MXPA97009271A - Diagnosis of, and vaccination against, in positive thread rna virus using an isolated polypeptide, do not process - Google Patents

Abstract

The unprocessed polyprotein translated from the genome of a positive strand RNA virus contains epitope configurations that are not retained in the processed proteins, the structural protein region, in particular, loses an epitopic configuration when processed at the site of separation between the region. genomic coding for the core protein and the genomic region that encodes the protein adjacent to the core protein, such as the protective protein in HCV. The compositions, methods and analyzes that relate to the diagnosis and detection of the presence of the positive strand RNA virus or antibodies to the positive strand RNA virus in a sample. The compositions and methods for the induction of immune responses in, and vaccination of, an animal. The combination of the unprocessed core region with a non-structural protein (such as an NSS or an unprocessed NS3-NS4 fusion of HC

Description

DIAGNOSIS OF, AND VACCINATION AGAINST, A POSITIVE THREAD RNA VIRUS USING AN ISOLATED, UNPROCESSED POLYPEPTIDE Technical field The present invention relates generally to methods and compositions for the highly sensitive, highly specific diagnosis of a positive strand RNA virus. The methods and compositions are also suitable for the extraction of an immune response in an animal, and for the vaccination of an animal, against a positive filament RNA virus.

BACKGROUND OF THE INVENTION The acquired immunodeficiency syndrome (AIDS) is caused by a group of retroviruses known as HIV (Barre-Sinoussi et al., Science 220-871, 1983; Gallo et al., Science 224: 500-503; Coffin et al., Science 232: 697, 1986). The first member of the group has been designated HIV-1 and is responsible for a majority of AIDS cases around the world. It is distinguished from HIV-2, an isolated Waf isolate (Clavel et al., Science 233: 343-346, 1986). Although HIV-2, like HIV-1, produces symptoms of immunodeficiency in man, it is also genetically distinct from HIV (Guyader et al., Nature 326: 662-669, 1987). The genomes of 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 normally 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 reported (Courouce et al., AIDS 2: 261-265, 1988). Therefore, the demand for sensitive and specific methods to detect HIV in blood or contaminated blood products is important. The AS, based on complete virus or viral lysate, have been developed for the detection of HIV. However, it has been found that the ElAs have an unacceptable non-specific reaction with specimens of 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-H IV-2 in samples, an ELISA has been developed and marketed by Abbott Laboratories for serological diagnosis of HIV infection using the HIV-1 core proteins and envelope proteins of HIV-1 and HIV-2. However, this ELISA has not provided the highly sensitive and highly specific detection necessary for the superior protection of the blood supply, or for early diagnosis of HIV in a patient.

In this way, in order to provide a better diagnosis of HIV in a patient, a need for products and methods capable of highly sensitive and highly specific detection of HIV remains unfulfilled. A need also remains for products and methods capable of extracting 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 strand RNA viruses also present significant health risks throughout the world. An example of such positive strand RNA viruses is Hepatitis C virus (HCV). HCV is distinguishable from other forms of liver diseases associated with viruses 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 (ie, hepatitis A, not B (NANBH)) accounts for up to 90% of these cases. An important problem that arises from this disease is the frequent progression of chronic liver damage (25-55%). Consequently, the demand for sensitive, specific methods to detect HCV in blood or contaminated 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 description of the HCV genome, and some of the cDNA sequences and polypeptides derived therefrom, as well as the methodologies that relate to such a subject, is provided in EP 0 318 216 Al in the name of Chiron Corporation. In particular, this disclosure provides a synthesized, C100-3 polypeptide, containing 363 virally encoded amino acids that can be used for the detection of a type of HCV antibody. At present, kits for detecting HCV antibodies at the base of the C100-3 antigen have been marketed by Abbott Laboratories. As suggested in EO 0 318 216 Al, HCV can 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 the hepatitis C virus protein is composed of structural proteins including a nucleocapsid (core) protein (C), two glycosylated envelope proteins (E1, E2) and several non-structural proteins (NS1-5). It has been confirmed that C100-3 described by Choo et al., Is a protein encoded by non-structural regions 3-4 of the HCV genome. It has been found that anti-C100-3 antibody is not detected in all cases of post-transfusion NANBH. Failure to detect anti-C100-3 antibody is possible due to hypermutation of the nucleotide sequence in the C100-3 region. In addition to working with the non-structural C100-3 antigen, an enzyme-linked immunosorbent assay (ELISA) has been developed for the 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. Nati, Acad. Sci. USA 88: 4641-4645, 1991). Thus, the Chiba et al. Trial used a non-glycosylated 22-kDa nucleocapsid protein (nucleus) in an effort to establish a sensitive, specific, antibody-based 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 strand RNA viruses, a need for products and methods capable of highly sensitive, highly specific detection of HCV remains unmet. It also remains unfulfilled, as with other positive strand RNA viruses, a need for products and methods capable of extracting an immune response to HCV, especially an immunoprotective immune response for HCV. The present invention provides these and other related advantages.

SUMMARY OF THE INVENTION The present invention is directed to the concept that a whole unprocessed polypeptide (s) (e.g., a polyprotein) or a partial polypeptide (s) unprocessed in the structural region and proteins of the non-structural region of filament positive ((+) - filament RNA viruses) can provide superior antigenicity and consequently improved detection and diagnosis of a positive filament RNA virus in a sample. The present invention also provides improved immunoactivation, including an improved immunoprotective response of an animal. In accordance with this, in a first aspect the present invention provides compositions derived from positive strand RNA viruses comprising a substantially complete, unprocessed polyprotein isolated from a positive strand RNA virus. In an alternative aspect, the present invention provides compositions derived from positive strand RNA viruses comprising the following: a) an isolated polypeptide comprising an antigen protein similar to the nucleus of positive strand RNA virus bound to an amino-terminal portion of an adjacent nucleic acid region of the positive strand RNA virus, wherein the amino-terminal portion of the adjacent nucleic acid region is sized such that the polypeptide has a specific epitope configuration for an adjacent nucleic acid region similar to the nucleus unprocessed positive strand RNA virus (this polypeptide is sometimes referred to herein as an "adjacent antigen protein similar to the nucleus"); and b) a non-structural protein isolated from the positive filament RNA virus. As discussed below, the full description of this application with respect to the adjacent nuclei-like antigen protein generally applies equally to a positive filament RNA virus env protein, whose env protein typically comprises at least one non-binding junction. process with an adjacent protein.

In the preferred embodiments that relate to each of the aspects of the present invention, the positive filament RNA virus is selected from the group consisting of Togaviridae, Coronoviridae, Retroviridae, Picornaviridiae, Caliciviridae and Flaviviridae, in addition to the group preference that consists of Hepatitis C virus, human immunodeficiency virus (HIV) and human T cell leukemia virus (HTLV). Unless otherwise specified, preferred embodiments refer to each of the aspects of the present invention. Alternatively, the positive filament RNA virus is any positive filament RNA virus other than HCV. In other preferred embodiments, the composition is produced by any suitable prokaryotic host cell, usually a bacterial, and preferably a BL21 from E. coli (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 for making a composition comprising a substantially complete, isolated, unprocessed polyprotein of a positive strand RNA virus. This aspect also provides a method for making multiple polypeptides obtained from a positive strand 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 a isolated polypeptide comprising an antigen protein similar to the nucleus of positive strand RNA virus bound to an amino-terminal portion of an adjacent nucleic acid region of the positive strand RNA virus, wherein the amino-terminal portion of the region of adjacent nucleic acid is sized such that the polypeptide has a specific epitope configuration for an adjacent nucleic acid region similar to the unprocessed nucleus of the positive strand RNA virusb) 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 a non-structural protein isolated from the positive filament RNA virus, e) incubating the second host cell under conditions suitable for the nucleic acid molecule to produce the non-structural protein, f) purifying the non-structural protein to provide a purified non-structural protein, and then g) combining the purified polypeptide and the purified non-structural protein in the composition. In a preferred embodiment, the method further comprises a) introducing into an appropriate host cell an expression vector capable of expressing a first nucleic acid molecule encoding an isolated polypeptide comprising an antigen protein similar to the RNA core of positive filament RNA bound to an amino-terminal portion of an adjacent nucleic acid region of the positive strand RNA virus, wherein the amino-terminal portion of the adjacent nucleic acid region is sized such that the polypeptide has a specific epitope configuration for a adjacent nucleic acid region similar to the unprocessed nucleus of the positive filament RNA virus, b) incubate the host cell under conditions suitable for the expression vector to produce the polypeptide and the non-structural protein, and c) purify the polypeptide and the protein non-structural to provide a purified polypeptide and a protein a non-structural purified. In another preferred embodiment, the method further comprises ligating the inventive polypeptide (s) to a solid substrate. In a further aspect, the present invention provides a composition comprising the substantially complete, isolated, unprocessed polyprotein of a positive strand RNA virus wherein the polyprotein is bound to a solid substrate. Alternatively, the composition comprises the adjacent antigen-like protein to the core linked to a solid substrate, preferably further comprising a non-structural protein of the positive strand RNA virus linked to the solid substrate. In another preferred embodiment, an assay for the detection of a positive filament RNA virus is a sample, comprising: a) providing an isolated polypeptide comprising an adjacent protein of antigen similar to the positive strand RNA virus core, b) placing contacting the isolated polypeptide with the sample under suitable conditions and for a time sufficient for the polypeptide to bind to one or more antibodies specific for the positive filament RNA virus present in the sample, to provide an antibody-bound polypeptide, and ) detecting the antibody-bound polypeptide, from there determining that the sample contains RNA virus of positive filament. In an alternative embodiment, the method comprises, a) providing an isolated polypeptide comprising a substantially complete, isolated, unprocessed polyprotein of a positive filament RNA virus, b) contacting the isolated polypeptide with the sample under suitable conditions and for a sufficient time for the pellipeptide to bind to one or more antibodies specific for the positive filament RNA virus present in the sample, to provide an antibody-bound polypeptide, and c) to detect the antibody-bound polypeptide, and thereby determine that the sample contains RNA virus of positive filament. In a preferred embodiment, the method further comprises a) in step a), providing a non-structural protein of the positive filament RNA virus bound to the solid substrate, b) in step b), contacting the non-structural protein with sample under suitable conditions and for a sufficient time for the non-structural protein to bind to one or more antibodies specific for the positive filament RNA virus present in the sample, to provide a non-structural protein of antibody-bonded positive-strand RNA virus, and c) in step c), to detect one or both of the antibody-bound polypeptide or the non-structural antibody-linked protein, and thereby determine that the sample contains positive filament RNA virus. In another preferred embodiment, the assay further comprises the step of ligating the isolated polypeptide, the non-structural 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. In still other preferred embodiments, the assay is selected from the group consisting of a counter-current immunoelectrophoresis (CIEP) assay, a radioimmunoassay, a radioimmunoprecipitation, an enzyme-linked immunosorbent assay (ELISA), a dot blot assay, a inhibition or competition assay, a "sandwich" assay, an immunoadhesion test ("dip-stick"), a simultaneous assay, an immunochromatographic assay, an immunofiltration assay, a latex bead agglutination assay, an immunofluorescence assay, a biosensor assay, and a low light detection assay. Moreover, the preference test is not a "western blot" test. In yet a further aspect, the present invention provides a method for producing an antibody, comprising the following steps: a) administering to an animal an isolated polypeptide comprising an antigen protein similar to the nucleus of positive strand RNA virus bound to a portion Amino-terminal of an adjacent nucleic acid region of the positive strand RNA virus, wherein the amino-terminal portion of the adjacent nucleic acid region is sized such that the polypeptide has a specific epitope configuration for a nucleic acid region adjacent to the unprocessed nucleus of the positive filament RNA virus; and b) isolating the antibodies to the polypeptide. Alternatively, the invention provides a method for producing an antibody, comprising the following steps: a) administering to an animal an isolated polypeptide comprising a substantially complete, unprocessed polyprotein isolated from a positive strand RNA virus, and b) isolating the antibodies to the polyprotein.

The present invention also provides the antibody produced according to any of the above methods, as well as an antibody to the other proteins described herein (such as non-structural proteins). Preferably, the antibodies are ligated to a solid substrate. In still another aspect, the present invention provides an assay for the detection of a positive filament RNA virus in a sample, comprising: a) contacting the sample with one or more of the antibodies described above under suitable conditions and for a period of time Sufficient time for the given antibody to bind its antigen protein, to provide a bound antibody, and b) detect the bound antibody, and hence determine that the sample contains RNA virus of positive filament. In a preferred embodiment, the sample is an unpurified sample, typically from an animal, and preferably from a human. In still other preferred embodiments, the assay is selected from the group consisting of a counter-current immunoelectrophoresis (CIEP) assay, a radioimmunoassay, a radioimmunoprecipitation, an enzyme-linked immunosorbent assay (ELISA), a dot blot assay , an inhibition or competition test, a "sandwich" test, an immunoadhesion test (dip-stick), 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. Moreover, the preference test is not a "western blot" test. In yet a further aspect, the present invention provides a composition capable of extracting an immune response in an animal comprising an isolated polypeptide comprising an adjacent antigen protein similar to the nucleus of positive filament RNA virus, in combination with a pharmaceutically carrier or diluent. acceptable. Preferably, the composition further comprises a non-structural protein of the positive filament RNA virus. In an alternative aspect, the composition capable of extracting an immune response in an animal comprises a substantially complete, isolated, unprocessed polyprotein of a positive filament RNA virus, in combination with a pharmaceutically acceptable carrier or diluent. Preferably, for each of the immuno-active aspects (as well as the other aspects) of the invention, the animal is a human being. In yet a further aspect, the present invention provides a vaccine against a positive filament RNA virus comprising an isolated polypeptide comprising an adjacent antigen protein similar to the nucleus of positive strand RNA virus, in combination with a pharmaceutically carrier or diluent. acceptable. Preferably, the composition further comprises a non-structural protein of the positive filament RNA virus. In an alternative aspect, the vaccine against a positive filament RNA virus comprises an unprocessed polyprotein, substantially complete of a positive filament RNA virus, in combination with a pharmaceutically acceptable carrier or diluent. The present invention also provides a method for inducing an immune response in an animal comprising administering to the animal an isolated polypeptide comprising an adjacent protein of antigen similar to the nucleus of positive strand RNA virus, in combination with a pharmaceutically acceptable carrier or diluent. Preferably, the method further comprises administering a non-structural protein of the positive strand RNA virus. In an alternative aspect, the method for inducing an immune response in an animal comprises administering to the animal, a substantially complete, unprocessed polyprotein isolated from a positive strand RNA virus, in combination with a pharmaceutically acceptable carrier or diluent. The present invention further provides a method for vaccinating an animal comprising administering to the animal an isolated polypeptide comprising an adjacent protein of antigen similar to the core of RNA virus of positive filament., in combination with a pharmaceutically acceptable carrier or diluent. Preferably, the method further comprises administering a non-structural protein of the positive strand RNA virus. In an alternative aspect, the method of vaccinating an animal comprises administering to the animal a substantially complete, isolated, unprocessed polyprotein of a positive strand RNA virus, in combination with a pharmaceutically acceptable carrier or diluent.

