. The animal may then be given a final boost of the desired protein or peptide, and three to four days later sacrificed. At this time, the spleen and lymph nodes may be harvested and disrupted into a single cell suspension by passing the organs through a mesh screen or by rupturing the spleen or lymph node membranes which encapsulate the cells. Within one embodiment the ed cells are subsequently ysed by the addition ofa hypotonic solution, followed by S. immediate return to isotonicity.

Within another embodiment, suitale cells for preparing monocona antibodies are obtained through the use of in vitro immunization techniques Briefly, an animal is scrificed, ad teseenadm uni z ati on techniques. Briefly, an animal is sacrificed, and the spleen and lymph node cells are removed as described above A single cell suspension is prepared, and the cells are placed into a culture ontaining a form of the protein or peptide of interest that is suitable for generating an immune response as described above. Subsequently, the lymphocytes are harvested and fused as described below.

Cells that are obtained through the use of in vitro immunization or from an immunized animal as described above may be immortalized by transfection with a virus such as the Epstein-Ba- Virus (EBV). (See Glasky and Reading, Hybridoma 8(4):377-389, 1989.) Alternatively, within a preferred embodiment, the harvested opleen and/or lymph node cell suspensions are fused with a suitable myeloma cell in order to create a "hybridoma" which secretes monoclonal antibodies. Suitable myeloma lines are preferably defective in the construction or expression of antibodies, and are additionally syngeneic with the cells from the immunized animal. Many such myeloma cell lines are well known in the art and may be obtained from sources such as the American Type Culture Collection (ATCC), Rockville, Maryland (see Catalogue of Cell Lines Hybridomas, 6th ed., ATCC, 1988). Representative myeloma lines include: for humans, UC 729-6 (ATCC No. CRL 8061), MC/CAR-Z2 (ATCC No. CRL 8147), and SKO-007 (ATCC No. CRL 8033); for mice, SP2/0-Agl4 (ATCC No. CRL 1581), and P3X63Ag8 (ATCC No. TIB and for rats, Y3-Agl.2.3 (ATCC No. CRL 1631), and (ATCC No. CRL 1662). Particularly preferred fusion lines include NS-1 (ATCC No. TIB 18) and P3X63 Ag 8.653 (ATCC No. CRL 1580), which may be utilized for fusions with either mouse, rat, or human cell lines. Fusion between the myeloma cell line and the cells from the immunized animal can be accomplished by a variety of methods, including the use of polyethylene glycol (PEG) (see Antibodies: A Laboratory Manual, supra) or electrofusion (see Zimmerman and Vienken, J. Membrane Biol.

S: 67:165-182, 1982).

Following the fusion, the cells are placed into culture plates containing a Ssuitable medium, such as RPMI 1640 or DMEM (Dulbecco's Modified Eagles Medium, S: 15 JRH Biosciences, Lenexa, Kan.). The medium may also contain additional ingredients, such as Fetal Bovine Serum (FBS, from Hyclone, Logan, Utah, or JRH Biosciences), thymocytes that were harvested from a baby animal of the same species as was used for immunization, or agar to solidify the medium. Additionally, the medium should contain a reagent which selectively allows for the growth of fused spleen and 20 myeloma cells. Particularly preferred is the use of HAT medium (hypoxanthine, aminopterin, and thymidine) (Sigma Chemical Co., St. Louis, After about seven days, the resulting fused cells or hybridomas may be screened in order to determine the presence of antibodies which recognizes the core-envelope region of said HCV or the HCV nonstructural protein. Following several clonal dilutions and reassays, hybridoma producing antibodies that bind to the protein of interest can be isolated.

Other techniques can also be utilized to construct monoclonal antibodies.

(See Huse et al., "Generation of a Large Combinational Library of the Immunoglobulin Repertoire in Phage Lambda," Science 246:1275-1281, 1989; see also Sastry et al., "Cloning of the Immunological Repertoire in Escherichia coli for Generation of Monoclonal Catalytic Antibodies: Construction of a Heavy Chain Variable Region- Specific cDNA Library," Proc. Natl. Acad Sci. USA 86:5728-5732, 1989; see also Alting-Mees et al., "Monoclonal Antibody Expression Libraries: A Rapid Alternative to Hybridomas," Strategies in Molecular Biology 3:1-9, 1990; these references describe a commercial system available from Stratacyte, La Jolla, California, which enables the production of antibodies through recombinant techniques.) Briefly, mRNA is isolated from a B cell population and utilized to create heavy and light chain immunoglobulin cDNA expression libraries in the XIMMUNOZAP(H) and 28 XIMMUNOZAP(L) vectors. These vectors may be screened individually or co-expressed to form Fab fragments or antibodies (see Huse et al., supra; see also Sastry et al., supra). Positive plaques can subsequently be converted to a non-lytic plasmid which allows high level expression of monoclonal antibody fragments from E.

coli.

Similarly, binding partners can also be constructed utilizing recombinant DNA techniques to incorporate the variable regions of a gene that encodes a specifically binding antibody. The construction of these binding partners can be readily accomplished by one of ordinary skill in the art given the disclosure provided herein.

(See Larrick et al., "Polymerase Chain Reaction Using Mixed Primers: Cloning of Human Monoclonal Antibody Variable Region Genes From Single Hybridoma Cells," Biotechnology 7:934-938, 1989; Riechmann et al., "Reshaping Human Antibodies for 9" Therapy," Nature 332:323-327, 1988; Roberts et al., "Generation of an Antibody with Enhanced Affinity and Specificity for its Antigen by Protein Engineering," Nature 328:731-734, 1987; Verhoeyen etal., "Reshaping Human Antibodies: Grafting an Antilysozyme Activity," Science 239:1534-1536, 1988; Chaudhary etal., "A Recombinant Immunotoxin Consisting of Two Antibody Variable Domains Fused to Pseudomonas Exotoxin," Nature 339:394-397, 1989; see also U.S. Patent No.

5,132,405 entitled "Biosynthetic Antibody Binding Sites".) Briefly, in one embodiment, DNA segments encoding the desired protein or peptide interest-specific antigen binding domains are amplified from hybridomas that produce a specifically binding monoclonal antibody, and are inserted directly into the genome of a cell that produces human antibodies. (See Verhoeyen et al., supra; see also Reichmann et al., supra.) This technique allows the antigen-binding site of a specifically binding mouse or rat monoclonal antibody to be transferred into a human antibody. Such antibodies are preferable for therapeutic use in humans because they are not as antigenic as rat or mouse antibodies.

In an alternative embodiment, genes that encode the variable region from a hybridoma producing a monoclonal antibody of interest are amplified using oligonucleotide primers for the variable region. These primers may be synthesized by one of ordinary skill in the art, or may be purchased from commercially available sources. For instance, primers for mouse and human variable regions including, among others, primers for VHa, VHb, VHc, VHd, CHI, VL and CL regions, are available from Stratacyte (La Jolla, Calif.). These primers may be utilized to amplify heavy or light chain variable regions, which may then be inserted into vectors such as IMMUNOZAPTM(H) or IMMUNOZAP

T

(Stratacyte), respectively. These vectors may then be introduced into E. coli for expression. Utilizing these techniques, large amounts of a single-chain protein containing a fusion of the VH and VL domains may be produced (see Bird et al., Science 242:423-426, 1988).

Monoclonal antibodies and binding partners can be produced in a number of host systems, including tissue cultures, bacteria, eukaryotic cells, plants and other host systems known in the art.

Once suitable antibodies or binding partners have been obtained, they may be isolated or purified by many techniques well known to those of ordinary skill in the art (see Antibodies: A Laboratory Manual, Harlow and Lane Cold Spring Harbor Laboratory Press, 1988; U.S. Patent No. 4,736,110; and U.S. Patent No.

4,486,530). Suitable isolation techniques include peptide or protein affinity columns, HPLC or RP-HPLC, purification on protein A or protein G columns, or any combination of these techniques. Within the context of the present invention, the term "isolated" as used to define antibodies or binding partners means "substantially free of other blood components." 15 The antibodies and binding partners of the present invention have many uses. As discussed further below, the antibodies and binding partners of the present invention are particularly useful for the detection and diagnosis of the positive-stranded RNA virus. Other uses include, for example, flow cytometry to sort cells displaying one more of the proteins of the present invention. Briefly, in order to detect the protein or 20 peptide of interest on cells, the cells are incubated with a labeled monoclonal antibody Po. which specifically binds to the protein of interest, followed by detection of the presence of bound antibody. These steps may also be accomplished with additional steps such as washings to remove unbound antibody. Labels suitable for use within the present invention are well known in the art including, among others, flourescein isothiocyanate (FITC), phycoerythrin horse radish peroxidase (HRP), and colloidal gold.

Particularly preferred for use in flow cytometry is FITC, which may be conjugated to purified antibody according to the method of Keltkamp in "Conjugation of Fluorescein Isothiocyanate to Antibodies. I. Experiments on the Conditions of Conjugation," Immunology 18:865-873, 1970. (See also Keltkamp, "Conjugation of Fluorescein Isothiocyanate to Antibodies. II. A Reproducible Method," Immunology 18:875-881, 1970; Goding, "Conjugation of Antibodies with Fluorochromes: Modification to the Standard Methods," J. Immunol. Methods 13:215-226, 1970.) Assays For The Detection Of A Positive-Stranded RNA Virus in A Sample As noted above, the invention provides a polypeptide comprising an unprocessed, substantially complete polyprotein from a positive-stranded RNA virus.

The invention also provides a polypeptide comprising a core-like antigen-adjacent protein and certain nonstructural proteins. The present invention further provides methods for detecting such polypeptides; in a sample. The assays are typically based on the detection of antigens displayed by the positive-stranded RNA virus or antibodies produced against the positive-stranded RNA virus, but may also include nucleic acid based assays (typically based uponl hybridization), as known in the art. The methods are characterized by the ability of the polypeptides of the present invention to be bound by antibodies against the positive-stranded RNA virus, and the ability of antibodies produced against the proteins of the present invention to bind to antigens of the positive-stranded RNA virus in a sample.

Surprisingly, the unprocessed polypeptides; of the present invention provide significantly better and more sensitive detection of the positive-stranded

RNA

virus. For example, with reference to IICV, the unprocessed core antigen-envelope protein provides significantly better detection of HCV in a sample than processed core :protein (sometimes referred to as p22) or fragments of the core protein, alone. Also :15 surprisingly, the use of both an unprocessed core-like antigen-adjacent protein and a nonstructural protein of the positive-stranded RNA virus in the assay provides a synergistic effect that permits significantly more sensitive detection of the positivestranded RNA virus than when either the unprocessed core-like antigen-adjacent protein or nonstructural protein is utilized alone.

*.20 A preferred assay for the detection of the positive-stranded RNA virus is a sandwich assay such as an enzyme-linked immunosorbent assay (ELISA). In one preferred embodiment, the ELISA comprises the following steps: coating a core antigen-envelope prti fthe pentinvention onto a solid phase, incubating a sample suspected of containing HCV antibodies with the polypeptide coated onto the solid phase under conditions that allow the formation of an antigen-antibody complex, adding an anti-antibody (such as anti-IgG) conjugated with a label to be captured by the resulting antigen-antibody complex bound to the solid phase, and measuring the captured label and determining therefrom whether the sample has HCV antibodies. Although a preferred assay is set forth above, a variety of assays can be utilized in order to detect antibodies that specifically bind to the desired protein from a sample, or to detect the desired protein bound to one or more antibodies from the sample. Exemplary assays are described in detail in Antibodies.- A Laboratory Manual, Harlow and Lane Cold Spring Harbor Laboratory Press, 1988. Representative examples of such assays include: countercurrent inimuno-electrophoresis

(CIEP),

radioimmunoassays, radioimnmunoprecipitations, enzyme-linjced immunosorbent assays (ELISA), dot blot assays, inhibition or competition assays, sandwich assays, ixnnunostick (dip-stick) assays, simultaneous assays, immunochromatographic assays, 31 ixnmunofiltration assays, latex bead agglutination assays, irnmunofluorescent assays, biosensor assays, and low-light detection assays (see U.S. Patent Nos. 4,376,110 and 4,486,530; WO 94/25597; WO/25598; see also Antibodies: A Laboratory Manual supra).

A fluorescent antibody test (PA-test) uses a fluorescently labeled antibody able to bind to one of the proteins of the invention. For detection, visual determinations are made by a technician using fluorescence microscopy, yielding a qualitative result. In one embodiment, this assay is used for the examination of tissue samples or histological sections.

In latex bead agglutination assays, antibodies to one or more of the proteins of the present invention are conjugated to latex beads. The antibodies cojgae to telatex basare the cotate ih a sapeunder conditUiLons permitting the antibodies to bind to desired proteins in the sample, if any. The results are then read visually, yielding a qualitative result. In one embodiment, this format can :15 be used in the field for on-site testing.