In still another aspect, the present invention provides an assembly for the detection of a positive filament RNA virus, the assembly comprising a) an isolated polypeptide comprising an antigen protein similar to the nucleus of positive strand RNA virus bound to a portion Amino-terminal of an adjacent nucleic acid region of the positive strand RNA virus, wherein the amino-terminal portion of the adjacent nucleic acid region is sized such that the polypeptide has a specific epitope configuration for a nucleic acid region adjacent to the unprocessed nucleus of the positive strand RNA virus, bound to a solid substrate, and b) means to detect the isolated polypeptide. Preferably, the set comprises a non-structural protein of the positive strand RNA virus and means for detecting the non-structural protein. Alternatively, the set for the detection of a positive strand RNA virus comprises a) a substantially complete, unprocessed polyprotein isolated from a positive strand RNA virus, bound to a solid substrate, and b) means to detect the polyprotein isolated. In an alternative aspect, the present invention provides an assembly for the detection of a positive filament RNA virus comprising: a) one or more of the antibodies discussed above, and b) means for detecting the antibody (s). The assemblies may also comprise a) the composition capable of extracting 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 composition derived from positive filament RNA virus comprising the following: a) an isolated polypeptide comprising an antigen protein similar to the nucleus of positive filament RNA virus bound to an acid region adjacent nucleic of the positive strand RNA virus, wherein the adjacent nucleic acid region is sized such that the polypeptide has a specific epitope configuration for an adjacent nucleic acid region similar to the nucleus of the positive strand RNA virus; and b) a second protein capable of cooperatively interacting with the antigen protein similar to the nucleus of positive strand RNA virus bound to the adjacent nucleic acid region of the positive strand RNA virus to increase the antigenicity of the antigen protein similar to the nucleus of positive filament RNA virus bound to the adjacent nucleic acid region of the positive filament RNA virus. The present invention also provides a method for making such a composition comprising multiple polypeptides, including one or both of the polypeptides described above; the proteins can be derived from the same or different positive strand RNA viruses. The present invention also provides a composition comprising a first protein isolated from the positive filament RNA virus and a second protein isolated from the positive filament RNA virus (preferably from the same positive filament RNA virus), wherein the first and second proteins are selected, according to methods set forth below for other embodiments of the claimed invention, so that the first and second proteins provide a synergistic effect for the detection of positive filament RNA virus and / or immunointensification of an animal against the RNA virus of positive filament. The invention also provides the isolated polypeptide comprising an antigen protein similar to the nucleus of positive strand RNA virus linked to an adjacent nucleic acid region of the positive strand RNA virus, wherein the adjacent nucleic acid region is sized in a manner that the polypeptide has a specific epitope configuration for an adjacent nucleic acid region similar to the unprocessed nucleus of the positive strand RNA virus, bound to a solid substrate, either alone or in combination with a second protein capable of cooperatively interacting with the antigen protein similar to the nucleus of RNA virus. positive strand attached to the adjacent nucleic acid region of the positive strand RNA virus to increase the antigenicity of the antigen protein similar to the nucleus of positive strand RNA virus bound to the adjacent nucleic acid region of the strand RNA virus positive of the positive filament RNA virus bound to the solid substrate. In still another aspect, the present invention provides an assay for the detection of a positive filament RNA virus in a sample, comprising: a) providing an isolated polypeptide comprising an antigen protein similar to the nucleus of positive filament RNA virus linked to the adjacent nucleic acid region of the positive strand RNA virus, wherein the adjacent nucleic acid region is sized such that the polypeptide has a specific epitope configuration for an adjacent nucleic acid region similar to the nucleus of the RNA virus of positive filament, b) contacting the isolated polypeptide with the sample under suitable conditions and for a sufficient time for the polypeptide to bind to one or more antibodies specific for the positive filament RNA virus present in the sample, to provide an antibody-bound polypeptide, and c) detecting the antibody-bound polypeptide , and from there determine that the sample contains positive filament RNA virus. The assay may also comprise, a) in step a), providing a second protein capable of cooperatively interacting with the antigen protein similar to the nucleus of positive strand RNA virus bound to the adjacent nucleic acid region of the RNA of positive filament to increase the antigenicity of the antigen protein similar to the nucleus of positive filament RNA virus bound to the adjacent nucleic acid region of the positive filament RNA virus, bound to the solid substrate, b) in step b ), contacting the second protein with the sample under suitable conditions and for a time sufficient for the second protein to interact cooperatively with the antigen protein similar to the nucleus of positive strand RNA virus bound to the nucleic acid region adjacent to the positive filament RNA virus, and c) in step c), detect bound antibodies, and from there determine that the sample with It has positive filament RNA virus. In a preferred embodiment, the assay further comprises the step of ligating the isolated polypeptide or the second protein to the solid substrate.

In addition, preferably, the assay is selected from the group consisting of a counter-current immuno-electrophoresis (CIEP) assay, a radioimmunoassay, a radioimmunoprecipitation, an enzyme-linked immunosorbent assay (ELISA), a dot blot assay, an inhibition or competition test, a "sandwich" test, an immunoadhesion test (dip-stick), 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 it is not a "western blot" assay. The present invention also provides a method for producing an antibody, comprising a) administering to an animal an isolated polypeptide comprising an antigen protein similar to the nucleus of positive strand RNA virus linked to an adjacent nucleic acid region of the RNA virus. of positive strand, wherein the adjacent nucleic acid region is sized such that the polypeptide has a specific epitope configuration for an adjacent nucleic acid region similar to the unprocessed nucleus of the positive strand RNA virus, and b) isolate the antibodies for the polypeptide. The method may further comprise administering to the animal a second protein capable of interacting cooperatively with antigen protein similar to the positive strand RNA virus nucleus attached to the adjacent nucleic acid region of the positive strand RNA virus to increase antigenicity of the antigen protein similar to the nucleus of positive strand RNA virus bound to the adjacent nucleic acid region of the positive strand RNA virus. The present invention features an antibody produced as above, which antibodies can be ligated to a solid substrate. The antibodies can also be used in assays, also as described above. In yet another aspect, the present invention provides a composition capable of extracting an immune response in an animal comprising an isolated polypeptide comprising an antigen protein similar to the nucleus of positive strand RNA virus linked to an adjacent nucleic acid region of a virus. RNA of positive filament, wherein the adjacent nucleic acid region is sized such that the polypeptide has a specific epitope configuration for an adjacent nucleic acid region similar to the unprocessed nucleus of positive strand RNA virus, in combination with a diluent or pharmaceutically acceptable carrier. The composition may further comprise a second protein capable of cooperatively interacting with the antigen protein similar to the nucleus of positive strand RNA virus bound to the adjacent nucleic acid region of the positive strand RNA virus to increase the antigenicity of the antigen protein similar to the nucleus of positive strand RNA virus bound to the adjacent nucleic acid region of the positive strand RNA virus. Preferably, the composition is a vaccine. The present invention also provides a method for inducing an immune response in an animal comprising administering to the animal an isolated polypeptide comprising an antigen protein similar to the nucleus of positive strand RNA virus bound to an adjacent nucleic acid region of an RNA virus. positive strand, wherein the adjacent nucleic acid region is sized such that the polypeptide has a specific epitope configuration for an adjacent nucleic acid region similar to the unprocessed nucleus of positive strand RNA virus, in combination with a diluent or carrier pharmaceutically acceptable. Preferably, the method further comprises administering a second protein capable of interacting cooperatively with the antigen protein similar to the positive strand RNA virus nucleus attached to the adjacent nucleic acid region of the positive strand RNA virus to increase the antigenicity of the antigen protein similar to the nucleus of positive filament RNA virus bound to the adjacent nucleic acid region of the positive filament RNA virus. More preferably, the method comprises a vaccination. In another aspect, the present invention provides an assembly for the detection of a positive strand RNA virus comprising: a) an isolated polypeptide comprising an antigen protein similar to the nucleus of positive strand RNA virus bound to an acid region adjacent nucleic of the positive strand RNA virus, wherein the adjacent nucleic acid region is sized such that the polypeptide has a specific epitope configuration for an adjacent nucleic acid region similar to the core of the positive strand RNA virus, linked 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 antigen protein similar to the nucleus of positive strand RNA virus linked to the adjacent nucleic acid region of the positive strand RNA virus to increase antigenicity of the antigen protein similar to the nucleus of RNA virus strand positive to the adjacent nucleic acid region of the positive strand RNA virus and means to detect the second protein. Alternatively, the set for the detection of a positive filament 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 apparent from the reference to the following detailed description and accompanying drawings. In addition, as noted above, several references are set forth through the present specification that describe in greater detail certain procedures or compositions (e.g., plasmids, etc.); such references are incorporated by reference in their entirety.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1A shows the nucleotide sequence of a nucleic acid molecule encoding a polypeptide comprising an HCV core antigen protein linked to an amino-terminal portion of an HCV envelope region. Figure 1 B shows the amino acid sequence encoded by the nucleotide sequence shown in Figure 1A. Figure 2 shows the structure of the expression vector pEN-2, which was constructed by inserting a cDNA encoding an HCV core antigen protein to an amino-terminal portion of an envelope region of HCV in a plasmid. The figure also shows a restriction map that illustrates certain important features of the pEN-2 vector.

Figure 3A shows the nucleotide sequence of a nucleic acid molecule encoding a polypeptide comprising a non-structural region of NS5. Figure 3B shows the amino acid sequence encoded by the nucleotide sequence shown in Figure 3A. Figure 4 shows the structure of the expression vector pEN-1, which was constructed by inserting a cDNA encoding a non-structural region NS5 into a plasmid. The figure also shows a restriction map that illustrates certain important characteristics 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 strand RNA virus contains epitope configurations that are not retained in the processed proteins. In particular, the region of core protein (or another protein encoded by the viral genome that serves the equivalent purpose as the "core" protein) loses an epitopic configuration upon processing at the site of cleavage between the genomic region (e.g. gene) encoding the core protein and the genomic region encoding the protein adjacent to the amino-terminal end of the core protein, such as the envelope protein in HCV. As discussed below in the part of the Examples of the present disclosure, the unprocessed epitope configuration of the core region provides a surprisingly improved ability to detect the presence of positive filament RNA virus, or antibodies to the RNA virus. of positive filament, in a sample, including an unpurified sample or a very small volume sample (which can be particularly useful when testing a sample from an infant or another person who has very little blood (or other suitable material) available for evaluation Even more surprising, combining the unprocessed core region with a non-structural protein (such as an NS5 protein or an unprocessed HCV NS3-NS4 fusion protein) resulted in a synergistic effect that greatly enhances the already improved sensitivity and specificity provided by the unprocessed core region, these important advantages in antigenicity and epitope configuration also provides surprisingly intensified 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 a substantially complete, unprocessed polyprotein isolated from a positive strand RNA virus. The present invention also features compositions and methods utilizing an isolated polypeptide comprising the positive strand RNA virus core antigen protein linked to an amino-terminal portion of the adjacent positive strand RNA virus protein, wherein the Amino-terminal envelope region of the positive filament RNA virus is sized such that the polypeptide has a specific epitope configuration for an adjacent unprocessed protein-core region of the positive strand RNA virus. The present invention further characterizes the combination of such an unprocessed adjacent protein-core region in a composition or method with a non-structural protein., thereby providing surprisingly sensitive and specific interactions with the given positive filament RNA virus. The present invention provides the first discovery that the entire polyprotein has unique configurations, and that such configurations result in antigenically important differences. The present invention also provides the first discovery that a lost epitope configuration occurs in the adjacent protein-core protein region. A "substantially complete, isolated, unprocessed polyprotein" of a positive strand RNA virus is the polyprotein that is initially translated from the genome of the positive strand RNA virus. Such polyprotein has not been subjected to processing, and therefore, the processing sites between the proteins of the polyprotein are not cut. The polyprotein is also isolated, which means that the polyprotein has been separated from its coding genome. The polyprotein is substantially complete when it retains all of the functional elements necessary to provide the immunoactive characteristics of the present invention, particularly the epitopic configuration (s) that are present only in the polyprotein and not in processed protein or subunits that they 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 substitutions of conservative amino acids, or insignificant amino acid additions, modifications or deletions, that can change the amino acid sequence of the protein but not significantly alter the functioning of the protein (ie, the unprocessed epitopic configuration is retained). However, such modifications may, when desired, suppress signals and / or protein processing sites. These modifications are discussed later. Integrity can also be determined by using the polyprotein in question in one or more of the assays discussed below and detecting effects of epitope-specific configurations of 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 of 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 exhibits the antigenicity of the core-like protein. Although alteration of the epitope configuration on processing was not known in the art, generally similar core proteins, and regions of core-like proteins that may be important for antigenicity, are well known in the art (see example, Okamoto, et al., J. Virol. 188: 331, 1992; Wang, U.S. Patent No. 5, 106,726). An antigen protein similar to the nucleus can be determined for a desired positive filament RNA virus, for example, by ELISA or "western blotting" or both, for a traditional nucleic type antigenic reactivity, as is well known in the art. The antigen protein similar to the nucleus can also be determined by SDS-PAGE followed by amino acid sequencing. Typically, the core-like antigen protein binds to an amino-terminal portion of the adjacent peptide or protein region of the positive strand RNA virus to provide an "adjacent protein-antigen similar to the nucleus" 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 linked to a carboxy-terminal portion of the adjacent protein of the positive strand RNA virus in unprocessed form to provide the inventive adjacent protein-antigen similar to the unprocessed core of the invention. In unprocessed form it means that the region similar to the nucleus and the adjacent region are normally, and preferably, maintained precisely as they are bound (ie, encoded) in a natural positive filament RNA virus. As with the polyprotein and other proteins herein, the antigen protein similar to the nucleus can be modified insignificantly without changing the inventive function of the antigen protein similar to the nucleus. The portion of the "adjacent protein" that is adjacent to the antigen protein similar to the nucleus is sized so that the fusion protein has a specific epitope configuration for an antigen protein similar to the nucleus of the positive strand RNA virus. Therefore, typically, the amino-terminal portion of the adjacent protein region must be of sufficient length to allow the fusion protein to exhibit the epitope-specific transient configuration for the unprocessed core-like region. In addition to core-like proteins, the env protein of a positive filament RNA virus can also provide the surprisingly enhanced antigenic conformations and interactions exhibited by the adjacent proteins-core-like antigens described herein. This is particularly true when the envelope protein is used in combination with a second protein, also as described herein. Preferably, the envelope protein includes a raw link to an adjacent protein (which may itself be an adjacent envelope protein, such as gp120 and gp41 in HIV), similar to that found with the adjacent protein-like antigen to the core described in the present. Additionally, the second protein can be a core-like protein, such as the HIV gag protein. Because the envelope region provides similar intensified detection and immunoinduction shown by the adjacent protein-antigen similar to the core of the present invention, unless stated otherwise or is otherwise clear from the context, the reference in the present the adjacent protein-antigen similar to the nucleus is equally applied to envelope proteins and / or envelope-adjacent proteins. The determination of whether a given envelope or envelope-envelope protein exhibits such enhanced detection and immunoinduction can be effected by testing as with an adjacent protein-core-like antigen, as discussed below. The determination of whether a given polypeptide exhibits the epitopic configuration of the adjacent protein-antigen similar to the inventive nucleus can be performed as follows. An adjacent protein-antigen similar to the nucleus in question can be included in a panel of adjacent proteins-antigens similar to the nucleus comprising an adjacent protein-antigen similar to the established nucleus, such as EN-80-2. The panel is placed in a series of cavities in a microtiter plate. The panel may also include other adjacent proteins-core-like antigens that have different lengths of adjacent protein. In a separate cavity is placed a non-structural protein, or other, established capable of a synergistic cooperation with the adjacent protein-antigen similar to the nucleus, such as EN-80-1. An antiserum is selected so that the adjacent protein-antigen similar to the established nucleus reacts weakly with the adjacent protein-antigen similar to the established nucleus and that is also non-reactive with the established non-structural 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 an adjacent protein-antigen similar to the appropriate nucleus and the established non-structural protein are present in the sample. Many examples of such antiserum, such as G614 (diluted 8 times), G614 (diluted 16 times, G615 (diluted 8 times), G615 (diluted 16 times), and 8-5 are discussed below in the Examples. introduced to the sample proteins under conditions suitable for the extraction and detection of a reaction between the antiserum and the given protein, and detects and measures such response.The established non-structural protein is then combined with an additional sample from each panel member adjacent protein-antigen similar to the nucleus, then the antiserum is introduced to the combined proteins under conditions suitable for the extraction and detection of a reaction between the antiserum and the proteins, and such response is detected and measured.Adjacent proteins-similar antigens the core which provides a cooperative effect are suitable for use in the present invention, preferably the antiserum will react at least approximately 1.25 or 1.5 times as strongly with the combined proteins when compared to the addition reaction of the antiserum with each protein, alone. More preferably, the antiserum will react at least about twice as strongly. Each of the aforementioned steps is routine in the art, in light of the present specification. The adjacent protein-antigen similar to the nucleus is preferably isolated, which means that the adjacent protein-antigen similar to the nucleus is separated from the rest of the polyprotein originally translated from the genome of the positive filament RNA virus. The adjacent protein-antigen similar to the nucleus is also preferably separated from its coding nucleic acid molecule. In a preferred embodiment, the adjacent protein-antigen similar to the nucleus of the present invention is used in combination with a second protein. The second protein is preferably of a positive filament RNA virus, is more preferably of the same positive filament RNA virus as the adjacent protein-antigen similar to the nucleus, and is most preferably a non-structural protein of an RNA virus. positive filament (preferably of the same positive filament RNA virus as the adjacent protein-antigen similar to the nucleus).