Enzyme immunoassays (EIA) include a number of different assays able to.

utilize the antibodies provided by the present invention. For example, a heterogeneous indirect ETA uses a solid phase coupled with an antibody of the invention and an affinity purified, anti-IgG immunoglobulin preparation. Preferably, the solid phase is a 20 polystyrene microtiter plate. The-. antibodies and immunoglobulin preparation are then contct. with tesample under conditions permitting antibody binding, which conditions are well known in the art. The results of such an assay can be read visually, 9#*ea*but are preferably read using a spectrophotometer, such as an ELISA plate reader, to a yield a quantitative result. An alternative solid phase ETA format includes plastic-coated ferrous metal beads able to be moved during the procedures of the assay by means of a magnet. Yet another alternative is a low-light detection immunoassay format. In this highly sensitive format, the light emission produced by appropriately labeled bound antibodies are quantitated automatically. Preferably, the reaction is performed using microtiter plates.

In an alternative embodiment, a radioactive tracer is substituted for the enzyme mediated detection in an ETA to produce a radioimnmunoassay

(RIA).

In a capture-antibody sandwich enzyme assay, the desired protein is bound between an antibody attached to a solid phase, preferably a polystyrene nucrotiter plate, and a labeled antibody. Preferably, the results are measured using a spectrophotometer, such as an ELISA plate reader. This assay is one preferred embodiment for the present invention.

In a sequential assay format, reagents are allowed to incubate with the capture antibody in a step wise fashion. The test sample is first incubated with the capture antibody. Following a wash step, an incubation with the labeled antibody occurs. In a simultaneous assay, the two incubation periods described in the sequential assay are combined. This eliminates one incubation period plus a wash step.

A dipstick/iminunostick format is essentially an immunoassay except that the solid phase, instead of being a polystyrene microtiter plate, is a polystyrene paddle or dipstick. Reagents are the same and the format can either be simultaneous or sequential.

In a chromatographic strip test format, a capture antibody and a labeled antibody are dried onto a chromatographic strip, which is typicay nitrocellulose or nylon of high porosity bonded to cellulose acetate. -The capture antibody is usually srydried as a line at one end of the strip. At this end there isanbsretmeil that is in contact with the strip. At the other end of the strip the labeled antibody is deposited in a manner that prevents it from being absorbed into the membrane. Usually, *15 the label attached to the antibody is a latex bead or colloidal gold. The assay may be initiated by applying the sample immediately in front of the labeled antibody.

Imnunofiltrationimmpunoconcentration formats combine a large solid phase surface with directional flow of sample/reagents, which concentrates and accelerates the binding of antigen to antibody. In a preferred format, the test sample is **20 preincubated with a labeled antibody then applied to a solid phase such as fiber filters or *3O* nitrocellulose membranes or the like. The solid phase can also be precoated with latex or glass beads coated with capture antibody. Detection of analyte is the same as stnarlmmunoassay. heflow of sample/reagents can be modulated by either Svacuum or the wicking action of an underlying absorbent material.

A threshold biosensor assay is a sensitive, instrumented assay amenable to screening large numbers of samples at low cost. In one embodiment, such an assay comprises the use of light addressable potentiometric sensors wherein the reaction involves the detection of a pH change due to binding of the desired protein by capture,.

antibodies, bridging antibodies and urease-conjugated antibodies. Upon binding, a pH change is effected that is measurable by translation into electrical potential (jivolts). The assay typically occurs in a very small reaction volume, and is very sensitive. Moreover, the reported detection limit of the assay is 1,000 molecules of urease per minute.

Composit .ons And Methods For The Elicitation o An mmune Resonse

T

The present invention also provides compositions and methods for the elicitation of an immune response to the positive stranded RNA virus, which may be either humoral, cellular, or both. Preferably, the immune response is induced by a vaccine against the positive stranded RNA virus, and is therefore an immunoprotective immune response. These compositions and methods typically involve an immunogen comprising an unprocessed polypeptide of the present invention in combination with a pharmaceutically acceptable carrier or diluent. In a preferred embodiment, the compositions and methods comprise both an unprocessed core antigen-envelope protein of HCV and a nonstructural protein of HCV, further preferably an NS5 nonstructural protein or a NS3-NS4 nonstructural protein. The compositions and methods may also include an inactivated preparation or an attenuated preparation comprising the proteins of the invention.

Accordingly, another aspect of the present invention provides isolated V. antigens capable of eliciting an immune response, preferably immunogens capable of immunizing an animal. In a preferred embodiment, comprising amino acid sequences or molecules shown in or derived from the sequences shown in Figures IA, IB, 3A or 3B or substantial equivalents thereof. As will be understood by one of ordinary skill in the art, with respect to the polypeptides of the present invention, slight deviations of the .amino acid sequences can be made without affecting the immunogenicity of the immunogen. Substantial equivalents of the above proteins include conservative substitutions of amino acids that maintain substantially the same charge and 20 hydrophobicity as the original amino acid. Conservative substitutions include replacement of valine for isoleucine or leucine, and aspartic acid for glutamic acid, as well as other substitutions of a similar nature (See Dayhoff et al. "Atlas of Protein Sequence and Structure," Natl. Biomed Res. Fdn., 1978).

As will be evident to one of ordinary skill in the art, the immunogens listed above, including their substantial equivalents, may stimulate different levels of response in different animals. The immunogens listed above, including their substantial equivalents, can be tested for effectiveness as a vaccine. These tests include T-cell proliferation assays, determination of lymphokine production after stimulation, and immunoprotection trials. Briefly, T-cell proliferation assays can be utilized as an indicator of potential for cell-mediated immunity. Additionally, evidence of lymphokine production after stimulation by an immunogen can be utilized to determine the potential for protection provided by an immunogen.

Finally, as described below, actual immunoprotection trials can be performed in order to determine protection in animals. In the case of humans, however, instead of immunoprotection trials it is preferred to first screen peripheral blood lymphocytes (PBLs) from patients infected with HCV in the following manner. Briefly, PBLs can be isolated from diluted whole blood using Ficoll density gradient centrifuigation and utilized in cell proliferation studies with 3 HI-thymidine as described below. Positive peptides are then selected and utilized in primate trials.

The iinmunogens, or polypeptides, of the present invention can be readily produced utilizing many other techniques well known in the art (see Sambrook et al., supra, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1989).

The inmunogens; comprising a polypeptides of the present invention in combination with a pharmaceutically acceptable carrier or diluent can be administered to a patient in accordance with a number procedures known in the art See WO 94/25597 and WO/25598.

For purposes of the present invention, warm-blooded animals include, *among others, humans and primates.

Many suitable carriers or diluents can be utilized in the present invention, including among others saline, buffered saline, and saline mixed with nonspecific serum albumin. The pharmaceutical composition may also contain other excipient ingredients, including adjuvants, buffers, antioxidants, carbohydrates such as glucose, sucrose, or *dextrins, and chelating agents such as EDTA. Within a particularly preferred embodiment, an adjuvant is utilized along with the irnmunogen. Examples of such adjuvants include alum or aluminum hydroxide for humans.

Te aountandfrequency oadisttoncan bedetermined in clinical trials, and may depend upon such factors as the positive stranded RNA viral species against which it is desired to protect, the particular antigen used, the degree of protection required, and many other factors. In a preferred embodiment, immunizations ~:will involve oral administration. Alternatively, the vaccine can be parenterally administrated via the subcutaneous route, or via other routes. Depending upon the application, quantities of injected immunogen will vary from 50 jig to several milligrams in an adjuvant vehicle and preferably about 100 g.g to I mg, in combination with a physiologically acceptable carrier or diluent. Booster immunizations can be given from 4-6 weeks later.

The present invention also includes the administration of a nucleic acid vector capable of expressing the unprocessed core antigen-envelope protein or nonstructural protein of HCV (or both) into an animal, wherein the nucleic acid molecule can elicit an immune response in, and preferably immunize, an animal against the expressed protein expressed from the nucleic molecule, and therefore HCV. In one embodiment of this procedure, naked DNA is introduced into an appropriate cell, such as a muscle cell, where it produces protein that is then displayed on the surface of the cell, thereby eliciting a response from host cytotoxic T-lymphocytes (CTLs). This can provide an advantage over traditional immunogens wherein the elicited response comprises specific antibodies. Specific antibodies are generally strain-specific and cannot recognize the corresponding antigen on a different strain. CTLs, on the other hand, are specific for conserved antigens and can respond to different strains expressing a corresponding antigen (Ulmer et al., "Heterologous protection against influenza by injection of DNA encoding a viral protein," Science 259:1745-1749, 1993; Lin et al., "Expression of recombinant genes in myocardium in vivo after direct injection of DNA," Circulation 82:2217-21, 1990); Wolff et al., "Long-term persistence of plasma DNA and foreign gene expression in mouse muscle," Human Mol. Gen. 1:363-69, 1992) Upon introduction of the naked vector construct into the animal's cell, the construct is then able to express the nucleic acid molecule (typically a gene) that it carries, which gene preferably comprises one (or more) of the unprocessed core antigen- *envelope protein or nonstructural protein of HCV. Accordingly, upon expression of the desired peptide, an immune response is elicited from the host animal. Preferably, the 15 immune response includes CD8+ CTLs able to respond to different strains that exhibit a form of the desired peptide.

SKits For Implementation Of The Various Asnects Of The Claimed Invention The present invention further provides kits for analyzing samples for the se -presence of antigens or antibodies from the positive-stranded RNA virus. The kits comprise a polypeptide or antibody of the invention and an appropriate solid phase.

Preferably, the polypeptide is bound to the solid phase. The kits can also provide one or S* more reagents and/or devices for the detection of the HCV antigens or antibodies.

A

variety of formats, reagents and devices for inclusion within the kits, including means for detecting the antigens or antibodies, are discussed herein.

The present invention also provides kits for the induction of an immune response. The kits comprise compositions comprising a polypeptide of the invention in combination with an pharmaceutically acceptable carrier or diluent, and can also provide devices for administering or assisting in the administration of the composition.

Other kits suitable for use-with the features of the present invention are also provided herewith.

The following Examples are offered by way of illustration, and not by way of limitation.

EXAMPLES

The following examples are separated into three groupings. First, are Examples relating to the isolation and production of a suitable core-like antigen-adjacent protein, namely an HCV unprocessed core antigen-envelope fusion protein, and uses thereof without a nonstructural protein. Second, are Examples relating to the isolation and production of an appropriate second protein for use with the core-like antigenadjacent protein, namely an HCV nonstructural protein, and uses thereof without the HCV core antigen-envelope fusion protein. Third, are Examples relating to the combination and use of the core-like antigen-adjacent protein with second proteins such as an HCV NS5 protein, an HCV NS3-NS4 protein, an HIV envelope protein and an HTLV-I envelope protein. Fourth, is an Example of the production of monoclonal antibodies to a core-like antigen-adjacent protein. Fifth, are Examples relating to the SG use of a suitable core-like antigen-adjacent protein, namely an HCV unprocessed core 15 antigen-envelope fusion protein to induce an immune response in an animal.

~THE ISOLATION AND PRODUCTION OF A CORE-LIKE ANTIGEN-ADJACENT

PROTEIN

1. Cloning of an HCV cDNA 20 The plasma of patients infected with the Hepatitis C virus was collected "'"and ultracentrifuged at 4°C and then the viral particles were obtained. Viral nucleic acid (RNA) was then extracted and purified from the viral particles using guanidine Sisothiocyanate and acidic phenol (Chomczynski et al., Anal. Biochem. 162:156-159, 1987).

The following oligonucleotide sequences: 5'-GGATCCATGAGCACAAATCCTAAACCT- 3 (SEQ

ID

No. 1) and (ii) 5'-GAATTCGGTGTGCATGATCATGTCCGC- 3 (SEQ

ID

No. 2) were used as primers in the cloning of cDNA. A single-stranded DNA molecule was produced using random primers, reverse transcriptase, and the RNA template. The double-stranded DNA molecule containing the HCV core-envelope region sequence was amplified by the PCR method using Taq polymerase and primers and (ii).

The cloned DNA molecule was subjected to sequence analysis for identification. The obtained molecule was designated EN-80-2. The DNA sequence of the molecule EN-80-2 is given in Fig. 1A (SEQ ID No. The DNA molecule was derived from the HCV core and envelope regions and has 669bp.

2. Construction of a Plasmid Containing an HCV cDNA The molecule EN-80-2 was treated with restriction endonucleases Barn HI and EcoRI to obtain a DNA fragment containing the desired HCV cDNA. The obtained DNA fragment was inserted into a vehicle plasmid which had been first cleaved with the restriction endonucleases Barn HI and EcoRI, to obtain an expression plasmid, designated pEN-2. The expression of the HCV cDNA is under the control of a 77 S.promoter. The structure of the expression plasmid pEN-2 and a restriction map are depicted in Fig. 2.

3. Transformation ofE. coli The expression plasmid pEN-2 were transformed into E. coli BL21 (DE3), spread into an ampicillin-agar plate and placed in a 37°C incubator overnight.

E coli colonies producing HCV core antigen protein were selected by screening their expression products by SDS-PAGE and Western Blotting.

4. Production Of The Unrocessed Core Anrt. ve 4 ocs re Antien-Envelope Protein The transformed E. coli colonies were incubated in a conditioned culture 'i medium. The colonies were centrifuged and lysed by freezing-thawing cycles and Slysozyme-digestion. The unprocessed core antigen-envelope protein product was released by the lysed cells and purified by column chromatography. The polypeptide was more than 90% pure.

The unprocessed core antigen-envelope protein has a molecular weight of about 25,000 daltons as measured by electrophoresis through a sodium dodecyl sulfate-polyacrylamide gel.

Immunological Reactivity of HCV Core Antien With HCV Antidie by Western Blotting The purified unprocessed core antigen-envelope protein was subjected to an SDS-PAGE electrophoresis using standard procedures. The SDS-PAGE gel was washed with deionized water at 4°C for 15 minutes and washed with Blotting Buffer (0.1SM sodium phosphate buffer, pH 6.7) at 4 0 C for 20 minutes. The polypeptide on the gel was then electroblotted onto nitrocellulose membrane under the Blotting Buffer at 1.3A for 1-1.5 hours. The membrane was washed with Wash Buffer (PBS-Tween pH 7.4) and blocked with Blocking Buffer (0.1M NaCI, 5mM EDTA, 50mM Tris, pH 7.2-7.4, 0.2% bovine serum albumin, 0.05% Nonidet p-40, IM urea) overnight.

The membrane was reacted with the sera of the persons infected with/without hepatitis C, which were first diluted with 40% Newborn Bovine Serum/Tris-HCI (pH 10X, at 40 0 C for 2 hours. After the reaction, the membrane was washed with Wash Buffer three times. The membrane was reacted with an antihIgG:HRPO conjugate (which was prepared as described hereafter) at 40 0 C for 2 hours.

After the reaction, the membrane was washed with Wash Buffer three times and then .reacted with 10 ml Substrate Solution (0.01% 4 -chloro-1-naphthol, 18% methanol, S0.04M Tris, pH 7.2-7.4, 0.IM NaCI and 0.01% H202) Por 20 minutes. The unprocessed core antigen-envelope protein of the core e pe protein of the present invention was reactive with the sera of HCV patients but not reactive with the sera of healthy persons.

6. ELISA for HCV Antibodies Treatment ofMicrotiter Plate A microtiter plate was coated with the purified unprocessed core antigenenvelope protein of the invention at appropriate concentrations and blocked with a 20 buffer containing bovine serum albumin. The treated microtiter plate was stored at 2horse radish perodase (RpO) using NaI4 to obtain the anti-IgwasPO conjugate.

S

The conjugate was purified by chromatography.

Components ofReagent Wash Solution: Phosphate Buffer containing 0.90 NaCI and Thimerosal.

Anti-hIgG:HRPO Conjugate Solution: the anti-hIgG:HRPO conjugate prepared as described above dissolved in Tris Buffer containing a proteineous stabilizer and antiseptics.

Sample Diluent: Tris Buffer containing a proteineous stabilizer and antiseptics.

39 OPD Substrate Solution: o-phenylene diamnine (OPD) dissolved in citrate-phosphate buffer containing

H

2 0 2 (If the solution becomes orange, it means that the solution has been contaminated and cannot be used any more.) Stopping Solution: 2N H2SO4 solution.

Positive/Negative controls: the serum samples of persons infected with/without hepatitis C diluted with phosphate buffer containing a proteineous stabilizer and antiseptics at an appropriate concentration.

Procedure: One hundred and fifty microliters (ul) of the test samples were iluted with Sample Diluent l0), and Positive/Negative Controls were added into the wells of the treated microtiter plate.

15 Some wells were retained as substrate blanks The plate was gently mixed by shaking and incubated at 37-40 0

C

for 1 hour.

S(c) The plate was washed three times with 0.3 mnl of Wash solution per well.

One hundred gl of anti-hIgG:HRPO Conjugate Solution was ""added to each well.

The plate was gently mixed by shaking and incubated at 37-40 0

C

for 30 minutes.

The plate was washed five times.

One hundred gil of OPD Substrate Solution was added to each well and the plate was incubated at 15-30*C in the dark for minutes.

One hundred tl of Stopping Solution was added to each well and gently mixed to stop the reaction.

The OD value per well was measured at 492 nm in a spectrophotometer.

E

D&"emW&6QD.

The OD 492 n m value per well subtracts the mean of the readings of the blanks (backgrounds). The difference (PCx-NCx) between the mean of the readings of the positive controls (PCx) and that of the negative controls (NCx) is equal to or more than The Cut-off value (CO) is calculated by the following formula: CO PCx X 0.15 NCx When the readings from test samples were less than the CO value, the samples were considered negative HCV antibodies could not be detected in the samples).

When the readings of test samples were equal to or more than the CO value, the samples were expected to be positive; however, it is preferred to repeat the .assay for the samples in duplicate. If the readings of either of the duplicate samples were less than the CO value, the samples were considered to be negative. If the duplicate samples were both more than or equal to the Cut-off value, the samples were 15 considered to be positive.

When the readings of test samples are more than NCx -but less than the CO value by 20%, the samples should be regarded as questionable samples and the assay has to be repeated for those samples.

Twenty-seven samples were tested by the ELISA according to the 20 invention. At the same time, the samples were also tested with the Abbott's kit (II) HCV antibody assay, which kit contains both structural and nonstructural proteins (i.e.

core (amino acids: 1-150), NS-3 and NS-4). The comparison between the test results of Abbott's kit (II) and those of the assay of the present invention is given in Table 1. It is noted that the results of Sample G 229 were negative according to Abbott's kit (II), but were positive according to the assay of the present invention. Sample G 229 was confirmed to be positive for HCV.

TABLE 1 Sample No. OD492am Results References Abbott's kit (II) TSGH 56 2.0 positive positive TSGH 57 2.0 positive positive G 23 1.469 positive positive G 30 2.0 positive positive G 32 2.0 positive positive G 49 2.0 positive positive G 56 2.0 positive positive G 58 2.0 positive positive G 114 1.559 positive positive G 128 2.0 positive positive G 186 2.0 positive positive G 208 2.0 positive positive G 214 2.0 positive positive G 231 2.0 positive positive G 250 2.0 positive positive Y I 2.0 positive positive USB 9 2.0 positive positive USB 19 2.0 positive positive *USB 20 2.0 positive positive U S 2 30 9 2p s ti ep s t v USB 23 0.952 positive positive G 11 0.147 negative negative *G 12 0.077 negative negative G 13 0.061 negative negative G 14 0.116 negative negative G 15 0.139 negative negative G 229 0.5 17 positive negative THE ISOLATION AND PRODUCTION OF A SUITABLE SECOND PROTEIN, AN HCV NONSTRUCTURL

PROTEIN

7. Cloning of an HCV cDNA Encoding The NS5 Nonstructural Protein The plasma of patients infected with Hepatitis C virus was collected and ultracentrifliged at 4*C and then the viral particles were obtained. Subsequently, the viral nucleic acid (RNA) was extracted and purified from the viral particles usinig guanidine isothiocyanate and acidic phenol (Chomczynski et al., Anal Biochem.

162:156-159, 1987).

5'-GGATCCCGGTGGAGGATGAGAGG(JATATCCG.3# (SEQ ID No. 3) and S'-GAATTCCCGGACGTCCTTCGCCCCGTAGCCAAATTT..3' (SEQ ID No. 4) 42 were used as primers in the cloning of cDNA. A single-stranded DNA molecule was produced using random primers, reverse transcriptase, and the RNA template. The double-stranded DNA molecule containing the NS-5 sequence was amplified by the PCR method using Taq polymerase and primers and (ii).

The cloned DNA molecule was subjected to sequence analysis for identification. The obtained molecule was designated EN-80-1. The DNA sequence of the molecule EN-80-1 is given in Figure 3A, and the amino acid sequence encoded by the molecule is given in Figure 3B. The DNA molecule was derived from the genome of HCV nonstructural region 5 and has 803 bp (SEQ ID No. The amino acid sequence of the molecule EN-80-1 is given in Fig. 3B (SEQ ID No. 10), and has 267 residues.

8. Construction of a Plasmid Containing an HCV cDNA The molecule EN-80- 1 was treated with restriction endonucleases Barn 15 HI and EcoRI to obtain a DNA fragment containing said HCV cDNA. The resulting DNA fragment was inserted into a vehicle plasmid which had been first cleaved with restriction endonucleases Ban HI and EcoRI, to obtain an expression plasmid, designated pEN-1. The expression of the HCV cDNA is under the control of a 77 promoter. The structure of the expression plasmid pEN-1 and restriction map are given 20 in Fig. 4.

e

S

9. Transformation ofE. coi The expression plasmid pEN-1 were transformed into E. coli BL21 (DE3), spread onto an ampicillin-agar plate and placed at 37°C incubator for overnight.

E coli colonies producing the HCV nonstructural protein were selected by screening their expression products by SDS-PAGE and Western Blotting.

Production of The NS5 Nonstructural Protein The transformed E. coli colonies were incubated in a conditioned culture medium. The colonies were centrifuged and lysed by freezing-thawing cycles and lysozyme-digestion. The protein product was released by the lysed cells and purified by column chromatography. The resulting polypeptide was more than 90% pure.

The polypeptide has a molecular weight of about 29,000 daltons as measured by electrophoresis through a sodium dodecyl sulfate-polyacrylamide gel.

43 Immnolo al Reactiv Th NturalProin With Antibodies by Western Bottin The purified polypeptide was subjected to SDS-polyacrylamide gel electrophoresis using standard procedures. The SDS-PAGE gel was washed with deionized water at 4 C for 15 minutes and washed with Blotting Buffer 15M sodium phosphate buffer, pH 6.7) at 40C for 20 minutes. The polypeptide on the gel was then electroblotted onto a nitrocellulose membrane under the Blotting Buffer at 1.3A for hours. The membrane was washed with Wash Buffer (PBS-Tween 20, pH 7.4) and blocked with Blocking Buffer 1M NaCI, 5mM EDTA, 50mM Tris, pH 7.2-7.4, 0.2% bovine serum albumin, 0.05% Nonidet p-40, 1M urea) overnight.

The membrane was reacted with the sera of the persons infected with/without hepatitis C, which were first diluted with 40% Newborn Bovine Serum/Tris-HCl (pH 10X, at 400C for 2 hours. After the reaction, the membrane was washed with Wash Buffer three times. The membrane was then reacted with an anti-hgG conjugate (which is prepared as described hereafter) at 40C for 2 hours. After the reaction, the paper was washed with Wash Buffer three times and then reacted with 10 ml Substrate Solution (0.01% 4 -chloro-l-naphthol, 18% methanol, 0.04M Tris, pH 7.2-7.4, 0.IM NaCI and 0.01%

H

2 0 2 for 20 minutes. The polypeptide of the present invention was reactive with the sera of HCV patients but was not reactive with the sera of healthy persons.

.12. ELISA for HCV Antiodies Treatment of Microtiter Plate A microtiter plate was coated with the NS5 nonstructural protein of the invention at appropriate concentrations and blocked with a buffer containing bovine serum albumin. The treated microtiter plate was stored at 2-8C.

Preparation of Anti-hG:HRP O C onjugae The purified anti-human Immunoglobulin G (anti-hIgG) was conjugated with horse radish peroxidase (HRPO) using NaI0 4 to obtain the anti-IgG:HRPO conjugate. The conjugate was purified by chromatography.

Components of ReaRent Wash Solution: Phosphate Buffer containing 0.9% NaCI and Thimerosal.

Anti-hIgG:HRPO Conjugate Solution: the anti-hIgG:HRPO conjugate prepared as described above dissolved in Tris Buffer containing a proteineous stabilizer and antiseptics.

Sample Diluent: Tris Buffer containing a proteineous stabilizer and antiseptics.

OPD Substrate Solution: o-phenylene diamine (OPD) dissolved in citrate-phosphate buffer containing H202. (If the solution becomes orange, it means that the solution has been contaminated and cannot be used any more.) Stopping Solution: 2N H 2 S0 4 solution.

Positive/Negative controls: the serum samples of persons infected with/without hepatitis C diluted with phosphate buffer containing a proteineous stabilizer and antiseptics at an appropriate concentration.

Procedure: One hundred and fifty microliters (pl) of test samples diluted with Sample Diluent (1:10) and Positive/Negative Controls were added to the wells of the treated microtiter plate. Some wells 20 were retained as substrate blanks.

The plate was gently mixed by shaking and incubated at 37-40 0

C

for 1 hour.

The plate was washed three times with 0.3 01 of Wash Solution per well.

One hundred pl of anti-hIgG:HRPO Conjugate Solution was added to each well.

The plate was gently mixed and incubated by shaking at 37-40°C for 30 minutes.

The plate was washed five times.

One hundred pl of OPD Substrate Solution was added to each well and the plate was incubated at 15-30°C in the dark for minutes.

One hundred ul of Stopping Solution was added to each well and gently mixed to stop the reaction.

The OD value per well was measured at 492 nm in a spectrophotometer.