In a preferred embodiment, the second protein is a non-structural protein. In positive strand RNA viruses other than HCV, non-structural proteins can be referred to by other names, as is well known in the art. For the purposes of the present specification, all such similar non-structural proteins should be referred to herein as "non-structural proteins". As noted above, non-structural coding regions of positive strand RNA viruses are well known in the art. The determination of a second appropriate protein that is suitable for use with the adjacent protein-core-like antigen, whose second protein may include portions of non-structural coding regions comprising more than one non-structural protein (or less than all of a non-protein). structural), can be done as follows. A second protein in question can be included in a panel of second proteins comprising a second established protein, such as EN-80-2. The panel is placed in a series of cavities in a microtiter plate. The panel may also include other second proteins that have different lengths of adjacent protein. In a separate cavity an adjacent protein-antigen similar to the established core capable of synergistic cooperation with the second protein, such as EN-80-1, is placed. An antiserum is selected for the second established protein that reacts weakly with the second established protein and that is also non-reactive with the adjacent protein-antigen similar to the established nucleus. 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 adjacent protein-antigen similar to the established nucleus are present in the sample. Many examples of such antiserum are discussed below in the Examples. The antiserum is introduced to the sample proteins under conditions suitable for the extraction and detection of a reaction between the antiserum and the given protein, and such response is detected and measured. The adjacent protein-antigen similar to the established nucleus is combined with each member of the second protein panel. Then, the antiserum is introduced to the combined proteins under conditions suitable for the extraction and detection of a reaction between the antiserum and the proteins, and such response is detected and measured. These second proteins that provide a cooperative effect are suitable for use in the present invention. Each of the aforementioned steps is routine in the art, in light of the present specification. The present invention also provides antibodies, preferably monoclonal antibodies, for the substantially complete polyprotein, the adjacent protein-core-like antigen, and / or non-structural 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 filament RNA virus in a sample. Moreover, the present invention provides compositions and methods for the extraction of an immune response in an animal (either humoral, cellular, or both). Still further, the compositions and methods can vaccinate an animal against the positive filament RNA virus. Preferably, the methods and compositions of the present invention, including those for the detection, immune response and vaccination, are applied to a human being. An example of the present invention is Hepatitis C virus (HCV). The following discussion generally focuses on HCV, and even more so on the HCV core antigen protein bound to an amino-terminal portion of an HCV envelope region. The discussion also focuses on such an envelope-core antigen region in combination with a non-structural HCV protein (particularly the non-structural proteins NS3-NS4 and HC5 NS5), or in combination with a second protein of another positive filament RNA virus (particularly the envelope protein of HIV and the envelope protein HTLV-I). As noted above, the discussion predicts the results to be obtained with adjacent proteins-antigens similar to the nucleus of positively-strand RNA virus in a general manner. The discussion also predicts the results to be obtained with the substantially complete polyprotein of the positively-strand RNA viruses in general, and the substantially complete polyprotein of HCV in particular.

Nucleic acid molecules encoding the unprocessed polypeptides and other polypeptides of the invention As noted above, the present invention includes a nucleic acid molecule that encodes a polypeptide comprising a substantially complete positive strand RNA virus polyprotein. The present invention also provides a nucleic acid molecule encoding a polypeptide comprising an adjacent protein-antigen similar to the nucleus, such as the HCV core antigen protein linked to an amino-terminal portion of the HCV envelope region. The present invention also provides a nucleic acid molecule encoding a polypeptide comprising a non-structural protein of such a positive strand 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 the nucleic acid sequences present in the plasma of a patient infected with HCV. As discussed below, the isolation of the molecule includes 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 technique. (PCR) The primaries used for the cloning were as follows: (i) 5'-GGATCCATGAGCACAAATCCTAAACCT-3 '(SEQ ID No.1) and (ii) 5'-GAATTCGGTGTGCATGATCATGTCCGC-3' (SEQ ID No.2) The cloned DNA molecule was sequenced in order to confirm its identity. The molecule obtained in this way was named EN-80-2. The DNA sequence of the EN-80-2 molecule is given in Figure 1a (SEQ ID No. 7), and has 669 base pairs. The amino acid sequence of the molecule EN-80-2 is given in Figure 1 B (SEQ ID No. 8), and it has 223 residues. The molecule EN-80-2, in the species of E. coli BL21 (DE3), was deposited with the American Type Culture Collection (ATCC) Rockville Maryiand 20852, on July 14, 1993, and has been granted the designation of ATCC 55451 The crop has been deposited under the conditions of the Budapest Treaty. In another preferred embodiment, the nucleic acid molecule is a DNA molecule that encodes a non-structural HCV NS5 protein that was isolated from nucleic acid sequences present in the plasma of an HCV infected patient. As with the isolation of the envelope protein-unprocessed core antigen discussed above (albeit with a 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 clone the desired DNA molecule via a Polymerase Chain Reaction (PCR) technique. The primaries used in the PCR were the following: (i) S'-GGATCCCGGTGGAGGATGAGAGGGAAATATCCG-S '(SEQ ID No. 3) and (ii) 5'-GAATTCCCGGACGTCCTTCGCCCCGTAGCCAAATTT-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 named EN-80-1. The DNA sequence of the EN-80-1 molecule is given in Figure 3A (SEQ ID No. 9) and has 803 base pairs. The amino acid sequence of the molecule EN-80-1 is given in Fig. 3B (SEQ ID No. 10), and it has 267 residues. The molecule EN-80-1, in the species £. coli BL21 (DE3), was deposited with American Type Culture Collection (ATCC) Rockville Maryland 20852, on July 14, 1993, and has been granted the designation of ATCC 55450. The crop has been deposited under the conditions of the Budapest Treaty. Figure 2 shows an expression plasmid, pEN-2, containing the DNA molecule encoding the unprocessed core-antigen envelope protein isolated using the primers of SEQ ID Nos. 1 and 2, discussed above. Figure 4 shows an expression plasmid, pEN-1, containing the DNA molecule encoding the NS5 non-structural protein isolated using the primers of SEQ ID Nos. 1 and 2, discussed above.

This general procedure has also been used to isolate a nucleic acid molecule representative of the non-structural region NS3-NS4 of HCV. See also Simmonds, Lancet 336: 1469-1472, 1990. The primaries used for cloning were as follows. (i ("ED3")) 5'-CACCCAGACAGTCGATTTCAG-3 '(SEQ ID No. 5) and (ii ("ED4")) 5'-GTATTTGGTGACTGGGTGCGTC-3' (SEQ ID No. 6) The molecule thus obtained was named EN-80-4. The polypeptide encoded by the isolated molecule has a molecular weight of approximately 20,000 Daltones as measured by electrophoresis through SDS-PAGE. Additional examples of polypeptides useful as the second protein include the envelope protein of HIV (molecular weight of approximately 18,000 daltons) and the envelope protein of HTLV (molecular weight of approximately 18,000 daltons). The present invention is responsible for the manipulation and expression of the nucleic acid molecules described above by culturing host cells containing a construct capable of expressing the genes described above. 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 refer typically to a DNA molecule, or to a clone of such a molecule (either single filament or double filament), which has been modified through human intervention to contain DNA segments combined and juxtaposed in a way that as a whole would not exist otherwise in nature. The vector constructs of the present invention comprise a first segment of DNA encoding one or more of an adjacent protein-antigen similar to the unprocessed core and a non-structural protein of a positive strand RNA virus feasibly 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 also include enhancers and other elements. See WO 94/25597 and WO / 25598. Mutations in nucleotide sequences constructed by expression of the inventive proteins preferably preserve the reading frame of the coding sequences. In addition, the preferred mutations will not create complementary regions that can hybridize to produce secondary mRNA structures, such as turns or closed curves, that could adversely affect mRNA translation. 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 the optimal characteristics of mutants at a given site, random mutagenesis at the target codon can be conducted and the expressed mutants classified by indicative biological activity.

Mutations can be introduced at particular sites by synthesizing oligonucleotides containing a sequence of mutants, flanked by restriction sites that allow ligation to fragments of the natural sequence. Following the ligation, the resulting reconstructed sequence encodes a derivative having the desired amino acid insertion, substitution or deletion. Alternatively, site-specific mutagenesis procedures, directed to oligonucleotides, can 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 discussed above are described by Walder et al (Gene 42: 133, 1986); Bauer et al (Gene 37:73, 1985); Craik (BioTechniques, January 1985, 12-19); Smith et al. . { Genetic Engineering: Principles and Methods, Plenum Press, 1981); and Sambrook et al (supra). The amino acid structure of the above-described proteins can also be modified by forming added or covalent conjugates with other chemical moieties, such as glycosyl groups, lipids, phosphate, acetyl groups or other proteins or polypeptides, provided that such modifications do not interfere with antigenicity of 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 for the adjacent protein-antigen similar to the nucleus.

A preferred type of the 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 a non-structural region NS5 of HCV and an envelope protein-antigen of HCV core, 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 the expression of the gene sequences of the present invention is a prokaryotic host cell, more 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 to express cloned DNA sequences in bacterial hosts generally contain a selectable marker, such as a gene for antibiotic resistance, and a promoter that functions in the host cell. Suitable promoters include trp (Nichols and Yanofsky, Meth Enzymol 101: 155-164, 1983), lac (Casadaban et al., J. Bacteriol 143: 971-980, 1980), and phage promoter systems? (Queen, J. Mol, Appl. Genet, 2: 1-10, 1983). The expression units may also include a transcriptional terminator. Plasmids useful for transforming bacteria include the pUC plasmids (Messing, Meth, Enzymol 101: 2078, 1983, Vieira and Messing, Gene 19: 259-268, 1982), pBR322 (Bolivar et al., Gene 2: 95-113). , 1977), pCQV2 (Queen, ibid.), And derivatives thereof. The plasmids can contain both viral and bacterial elements. In another embodiment, the host cell can be a prokaryotic cell, provided that either the host cell has been modified so that the host cell can not process, for example, the adjacent protein-antigen similar to the unprocessed core or the non-target region. unprocessed structural (such as the NS3-NS4 non-structural protein), or the processing signals and / or processing sites in the unprocessed polypeptide have been modified so that the protein is no longer susceptible to processing (such modifications should not be affect the antigenicity of the unprocessed protein). Eukaryotic host cells suitable for use in the practice of the present invention include mammalian cells, birds, plants, insects and fungi. Preferred eukaryotic cells include cultured mammalian cell lines (e.g., rodents or human cell lines), insect cell lines (e.g., Sf-9) and fungal cells, including yeast species (e.g., Saccharomyces spp. ., particularly S. cerevisiae, Schizosaccharomyces spp., or Kluyveromyces spp.) or filamentous fungi (for example, Aspergillus spp., Nuerospora spp.). Techniques for transforming these host cells, and methods for expressing foreign DNA sequences cloned thereon, are well known in the art (see, for example, 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 (ed.), 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., (eds.), 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 can be introduced into the host cell can be minimized and the 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 apparent to those skilled in the art. The 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 the 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, for example, growth factors or serum, which may be required by the particular host cells. The growth medium will generally be selected for cells containing the DNA construct (s) by, for example, drug selection or deficiency in an essential nutrient which is complemented by the selectable marker in the DNA construct or co- transfected with the DNA construct.