Determination: The OD 492 n m value per well subtracts the mean of the readings of the blanks (backgrounds). The difference (PCx-NCx) between the mean of the readings of the positive controls (PCx) and that of the negative controls (NCx) is equal to or more than The Cut-off value (CO) is calculated by the following formula: CO=PCx X 0.15+NCx When the readings of test samples were less than the CO value, the samples were considered to be negative HCV antibodies could not be detected in the samples). When the readings of test samples were equal to or more than the CO value, the samples were expected to be positive; however, it is preferred to repeat the assay for the samples in duplicate. If the readings of either of the duplicate samples were less than the CO value, the samples will be negative. If the duplicate samples were both more than or equal to the CO value, the samples were considered to be positive.

When the readings of the test samples are more than NCx but less than the CO value by 20%, the samples should be regarded as questionable samples and the assay has to be repeated for the samples.

Eighteen samples were tested by the ELISA according to the invention.

At the same time, the samples were also tested with the Abbott's kit HCV antibody assay, which kit contains the nonstructural protein C100-3, and with the Abbott's kit (II) HCV antibody assay, which kit contains both structural and nonstructural proteins. The comparison between the test results of the Abbott's kits and those-of the assay of the invention is given in Table 2. It is noted that the results of Sample G 30 and Sample G 128 were negative according to Abbott's kit but were positive according to the assay of the present invention. These samples were confirmed to be positive for HCV.

TABLE 2 Sample No.

OD

49 2 m Results References Abbott's kit

(I)

TSGH 56 2.0 positive positive positive G 23 0.813 positive positive positive G 26 1.607 positive positive positive

G

G

G

G

G

G

G

G

y

USB

USB

USB

G

G

G

30 32 56 128 186 208 214 231 1 9 19 20 201 202 211 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 0.062 0.072 0.059 positive positive positive positive positive positive positive positive positive positive positive positive negative negative negative negative positive positive negative positive Positive positive Positive positive positive positive positive positive positive positive positive positive negative negative negative 00..

000* 00 0 0 000* 0 0 0 0 0 0 0 a 00 0 0 00 0009 0*00 DETECTION USING BOTH A CORE-LK ANTIGENADJACET

PROTEIN

AND) A SECOND

PROTEIN

13. ELIS ks Fo r HCV' Usin I Bothi nrocessed Core Antien -nvelope Potein And An NS5 Nonstructural -Protin A. ASSAYS CMA ING THEF ORE~ ATIGENOYENVL R TEINryT AND THE NS5 OS thCRLPROTEIN WITHABBO-4Tr HCV ASSAYS I. FiRsT ASSAY The method was analogous to the ELISAs described above, except that unprocessed core antigen-envelope protein was combined with an NS5 nonstructural protein 1) (known as the EverNew Anti-H CV EJA In a first assay, twenty-four samples were tested by the above-described method. At the same time, the samples were also tested by Abbott's kit (If1). The results are given in Table 3. In this assay, the results of the Abbott's kit (1H) were the same as the assay using the antigens of the present invention.

TABLE 3 Sample No.

Results a. a.

S

a a..

a a.

a. a a a.

a.

a.

TSGH 56 TSGH 57 G 23 G 26 G 30 G 32 G 49 G 56 G 58 G 114 G 128 G 186 G 214 G 231 G 250 yI USB 9 USB 19 USB 20 USB 23 USB 27 G 92 G 93 G 94 2.0 2.0 1.469 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 0.03 8 0.056 0.071 positive positive positive positive positive positive positive positive positive positive positive positive positive positive positive positive positive Positive positive positive positive negative negative negative References Abbott's kit (HI) positive positive Positive positive positive positive Positive positive positive Positive Positive Positive positive positive Positive positive Positive positive positive positive positive negative negative I. SECOND

ASSAY

The clinical trial report of blood donors for EverNew Anti -HCV EJA is shown in TABLE 4: Hospital: Taipei Thi-Service General Hospital Sample Source: Collected from Blood Bank Classification of Sample: Volunteer Blood Donors Reference Kit: Abbott's Kit (11) Results: TABLE 4

ABBOTT

Total 5 6 EverNew 193 194 (97%) total 6 194 200 (100%) The results in Table 4 indicate that both assays provided the same detection.

m. THIRD ASSAY The clinical trial report of high risk patients for EverNew Anti-HCV EIA is shown in TABLE Hospital: Taipei Veteran General Hospital Sample Source: Collected from Department of Clinical Virology Classification: NANB, sporadic NANB, PHT 12 HCC Liver cirrhosis 9 Chronic hepatitis B and carrier Biliary tract stones 4 Alcoholic liver disease 3 Fatty liver 2 Acute hepatitis, etiology? 2 Schistosomiasis of liver 1 Hepatic cysts 1 Cholangio-CA 1 Non-hepatobiliary disease 6 No data 2 Total 88 Reference Kit: ABBOTT HCV EIA 2nd generation Results: TABLE

ABBOTT

-Total 54 (61.36%) 54 (61.36%) EverNew 1 33 34 (38.64%) total 55 33 88 (100%) 49 HCV RT/PCR Method: Negative The clinical data and the HCV RT/PCR results indicate that the efficiency of the EverNew Anti-HCV EIA for HCV antibody detection was better than the ABBOTT HCV EIA 2nd generation licensed by the U.S. FDA.

B. ASSAYS SHOWING THE SYNERGISTIC COOPERATION OF CORE-LIKE ANTIGEN-ADJACENT PROTEINS AND A VARIETY OF SECOND PROTEINS. AND COMPARISON OF AN HCV CORE ANTIGEN- ENVELOPE PROTEIN WITH AN HCV PARTIAL CORE PROTEIN I. FIRST ASSAY This assay shows the results of an ELISA similar to those set forth above, and shows cooperative interaction between EN-80-2 and EN-80-1 proteins of HCV. The protocol for the ELISA is as follows: Coating buffer: 0.05 M Tris-HCl/0.15 N NaC/6 M Urea pH: 7.4 0.2.

Washing buffer: PBS with 0.05% Tween Postcoating buffer: PBS buffer with 1% BSA.

Coating procedure: EN-80-1 and EN-80-2 proteins were added into coating buffer (final concentration: about 1.5 gg/ml) and mixed at room temperature for minutes. After mixing, the diluted EN-80-1 and EN-80-2 proteins were added into microtiter wells, 100 pl/well, and incubated in a 40°C incubator for 24 hours. The microtiter wells were then washed, and postcoating buffer was added into wells. The microtiter wells were then let stand at 4 0 C for overnight. After postcoating, the coated 25 microtiter wells can be used for anti-HCV antibody detection.

Sample diluent: 0.1 M Tris-HCl pH: 7.4 0.2 with 40% NBBS, 1% BSA and 2% mouse serum.

Conjugate: anti-human IgG monoclonal antibody coupled with HRPO using NaIO 4 After coupling, the anti-human IgG:HRPO conjugates were purified by S-200 gel filtration and were diluted in sample diluent.

OPD tablets: purchased from Beckman.

Substrate diluent: citrate-phosphate buffer containing H 2 0 2 Stopping Solution: 2N H 2 S0 4 Positive control: anti-HCV positive serum diluted in sample diluent.

Negative control: recalcified human serum, which is non-reactive for HBV markers, anti-HIV, anti-HTLV I and anti-HCV.

Assay procedure: 100 pl sample diluent was added into each well.

pl sample, positive control and negative control was added into appropriate wells.

Sample incubation: incubated at 40 I°C for 30 2 minutes.

Sample wash: the wells were washed 3 times using washing buffer.

100 pl anti-human IgG:HRPO conjugate was added into each well.

Conjugate incubation: incubated at 40 1°C for 30 2 minutes.

Conjugate wash: the wells were washed 6 times using washing buffer.

After washing, 100 p substrate solution was added (the substrate solution was prepared by dissolving one tablet OPD in 5 ml substrate diluent), then the .e mixture was allowed to stand at room temperature for 10 minutes. In order to prevent light, the microtiter wells were covered with a black cover.

100 p] stopping solution was added into each well. Gently mix.

Evaluation: The OD value per well was measured at 492 nm in a spectrophotometer.

Interpretation: Determination of cutoff value: cutoff value PCx X 0.25 NCx.

An absorbance equal to or greater than cutoff value indicated that a 20 reaction was considered to be positive, which means reactive for anti-HCV antibody An absorbance less than the cutoff value was considered to be negative, which means non-reactive for anti-HCV antibody.

The sample sources for the assay reflected in Table 6 were as follows: Sample source I: G83, G191, G205 and G235 were GPT abnormal samples that were anti-HCV antibody negative and were collected from Taipei blood donation center.

Sample source II: G614 and G615 were anti-HCV antibody positive and were purchased from the U.S.A.

Sample source III: 8-5 was anti-HCV antibody positive and was collected from the Taichung blood donation center.

Sample source IV: N345 was a patient serum.

IABLE-6 Sample EN-80-1 EN-80-2 EN-80-I EN-80-2 G83 0.027@ 0.047 0.055 G191 0.071 0.209 0.056 G205 0.027 0.034 0.039 G235 0.025 0.044 0.043 G614 8X# 0.066 0.831 1.894 G614 16X 0.059 0.348 0.848 G615 8X 0.048 0.495 1.592 G615 16X 0.053 0.209 0.740 8-5 0.059 0.352 0.690 N345$ 0.008 0.420 0.730 Absorbance at 492 rim.

Samples were diluted with recalcified human serum, which is non-reactive for HBV, HCV and HIV.

Abbott's kit (II) found this sample to be negative.

These data demonstrate that when the EN-80-2 and EN-80-1 proteins were combined, the absorbance at 492 nm for anti-HCV positive samples was synergistic, not additive. Thus, cooperative interactions between EN-80-2 and EN-80-1 proteins of HCV were found. One benefit of this synergism is shown, for example, with sample N345, which was found to be HCV negative by Abbott's kit but due to the synergistic effect was found to be positive by the present invention. These data also demonstrate that the synergistic effect is helpful in screening for anti-HCV antibodies in samples, particularly in early detection situations.

IT. SECOND

ASSAY

This assay was conducted as set forth in the First Assay, above,'and included the provision in a single well of a core-envelope fusion protein of the invention in combination with an NS3-NS4 protein identified as EN-80-4. The results of the ELISA are set forth in Table 7.

TABLE 7 Sample jEN-80-2 EN-80-4 EN-80-2 I II EN-80-4 G83 0.047@ 0.032 0.049 G191 0.209 0.103 0.102 G205 0.034 0.045 0.046 G235 1 0.044 0.064 0.068 G58 2 1 X# 0.561 0.041 1.729 G612 161X 1.298 0.218 >2.o 1_G613 40X %0.202 0.243 0.708 Absorbance at 492 arn.

Sam~es erediluted with recalcified human serum, which is non-reactive for HBV, HCV and HV The data in Table 7 demonstrate that when the EN-80-2 and EN-80-4 *9 *proteins were combined, the absorbance at 492 rn for anti-HCV positive samples **showed a synergistic effect, not merely an additive effect. Thus, cooperative interactions between EN-80-2 and EN-80-4 proteins of HCV were found.

II. THID ASSAY VO This assay was conducted as set forth in the First Assay, above, and included the provision in a single well of a core-envelope fusion protein of the invention in combination with an IV envelope protein. The results of the ELISA are set forth in .*15 Table 8.

53 TABLE 8 Samples EN-80-2 HIV EN-80-2 envelope lilY enIvelope Recalcified human 0.030@ 0.056 0.093 serum G614 30.0 X# 0.116 0.064 0.250 G614 15.0 X 0.221 0.055 0.411 G614 9.9 X 0.403 0.054 0.798 G614 7.5X 0.598 0.046 1.061 G614 6.0 X 0.821 0.045 1.282 0614 5.0 X 1.022 0.040 1.656 0614 4.3 X 1.445 0.042 1.889 1. _j Absorbance at 492 nm.

W: Samples were diluted with recalcified human serum, which is non-reactive for HBV, HCV and HIV.

The data in Table 8 demonstrate that when the EN-80-2 protein 4" core-envelope fusion protein) of HCV and an HV envelope protein were combined, the absorbance at 492 nm for anti-HCV positive samples showed a synergistic effect, not Smerely an additive effect. Thus, cooperative interaction between the EN-80-2 protein of HCV and the HIV envelope protein were found.

IV. FouRTH ASSAY This assay was conducted as set forth in the First Assay, above, and included the provision in a single well of a core-envelope fusion protein of the invention in combination with an HTLV-I envelope protein. The results of the ELISA are set forth in Table 9.

54 IABLE9 Samples EN-80-2 HTLV-I EN-80-2 envelope

HTLV-I

Recalcified human 0.030@ 0.035 0.084 serum G614 30.0 X# 0.116 0.031 0.375 G614 15.0 X 0.221 0.027 0.561 G614 9.9 X 0.403 0.034 1.017 G614 7.5 X 0.598 0.033 1.303 G614 6.0 X 0.821 0.025 1.502 G614 5.0 X 1.022 0.017 G614 4.3 X 1.445 0.021 Absorbance at 492 rnm.

Samples were diluted with recalcified human seru, which is non-reactive for HBV, 5 HCV and HIV.

9 The data in Table 9 demonstrate that when the EN-80-2 protein of HCV and an HTLV-I envelope protein were combined, the absorbance at 492 nm for anti- HCV samples showed a synergistic effect, not merely an additive effect. Thus, cooperative interactions between the EN-80-2 protein of HCV and the HTLV-I envelope protein were found.

V. FIFTH ASSAY This assay was conducted as set forth in the First Assay, above, and included the provision in a single well of a core-envelope fusion protein of the invention in combination with an HTLV-I pol protein. The results of the ELISA are set forth in Table 10.

Samples EN-80-2 IHTLV-I E-8ol& HTL V-I ip01 Recalcified human 0.027@ 0.039 0.073 serum 0614 1I.0X 0.167 0.057 0.379 G614 9.9 X 0.288 0.047 0.543 0614 7.5 X 0.418 0.060 0.805 G614 6.0OX 0.600 0.053 1.188 0614 H .O 0.706 0.040 1.568 G614 4.3 X 0.867 0.047 1.644 8-5 1 0.436 0.052 0.779p.

S

*5 p p p *5 p p Th le approxdiate molecular weight of HTLV-I pol protein is 16,000 daltons.

Absorbance at 492 rn.

5 Samiples were diluted with recalcified humnan serum, which is non-reactive for HBV, HCV and MV.

The data in Table 10 demonstrate that when the EN-80-2 Protein of HCV and an HTLV-I pol protein were combined, the absorbance at 492 n for anti- HCV samples showed a synergistic effect, not merely an additive effect. Thus, 10 cooperative interactions between the EN-80-2 protein of HCV and the HTLV-I pol protein were found.

VI. S=~r ASSAY Table I11 depicts the results of an assay that was similar to that in the Fifth Assay and shows that there were no cooperative interactions between the HBV antigens HBsAg and HBcAg and the EN-80-1 protein of HG V.

HBsAg: purified from HBsAg positive human plasma.

HBcAg: derived from HBV cDNA fragment.

Sample source 1: G30 and G49 were OPT abnormal samples,. which were anti-HCV antibody positive and were collected fro the Taipei Blood Donation Center.

Sample source II: 0612, G613, 0614 and 0615 were anti-HCV antibody positive and were purchased from the United States of America.

56 0 6 1 2 8 0 4 E 0 1 0.1 1 1 0 1 4 5 E 0 1 8 E 0 1 7 0 102X@ 0.0885 0.117 2.3 0.232 0.239 G614 16X 0.059 0.124 0.12 011 0.11 615 16X 0.053 0.-107 0.13 015 0.232 Samples were serially diluted with recalcifled hmnsrn wihwsnnratv for HBV, HCV, and HIV.

;5 Absorbance at 492 nm.

The data in Table I11 demonstrate that when the IHBsAg or the HBcAg were coated together with the EN-80- 1 (NS5) protein, the absorbance of anti-HCv Positive samples was not synergistic. NO apparent interactions between the HBsAg and the EN-80-1 protein, or the HBcAg and the EN-80-1 Protein, were found.

9VII. SEVENTH

ASSAY

Table 12 shows a comparison of the detection of anti-HCV antibodies between the EverNew Anti-HCV ETA and the Abbott's kit The samples for the test *15 were obtained from the following sources: Sample source 1: 023, G26, 030, G32, 049, G58, GI 14, G128, GI 86, G23 1, 0250 and G262 were GPT abnormal samples, which were anti- HCV antibody positive and were collected from Taipei blood donation center.

Sample source UI: 0612, G613, 0614 and 0615 were anti-HCIV antibody positive and were purchased from U. S.X_ Sample source III VGH7, VGHI 1, VGH12, VGH13, VGHI6, VGH26, VGH27, VGH29, VGH3o, VGR32, VGH33, VGH4O, VGH43, VGH46 and VGH52 were anti-HCV antibody positive and were collected from Taipei Veteran General Hospital.

Classification for the samples from source 111.

VGH7 HMD stones VGHI 1 NANB, sporadic VGH12 NANB, sporadic VGH13 NANB,

PTH

VGH16

HCC

VGH26 Liver cirhsis VGE27 NANB, sporadic VGH29 ill) stone Schistosomfiasis of liver VGH32 NANB, sporadic VGH33 Liver cirrhosis VGH40 No data VGH43 NANB, sporadic VGH46 Liver cirrhosis with HCC VGH52 NANB, sporadic *9 .9 9 9* 9 9 9 9**9 9 .9.

9 9* C) 9 9 9*99** 4 9* .9

C

9 Control: Recalcified human Serum (nnnrPa,-+;. L xxn vvi, Yi Ufti-tlV 5 and HI[V). This human serum was also used to dilute the above-mentioned anti-HCV positive samples.

10 antigen.

antigen.

Tested Kits: EverNew Anti-HCV EIA Microtiter wells coated with EN-80-1 EverNew Anti-HCV ELA Mcrotiter wells coated with EN-80-2 EverNew Anti-HCV EIA Mcrotiter wells coated with EN-80- 1 and EN-80-2 antig, Reference Kit: Abbott's kit (II).

Results: IABLE 12 EN-80-1 Sample Dilution EN-80-1 EN-80-2 EN-80-2

ABBOTT

Recalcified n/a negative negative negative ngtv human serum eatv (Control) G23 20Xt) no- .1VV' negative positive negative positive positive positive positive 026 G32 G49 555* is..

*s 5 5

S

5*5* 5 *55* 5.

S

*s .ss 5

S

5ss* Sins

S

55* S 5* 5 is ews.

G58 GI 14 G128 G1 86 0231 8X 1 6X 51X 1 02X 51X 1 02X 21X 42X 1 6X 32X lox 20X 1 20X 240X 42X 84X 336X 672X 1 68X 336X 84X 1 68X 402X 804X 26X 52X 8X 1 6X negative negative negative negative Positive negative negative negative negative negative negative negative negative negative negative negative negative negative negative negative negative negative negative negative negative negative negative negative negative negative positive negative negative negative negative negative negative negative positive negative positive negative negative negative negative negative negative negative negative negative positive negative negative negative negative negative Positive negative Positive negative Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive positive Positive Positive Positive Positive negative Positive positive positive positive positive Positive positive Positive positive positive positive positive positive positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive negative Positive negative Positive negative Positive Positive Positive positive Positive negative Positive Positive positive positive Positive Positive 0250 G262 G6 12 G6 13 G6 14 G61 5 8X 16X a. a.

a a

C,

VGH7

VGHII

VGH12 VGH13 VGH1 6 VGH26 VGH27 VGH29 VGH3O VGH32 VGH33 VGH43 VGH46 VGH52 42X 84X 126X 252X 252X 504X 252X 504X 252X 504X 84X 1 68X 42X 84X 42X 84X 42X 84X 504X 1 008X 84X 168X 9x 1 8X 9X 1 8X 9X 12X 126X 252X negative negative positive negative negative negative negative negative negative negative negative negative negative negative negative negative positive negative negative negative negative negative negative N.D. negative

N.D.

negative

N.D.

negative negative positive negative negative negative negative negative positive negative negat ive negative negative negative negative negative positive negative negative negative negative negative positive negative negative

N.D-

negative

N.D.

negative

N.D.

negative negative positive positive positive positive positive positive positive positive positive positive positive positive positive positive positive positive positive positive positive positive positive positive positive negative positive positive positive positive positive negative positive positive positive positive positive positive positive positive positive positive positive positive negative negative positive negative positive negative negative negative negative negative negative negative negative negative positive positive positive negative a.

a a a. a Samples were serially diluted with recalcified human serum which was nonreactive with HBV, anti-HCV and HIV.

negative non-reactive with anti-HCV antibody.

positive reactive with anti-HCV antibody.

N.D. not done.

The data in Table 12 in bold show instances of synergy between the core antigen-envelope protein and the nonstructural (NS5) region of HCV. The data in bold also demonstrate instances where the invention provided better detection than the reference Abbott's kit (IH) HCV detection kit. These data indicate that the detectability of the microtiter wells coated with EN-80-1 and EN-80-2 antigens was more efficient than the microtiter wells coated with either EN-80-1 antigen or EN-80-2 antigen alone.

Furthermore, anti-HCV antibody in samples G128 240X, G231 672X, G612 804X, VGH27 42X, VGH27 84X, VGH29 84X, VGH30 84X, VGH32 504X, VGH32 1008X, VGH33 84X, VGH33 168X, VGH40 9X, VGH43 9X and VGH43 18X could be detected by using EverNew Anti-HCV EIA (microtiter wells coated with EN-80-1 and EN-80-2 antigens) but was not detected using the Abbott's kit (II).

VIII. EIGHTH ASSAY This assay shows the results of an ELISA performed according to the protocol set forth in the First Assay, above, wherein a partial core protein was combined with the EN-80-1 (NS5) protein of HCV. The partial core protein consisted of amino acids I through 120, and was a gift from the Development Center of Biotechnology (DCB) in Taiwan.

Sample source I: G235 was a GPT abnormal sample, which was anti- HCV antibody negative and was collected from the Taipei blood donation center.

Sample source II: G614 and G615 were anti-HCV positive samples and were purchased from the U.S.A.

TABLE 13 Absorbance at 492 nm.

The data in Table 13 demonstrate that when the partial core (amino acids I through 120) and EN-80-1 proteins were coated together, the absorbance at 492 nm of anti-HCV positive samples was not synergistic. No cooperative interaction between partial core and NS5 proteins of HCV were found.

IX. NINM ASSAY Table 14 confirms the above-presented results and shows an enzyme immunoassay comparison of the detection of anti-HCV antibodies using partial core (EN-80-5 antigen, which is an HCV partial core antigen having a molecular weight of about 15,000 daltons as measured by electrophoresis through an SDS-polyacrylanide .gel), core antigen-envelope protein (EN-80-2 antigen) and/or an HCV nonstructural protein (NS5; the EN-80-1 antigen discussed above). The samples for the assay were anti-HCV positive samples nos. N8, N81, N89, N12 and N302, and anti-HCV negative samples nos. N202, N203 and N302. The positive samples were diluted between and 672X with 0.1M Tris-HCI, pH 7.4 0.2) with 40% new born bovine serum, 1% BSA and 2% mouse serum. The samples were assayed in microtiter wells with a monoclonal anti-human IgG:HRPO conjugate solution, in combination with the following antigens or combinations of antigens: NS5; core antigen-envelope protein; partial core protein; NS5 and core antigen-envelope protein; NS5 and partial core protein; f) core antigen-envelope protein and partial core; and, core antigen-envelope protein, and partial core.

The following results were obtained: St Table 14 Sample NS5 core-env core NS5 NS5 core NS5 ID core-env core core-env core core-en N8 50X 0.098* 1.009 0.952 2.0 0.535 2.0 0.047 0.473 0.400 0.869 0.228 0.781 0.781 N81 336X 0.018 1.572 1.778 2.0 0.696 2.0 672X 0.019 0.697 0.633 0.742 0.344 0.912 0.982 N89 336X 0.083 2.0 2.0 2.0 1.918 2.0 672X 0.040 1.301 0.794 1.671 0.589 1.321 1.694 N12 25X 0.019 1.848 2.0 2.0 0.676 2.0 62 0.013 0.775 0.898 1.587 0.278 1.297 0.966 100X 0.009 0.333 0.317 0.566 0.092 0.390 0.435 N302 168X 0.188 >2.0 2.0 2.0 2.0 2.0 336X 0.078 1.161 1.968 1.645 1.660 >2.0 672X 0.046 0.496 0.819 0.829 0.612 0.805 1.025 *07 To8--0oT- -o07- N202 0.043 0.081 0.069 0.077 0.048 0.081 0.075 N203 0.100 0.208 0.124 0.185 0.117 0.189 0.169 N209 0.023 0.033 0.054 0.036 0.037 0.045 0.042 Sample 0.018 0.028 0.018 0.021 0.025 0.028 002 diluent 0.028 0.027 Anti-HCV positive serum diluted with sample diluent. Absorbance at 4 92nm.

Sample diluent: 0.1 M Tris-HCI pH: 7.4+0.2 with 40% new born bovine S::.erum, 1% BSA and 2% mouse serum.

X. TENTH ASSAY The tenth assay was an enzyme immunoassay directed to the use of an 1 HV gag protein in combination with an HIV env protein to detect the presence of anti- 0 HIv-I antibodies in human sera.

,The antigens used for the assay were as follows: First, a recombinant fusion protein comprising the amino-terminaj fragment of 1-galactosidase (377 a.a.) fused to gag-17 15-132) followed by gag p24 133-363) followed by gag 364-437). This protein had a Mw of 92.8 kDa, 831 a.a. (including spacer amino acids), and was entitled the EN-I-5 antigen. The protein used for the assay was purified from E. coli to greater than 90% purity and was non-glycosylated. Second, a recombinant fusion protein comprising the amino-terminal fragment of 0-galactosidase (311 fused to amino acids 474-863 of env, i.e, gpl60. This protein had a Mw 80.7 kDa; 705 aa. (including spacer amino acids), and was entitled the EN-I-6 antigen. The envelope cleavage site within gp160 is found between amino acids nos. 491 and 492, according to Ratner et al., Aids Res. And Human Retroviruses 3(1):57-69, 1987. Thus the EN-I-6 antigen includes both the carboxyl-terminal of gp120 and the amino-terminal of gp4 1. The protein used for the assay was purified from E. coli to greater than purity and was non-glycosylated.