Polypeptides comprising the unprocessed polypeptides of the invention As noted above, the invention provides a polypeptide comprising a substantially complete, unprocessed polyprotein of a positive strand RNA virus. The invention also provides a polypeptide comprising an antigen protein similar to the nucleus, such as the HCV core protein, attached to an amino-terminal portion of an adjacent protein, such as the envelope region of HCV. The present invention also provides certain non-structural proteins. In a preferred embodiment, the amino acid sequence of the antigen protein similar to the nucleus is that shown in Fig. 1 B (SEQ ID No. 8). In such a preferred embodiment, the polypeptide has a molecular weight of approximately 25,000 daltons as measured by electrophoresis through a sodium dodecyl sulfate-polyacrylamide gel and has been deduced as having approximately 223 amino acids.

The unprocessed polypeptide of the positive strand RNA virus is capable of binding antibodies specified for the positive strand RNA virus. In the case of HCV, this has been confirmed by Western Blotting and by an enzyme-linked immunosorbent assay (ELISA). It has been found that the unfolded core antigen-envelope protein is specifically reactive with the serum of patients with HCV, and therefore is not reactive with the serum of people without HCV. The unprocessed polypeptide of the positive filament RNA virus is also capable of detecting the presence of antibodies in specific samples for the positive filament RNA virus, and is therefore useful for the detection and diagnosis of the positive filament RNA virus. in a patient, particularly in a human being. The present invention also provides a polypeptide comprising a non-structural protein of the positive filament 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 sulfate-polyacrylamide gel (SDS-PAGE) and it has been deduced that it is approximately 267 amino acids. The non-structural protein of the present invention is capable of binding antibodies specific for the positive filament RNA virus, which in the case of HCV has been confirmed by Western Blotting and (ELISA) for both non-structural proteins NS5 and NS3-NS4 described at the moment. The non-structural protein of the present invention is specifically reactive with the serum of patients infected with the positive filament RNA virus, and therefore is not reactive with the serum of people without the positive filament RNA virus. The non-structural protein is also capable of detecting the presence of antibodies specific for the filament-positive RNA virus under the conditions of the Budapest Treaty, and on samples, and is therefore useful for the diagnosis of RNA virus of positive filament in a patient, particularly a human being. When the protein of the present invention is encoded by a portion of a natural gene, a derivative of a natural gene, or has been modified in another way, the protein substantially maintains the same biological activity as the natural protein. For example, the structure of proteins corresponding to the substantially complete, unprocessed polyprotein of a positive strand RNA virus, the adjacent protein-antigen similar to the nucleus, or the non-structural protein can be predicted from the primary translation products of the same using the hydrophobicity graph function, for example P / C Gene or Intelligenetic Suite (Intelligenetics, Mountain View, Calif.), or according to the methods described by Kyte and Doolittle (J. Mol. Biol., 157: 105-132, 1982). In a preferred embodiment, the present invention provides isolated proteins. The proteins can be isolated by, among other methods, culturing the appropriate host and vector systems to produce the recombinant translation products of the present invention. Supernatants of such cell lines, or protein inclusions or whole cells where the protein is not excreted or secreted in the supernatant, can 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 a Pellicon Millipore or Amicon ultrafiltration unit. Following the concentration, the concentrate can be applied to a suitable purification matrix such as, for example, an anti-protein antibody bound to a suitable support. Alternatively, the anion or cation exchange resins can be used for the purpose of purifying the protein, as a further alternative, one or more steps of high performance reverse phase liquid chromatography (RP-HPLC) can be employed to further purify the protein. Other methods for 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 considered to be "isolated" within the context of the present invention if no other (unwanted) protein is detected according to the SDS-PAGE analysis followed by "coomasie" blue dye. Within other embodiments, the desired protein may be isolated such that no other (unwanted) protein, and preferably no lipopolysaccharide (LPS), is detected according to the SDS-PAGE analysis followed by silver staining. Still within other embodiments, the protein is isolated if no other protein having significant antigenic activity that interferes significantly with detection assays or immunological events is included with the protein.

Ligation partners to the unprocessed polypeptides of the invention The present invention also provides monoclonal and polyclonal antibodies directed against the unprocessed positive filament RNA virus polyprotein, the adjacent protein-antigen similar to the nucleus of a positive filament RNA virus, the non-structural protein of such an RNA virus. positive filament or other proteins of the invention. 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 positive filament RNA virus in a sample, preferably a sample from a human. See WO 94/25597 and WO / 25598. Polyclonal antibodies can be easily generated by someone with 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 used to immunize the animal, usually through intraperitoneal, intramuscular, intraocular or subcutaneous injections. The immunogenicity of the protein or peptide of interest can be increased through the use of an adjuvant such as Freund's complete or incomplete adjuvant. Following several booster immunizations, small samples of the serum are collected and tested for the reactivity of the desired protein or peptide. Once the titer of the animal has reached a stabilization in terms of its reactivity to the protein, larger amounts of the polyclonal antiserum can be easily obtained either by weekly bleeding or by exsanguination of the animal. Monoclonal antibodies can also be easily generated using well known techniques (see US Patent Nos. Re 32,011, 4,902,614, 4,543,439, and 4,41 1, 993; see also Monoclonal Antibodies, Hybridombas: A New Dimension in Biological Analyzes, Plenum Press, Kennett , McKearn, and Bechtol (eds.), 1980, and Antibodies: A Laboratory Manual, supra). Briefly, in one embodiment, an objective animal such as a rat or mouse is injected with a desired protein or peptide. If desired, several techniques can be used in order to increase the resultant immune response generated by the protein, in order to develop greater antibody reactivation. For example, the desired protein or peptide may be coupled with another protein such as ovalbumin or keyhole limpet hemocyanin (KLH), or through the use of auxiliaries such as complete or incomplete Freund's aids. The initial extraction of an immune response can be through intraperitoneal, intramuscular, intraocular or subcutaneous routes. Between one and three weeks after the initial immunization, the animal can be reinmunized with booster immunization. The animal can then be bled for testing and the serum tested for binding to the unprocessed polypeptide using assays as described above. Additional immunizations can also be made until the animal has reached a stability in its reactivity to the desired protein or peptide. The animal may then be provided with a final boost of the desired protein or peptide, and three or four days after it is sacrificed. At this time, the spleen and lymph nodes can be collected and dissolved in a single cell suspension by passing the organs through a mesh classification or by breaking the membranes of the lymph node or spleen which encapsulate the cells. Within one modality the red cells are subsequently used by the addition of a hypotonic solution, followed by an immediate return to isotonicity. Within another embodiment, cells suitable for preparing monoclonal antibodies are obtained through the use of in vitro immunization techniques. Briefly, an animal is sacrificed, and the cells of the lymph nodes and the spleen are removed as described above. A single cell suspension is prepared, and the cells are placed in a culture containing a form of the protein or peptide of interest that is suitable to generate an immune response as described above. Subsequently, the lymphocytes are harvested and fused as described above. Cells that are obtained through the use of in vitro immunization or an immunized animal as described above can be immortalized by transfection with a virus such as Epstein-Barr Virus (EBV). (See Glasky and Reading, Hybridoma 8 (4): 377-389, 1989). Alternatively, within a preferred embodiment, suspensions of collected lymph node and / or spleen cells 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 further syngeneic with the cells of the immunized animal. Many of those myeloma cell lines are well known in the art and can be obtained from sources such as American Type Culture Collection (ATCC), Rockville, Maryland (see Catalog 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-Ag14 (ATCC No. CRL 1581), and P3X63Ag8 (ATCC No. TIB 9); and for rats, Y3-Ag1.2.3 (ATCC No. CRL 1631), and YB2 / 0 (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 can be used for fusion with either human, mouse, or rat cell lines . The fusion between the myeloma cell and the cells of the immunized animal can be performed 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. 67: 165-182, 1982).

Following the fusion, the cells are placed in culture dishes containing a suitable medium, such as RPMI 1640 or DMEM (Dulbecco's Modified Eagles Medium, JRH Biosciences, Lenexa, Kan.). The medium may also contain additional ingredients, such as Fetal Bovine Serum (FBS, for example, from Hyclone, Logan Utah, or JRH Biosciences), thymocytes that were collected from a baby animal of the same species that were used for immunization, or agar to solidify the medium. Additionally, the medium should contain a reagent which selectively allows the growth of fused myeloma and spleen cells. Particularly preferred is the use of the HAT medium (hypoxanthine, aminopterin and thymidine) (Sigma Chemical Co., St. Louis, Mo.). After about seven days, the resulting hybridomas or fused cells can be classified in order to determine the presence of antibodies that recognize the core-shell region of said HCV or the non-structural HCV protein. After several clonal dilutions and re-assays, the hybridoma that produces antibodies that bind to the protein of interest can be isolated. Other techniques can also be used to construct monoclonal antibodies. (See Huse et al., "Generation of a Large Combinatorial 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. Nati Acad. Sci. USA 86: 5728-5732, 1989; see also Alting-Mees et al.," Monoclonal Antibody Expression Libraries. Rapid Altemative to Hybridomas, "Strategies in Molecular Biology 3: 1-9, 1990, these references describe a commercial system available from Stratacyte, La Jolla, California, that allows the production of antibodies through recombinant techniques.) Briefly, mRNA is isolated from a population of B cells and used to create light and heavy chain immunoglobulin cDNA expression libraries in vectors lMMUNOZAP (H) and? lMMUNOZAP (L). These vectors can be individually classified or co-expressed to form antibodies or Fab fragments (see Huse et al., Supra, see also Sastry et al., Supra). Positive plaques may subsequently be converted to a non-lytic plasmid which permits high level of expression of E. coli monoclonal antibody fragments. Similarly, ligation partners can also be constructed using recombinant DNA techniques to incorporate the variable regions of a gene encoding a specifically binding antibody. The construction of these ligation partners can be easily 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 Therapy, "Nature 322: 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 et al.," Reshaping Human Antibodies: Grafting an Antilysozime Activity, "Science 239: 1534-1536, 1988; Chaudhary et al.," A Recombinant Immunotoxin Consisting of Two Antibody Variables 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, the DNA segments encoding the desired antigen-specific protein or peptide binding domains of interest are amplified from the hybridomas they produce a specifically ligation monoclonal antibody, and they 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 ligation-antigen site of a rat monoclonal antibody or ligation mouse specifically to be transferred to a human antibody. Such antibodies are preferably for therapeutic use in humans because they are not as antigenic as rat or mouse antibodies. In an alternative embodiment, the genes encoding the variable region of a hybridoma that produces a monoclonal antibody of interest are amplified using primers of oligonucleotides for the variable region. These primaries can be synthesized by someone of ordinary skill in the art, or can be purchased from commercially available sources. For example, primaries for variable regions of human and mouse including, among others, primaries for regions VHa, VHb, VHc > VHd, CH ?, V and C, are available from Stratacyte (La Jolla, Calif.). These primaries can be used to amplify heavy or light chain variable regions, which can be inserted into vectors such as IMMUNOZAP ™ (H) or IMMUNOZAP ™ (L) (Stratacyte), respectively. These vectors can then be introduced into E. coli by expression. Using these techniques, large amounts of a single chain protein containing a fusion of the VH and V domains can occur (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 can be isolated or purified by many techniques well known to those of ordinary skill in the art (see Antibodies: A Laboratory Manual, Harlo and Lane (eds.), 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 protein or peptide 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." The antibodies and binding partners of the present invention have many uses. As discussed below, the antibodies and binding partners of the present invention are particularly useful for the detection and diagnosis of the positive filament RNA virus. Other uses include, for example, flow cytometry to order cells that display one or more of the proteins of the present invention. Briefly, in order to detect the protein or peptide of interest in the cells, the cells are incubated with a labeled monoclonal antibody which specifically binds the protein of interest, followed by detection of the presence of the bound antibody. These steps can also be performed with additional steps such as washes to remove the unbound antibody. Labels suitable for use within the present invention are well known in the art including, among others, fluorescein isothiocyanate (FITC), phycoerythrin (PE), horseradish peroxidase (HRP), and colloidal gold. Particularly preferred for use in flow cytometry is FITC, which may be conjugated to an antibody purified according to the Keltkamp method 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 detecting a positive filament RNA virus in a sample As noted above, the invention provides a polypeptide comprising a substantially complete polyprotein of a positive filament RNA virus. The invention also provides a polypeptide comprising an adjacent protein-antigen similar to the nucleus and certain non-structural proteins. The present invention further provides methods for detecting such polypeptides in a sample. Assays are usually based on the detection of antigens exhibited by the positive strand RNA virus or antibodies raised against the positive strand RNA virus, but may also include nucleic acid based assays (typically based on hybridization), as is known in the technique. The methods are characterized by the ability of the polypeptides of the present invention to be ligated by antibodies against the positive filament RNA virus, and the ability of antibodies raised against the proteins of the present invention to bind antigens of the RNA virus of positive filament in a sample. Surprisingly, the unprocessed polypeptides of the present invention provide significantly better and more sensitive detection of the positive strand RNA virus. For example, with reference to HCV, the unfolded core-antigen protein provides significantly better detection of HCV in a sample than the processed core protein (sometimes referred to as p22) or fragments of the core protein alone. Also surprisingly, the use of both an adjacent protein-antigen similar to the unprocessed nucleus and a non-structural protein of the positive strand RNA virus in the assay provides a synergistic effect that allows a significantly more sensitive detection of the RNA virus. positive filament that when either the adjacent protein-antigen similar to the unprocessed nucleus or nonstructural protein is used alone. A preferred assay for the detection of positive strand RNA virus is a "sandwich" assay such as an enzyme-linked immunosorbent assay (ELISA). In a preferred embodiment, the ELISA comprises the following steps: (1) covering an envelope-core antigen protein of the present invention on a solid phase, (2) incubating a sample suspected of containing HCV antibodies to the polypeptide covered on the solid phase under conditions that allow the formation of an antigen-antibody complex, (3) adding an anti-antibody (such as anti-IgG) conjugated to a label to be captured by the resulting antigen-antibody complex bound to the solid phase, and (4) measure the captured label and determine from there if the sample has HCV antibodies. Although a preferred assay is discussed above, a variety of assays can be used in order to detect antibodies that specifically bind to the desired protein in a sample, or to detect the desired protein linked to one or more antibodies in the sample. Exemplary assays are described in detail in Antibodies: A Laboratory Manual, Harlow and Lane (eds.), Cold Spring Harbor Laboratory Press, 1988. Representative examples of such assays include: counter-current immuno-electrophoresis (CIEP), radioimmunoassays, radioimmunoassay precipitation, enzyme-linked immunosorbent assays (ELISA), dot blot assays, inhibition or competition assays, sandwich assays, immunoadhesion assays (dip-stick), simultaneous assays, immunochromatographic assays, immunofiltration assays, pearl agglutination assays latex, immunofluorescent assays, biosensing assays, and low light detection assays (see US 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 (FA-test) uses a fluorescently labeled antibody capable of binding to one of the proteins of the invention. For detection, visual determinations are made by a technician using fluorescence microscopy, producing a qualitative result. In one embodiment, this assay is used for the determination of tissue samples or histological sections. In the latex bead agglutination assays, antibodies to one or more of the proteins of the present invention are conjugated to latex beads. The antibodies conjugated to the latex beads are then contacted with a sample under conditions that allow the antibodies to bind to the desired proteins in the sample, if any. The results are then read visually, producing a qualitative result. In one modality, this format can be used in the field for testing on the site. Enzyme immunoassays (EIA) include a number of different assays capable of using the antibodies provided by the present invention. For example, a heterogeneous indirect EIA uses a solid phase coupled with an antibody of the invention and a preparation of purified affinity anti-IgG immunoglobulin. Preferably, the solid phase is a polystyrene microtiter plate. The antibodies and the immunoglobulin preparation are then contacted with the sample under conditions that allow the ligation of the antibody, the conditions of which are well known in the art. The results of such an assay can be read visually, but are preferably read using a spectrophotometer, such as an ELISA plate reader, to produce a quantitative result. An alternative solid phase EIA form includes ferrous metal beads coated with plastic capable of being moved during the assay procedures by means of a magnet. Yet another alternative is a low light detection immunoassay format. In this highly sensitive form, the emission of light produced by appropriately labeled bound antibodies is quantified automatically. Preferably, the reaction is performed using microtiter plates. In an alternative embodiment, a radioactive indicator is replaced by enzyme-mediated detection in an EIA to produce a radioimmunoassay (RIA). In an antibody-capture "sandwich" enzyme assay, the desired protein is ligated between an antibody bound to a solid phase, preferably a polystyrene microtiter plate, and a labeled antibody. Preferably, the results are measured using a spectrophotometer, such as an ELISA plate reader. This test is a preferred embodiment of the present invention.

In a sequential assay format, the reagents are allowed to incubate with the capture antibody in a step in a known manner. The test sample is incubated first with the capture antibody. Following a wash step, incubation occurs with the labeled antibody. In a simultaneous assay, the two incubation periods described in the sequential assay are combined. This eliminates an incubation period plus a washing step. A dipstick / immunoadhesion format is essentially an immunoassay except that the solid phase, instead of being a polystyrene microtiter plate, is a polystyrene or dipstick paddle. The reagents are the same and the format can be either simultaneous or sequential. In a chromatographic strip test format, a capture antibody and a labeled antibody are dried on a chromatographic strip, which is usually nitrocellulose or high porosity nylon bound to cellulose acetate. The capture antibody is usually spray dried as a line at one end of the strip. At this end there is an absorbent material that is in contact with the strip. At the other end of the strip the labeled antibody is deposited in a way that prevents it from being absorbed into the membrane. Usually, the label attached to the antibody is a latex bead or colloidal gold. The assay can be started by applying the sample immediately in front of the labeled antibody. The immunofiltration / immunoconcentration formats combine a large solid surface with directional sample / reagent flow, which concentrates and accelerates the binding of the antigen to the antibody. In a preferred format, the sample is pre-incubated with a labeled antibody when applied to a solid phase such as fiber filters or nitrocellulose membranes or the like. The solid phase can also be precoated with latex or glass beads coated with capture antibody. The detection of the analyte is the same as a standard immunoassay. The flow of sample / reagents can be modulated by either vacuum or the wicking action of an implicit absorbent material. A threshold biosensor assay is an instrumented, sensitive receptive assay for classifying large numbers of samples at a low cost. In one embodiment, such assay comprises the use of light-directional potentiometric sensors wherein the reaction involves the detection of a pH change due to the ligation of the desired protein by capture antibodies, bridging antibodies and urease-conjugated antibodies. Upon ligation, a pH change is effected that is measurable by translation in electric potential (μvolts). The assay typically occurs in a very small reaction volume, and is very sensitive. Furthermore, the reported detection limit of the assay is 1,000,000 urease molecules per minute.

Compositions and methods for the extraction of an immune response for HCV The present invention also provides compositions and methods for the extraction of an immunoresponse to the positive filament RNA virus, which may be either humoral, cellular or both. Preferably, the immune response is induced by a vaccine against the positive filament RNA virus, and 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 diluent or carrier. In a preferred embodiment, the compositions and methods comprise both a non-processed core HCV envelope-antigen protein and a non-structural HCV protein, more preferably a non-structural protein NS5 or a non-structural protein NS3-NS4. 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 antigens capable of extracting 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 1A, 1 B, 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 variations of the amino acid sequences can be made without affecting the immunogenicity of the immunogen. Substantial equivalents of the above proteins include conservative amino acid substitutions that maintain substantially the same charge and hydrophobicity as the original amino acid. Conservative substitutions include the replacement of valine by isoleucine or leucine, and aspartic acid by glutamic acid, as well as other substitutions of a similar nature (See Dayhoff et al. (Ed.), "Atlas of Protein Sequence and Structure," Nati. Biomed, Res. Fdn., 1978). As will be apparent to one of ordinary skill in the art, the immunogens listed above, including their substantial equivalents, can 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 assays. Briefly, T-cell proliferation assays can be used as an indicator of the potential for cell-mediated immunity. Additionally, evidence of lymphokine production after stimulation by an immunogen can be used to determine the protection potential provided by an immunogen. Finally, as described below, current immunoprotection assays can be performed for the purpose of determining protection in animals. In the case of humans, however, instead of immunoprotection assays it is preferable to first classify 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 centrifugation and used in cell proliferation studies with [3H] -thymidine as described below. The positive peptides are then selected and used in trials with primates. The immunogens, or polypeptides, of the present invention can be readily produced using many other techniques well known in the art (see Sambrook et al., Supra, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1989). Immunogens comprising polypeptides of the present invention in combination with a pharmaceutically acceptable diluent or carrier can be administered to a patient according to a number of procedures known in the art. See WO 94/25597 and WO / 25598. For purposes of the present invention, warm-blooded animals include, but are not limited to, humans and primates. Many suitable carriers or diluents can be used 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 auxiliaries, buffers, antioxidants, carbohydrates such as glucose, sucrose or dextrins and chelating agents such as EDTA. Within a particularly preferred embodiment, an auxiliary is used together with the immunogen. Examples of such auxiliaries include aluminum hydroxide or alum for humans.

The amount and frequency of administration can be determined in clinical trials, and may depend on factors such as the viral strains of positive filament RNA 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 administered parenterally via the subcutaneous route or via other routes. Depending on the application, the amounts of injected immunogen will vary from 50 μg to several milligrams in an auxiliary vehicle and preferably approximately 100 μg to 1 mg, in combination with a physiologically acceptable carrier or diluent. Booster immunizations can be provided from 4-6 weeks later. The present invention also includes the administration of a nucleic acid vector capable of expressing the raw core antigen-envelope protein or the non-structural HCV protein (or both) in an animal, wherein the nucleic acid molecule can extract an immune response to, and preferably immunizing, an animal against the expressed protein of the nucleic molecule, and thus of HCV. In one embodiment of this method, pure 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 extracting a response from host cytotoxic T lymphocytes ( CTLs). This may provide an advantage over traditional immunogens wherein the extracted response comprises specific antibodies. The specific antibodies are generally species-specific and can not recognize the corresponding antigen in a different species. CTLs, on the other hand, are specific for conserved antigens and can respond to different species that express a corresponding antigen (Ulmer et al., "Heterologous protection against influenza by injection of DNA encoding to 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) On the introduction of the pure vector construct into the animal's cell, the construct is then able to express the nucleic acid molecule (usually a gene) it carries, whose gene preferably comprises a (or more) of the raw protein shell-antigen protein or non-structural HCV protein. According to this, on the expression of the desired peptide, an immune response is extracted from the host animal. Preferably, the immune response includes CD8 + CTLs capable of responding to different species that exhibit a desired peptide form.

Sets for the implementation of several aspects of the claimed invention The present invention further provides kits for analyzing samples for the presence of antigens or antibodies of the positive filament RNA virus. The sets comprise a polypeptide or antibody of the invention and an appropriate solid phase. Preferably, the polypeptide is linked to the solid phase. The assemblies may also provide one or more reagents and / or devices for the detection of antibodies or HCV antigens. A variety of formats, reagents and devices for inclusion within the sets, including means for detecting antigens or antibodies, is discussed herein. The present invention also provides kits for the induction of an immune response. The sets comprise compositions comprising a polypeptide of the invention in combination with a pharmaceutically acceptable diluent or carrier, and may also provide devices to administer or assist in the administration of the composition. Other assemblies suitable for use with the features of the present invention are also provided herein. The following Examples are offered as a form of illustration, and not as a form of limitation.

EXAMPLES The following examples are separated into three groupings. First, they are Examples that relate to the isolation and production of an adjacent protein-antigen similar to the proper nucleus, namely a shell fusion protein-unprocessed HCV core antigen, and uses thereof without a structural protein. Second, they are Examples that relate to the isolation and production of a second protein suitable for use with the adjacent protein-core-like antigen, namely a non-structural protein of HCV, and uses thereof without the envelope fusion protein. - HCV core antigen. Third, they are Examples that relate to the combination and use of the adjacent protein-antigen similar to the nucleus with second proteins such as an NS5 protein of HCV, an NS3-NS4 protein of HCV, an envelope protein of HIV and a protein of HTLV-I envelope. Fourth, it is an example of the production of monoclonal antibodies to an adjacent protein-antigen similar to the nucleus. Fifth, are Examples that relate to the use of an adjacent protein-antigen similar to the appropriate nucleus, namely an envelope fusion protein-unprocessed HCV core to induce an immune response in an animal.

THE ISOLATION AND PRODUCTION OF A ADYACENT-ANTIGEN PROTEIN SIMILAR TO THE NUCLEUS 1 - . 1 - Cloning of an HCV cDNA Plasma from patients infected with Hepatitis C virus was collected and ultracentrifuged at 4 ° C and then the viral particles were obtained. The viral nucleic acid (RNA) was then extracted and purified from the viral particles using guanidine isothiocyanate and phenol acid (Chomczynski et al., Anal. Biochem. 162: 156-159, 1987). The following oligonucleotide sequences: (i) 5'-GGATCCATGAGCACAAATCCTAAACCT-3 '(SEQ ID No. I) and (ii) S'-GAATTCGGTGTGCATGATCATGTCCGC-S' (SEQ ID No. 2) They were used as primaries in the cloning of cDNA. A simple filament DNA molecule was produced using random primaries, reverse transcriptase, and the RNA model. The double-stranded DNA molecule containing the HCV core-envelope region sequence was amplified by the PCR method using Taq polymerase and the primaries (i) and (ii). The cloned DNA molecule was subjected to sequence analysis for identification. The molecule obtained was designated EN-80-2. The DNA sequence of the EN-80-2 molecule is given in Fig. 1A (SEQ ID No. 7). The DNA molecule was derived from the envelope and core regions of HCV and has 669 base pairs. 2. Construction of a plasmid containing an HCV cDNA The EN-80-2 molecule was treated with restriction endonucleases Bam Hl 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 cut first with restriction endonucleases Bam Hl and EcoRI, to obtain an expression plasmid, designated pEN-2. The expression of the HCV cDNA is under the control of a T7 promoter. The structure of expression plasmid pEN-2 and a restriction map are shown in Fig. 23. Transformation of E. coli The expression plasmid pEN-2 were transformed into E. coli BL21 (DE3), sprayed on an ampicillin-agar plate and placed in an incubator at 37 ° C overnight. The colonies of E. coli that produce HCV core antigen protein were selected by classifying their expression products by SDS-PAGE and Western Blotting. 4. Production of unfolded core-antigen envelope protein The transformed E. coli colonies were incubated in a conditioned culture medium. The colonies were centrifuged and used by freeze-thaw cycles and lysozyme digestion. The unfolded core-antigen envelope protein product was released by the used cells and purified by column chromatography. The polypeptide was more than 90% pure. The unreacted core-antigen envelope protein has a molecular weight of approximately 25,000 daltons as measured by electrophoresis through a sodium dodecyl sulfate-polyacrylamide gel.

. Immunological Reactivity of HCV Core Antigen with HCV Antibodies by Western Blottine The purified untreated core-antigen envelope protein was subjected to 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" cushion (sodium phosphate buffer 0. 15M, pH 6.7) at 4 ° C for 20 minutes. The polypeptide in the gel was then "electroblotted" (electroblotted) on nitrocellulose membrane under the Blotting Buffer at 1 .3a for 1-1.5 hours. The membrane was washed with Wash Buffer (PBS-Tween 20, pH 7.4) and blocked with Blocking Buffer (0.1 M NaCl, 5 mM EDTA, 50 mM Tris, pH7.2-7.4, 0.2% serum albumin of bovine, 0.05% of Nonidet p-40, 1 M of urea) overnight. The membrane was reacted with the serum of the infected persons with / without hepatitis C, which was first diluted with 40% of newborn bovine serum / Tris-HCl (pH 7.4), 10X, at 40 ° C for 2 hours . After the reaction, the membrane was washed with Wash Buffer three times. The membrane was reacted with an anti-hlgG: HRPO conjugate (which was prepared as described below) at 40 ° C for 2 hours. After the reaction, the membrane was washed with Wash Buffer three times and then reacted with 10 ml of Substrate Solution (0.01% of 4-chloro-1-naphthol, 18% of methanol, 0.04M of Tris, pH 7.2-7.4, 0.1 M NaCl and 0.01% H2O2) for 20 minutes. The unfolded core antigen-envelope protein of the present invention was reactive with the serum of HCV patients but was not reactive with the serum of healthy persons. 6. ELISA for HCV antibodies (A) Microtiter plate treatment A microtiter plate was coated with the purified untreated core antigen envelope protein of the invention at appropriate concentrations and blocked with a buffer containing bovine serum albumin. . The treated microtitre plate was stored at 2-8 ° C.

(B) Preparation of anti-hlgG conjugate: HRPO Purified anti-human immunoglobulin G (anti-hlgG) was conjugated to horseradish peroxidase (HRPO) using NalO4 to obtain the anti-IgG: HRPO conjugate. The conjugate was purified by chromatography.