63 The positive samples for the assay were obtained from clinically proven HIV positive human beings, therefore were proven anti-HIV-1 antibody positive sera, and were numbered TI, T2, T3, T4, T5, T6, P1, P2 and P3. The control sample was numbered NC and was an anti-HIV-1 antibody negative serum. The samples were assayed in microtiter wells with a monoclonal anti-human IgG:HRPO conjugate solution, in combination with the following antigens or combinations of antigens: a.) antigen (1 jAg/ml, 0.1 ml/well); EN-I-6 antigen (1 pg/ml, 0.1 ml/well); and, EN-I-5 and En-I-6 (both I 1ig/mL, antigens, 0.1 mil/well).

The following results were obtained: Table 15 Samples HV gag HIV env HIV gag HIV env p1 0.043 0.942 1.586 p2 0.031 0.698 1.142 p3 0.019 0.342 0.468 TI'24X 0.007 0.957 1.520 TI 72X 0.000 0.440 0.863 T2 72X 0.000 0.407 0.644 T T3 8X 0.000 0.350 0.548 T4 648X 0.001 0.319 0.488 T5 72X 0.000 0.227 0.353 T6 72X 0.005 0.560 0.799 e NC 0.019 0.028 0.030 NC 4X 0.012 0.027 0.025 the absorbance of 4 9 2nm.

samples diluted with sample diluent.

Table 15 indicates, surprisingly, that synergistic interactions are found between an HIY- 1 gag and env protein.

XI. ELEVENTH

ASSAY

The eleventh assay was an enzyme immunoassay directed to the use of the HiV env protein in combination with other, second proteins to detect the presence of anti-HIV-1 antibodies in human sera.

0 0 *0 0 0 *00 0 00 00 0 0*00*0 0* 0 0 The antigens used for the assay were an FHV env protein (the EN-1-6 antigen, described above), an HCV NS5 Protein (the EN-80-1 antigen, described above) and an HCV core-Eike antigen-adjacent protein (the EN-80-2 antigen, also described above). The positive samples for the assay were TI, T2, T3, T4, T5 and T6, which were anti-HJV-1 antibody positive sera; and the control samples were N639, N626, N634, N632 and N637, which were anti-EICV and anti-Hfv- I antibody negative sera.

The samples were assayed in microtiter wells with a monoclonal antihuman IgG:HRPO conjugate solution, using the antigens or combinations of antigens set forth below in Table 16. The results of the assays are also set forth in Table 16.

Table 16 HCV NS5 HCV core-env Samples: HCV NIS5 11WV env HIV env MIV env HCV core-env TI 24X 0.048* 0.833 0.602 0.930 0.038 72X 0.057 0.599 0.460 0.679 0.048 Z16X 0.055 0.278 0.213 0.3 14 0.039 N639 0.077 0.092 0.097 0.110 0.069 T2 24X 0.048 0.876 0.512 0.947 0.026.

72X 0.052 0.520 0.3 77 0.697 0.031 216X 0.069 0.228 0.191 0.284 0.037 Sample 0.069 0.052 0.029 0.040 0.047 Diluent T3 4X 0.029 0.503 0.492 0.579 0.030 8X 0.023 0.374 0.319 0.443 0.030 24X 0.023 0.170 0138 0.187 0.024 N626 0.065 0.079 0.073 0.101 0.094 T4 72X 0.031 1.424 1.293 1.666 0.084 216X 0.051 1.065 1.008 1.259 0.109 648X 0.035 0.724 0.641 0.233 0.099 N634 0.076 0.054 0.059 0.108 0.155 24X 0.021 0.518 0.423 0.556 0.016 72X 0.016 0.262 0.204 0.297 0.006 216X 0.014 0.094 0.074 0.094 0.017 N632 0.034 0.0316 0.53 0.041 0.051 T6 24X 0.021 0.864 0.783 1.048 0.023 HCV NS5 HCV c rP.. HCVSS Icor--, Samples: HCV NS5 HTfV env FHV env MIV env HCV core-env rYI A 1o .L .Z .7 .5 .1 Jy.: 0.475 0.659 0.015 216X 0.016 0.272 0.202 0.284 0.026 N637 0.051 0.050 0.042 0.052 0.072 Anti-HIV- 1 positive samples diluted with sample diluent (0.1 M Tris-HC1, pH: 7.4 0.2 with 40% new born bovine serum, 1% BSA and 2% mouse serum).

Absorbance at 492 nm.

These results indicate that the HIV env protein is capable of synergistic interactions with a second protein, similar to the synergistic interaction that has been shown with the HCV core-env protein discussed above.

THE PRODUCTION OF MONOCLONAL ANTIBODIES TO A CORE- LIKE ANTIGEN-ADJACENT

PROTEIN

14. Preparation of Antibodies Against HCV Antibodies against unprocessed core antigen-envelope protein and the nonstructural protein were produced according to a standard procedure for 10 producing monoclonal antibodies. In particular, a BALB/c mouse was immunized with a.the purified proteins described above in Examples 2 and 10 mixed with an adjuvant; and then the spleen cells were fused with mouse myeloma cells (FO cells line) using polyethylene glycol to form hybridoma cells. The desired clones producing desired monoclonal antibodies was obtained by screening the titer of the antibodies produced by the hybridoma clones so prepared. In one embodiment of the invention, a hybridoma clone was designated EN-80-1-99.

THE USE OF AN HCV CORE-LIKE ANTIGEN-ADJACENT PROTEIN TO INDUCE AN IMM1UNE RESPONSE Administration OfAn HCV Core-Like Antigen-Adjacent Protein A core antigen-envelope protein (EN-80-2) was administered intramuscularly to ICR mice at 6-8 weeks of age. The first administration, boost and sampling schedule was as follows: Negative Control Group: (ID nos. 0-1 and 0-2) Day 0: no immunization.

Day 13: 1 st bleeding Day 28: 2nd bleeding 66 Test Group 1: (ID nos. 1-1, 1-2, 1-3, 1-4, 1-5 and 1-6) Day 0: 50 g/mouse of EN-80-2 protein using complete Freund's adjuvant (CFA) (Gaithersburg, MD, USA, 20877).

Day 13: 1st bleeding Day 28: 2nd bleeding Day 39: 3rd bleeding Test Group 2: (ID nos. 2-1, 2-2, 2-3, 2-4, 2-5 and 2-6) Day 0: 50 jig/mouse of EN-80-2 protein using complete Freund's adjuvant (CFA), GIBCO.

Day 13: 1st boost, with 80 jg/mouse of EN-80-2 protein using incomplete Freund's adjuvant (IFA), also from GIBCO (Gaithersburg, MD, USA, 20877).

Day 28: 1st bleeding Day 39: 2nd bleeding Test Group 3: (ID nos. 3-1, 3-2, 3-3, 3-4, 3-5 and 3-6) Day 0: 50 jg/mouse of EN-80-2 protein using complete Freund's 20 adjuvant (CFA), GIBCO.

Day 13: 1st boost, with 80 jig/mouse of EN-80-2 protein using incomplete Freund's adjuvant (IFA), GIBCO.

Day 28: 2nd boost, with 80 ug/mouse of EN-80-2 protein, in PBS.

Day 39: 1st bleeding 16. Detection Of The Immune Response Induced By The Administration Of The Core Antigen-Envelope Protein The presence or absence of an immune response in the test animals was determined using two enzyme immunoassays (EIAs) similar to those described above.

In the first EIA, a rat anti-mouse:HRPO conjugate was added to the wells of a microtiter plate that had been coated a core antigen-envelope protein (EN-80-2) along with a rat anti-mouse:HRPO conjugate. The results of the first EIA are shown below in Table 17.

Table 17 Sample ID Day 13 Day 28 Day 39 Negative control: 0-1 50X@ 0.141 0.160 N.D. S 500X 0.058 0.060 N.D.

2500X 0.008 0.025 N.D.

12500X 0.000 0.010 N.D.

62500X 500X 2500X 12500X 6250OX a .gt e.g.

C. C.

C

C

C.

C

C

C

C a

C*

C

Group 1: 1-1 50X 500X 2500X 12500X 62500X 1-2 5OX 500X 250OX 12500X 6250OX 1-3 50X 500X 250OX 1250OX 62500X 1-4 50X 500x 2500X 1250OX 6250OX 50X 250OX 12500X 62500X 1-6 50X 500X 250OX 1250OX 62500X 0.000 0.188 0.048 0.000 0.000 0.000 0.720 0. 144* 0.018 0.000 0.000 0.257 0.062 0.004 0.000 0.000 0.213 0.042 0.000 0.000 0.000 0.259 0.050 0.002 0.000 0.000 0.580 0.111 0.010 0.000 0.000 0.443 0.161 0.026 0.000 0.000 0.012 0.160 0.050 0.018 0.013 0.009

N.D.

N.D.

N.D.

N.D.

N.D.

>2.0/>2.0 0.976 1.263 0.187 0.278 0.023 0.062 0.000 /0.018 2.0 0.424 0.058 0.000 0.000 >2.0 2.0 1.882 2.0 0.348 0.506 0.048/0.098 0.000 0.037 2.0 1.774 2.0 0.336 0.471 0.041 /0.097 0.000 0.030 0.341 0.191 0.071 0.025 0.016

N.D.

N.D.

N.D.

N.D.

N.D.

N.D.

N.D.

N.D.

ND.

N.D.

N.D.

0.560 0.132 0.027

N.D.

N.D.

N.D.

N.D.

N.D.

0.886 0.163 0.039 1.616 1.646 0.313 0.067 0.021

N.D.

N.D.

N.D.

N.D.

N.D.

C

Group 2: 2-1 2-2 50X 2500X 12500X 6250OX 250OX 1250OX 2.0 0.939 1.161 0.161 /0.200 0.032 0.038 0.016 0.017 2.0 2.0 >2.0/>2.0 1.092 1.316 0.232 0.267 1.478 0.280 0.059 0.022 1.158 0.250 62500X 500x 2500X 12500X 62500X 500X 2500X 12500X 62500X 2500X 12500X 62500X 50OX 2500X 12500X 6250OX 0.050 0.063 0.544 0.121 0.028 0.010 0.013 2.0 2.0 >2.0/>2.0 0.909 /1.209 0. 177 /0.232* 0.037 10.058 1.860 0.379 0.071 0.018 0.010 2.0 2.0 1.475 /1.780 0.33 3 0.383 0.066 /0.080 0.019 0.078 0.061

N.D.

N.D.

NJ.

N.D.

N.D.

0.794 0.156* 0.051 0.836 0.155 0.030 0.0 19 1.577 0.357* 0.075 0.025

S

Group 3:

SOX

2500X 12500X 62500X 5OX 500X 2500X 12500X 62500X 500X 2500X 12500X 625OOX 250OX 1250OX 62500X 500X 250OX 12500X 62500X 1.647 0.362 1.032 0.195 0.053 1.814 0.312 0.060 0.026 0.895 0.181* 0.048 0.701 0.146* 500X 2500X 12500X 0.726 62500X 0.172 Mouse serum diluted 50X, 500X, 2500X, 12500X and 62500X with 1%

BSA.

Absorbance at 492nm.

End point ofdetectability.

Assay not done because there was no serum for the assay.

In a second EIA, a rat anti-mouse:HRPO conjugate was added to the 10 wells of a microtiter plate that had been coated with the following antigens or combinations of antigens: NS5 (EN-80-1 antigen); core antigen-envelope protein (EN-80-2 antigen); partial core protein (EN-80-5 antigen); NS5 and core antigenenvelope protein; NS5 and partial core protein; core antigen-envelope protein and partial core; and, NS5, core antigen-envelope protein, and partial core. The samples .15 used in the second EIA were as follows: 0-2 (50X diluted, from day 28); 0-2 (500X diluted, from day 28); 2-2 (2500Xdiluted, from day 28); 3-1 (12500X diluted, from day 39); 3-4 (2500X diluted, from day 39); 3-5 (2500X diluted, from day 39); 3-6 (2500X diluted, from day 39); and, 3-6 (12500X diluted, from day 39).

*The results of the second EIA are shown below in Table 18.

Table 18 a Sample NS5 core-env core NS5+ NS5 core+ NS5 ID core-env core core-env core core-env Negative control: 0-2 50X 0.018@ 0.024 0.025 0.026 0.020 0.027 0.029 0-2 500X 0.008 0.010 0.011 0.014 0.014 0.022 0.019 Group I: 2-2 0.004 0.398 0.007 0.489 0.009 0.313 0.388 2500X Group II: 3-1 0.002 0.506 0.009 0.760 0.009 0.513 0.472 12500X 0.003 0.220 0.007 0.344 0.006 0.192 0.227 2500X 0.003 0.705 0.007 1.168 0.006 0.592 0.747 2500X 3-6 0.005 0.693 0.005 1.012 0.008 0.542 0.704 2500X 3-6 0.005 0.144 0.008 0.224 0.009 0.126 0.134 12500X Absorbance at 492nm.