(C) Reagent components (a) Washing solution: Phosphate buffer containing 0.9% NaCl and Thimerosal. (b) Anti-hlgG Conjugate Solution: HRPO: the anti-hlgG conjugate: HRPO prepared as described above dissolved in Tris Buffer containing a protein stabilizer and antiseptic. (c) Sample Diluent: Tris buffer containing a protein and antiseptic stabilizer, (d) OPD Substrate Solution: o-Phenylenediamine (OPD) dissolved in citrate-phosphate buffer containing H2O2. (If the solution turns orange, it means that the solution has been contaminated and can not be used anymore). (e) Stop Solution: H2SO 2N solution. (f) Positive / negative controls: serum samples from persons infected with / without hepatitis C diluted with phosphate buffer containing a protein stabilizer and antiseptic at an appropriate concentration.

(D) Procedure (a) One hundred fifty microliters (μl) of the test samples were diluted with Sample Diluent (1: 10), and Positive / Negative Controls were added into the wells of the treated microtiter plate. Some cavities were retained as substrate targets. (b) The plate was mixed gently by shaking and incubated at 37-40 ° C for 1 hour. (c) The plate was washed three times with 0.3 ml of Washing by cavity. (d) One hundred μl of Anti-hlgG conjugate solution: HRPO were added to each well. (e) The plate was mixed gently by shaking and incubated at 37-40 ° C for 30 minutes. (f) The plate was washed five times. (g) One hundred μl of OPD Substrate Solution were added to each well and the plate was incubated at 15-30 ° C in the dark for 30 minutes. (h) One hundred μl of Stop Solution were added to each well and mixed gently to stop the reaction. (i) The OD value per cavity was measured at 492nm in a spectrophotometer.

(E) Determination: The value OD492nm per cavity subtracts the meaning of the readings of the targets (foundations). The difference (PCx-NCx) between the meaning of the readings of the positive controls (PCx) and those of the negative controls (NCx) is equal to or more than 0.5. The cutoff value (CO) is calculated by the following formula: CO = PCx X 0.15 + NCx When the readings of the test samples were less than the CO value, the samples were considered negative (ie, HCV antibodies could not be detected in the samples). When the readings of the test samples were equal to or more than the CO value, the samples were expected to be positive; however, it is preferable to repeat the assay for duplicate samples. If the readings of any of the samples in duplicate were less than the CO value, the samples are considered negative. If the duplicate samples were both more than or equal to the cut-off value, the samples were considered positive.

When the readings of the test samples are more than NCx but less than the CO value by 20%, the samples should be considered as questionable samples and the test has to be repeated for those samples. Twenty-seven samples were tested by the ELISA according to the invention. At the same time, the samples were also tested with the HCV antibody assay of the Abbott (II) pool, which pool contains both structural and non-structural proteins (i.e., core (amino acids: 1-150), NS-3 and NS-4). The comparison between the results of the Abbott test (II) and those of the test of the present invention is given in Table 1. It is noted that the results of Sample G 229 were negative according to the Abbott set (II ) but were positive according to the assay of the present invention. Sample G 229 was confirmed positive for HCV.

TABLE 1 / Sample No. OD 92nm Results References Set of Abbott (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 1 14 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 And 1 > 2.0 Positive Positive SB 9 > 2.0 Positive Positive SB 19 > 2.0 Positive Positive SB 20 > 2.0 Positive Positive SB 23 0.952 Positive Positive SB 27 0.753 Positive Positive G 1 1 0.147 Negative Negative G 12 0.077 Negative Negative G 13 0.061 Negative Negative G 14 0.1 16 Negative Negative G 15 0.139 Negative Negative G 229 0.517 Positive Negative ISOLATION AND PRODUCTION OF AN ADEQUATE SECOND PROTEIN, A NON-STRUCTURAL PROTEIN OF HCV 7- Cloning of an HCV cDNA encoding the non-structural protein NS5 The plasma of patients infected with Hepatitis C virus was collected and ultracentrifuged at 4 ° C and then the viral particles were obtained. Subsequently, the viral nucleic acid (RNA) was extracted and purified from the viral particles using guanidine isothiocyanate and phenol acid (Chomczynski et al., Anal. Biochem. 162: 156-159, 1987). (i) 5 * -GGATCCCGGTGGAGGATGAGAGGGAAATATCCG-3 '(SEQ ID No. 3) and (ii) 5'-GAATTCCCGGACGTCCTTCGCCCCGTAGCCAAATTT-3' (SEQ ID No. 4) They were used as primaries in the cloning of cDNA. A simple filament DNA molecule was produced using random primaries, reverse transcriptase, and the RNA model. The double-stranded DNA molecule containing the sequence NS5 was amplified by the PCR method using Taq polymerase and the primaries (i) and (ii). The cloned DNA molecule was subjected to sequence analysis for identification. The molecule obtained was designated EN-80-1. The DNA sequence of the EN-80-1 molecule 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 the non-structural region 5 of HCV and has 803 base pairs (SEQ ID No. 9). The amino acid sequence of the molecule EN-80-1 is given in Fig. 3B (SEQ ID No. 10), and it has 267 residues. 8. Construction of a plasmid containing an HCV cDNA The EN-80-1 molecule was treated with restriction endonucleases Bam Hl and EcoRI to obtain a DNA fragment containing said HCV cDNA. The resulting DNA fragment was inserted into a vehicle plasmid which had been cut first with restriction endonucleases Bam Hl and EcoRI, to obtain an expression plasmid, designated pEN-1. The expression of the HCV cDNA is under the control of a T7 promoter. The structure of expression plasmid pEN-1 and a restriction map are given in Fig. 4. 9. Transformation of E. coli The expression plasmid pEN-1 were transformed into £. coli BL21 (DE3), sprayed on an ampicillin-agar plate and placed in an incubator at 37 ° C overnight. Colonies of £. coli that produce non-structural HCV protein were selected by classifying their expression products by SDS-PAGE and Western Blotting.

. Production of non-structural protein NS5 Colonies of £. Transfected coli were incubated in a conditioned culture medium. The colonies were centrifuged and used by freeze-thaw cycles and lysozyme digestion.

The protein product was released by the used cells and purified by column chromatography. The resulting polypeptide was more than 90% pure. The polypeptide has a molecular weight of approximately 29,000 daltons as measured by electrophoresis through a sodium dodecyl sulfate-polyacrylamide gel. 1 1. Immunological reactivity of NS5 non-structural protein with HCV antibodies by Western Blotting The purified polypeptide was subjected to 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" cushion (0.15M sodium phosphate buffer, pH 6.7) at 4 ° C for 20 minutes. The polypeptide in the gel was then "electromanufactured" on nitrocellulose membrane under the Blotting Buffer at 1.3A for 1-1.5 hours. The membrane was washed with Wash Buffer (PBS-Tween 20, pH 7.4) and blocked with Blocking Buffer (0.1 M NaCl, 5 mM EDTA, 50 mM Tris, pH7.2-7.4, 0.2% serum albumin of bovine, 0.05% of Nonidet p-40, 1 M of urea) overnight. The membrane was reacted with the serum of the infected persons with / without hepatitis C, which was first diluted with 40% of newborn bovine serum / Tris-HCl (pH 7.4), 10X, at 40 ° C for 2 hours . After the reaction, the membrane was washed with Wash Buffer three times. The membrane was then reacted with an anti-hlgG: HRPO conjugate (which was prepared as described below) at 40 ° C for 2 hours. After the reaction, the paper was washed with Wash Buffer three times and then reacted with 10 ml of Substrate Solution (0.01% of 4-chloro-1-naphthol, 18% of methanol, 0.04M of Tris, pH 7.2-7.4, 0.1 M NaCl and 0.01% H2O2) for 20 minutes. The polypeptide of the present invention was reactive with the serum of HCV patients but was not reactive with the serum of healthy persons. 12. ELISA for HCV antibodies (A) Microtiter plate treatment A microtiter plate was coated with the purified NS5 non-structural protein of the invention at appropriate concentrations and blocked with a buffer containing bovine serum albumin. The treated microtitre plate was stored at 2-8 ° C.

(B) Preparation of anti-hlgG conjugate: HRPO Purified anti-human immunoglobulin G (anti-hlgG) was conjugated to horseradish peroxidase (HRPO) using NalO4 to obtain the anti-IgG: HRPO conjugate. The conjugate was purified by chromatography.

(C) Reagent components (a) Washing solution: Phosphate buffer containing 0.9% NaCl and Thimerosal. (b) Anti-hlgG Conjugate Solution: HRPO: the anti-hlgG conjugate: HRPO prepared as described above dissolved in Tris Buffer containing a protein stabilizer and antiseptic. (c) Sample diluent: Tris buffer containing a protein and antiseptic stabilizer. (d) OPD Substrate Solution: o-Phenylenediamine (OPD) dissolved in citrate-phosphate buffer containing H2O2. (If the solution turns orange, means that the solution has been contaminated and can not be used anymore). (e) Stop Solution: 2N H2SO4 solution. (f) Positive / negative controls: serum samples from persons infected with / without hepatitis C diluted with phosphate buffer containing a protein stabilizer and antiseptic at an appropriate concentration.

(D) Procedure (a) One hundred fifty microliters (μl) of the test samples were diluted with Sample Diluent (1: 10), and Positive / Negative Controls were added into the wells of the treated microtiter plate. Some cavities were retained as substrate targets. (b) The plate was mixed gently by shaking and incubated at 37-40 ° C for 1 hour. (c) The plate was washed three times with 0.3 ml of Wash Solution per cavity. (d) One hundred μl of Anti-hlgG conjugate solution: HRPO were added to each well. (e) The plate was mixed gently and incubated by shaking at 37-40 ° C for 30 minutes. (f) The plate was washed five times. (g) One hundred μl of OPD Substrate Solution were added to each well and the plate was incubated at 15-30 ° C in the dark for 30 minutes. (h) One hundred μl of Stop Solution were added to each well and mixed gently to stop the reaction, (i) The OD value per well was measured at 492nm in a spectrophotometer.

(E) Determination: The value OD492nm per cavity subtracts the meaning of the readings of the targets (foundations). The difference (PCx-NCx) between the meaning of the readings of the positive controls (PCx) and those of the negative controls (NCx) is equal to or more than 0.5. The cutoff value (CO) is calculated by the following formula: CO = PCx X 0.15 + NCx When the readings of the test samples were less than the CO value, the samples were considered negative (ie, HCV antibodies could not be detected in the samples). When the readings of the test samples were equal to or more than the CO value, the samples were expected to be positive; however, it is preferable to repeat the assay for duplicate samples. If the readings of any of the samples in duplicate were less than the CO value, the samples will be negative. If the duplicate samples were both more than or equal to the cut-off value, the samples were considered positive. When the readings of the test samples are more than NCx but less than the CO value by 20%, the samples should be considered as questionable samples and the test has to be repeated for those samples. Eighteen samples were tested by the ELISA according to the invention. At the same time, the samples were also tested with the HCV antibody assay of the Abbott (I) pool, which contains the non-structural protein C100-3, and with the HCV antibody assay of the Abbott (II) pool , whose set contains both structural and non-structural proteins. The comparison between the results of the test of the Abbott (II) sets and those of the test of the present invention is given in Table 1. It is noted that the results of the Sample G 30 and the Sample G 128 were negative according to to the Abbott set (I) but were positive according to the assay of the present invention. These samples were confirmed positive for HCV.

TABLE 2 Sample No. OD 92np Results References Set of Abbott (I) (M) TSGH 56 > 2.0 Positive Positive Positive G 23 0.813 Positive Positive Positive G 26 1 .607 Positive Positive Positive G 30 > 2.0 Positive Positive Negative G 32 > 2.0 Positive Positive Positive G 56 > 2.0 Positive Positive Positive G 125 > 2.0 Positive Positive Negative G 186 > 2.0 Positive Positive Positive G 208 > 2.0 Positive - Positive G 214 > 2.0 Positive - Positive G 231 > 2.0 Positive - Positive And 1 > 2.0 Positive - Positive USB 9 > 2.0 Positive - Positive USB 19 > 2.0 Positive - Positive USB 20 > 2.0 Positive - Positive G 201 0.062 Negative ~ Negative G 202 0.072 Negative - Negative G 21 1 0.059 Negative - Negative DETECTION USING BOTH AN ADJACENT PROTEIN OF ANTIGEN SIMILAR TO THE NUCLEUS AS A SECOND PROTEIN 13. ELISAs for HCV using both unfolded core antigen-envelope protein and NS5 non-structural protein A. TESTS COMPARING THE ENVOLTATION PROTEIN- NUCLEO ANTIGEN AND NON-STRUCTURAL PROTEIN NS5 WITH ABBOTT HCV ESSAYS I. FIRST TEST The method was analogous to the ELISAs described above, except that the unfolded core antigen-envelope protein was combined with a nonstructural protein NS5 (9: 1) (known as EverNew Anti-HCV EIA). In the first trial, twenty-four samples were tested by the method described above. At the same time, the samples were also tested by the Abbott set (II). The results are given in Table 3. In this assay, the results of the Abbott (II) pool were the same as the assay using the antigens of the present invention.

TABLE 3 / Sample No. OD 92nm Results References Set of Abbott (II) TSGH 56 > 2.0 Positive Positive TSGH 57 > 2.0 Positive Positive G 23 1 .469 Positive Positive G 26 > 2.0 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 1 14 > 2.0 Positive Positive G 128 > 2.0 Positive Positive G 186 > 2.0 Positive Positive G 214 > 2.0 Positive Positive G 231 > 2.0 Positive Positive G 250 > 2.0 Positive Positive Y 1 > 2.0 Positive Positive USB 9 > 2.0 Positive Positive USB 19 > 2.0 Positive Positive USB 20 > 2.0 Positive Positive USB 23 > 2.0 Positive Positive USB 27 > 2.0 Positive Positive G 92 0.038 Negative Negative G 93 0.056 Negative Negative G 94 0.071 Negative Negative II. SECOND ASSAY The report of clinical blood donor tests for EverNew Anti-HCV EIA is shown in TABLE 4: Hospital: Taipei Tri-Service General Hospital Sample Source: Blood Bank Collection Sample Classification: Donors of blood volunteers Reference set: Abbott reference set (II) Results: TABLE 4 ABBOTT Total 5 (2.5%) 1 (0.5%) 6 (3%) EverNew 1 (0.5%) 193 (96.5%) 194 (97%) total 6 (3%) 194 (97%) 200 (100%) The results in Table 4 indicate that both assays provide the same detection. lll. THIRD TEST The report of clinical trials of high-risk patients for EverNew Anti-HCV EIA is shown in TABLE 5: Hospital: Taipei Veterans General Hospital Sample Source: Collected from the Department of Clinical Virology Classification: NANB, sporadic 20 NANB, PHT 12 HCC 15 Cirrhosis of liver 9 Chronic hepatitis B and carrier 10 Stones in the biliary tract 4 Liver disease of alcoholic 3 Fatty liver 2 Acute hepatitis, etiology? 2 Schistosomiasis of liver 1 Hepatic cysts 1 Colangio-CA 1 Non-hepatobiliary disease 6 No data 2 Total 88 Reference set: ABBOTT HCV EIA 2nd generation Results: TABLE 5 ABBOTT Total 54 (61.36%) 0 (0%) 54 (61.36%) EverNew 1 (1 .14.5%) © 33 (37.5%) 34 (38.64%) total 55 (62.5%) 33 (37.5%) 88 (100%) > : HCV RT / PCR method: Negative Clinical data and HCV RT / PCR results indicated that the efficiency of the EverNew Anti-HCV EIA for HCV antibody detection was better than the ABBOTT HCV EIA 2nd generation authorized by the US FDA.