Throughout this specification, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element or integer or group of elements or integers but not 5 the exclusion of any other element or integer or group of elements or integers.

Reference to any prior art in this specification is not, and should not be taken as, an acknowledgment or any form of suggestion that this prior art forms part of the S common general knowledge in Australia or any other country.

4 «*r

Claims (6)

1. A positive-stianded RNA virus-dcnivcd coinposiion cox~prising tbo following: a) an. isobacd polypeptido coiuprlsing a positivcotianded RNA virus cor=-lke antigen protein joined to an amnlno-terminal pordoi *Of an adJacent protein or said positive-strandod. RNA virus in unprocessed form, wbercin said amino-termjinal portion of said adjaccnt protin is sized such that czd* polypepticle hia., an epitopic configrtion corresponding to an uaprocessed core-ike antigen-adjacent protein of said :*.s0e Positive-stramded RNA virus;- and *se9b) au Isolated noaserun- protein of sad positivc-straded RNA virus. I The counpslion of claim I whcraiu said positive-str~Axidd RNA virus is selected from the group consisting of Togaviridae, Coronaviridas, Retroviridac, @009 Picarnavividae, Cal iciviridae, and 1Fuviviridar.

3. The comp~osition of c6aim 2 wherein said positiva-strmulad RNA virus is fielectrad from the group cotuisdri of Xluxoan 1mmnodeficiecncy virus (HflV) and 0**ee i-I unan T-call Leukemia virus aMTLV). @00054. The comuposition of claim I. wherein said isolated polypeptide is S producod by a suitable proksic host cell. The composition of cLaim I whczcin said isolated polypeptide is produced by a culcaryotic host ccli that is unable to process said isolated polypcptidc.

6. A method of making a composition comprising multiple polypeptdes obtained from a positive-stranded R~NA vi=u, comprising thte fbowong steps: a) introducing into a Enst host coli, a BMrs cxpression vector capalbic of expressing a nucleic acid molecule encoding an. isolatcd polypeptide comprising a posit vo-stRu~rded RNA vimu core-like antigen protein joinod to on aminkrmirud potionl of an adacent protzin of said positive-stranded RNA virus in unprocessed forni wh~ereint said amino-terminal portion off said adacent protein is sized such that said 72 polypeptide has an epitopic cwnfgurazion carrrsponding to an unprocssed care-like =dtgen-adjaccat proltain of said positive-Strandod. RNA vims b) inmibating said first host edl u~nder conditions suitable for -%aid cxpression vector to produce said polypeptide, purifyig said polypeptide to provida a purified polypeptide, and d) introducitia iMt a second host cell a 'second wqprssion vector capable -of =.~prssing a nucleic acid mnolecule encoding an isolated nons=rCrURZl PrOteh of said positive-strarded RNA virus, V'6.c) incubatIng Wad second host cel undcr conditions suitable for said nuclaic acid molecule to produce said nonstructural. protein, 0 purifying said nonstructural proten to provide an purificd nonstrutural protein, and then 9 combining said purified polypeptide and said purified nonstructural. protein to form said composition.

7. A mctlod of makig a composition complising m11tiplc polypeptides obtained from a posifive-tranded RNA virus, comprising ON. followings steps: a) iumoducing into a host cell an cxpression vector capablo of expresin a first nucleic acid mnolecule encodIng an isolated polypoptide conprisirig 'a poaltiv-srvde RNA virm cora-jjikc antigen protein joined to an aiiiotomnanW portion of an adacont pitottin of said positive-strwided R~NA virus in unprocessed form~, %whereirt said amino-terminal portion of said adjacent protein, is si;zd simh tha said polypeptide basa n cpitapic configuration corresponding to an unprocoed core-like antigen-4dacent protein of said positivo-'straded RNA virus, said expression vector also capable of expressin a second protdn comprising a non=4wual protein derived from said positive-strande-d R~NA virus, b) incubating s aid host ccll under conditions suitable for said expression vcctor to produce said polypeptide and said nonstructural protein. and c) ptuifying said polypeptide and sad nonstzucrral protein to provide a purified polypeptidc and a pded nonstucau-a protein. S. The method of claim 6 or 7 wherein said positive-=iandcd RNA vi=u is selected from the group conisting of Togaviridac, Coronavi-ridwe, Reiioviridac. Picornaviridayc, Caliciviridao and Flaviviridae.

71- 9. A composition CoMpnisiIIS an isolatcd, substanfialy complete, unprocesscd polyprotein from a positive-strandcd RNA virus bound to a solid subsumae. A com~positon comprising an Isolated poLypepti de comprising a poslive-strandcd RNA viras core-like anicn. protein joined to an amino-terminal pwiion or an adjacent protein of sad ;ositive-standed. RNA vuiris in unprocessed form, whcreizL said amio-terminal portion or said adjaoezt protein is sized such that said polypeptide has an ejpitopic configus-ation =ConSPOr14in to an unprocessed core-like antigen-adjacent protein of said positive-strald.~ RNA virus, bomnd to a solid substratc, 11. The composition of clairn 10 further comnprising a nonrucri~l protein of said positive-=nrnded UlNA virus bound to said solid subsirate. 12. An assay for the detection of a positive-smraded RNA virus in a samnple. cornpriuing: a) providing an. isolatod polypeptide conmprising a positivi-stranded lINA virus core-like antigen protein joined to an amino-=eminal portion of an. adjaczt protein of said posittve-stranded RNA virus in unprocessed form, whcrein said amino-tennina1 portion of said adacent pfotcin is sized such. that said polypepride has an *ptopic configuration crresponding to an unprocessed core-like antige-adjacent Protein or said positive-strandod RNA virus, b) contacting said isolated polypeptide with said samqple under conditions suftable and for a timne suffcient for said polypcptide to bind to one or =ore antibodies specific for 3aid positive-~trarided RNA virus pre-sent in said sample, to provide an antibody-bound polypeptid;, and c) dctecting Wad antibody-bound polypeptide and thcrfro-= determiaing that saia sample contains Positivc-mtrnded LNA virus. 13. The assay of claimn 12 furd= comprisin, a) in step providing a nonstruetuai protein of sad positive-strandcd RNA virus bound to said solid substrate, b) in step contacting said nonstructural protein with said samnple under conditions suitable and for s time suffiilanE for said nonstructural plotein to bind to orza of more antibodies specific for said positivc-straudcd RNA virus present in said sample, to provide an antibody-bound positive-strnded RNA virus nonstructural. protein and