B. TESTS SHOWING THE SYNERGISTIC COOPERATION OF PROTEINS ADJACENT-ANTIGENS SIMILAR TO THE NUCLEUS AND A VARIETY OF SECOND PROTEINS. AND COMPARISON OF A HCV NUCLEO ENVOLTAGE-BINDING PROTEIN WITH A PARTIAL NUCLEI PROTEI OF HCV FIRST TEST This test shows the results of an ELISA similar to those discussed above, and shows the cooperative interaction between the proteins EN-80-2 and EN-80-1 of HCV. The protocol for the ELISA is as follows: Coating buffer: 0.055 Tris-HCl / 0.15N NaCl / 6 M urea pH: 7.4 ± 0.2. Shock absorber: PBS with 0.05% Tween 29. Post-coating shock absorber: PBS buffer with 1% BSA. Coating procedure: proteins EN-80-1 and EN-80-2 were added to the coating buffer (final concentration: approximately 1.5 μg / ml) and mixed at room temperature for 30 minutes. After mixing, proteins EN-80-1 and EN-80-2 were added in microtitre cavities, 100 μl / well, and incubated in an incubator at 40 ° C for 24 hours. The microtiter cavities were then washed, and the post-coating buffer was added to the cavities. The microtiter cavities were then left at 4 ° C overnight. After post-coating, the coated microtitre cavities can be used for the detection of anti-HCV antibodies. Sample diluent: 0.1 M Tris-HCl pH: 7.4 ± 0.2 with NBBS, 1% BSA and 2% mouse serum. Conjugate: anti-human IgG monoclonal antibody coupled with HRPO using NalO. After coupling, the anti-human IgG: HRPO conjugates were purified by S-200 gel filtration and diluted in the sample diluent.

OPD tablets: purchased from Beckman. Substrate thinner: citrate-phosphate buffer containing H2O2. Stop solution: H2SO4 2N. Positive control, positive serum of anti-HCV diluted in the sample diluent. Negative control: recalcified human serum, which is not reactive for HBV, anti-HIV, anti-HTLV I and anti-HCV markers. Test procedure: 100 μl of sample diluent was added in each well. 50 μl of sample, positive control and negative control were added to the appropriate cavities. Incubation of the sample: incubated at 40 ± 1 ° C for 30 + 2 minutes. Sample wash: the cavities were washed 3 times using washing buffer: 100 μl of IgG conjugate: anti-human HRPO were added in each cavity. Incubation of the conjugate: incubated at 40 ± 1 ° C for 30 t 2 minutes. Washing of the conjugate: the cavities were washed 6 times using washing buffer. After washing, 100 μl of the substrate solution was added (the substrate solution was prepared by dissolving an OPD tablet in 5 ml of substrate diluent), then the mixture was allowed to stand at room temperature for 10 minutes. In order to avoid light, the microtiter cavities were covered with a black cover. 100 μl of stop solution were added in each cavity. It mixed gently. Evaluation: The OD value per cavity was measured at 492 nm in a spectrophotometer. Interpretation: Determination of the cut-off value: cut-off value = PCx X 0.25 + NCx. An absorbance equal to or greater than the cut-off value indicated that a reaction was considered positive, which means that it is reactive for the anti-HCV antibody. An absorbance lower than the cut-off value was considered negative, which means that it is not reactive for the anti-HCV antibody. The sources of the sample for the assay reflected in Table 6 were as follows: Sample source I: G83, G191, G205 and G235 were abnormal samples that were negative for the anti-HCV antibody and were collected from the donor donation center. blood of Taipei. Source of sample II: G614 and G615 were positive for the anti-HCV antibody and were purchased from the United States. Sample source l l l: 8-5 was positive for anti-HCV antibody and was collected from the Taichung blood donation center. Source of sample IV: N345 was a patient serum.

TABLE 6 j: Absorbance at 492 nm. *: The samples were diluted with recalcified human serum, which is not reactive for HBV, HCV and HIV. $: The Abbott group (II) found this negative sample.

These data demonstrate that when the EN-80-2 and EN-80-1 proteins were combined, the absorbance at 492 nm for the anti-HCV positive samples was synergistic, non-additive. Thus, cooperative interactions between proteins EN-80-2 and EN-80-1 of HCV were found. A benefit of this synergism is shown, for example, with sample N345, which was found to be negative for HCV by the Abbott (II) set, but due to the synergistic effect it was found positive by the present invention. These data also show that the synergistic effect is useful when classifying anti-HCV antibodies in samples, particularly in situations of early detection.

II. SECOND ASSAY This assay was conducted as set forth in the First Assay, above, and provision is included in a single cavity 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 shown in Table 7.

TABLE 7 @: Absorbance at 492 nm.

*: The samples were diluted with re-cyclic human serum, which is not reactive for HBV, HCV and HIV.

The data in Table 7 demonstrate that when the EN-80-2 and EN-80-4 proteins are combined, the absorbance at 492 nm for the anti-HCV positive samples showed a synergistic effect, not merely an additive effect. Thus, cooperative interactions between proteins EN-80-2 and EN-80-4 of HCV were found. lll. THIRD TEST This test was conducted as set forth in the first Test, above, and included the provision in a single cavity of a core-envelope fusion protein of the invention in combination with an HIV envelope protein. The results of the ELISA are shown in Table 8.

TABLE 8 @: Absorbance at 492 nm. #: The samples were diluted with recalcified human serum, which is not reactive for HBV, HCV and HIV.

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

IV. FOURTH TEST This test was conducted as discussed in the first Test, above, and included the provision in a single cavity of a core-envelope fusion protein of the invention in combination with an envelope protein of HTLV-I. The results of the ELISA were shown in Table 9.

TABLE 9 @: Absorbance at 492 nm. *: The samples were diluted with recalcified human serum, which is not reactive for HBV, HCV and HIV.

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

FIFTH ASSAY This assay was conducted as set forth in the First Assay, above, and included the provision in a single cavity of a shell-core fusion protein of the invention in combination with a HTLV-I protein poly. The results of the ELISA were shown in Table 10.

TABLE 10 & amp: The approximate molecular weight of the HTLV-I protein poly is 16,000 daltons @: Absorbance at 492 nm. *: The samples were diluted with recalcified human serum, which is not reactive for HBV, HCV and HIV.

The data in Table 19 demonstrate that when the EN-80-2 protein of HCV and the pol protein of HTLV-I 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 HCV EN-80-2 protein and the HTLV-I pol protein were found. SAW. SIXTH TEST Table 11 shows the results of an assay that was similar to that in the Fifth Trial (V), and shows that there were no cooperative interactions between the HBV antigens HbsAg and HbcAg and the HCV EN-80-1 protein. HbsAg: purified from human plasma positive for HbsAg. HbcAg: derived from the HBV cDNA fragment. Source of sample I: G30 and G49 were abnormal samples of GPT, which were positive for anti-HCV antibodies and were collected from the Taipei Blood Donation Center. Source of sample II: G612, G613, G614 and G615 were positive for anti-HCV antibody and were purchased in the United States.

TABLE 11 @: The samples were seriously diluted with recalcified human serum, which was not reactive for HBV, HCV and HIV. #: Absorbance at 492 nm.

The data in Table 11 show that when HbsAg or HbcAg were covered together with the protein EN-80-1 (NS5), the absorbance of the samples positive for anti-HCV was not synergistic. No apparent interaction between HbsAg and protein EN-80-1, or HbcAg and protein EN-80-1 was found.

Vile. SEVENTH TEST Table 12 shows a comparison of the detection of anti-HCV antibodies between the EverNew Anti-HCV EIA and the Abbott set (I I). Samples for the test were obtained from the following sources: Sample source I: G23, G265, G30, G32, G49, G58, G14, G128, G186, G231, G250 and G262 were abnormal GPT samples, which were positive for the anti-HCV antibody and were collected from the Taipei blood donation center. Source of sample II: G612, G613, G614 and G615 were positive for the anti-HCV antibody and were purchased in the United States.

Sample source III: VGH7, VGH1 1, VGH12, VGH13, VGH16, VGH26, VGH27, VGH29, VGH30, VGH33, VGH40, VGH43, VGH46 and VGH52 were positive for the anti-HCV antibody and were collected from the Veterans General Hospital from Taipei. Classification for the samples of the source lll: VGH7 Stones IHD VGH1 1 NANB, sporadic VGH12 NANB, sporadic VGH13 NANB, PTH VGH16 HCC VGH26 Liver cirrhosis VGH27 NANB, sporadic VGH29 Stone IHD VGH30 Liver schistosomiasis VGH32 NANB, sporadic VGH33 Cirrhosis of liver VGH40 No data VGH43 NANB, sporadic VGH46 Cirrhosis liver with HCC VGH52 NANB, sporadic Control: recalcified human serum (not reactive with HBV, anti-HCV and HIV). This human serum was also used to dilute the aforementioned anti-HCV positive samples.

Tested assemblies: EverNew Anti-HCV EIA - Microtiter cavities covered with EN-80-1 antigen. EverNew Anti-HCV EIA - Microtiter cavities covered with antigen EN-80-2. EverNew Anti-HCV EIA - Microtiter cavities covered with antigens EN-80-1 and EN-80-2. Reference Set: Abbott's set (II).

Results: TABLE 12 Sample Dilution EN-80-1 EN-80-2 EN-80-1 + ABBOTT EN-80-2 Serum hu- n / a Negative Negative Negative Negative hand re-calcified (control) G23 20X @ Negative $ Positive * Positive Positive 40X Negative Positive Positive Negative G26 8X Negative Positive Positive Positive 16X Negative Positive Positive Negative G30 51X Negative Negative Positive Positive 102X Negative Negative Positive Positive G32 51X Positive Negative Positive Positive 102X Negative Positive Positive Negative G49 21X Negative Positive Positive Negative 42X Negative Positive Positive Negative G58 16X Positive Positive Positive Negative 32X Negative Positive Positive Negative G114 10X Positive Positive Positive Negative 20X Negative Positive Positive Negative G128 120X Negative Positive Positive Negative 240X Negative Negative Positive Negative G186 42X Negative Positive Positive Negative 84X Negative Negative Positive Negative G231 336X Negative Positive Positive Negative 672X Negative Negative Positive Negative G250 168X Negative Positive Positive Negative 336X Negative Positive Positive Negative G262 84X Negative Positive Positive Positive 168X Negative Positive Positive Negative G612 402X Negative Positive Positive Negative 804X Negative Negative Positive Negative G613 26X Negative Positive Positive Negative 52X Negative Positive Positive Negative G614 8X Positive Positive Positive Negative 16X Negative Positive Positive Negative G615 8X Positive Positive Positive Negative 16X Negative Positive Positive Negative VGH7 42X Positive Positive Positive Negative 84X Negative Positive Positive Negative VGH 11 126X Positive Positive Positive Negative 252X Negative Positive Positive Negative VGH12 252X Negative Positive Positive Negative 504X Negative Positive Positive Negative VGH13 252X Negative Positive Positive Positive 504X Negative Positive Positive Negative VGH16 252X Negative Positive Positive Negative 504X Negative Positive Positive Negative VGH26 84X Negative Positive Positive Negative 168X Negative Positive Positive Negative VGH27 42X Negative Negative Positive Negative 84X Negative Negative Positive Negative VGH29 42X Positive Positive Positive Negative 84X Negative Negative Positive Negative VGH30 42X Positive Positive Positive Negative 84X Negative Negative Positive Negative VGH32 504X Negative Negative Positive Negative 1008X Negative Negative Positive Negative VGH33 84X Negative Positive Positive Negative 168X Negative Negative Positive Negative VGH40 9X Negative Negative Positive Negative 18X N.D. & N. D. Positive Negative VGH43 9X Negative Negative Positive Negative 18X N.D. N. D. Positive Negative VGH46 9X Negative Positive Positive Negative 12X N.D. N.D. Positive Positive VGH52 126X Negative Negative Positive Positive 252X Negative Negative Negative Negative @: The samples were seriously diluted with recalcified human serum which was not reactive with HBV, anti-HCV and HIV. $: negative - not reactive with anti-HCV antibody. #: positive - reactive with anti-HCV antibody. &: N.D. - unrealized.

The data in Table 12 in bold letters show the cases of synergy between the envelope protein-core antigen and the non-structural region (NS5) of HCV. The bold data also demonstrates cases where the invention provides better detection than the reference of the HCV detection set of the Abbott (II) set. These data indicate that the detectability of the microtiter cavities covered with the EN-80-1 and EN-80-2 antigens was more efficient than the microtiter cavities covered with either the EN-80-1 antigen or the EN- 80- antigen. 802 alone. In addition, the anti-HCV antibody in the 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 and VGH43 18X they could be detected when using EverNew Anti-HCV EIA (microtitre cavities covered with EN-80-1 and EN-80-2 antigens) but were not detected using the Abbott (II) set.

Vlll. EIGHT ASSAY This trial shows the results of an ELISA performed according to the protocol described in the First Essay, above, where a partial core protein consisted of amino acids 1 to 120, and was a gift from the Biotechnology Development Center (DCB) ) in Taiwan. Source of sample I: G235 was an abnormal sample of GPT, which was negative for the anti-HCV antibody and was collected from the Taipei blood donation center. Source of sample II: G614 and G615 were positive samples for anti-HCV and were purchased in the United States.

TABLE 13 Absorbance at 492 nm.