74- c) in stcp detecting one or both of said antibody-bound polypc-ptide or said antibody-bound nonstmumlra protein, and dmtrfr detcunining th~at said samnpto contains sad positiv-staz~ded RNA virus. 14. An assay for the detectionL of a Positive-shraned RNA virus in a SazPIpC, comprisins. a) providing an isolated poiypeptide coaqpi sing an isolated, substantially complete, Improcessed polypwoEdu from a pasitiv-str~mded RNA virus, b) contacing said isolated polypotide with said sample undcr conditions auitablo =d for a timie m fcia for said polypcp~ide to bind to ore or more antibodios specifc for said posjtivc-standd RNA virus prwset in said sample, to provide aa antibody-bound polypeptide, and cdetectin~g said antibody-bound polypeptide, and therefrom determining thaL -said samnple contaias. said Positive-stranded RNA virus. IS. The assy of claim 12, 13 or 14 further compiising th.e. step of binding said isolated polypeptjde, said norstructu-al prolain, or said polYProteimk to a solid substrate. 16. The assay of clam 12;. 13 or 14 wherein said zwmplc is an unpurified sample. 17. The assay of claim I2, 13 or 14 Aizthcr comprising. prior to said contacting, the step of obUIniag maid samaple from an anbnaL- 18. Theassay of Waim 1 7 wherein said animal is ahwun=bein. 19. The assay of claiin 12, 13 or 14 wherein said assay is selected from the group consisting of a c uatetcurrcnt immuno-olecixophorsis (ClIE?) assay. a radlioimmoassay, a radioimzn'noprecipitation, an czymc-1mked immuno-sorbant awsay (ELISA), a dot blot assay, an inhibiiion or competition assay, a sandwich assay. an irnmunostick (dip-stick) assays. a simultLneus assay, an inuncchromatographic assay. an imlmunofiltration assay, a Waex bead agglutia assay, an irnnmunafluorasccnl ssay, a biowensr assay, and a low-Light dEtection assay. 75 The amsy of claim 12, 13 ox 14 wherein said assay is riot a west=m blot assay. 21. A method of prodtwinig an azaibOdy, comprising the follOwinz' Steps: a) dmninisteing to an~ aumal an isolated polypeptide comprising a positive-svanided RNA vimu core-like wnigen protein joined to aw =iia-tenina1 portion of an adjaccal protein of said positiv-strmded RNA virus in unprceszed form, wherein said arnino..tczninal portion of sAid adjaenL, proten is sizod such thiat raid polypeptide has an apitopic; con±&guration corcpanding to an unproessed corc-Iike anxigen-adacent protein of said pos-itive-stranded RNA virus, and b) isolating said antibodies to said polypetide. 22. An antibody produced according to claim 21. 23. A method of producing an antibody, comaprising tbte following a) administering to an animai =u isolated polypeptide comprising am. isolated, substantially complete. unprocassed polyproteint from a positv-staded RNA virus, and isolating said antibodies to said polyprotein. 24. An antibody produced according to claim 23. The &itibodies of claim 22 or claim 24 whcreirn said antibodies axe bound to a solid substrate- 26. An assay for the datertion of a. poaitivc-3wanded RNA, virus in a sample, COMPrIsig: a) contaig said sample with the antibody of claim 22 under conditions suitable and for a tima suffcient for said antibody to bind said uriprocassod positive-stranded RNA virus core-like anti&=n protcin to provide abound antibady, and b) detecting said bournd andibody, and thcrfrotn determidning that sad sample coatains positivc-strwided RNJA virus. 27. The assay of claim 26 further comiprising, 76 a) in step contatig sa~id sample wkith a futher &utibody specifc far a Positivc-trandcd RNA virus =mostrucrural protein under conditions si~dtable id f-or a time sufIcicat for said further antibody Io bind said Posffive-straded RNA virus nonstructural Protein, to provide a bound further antibody, and b) in step dctectlIi one or both of said bound antibody or said bound Anrther antibody, and therehro detcminiiag that said sample contiins positive-stra~ded R~NA vi=u. 28. An assay for the dtection of a positive-stranded RNA virus in a A) contacting said vinple with the- antibody of Clam 2-4 =nder 9. conditions suitable and for a time sufficient for &aid enibody to bind an antigerL speciflo for said positiv I-stxunded RNA yinis, to provide a bound antibody, and 0) deeoctng Wad. bound antibody, and therefrom dotennining that sald samPle COfltans Positive-SUraded F1NA virus. 2.A composition capable: of eliciting an immune response in anl 3lO21 compriing an iSclatd polypeptide comprising a. positive-suan4cd RNA vi=~ .cOrc-Jile anlgca Protcin Joined to an amno-tzrninal ponion of an adjacent Protein of -said positive-tranded RNA virus in Unproccssed S) whexcin said wnino-teIminai ~POrion Of said 9awcnt Protein is sized such dwa said polypaptidc has an epitopic 9$ coufuratiOn corresponding to an unprocessecd czzrr.ik* =dtgen-adjacent protein of Raid PositivP-stranded RNA virus, in ombiztation with a, pbaznaeuticahly acceptable carrier or diluent. The composiTion of cLm 29 firthcr coumprising a zonstructural piotcin from said Positvc-stranded RNA virus. 31- A composition capable of eliciling an immune zcsponse in an ani=4a comprising an isolated substmitially Cotnplet4, unprocesscd potyprotein from a Positlvc-Strandcd RNA virus, in combination with a phunaceticafly aceptable carrier or dilu=n 32. The composition of claim 29, 30 or 31 wherein said animal is a human being- 77 33. A vaccine againt a positive-stranded XRNA virus comnprisinig an isolated polypeptide comprising positive-straded R~NA virus core-like. antigen protein joined to an amino-terminal portion of an adjacen protein of said Positive-stan-ded RNA virus in uuproccsscd fonn, wherein sad amino-tcrnina portion of said adjaccin protein is s&ed such that said polypeptide has an cpitopic configuration coirspondirig to an. unprocessed core-like aaitgen-adj scet protein of said positivie-stranded RNA virus, in cornbintioxi with a phamacctically acceptable carrier or diluent 34. A vaccine again~st a positivc-=taniied R~NA. vi=u comprising an isolated, substuntially cotoplete unproeasscd poInproiein from a positive-stranded RWA virus, jo. combination wit a pharroaceutizally =ceptable carrier or diluen. :35. The vaccine of claim 33 or 34 furthr comprising a nonstructumJ Protein from -said positive-stranded RNA vimus 36. A kit for the detection of a positivo-strandcd RNA *virus comprising; a) an isolatod polypeptide compeising a positive-stranded RNA virus core-like antigen protein joined to an azrinzo-terminal portion of an 4dacent pwozein of Said rosirlvc-Strndcd RNA virus in unprocessed fbm, wherein said =~in -te~ia Portion Of said adjacnt pirotmin is sized Such. that sgid polypcptide lhu an epitopic ~confivurntiori corresponding to an unproesed ooze-like antigen-adjcMe protein of said posifive-stranded RNA virus, bouind to a solid subsrate, and b) one or both of a. reagent or a device for dctecting said isolated Polypeptidc. 37. The kit of claim 36 furth= comrnpising a nonstructuzal protein fromn said positive-st-anded RINA virus and one or both of a reagent or a dovicc for detcctiag said nonstructural protein. 38. A kit for the dctection of a positive-stxandud RNA. virus Comprising: a) an isolaed substantially complmt, unproccised polyprotein from a positive-stranded RNA vimu, bound to a solid subscr, =md b) one or both of a reagent or a. device for detctting said isolated polyprotain. 78 39. A it for the dctccioa of ai pasitivce-st-andcd RNA v" uwi comprising: a) the antibody of dailm 22, and b) one or both of a rcag=n or a device for detecting said aaTibody. The kW of clai= 39 further coxnprisizng a &-a=he anibody Specilic for an~ IfCV nonmuncnirai protein and Oo of bath of a renat or a device for deecing s9ad finthe antibody. 41, A it for the detection of a pobitivo-stranded RNA virus :comprising: the antxbody of cluimn 24, arid b) one or both of a reagenrt or a davico for detctidng said antibody. 42. A Pozitive-Stazdcd RNA viru%-derived composition coxnprising the following: a) an isolatd palypeptide comprising a positive-straned RNA virus Core-like antigon Ptotein joined to an act protein of said poditivo-strandcd P~A virus in unproccssed foim, whcrein said adjaccn pzotcin is siwd such ihat said Polypeptide has an opiropir conliguration coxrsporiding to an utprocesed core-lI.ke axxtgeu-aac=n protein of wid Positive-stranded RNA virus; and b) a second. protein caprable of cooperatively intez'actng with said isolated polypeptidc to iucgease the antigenciiy of said isolated polypeptide. 43. A method of makinig a comnposition comzprisigg multiple polypcptides' Comprising the foll-owing Steps: a) inoducing I=t a fint host cell a fimt expression vector capabic of expressing a nuclec. acid molcule ancoding an isolated polyrpepd& compxising a Positive-standed RNA viius coro-like antien protein joincd to an adjacent prorain of said Positivo-straiidd RNA virus in unprocssed form, wberein said adjacent protcin is sized such that said polypeptide ha~s an epitopic configmrtion corrcspoiding to an unprocessed core-likc ant gen-adjaeeiiz protein, of said pasitivoeatrAnded RNA virus. b) incubating said first host cell under conditions suitable Ibr said exptession vcctor to produce said polypeptid.1e, a) prif'ing sold polypqptide io provide a purified polypcptide, and 79 d) ianrducing int a sccand host call a scond Oxression capable of expressing a nucleic acid molecule enicoding a second isolaed. protein capable of coaperadvely interacting with said isolated polypetide to increase the antigenicity r) F said isolated polypeptidc, e) incubating said second bost ccli under conditions suiLable, for said nucleic acid raolecule to produce said second protein, t)puriI~ing said second protein to provide an pored scond protein. and theit com~bining said purified polypeptide and said purified secon~d protein to form said composition. *44. A method of ma~king' a coMupositior comnprising mnultiple Plpcptios% ad ]cast one of~ whicb is obtained rom a positive-straded RNA virus, comnprising the following steps: a) introducing into a host ccIl an expression vcto capable of epessizag a first nucleir. acid molcecoding an iolated polypeiflide corisin a positive-stranded RNA vinus core-Like antigen protein joined to an adjacet protein of said positive-stranded RNA virus i tmrcse form, wlzmcin said anacent protein is ~Szedc such tha said polypeptide I=s ani epitopic coiuration corresponding to an unprocessed coEke antigen-adjaccat protein of said positive-stranded I~A virussi exrsin vector als capable ofepesn a secondL prUoteia caal of cooperilVy interacting with said isolated polypeptide to incras the antigenicity of said isolaied polypeptide. b) incubating said host cell tndr conditions suitable: for said expression vc-tor to produce said polypeptide aad said secnd prwc, and c) purifYirig said polypoptide a nd said second protein to provide a composition comprisig a purified polypeptide and a purified second proteiD. The method of claim 43 or 44 wherein said second prmrLa is detived from a positivc-stra~dd RNA virus. 46. A composition, comprising an isolated polypetide Qonmp~isirng a positive-stranded RNA virus core-like antigen protein joined to an. adjacent protein of said posidive-strandcd RNA virus in unprocessd form,~ wherein said adjacnt protein is sized such that said polypeptidc has an epitopic ccanflguration. coxrespon~ing to all 80 uoprocessed, coe.like anigen-adjacent prtein. of said pstvo-standed RNA vi=, bound to a solid substxate. 47. The composition of claim 46 I'urtber coqprising a scwnd proteirn capable of cooperatively iftcrating with -saicl isoLated polypeptide to increase tho andigwicity of said isolated polyetide. 48. ,An assay for &h detection of a positive-stande RNA virus in a sampe; comnprising: a) providing an. isolaWe polypaptidc comtprising a positive-stnded RNA vi=u cozo-4ike antigen protein joined to e4ae-at protein of said positive-sraded RNA vi=u in iprocessed form viberein said adja~cut proteix is sized such tha said PolYpeptide has an epitopic conflgurtion corresponding to an uapwwocsd coromlike anTigen-*dJaccat protein of maid positivc-s-tranded RNA viniS, b) contacting said isoltd polyptptdc -with sad sample under conditions su.itable and for a time sixfficianL ror said polypeptide to bind* to one or more arabodies specific for said positive-stradd RNA vizrz prsent in said sanple to provide ala awibody-bowid polypeptide, and C) dtc~ting Said antodyl-bound polypeptido, and Lherefront datmiwjing that said samnple contains said Positive-sou Rrna NA vinz. *aa49. The assay of claim 49 firther conmpnsmig, a) in step providing a second proten capable of cooperativey ntacing wit said isolated polypeptido to idcrase the antigenicity of said isolated polypeptide. b) in step Contactinig said second protein with said sample under conditions suitable and for a time Suxfficient for said second protein to cooperaiively intcract with said isolafed polypeptide, and in step detecting bound antibodies, and Lheccfrumn determining that said sam~ple contains said positive-sftnded RNA vinis. The- assay off cULn 48 or 49 fuxtlr con prisig the Step of binding said isolatcd polypcptido, or said second protein to a solid substrate. 51. A =nthod of prducing an antbody, comprising -thc foUowing steps: 81 9* a a a. a a a a. a a 9t*a a) admnistering to an animal an isolated polypeplide comprising a posifivc-stra.fdcd RNA virus core-Uikt aniigeu Protein -joined to an adjacent Prntein Of said positivc-sanwndd RNA virus in Uaprocelsd frmr, whmrin said adjaent protein is sized such that said pqlypcptide has an epitopir, configuratioa con7evpofdiftg to an unprocessed core-like antigen-saacmt Protein Of said positivc-sftnad~d RNA virus, and b) isolating said antibodies to said polYpepide. 52. The rrmhad of claim, 51 fultbrx comprising adinistering to said animal a second protein capable of eooperaiivelY intraitiDS with said isolated polypeplide to increase the antigenicity of said isolated polypetide. ,53. -An antibody producd according to claim 5 1 ccr52, 54. Antibodies produced according to claim 5 1 or 52 whercin said antibodies are bouad to a solid substrafe.. 55. An assay for the detection of a positive-stranded RNA vizus iM a sample, comrisinlg- a) contacting said sauple with an antibody produced accordinig to claim 51 or 52 under conditions suitable and for a time sufficken for said anibody to bind said unprocessed positive,-strandcd RN4A virus core-like antigen protein, to provide a bound antibody, and b) deiecting said bound n=tibody, and therefrom delerriin that said sample contains positive-standed RNA virus- 56. A composition capable of cliciting an immune response In an animal conmprising an isolated polypcptide comprising positive-strandod R~NA vitus core-lik, antigen prptein joined to aa adjacent protein of said positive-strandod RNA virus in unprocessed forma, wherei said adjacczit protein is sized such that said polyppptidc has an epitopic =ofiguration corresponding to an unprocessed core-lika anti~f.n-aAjaeerit protein of sald posifive-stranded RNA virus, in combination with a. pharmaceutically acceptable carrier or diluent. 51. The compositon ofc imr 56 further comaprising a second protcin. capable of cooperadwvly intmrcfing with said isolatod polypeptide to increase the antigenicity of Wad isolated polypcptide. 82 59. A vac agu~st, a Positiv.sraudcd RNA vini comprising an. isolaWe POlYpeptide comprising a positive-strandcd RNA virus ccre-like antigca plotiin joined to an adjucet protein of ,Aid positive-stnuided RNA virus in unprocessed form, Wherein said adjacent protein is sized such Tha said polypeptide has an epitopic con flgmvaioni corresponding to an unprocessed core-liko antigen-adjaccmt protein of said poaitive-3tranded RNA vinis, in combinalion. with a pbannacmuicAly acceptadble carricr or dilunL 59. The vaccinc of claim 58 further coutprsing a Secornd Protein ~CaPablc Of coOPCraivcly interacting with said isolated polypeptido to increase te antigenicity of sad 1SOJAted polypaptide. ,TI= composition of any one of claims 1-5. 9-11, 2-32, 42. 46. 471, a 56 or 57 for us* as an aclive I rapautic substancc. active therapeutic substanc. 62.- The composition Of azw ono of claims 1-5, 9-11, 29-32, 42, 46, 47, :56 or 57 for use in the anLfutwI*c of a radjaffe=l for inhibiing, preventina or treating **HCV ixiIection in an anim-al, 63. Tho vaccine of aay one or claims 33-35, 58 o~r 59 for use in the Mnufacturt Of a zncdicazent for iabibiting, preventing or frcartx RCV inftcion in an animal. 64. A kit for the detection, of a positive-sizjded RNA vitvs comaprimIng: A) an Jsolated polypeptide comprising a posttva.,stxanded RNA virus core-like =x~igen protein joined to an adjacont protain of maid posifive-sirandcd RNA vius wherein said adjacent protein is sized such that said polypeptide has an' epitopic configmation corresponding to an unprocessed co-flkc anigen-4d=cnt protein. of said positive-stranded RNA vinw, bound to a -solid substrate, and b) one or both of a reagent or a devie for dctcctin~g said isolated p olypeptide. 83 The it of claim 64 further comaprsng a seconld protein capable of cooperatively interacting with said isolated polypeptida to incrase !he antige' of said isolated polypeptido and oile or both of a reagent or a davice for detecting Saidt second protein. 66. A kit for the dmction of a posizlve-straded RNA viu's cmpftaig-, a) an anfibody produced according to clam 51 or 52, and b) one or both of a rcagcot or a devico for detecting said antibody. 67. The assay of claim 12. 13, 14, 48 or 49 whereia. the step of providing comprises providing at leasut two isolatcd potypeptides. at least two of which are obtained from different positive-stmnded.RNA viruses ieleced from the group ~consisting. of Hepatitis C virus (IICV), Huzna lImmodeiiciency virus (~1)axad Humnan T--cUl Leukemija virus (HTLV), and wberein the step of contacting comprises cortactiiag the isolazcd polypeptides with said satuple under conditions suilable and for a time suffcient for each of raid isolated polypeptides to bind to one or more antibodies specific therefor. thereby providing one or mome antibody-bound polypepd4.s. 68. The assay of claim 12, 13, 14, 48 or 49 wherefin the step of providing comprises providing at least thre isolated polypeptides, at Last three of which are fromn diffcret positive-straddRNA viruses selected from the group consisting of Ilepatitis C virus (HCV), kluman Immnunodeficiency virus (HiV) and Humann T-*~U Leukemia virus (W4TV), and wher~n the step of contciZcomrises contacting the isolated polypepti des with said sample under conditions suitablc and for a time sufficiet for each of said isolatod polypeptides to bond to one or more antibodies specific, Ihcrcfbr, thereby providing one or more antibody-bound polypoptides 69. The kit of claim 36, 37, 64 or 65 whczein said kit comprises a) at least two, of said isolated polypeprides fronx at 1cast two different positive-stranded RNA -84- A composition according to any one of Claims 1 to 5, 9 to 11, 29 to 32, 42, 46, 47, 56, 57, 60 and 62 or a method according to any one of Claims 6 to 8, 21, 43 to 45, 51 and 52 or an assay according to any one of Claims 12 to 20, 26 to 28, 48 to 67 and 68 or an antibody according to any one of Claims 22 to 25, 53 and 54 or a vaccine according to any one of Claims 33 to 35, 58, 59, 61 and 63 or a kit according to any one of Claims 36 to 41, 64 to 66 and 69 substantially as hereinbefore described with reference to the accompanying Figures and/or Examples. oo DATED this thirty-first day of August 2000. oo BioNova Corporation by DAVIES COLLISION CAVE Patent Attorneys for the Applicant ft ft. ft. *t ft *t 6