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

IX. NINTH ESSAY Table 14 confirms the results presented above and shows an immunoassay comparison by enzyme of the detection of anti-HCV antibodies using partial nucleus (antigen EN-80-5, which is an HCV partial core antigen that has a molecular weight of approximately 15,000 daltons as measured by electrophoresis through an SDS-polyacrylamide gel), envelope protein-core antigen (EN-80-2 antigen) and / or a non-structural HCV protein (NS5; -80-1 discussed before). The samples for the test were positive samples for anti-HCV nos. N8, N81, N89, N 12 and N302, and negative samples for anti-HCV nos. N202, N203 and N302. Positive samples were diluted between 25X and 672X with 0.1M Tris-HCl, pH 7.4 (+/- 0.2) with 40% newborn bovine serum, 1% BSA and 2% mouse serum. The samples were tested in microtitre cavities with a solution of IgG conjugate: monoclonal anti-human HRPO, in combination with the following antigens or combinations of antigens: a.) NS5; b.) envelope protein-core antigen; c.) partial core protein; d.) NS5 and envelope protein-core antigen; e.) NS5 and partial core protein; f.) envelope protein-core antigen and partial nucleus; and, g.) NS5, envelope and core antigen protein and partial nucleus. The following results were obtained: TABLE 14 Sample NS5 n core-core NS5 + NS5 + Core NS5 + I D env core- core + core + env core- core- env env N8 50X @ 0.098 * 1 .009 0.952 > 2.0 0.535 > 2.0 > 2.0 100X 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 > 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 > 2.0 672X 0.040 1,301 0.794 1 .671 0.589 1 .321 1,694N 12 25X 0.019 1 .848 > 2.0 > 2.0 0.676 > 2.0 > 2.0 50X 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 > 2.0 336X 0.078 1 .161 1 .968 1 .645 1 .660 > 2.0 > 2.0 672X 0.046 0.496 0.819 0.829 0.612 0.805 1 .025 N202 0.043 0.081 0.169 0.077 0.048 0.081 0.075 N203 0.100 0.208 0.124 0.185 0.1 17 0.189 0.169 N209 0.023 0.033 0.054 0.036 0.037 0.045 0.042 Diluent 0.018 0.028 0.018 0.021 0.025 0.028 0.027 sample * @: Serum positive anti-HCV diluted with sample diluent. *: Absorbance at 492 nm. #: Sample Diluent: 0.1 M Tris-Hcl pH: 7.4 ± 0.2 with 40% newborn bovine serum, 1% BSA and 2% mouse serum.

X. TENTH TEST The tenth assay was an enzyme immunoassay directed to the use of an HIV gag protein in combination with an HIV envelope protein to detect the presence of anti-HIV-1 antibodies in human serum. The antigens used for the assay were as follows: First, a recombinant fusion protein comprising the amino-terminal fragment of β-galactosidase (377 aa) fused to gag-17 (aa 15-132) followed by gag p24 (aa 133- 363) followed by gag p15 (364-437). This protein has a molecular weight of 92.8 kDa, 831 a.a. (including amino acid separators), and the EN-l-5 antigen was titrated. The protein used for the assay was purified from £. coli for greater purity than 90% and was non-glycosylated. Second, a recombinant fusion protein comprising the amino-terminal fragment of β-galactosidase (311 a.a.) fused to amino acids 474-863 of env, ie, gp160. This protein had a molecular weight of 80.7 kDa; 705 a.a. (including amino acid separators), and the EN-I-6 antigen was titrated. The envelope cut site within gp160 was found among amino acids nos. 491 and 492, according to Ratner et al., Aids Res. And Human Retroviruses 3 (1): 57-69, 1987. Thus, the antigen EN-l-6 includes both the carboxyl terminus of gp120 and the amino terminal of gp41. The protein used for the assay was purified from £. coli for greater purity than 90% and was non-glycosylated. The positive samples for the assay were obtained from clinically tested HIV positive humans, therefore they were positive sera of anti-HIV-1 antibody, and were numbered T1, T2, T3, T4, T5, T6, P1, P2 and P3. The control sample was numbered NC and was a negative serum of anti-HIV-1 antibody. The samples were tested in microtitre cavities with a conjugate solution of lgG: anti-human monoclonal HRPO, in combination with the following antigens or combinations of antigens: a.) EN-l-5 antigen (1 μg / ml, 0.1 ml /cavity); b.) EN-l-6 antigen (1 μg / ml, 0.1 ml / well); and c.) EN-l-5 and EN-l-6 (both 1 μg / ml, antigens, 0.1 ml / well). The following results were obtained: TABLE 15 Samples HlV 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 T1 '24X # 0.007 0.957 1.520 T1 72X 0.000 0.440 0.863 T2 72X 0.000 0.407 0.644 T3 8X 0.000 0.350 0.548! "4 648X 0.001 0.319 0.488 T5 72X 0.000 0.227 0.353 T6 72X 0.005 0.560 0.799 NC 0.019 0.028 0.030 NC 4X 0.012 0.027 0.025 @: The absorbance of 492 nm. #: samples diluted with sample diluent.

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

XI. ELEVENTH TEST 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 serum. The antigens used for the assay were an HIV env protein (the EN-l-6 antigen, described above), an NS5 HCV protein (the EN-80-1 antigen, described above) and an adjacent protein-antigen similar to HCV core (the EN-80-2 antigen, also described above). The positive samples for the test were T1, T2, T3, T4, T5 and T6, which were sera positive for anti-HIV-1 antibody; and the control samples were N639, N626, N634, N632 and N637, which were negative sera for anti-HIV-1 and anti-HCV antibodies.

The samples were tested in microtitre cavities with a conjugate solution of IgG: monoclonal anti-human HRPO, using the antigens or combinations of antigens discussed below in the Table 16. The results of the tests are also shown in Table 16.

TABLE 16 Samples: HCV NS5 HCV NS5 & HIV env HCV HCV HIV env core-env core-env & HIV env T1 24X @ 0.048 * 0.833 0.602 0.930 0.038 72X 0.057 0.599 0.460 0.679 0.048 216X 0.055 0.278 0.213 0.314 0.039 N639 0.077 0.092 0.097 0.1 10 0.069 T2 24X 0.048 0.876 0.512 0.947 0.026 72X 0.052 0.520 0.377 0.697 0.031 216X 0.069 0.228 0.191 0.284 0.037 Diluent 0.069 0.052 0.029 0.040 0.047 sample 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 0.138 0.187 0.024 N626 0.069 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 T5 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 0.036 0.053 0.041 0.051 T6 24X 0.021 0.864 0.783 1.048 0.023 72X 0.018 0.523 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 @: Positive samples for anti-HIV-1 diluted with sample diluent (0.1 M Tris-HCl, pH: 7.4 ± 0.2 with 40% newborn 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 env HCV core protein discussed above.

THE PRODUCTION OF MONOCLONAL ANTIBODIES FOR AN ADJACENT-ANTIGEN PROTEIN SIMILAR TO THE NUCLEUS 14. Preparation of antibodies against HCV Antibodies against the envelope protein-unprocessed core antigen and the NS5 non-structural protein were produced according to a standard procedure to produce monoclonal antibodies. In particular, a BALB / c mouse was immunized with the purified proteins described above in Examples 2 and 10 mixed with an auxiliary; and then the spleen cells were fused with mouse myeloma cells (FO cell line) using polyethylene glycol to form hybridoma cells. The desired clones producing the desired monoclonal antibodies were obtained by sorting the titer of the antibodies produced by the hybridoma clones thus prepared. In one embodiment of the invention, a hybridoma clone was designated EN-80-1-99.

THE USE OF AN ADJACENT-ANTIGEN PROTEIN SIMILAR TO THE NUCLEUS TO INDUCE AN IMMUNOSPOSIT . Administration of an Advacent protein-antigen similar to the HCV nucleus A core-antigen envelope protein (EN-80-2) was administered intramuscularly to ICR mice at 6-8 weeks of age. The first administration, booster and sample card were as follows: Negative control group: (ID nos. 0-1 and 0-2) Day 0: no immunization Day 13: 1st bleeding Day 28: 2nd bleeding Test Group 1: (ID Nos. 1-1, 1-2, 1-3, 1-4, 1-5 and 1-6) Day 0: 50 μg / mouse protein EN-80-2 using adjuvant Complete Freund (CFA) (Gaitherburg, MD, EU, 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 μg / mouse protein EN-80-2 using auxiliary Complete Freund (CFA) GIBCO. Day 13: 1st immunization, with 80 μg / mouse protein EN-80-2 using incomplete Freund's assistant (IFA), also from GIBCO (Gaithersburg, MD, EU, 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 μg / mouse protein EN-80-2 using auxiliary Complete Freund (CFA) (Gaitherburg, MD, EU, 20877). Day 13: 1st immunization, with 80 μg / mouse protein EN-80-2 using incomplete Freund's assistant (I FA), GIBCO Day 28: 2a immunization, with 80 μg / mouse protein EN-80-2, in PBS.

Day 39: 1st bleeding. 16. Detection of the immune response induced by the administration of the envelope protein-core antigen The presence or absence of an immune response in the test animals was determined using two enzyme immunoassays (ElAs) similar to those described above. In the first EIA, an anti-mouse conjugate: Rat H RPO was added to the cavities of a microtiter plate that had been coated with an envelope-core antigen protein (EN-80-2) together with a conjugate of anti-mouse: rat HRPO. 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. $ 500X 0.058 0.060 N. D. 2500X 0.008 0.025 N. D. 12500X 0.000 0.010 N. D. 62500X 0.000 0.012 N. D. 0-2 50X 0.188 0.160 N. D 500X 0.048 0.050 N. D 2500X 0.000 0.018 N. D 12500X 0.000 0.013 N.D. 62500X 0.000 0.009 N.D.

Group 1: 1-1 50X 0.720 N.D. N.D. 500X 0.144 * N.D. N.D. 2500X 0.018 N.D. N.D. 12500X 0.000 N.D. N.D. 62500X 0.000 N.D. N.D. 1-2 50X 0.257 * > 2.0 / > 2.0 > 2.0 500X 0.062 0.976 / 1.263 > 2.0 2500X 0.004 0.187 / 0.278 * 0.560 12500X 0.000 0.023 / 0.062 0.132 * 62500X 0.000 0.000 / 0.018 0.027 1-3 50X 0.213 * > 2.0 N.D. 500X 0.042 0.424 * N.D. 2500X 0.000 0.058 N.D. 12500X 0.000 0.000 N.D. 62500X 0.000 0.000 N.D. 1-4 50X 0.259 * > 2.0 / > 2.0 > 2.0 500X 0.050 1.882 / > 2.0 > 2.0 2500X 0.002 0.348 / 0.506 * 0.886 12500X 0.000 0.048 / 0.098 0.163 * 62500X 0.000 0.000 / 0.037 0.039 1-5 50X 0.580 > 2.0 / > 2.0 1.616 500X 0.111 * 1.774 / > 2.0 1.646 2500X 0.010 0.336 / 0.471 * 0.313 * 12500X 0.000 0.041 / 0.097 0.067 62500X 0.000 0.000 / 0.030 0.021 1-6 50X 0.443 0.341 N.D. 500X 0.161 * 0.191 * N.D. 2500X 0.026 0.071 N.D. 12500X 0.000 0.025 N.D. 62500X 0.000 0.016 N.D.

Group 2: 2-1 50X < 2.0 / > 2.0 > 2.0 500X 0.939 / 1.161 1.478 2500X 0.161 / 0.200 * 0.280 * 12500X 0.032 / 0.038 0.059 62500X 0.016 / 0.017 0.022 -2 50X > 2.0 / > 2.0 > 2.0 500X > 2.0 / > 2.0 > 2.0 2500X 1.092 / 1.316 1.158 12500X 0.232 / 0.267 * 0.250 * 62500X 0.050 / 0.063 0.061 2-3 50X 0.544 N. D. 500X 0.121 * N. D. 2500X 0.028 N. D. 12500X 0.010 N. D. 62500X 0.013 N. D. 2-4 50X > 2.0 / > 2.0 > 2.0 500X > 2.0 / > 2.0 > 2.0 2500X 0.909 / 1,209 0.794 12500X 0.177 / 0.232 * 0.156 '62500X 0.037 / 0.058 0.051 2-5 50X 1 .860 > 2.0 500X 0.379 * 0.836 2500X 0.071 0.155 * 12500X 0.018 0.030 62500X 0.010 0.019 2-6 50X < 2.0 / < 2.0 > 2.0 500X 1.475 / 1.780 1 .577 2500X 0.333 / 0.383 * 0.357 * 12500X 0.066 / 0.080 0.075 62500X 0.019 / 0.078 0.025 Group 3: -150X > 2.0 500X > 2 0 2500X 2.0 12500X 1 .647 62500X 0.362 * -2 50X > 2.0 500X > 2.0 2500X 1032 12500X 0.195 * 62500X 0.053 -3 50X > 2.0 500X 1-814 2500X 0.312 * 12500X 0.060 62500X 0.026 -4 50X > 2.0 500X > 2.0 2500X 0.895 12500X 0.181 * 62500X 0.048 -5 50X > 2.0 500X > 2.0 2500X > 2.0 12500X 0.701 62500X 0.146 * 3-6 50X > 2.0 500X > 2.0 2500X > 2.0 12500X 0.726 62500X 0.172 * @: Diluted mouse serum 50X, 500X, 2500X, 12500X and 62500X with 1% BSA. #: Absorbance at 492 nm. *: Endpoint of detectability. $: N.D .: Test not performed because there was no serum for the test.

In a second EIA, an anti-mouse conjugate: Rat HRPO was added to the cavities of a microtiter plate that had been covered with the following antigens or combinations of antigens: a.) NS5 (antigen EN-80-1); b.) envelope protein-core antigen (antigen EN-80-2); c.) partial core protein (antigen EN-80-5); d.) NS5 and envelope protein-core antigen; e.) NS5 and partial core protein; f.) envelope protein-core antigen and partial nucleus; and g.) NS5, envelope protein-core antigen, and partial core. The samples used in the second EIA were as follows: 0-2 (diluted 50X, from day 28); 0-2 (diluted 500X from day 28); 2-2 (diluted 2500X from day 28); 3-1 (diluted 12500X from day 39; 3-4 (diluted 2500X from day 39); 3-5 (diluted 2500X from day 39); 3-6 (diluted 2500X from day 39); and 3-6 (diluted 12500X) of day 39) The results of the second EIA are shown below in Table 18.

TABLE 18 Sample NS5 core-core NS5 + NS5 + core NS5 + env ID core-core + core env core- + env 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 0.01 1 0.014 0.014 0.022 0.019 Group II: 2-2 0.004 0.398 0.007 0.489 0.009 0.313 0.388 2500X Group III: 3-1 0.002 0.506 0.009 0.760 0.009 0.513 0.472 12500X 3-4 0.003 0.220 0.007 0.344 0.006 0.192 0.227 2500X 3-5 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 492 nm.

Claims (72)

1. CLAIMS A composition derived from positive filament RNA virus comprising the following: a) an isolated polypeptide comprising an antigen protein similar to the nucleus of positive strand RNA virus bound to an amino-terminal portion of an adjacent protein of said RNA virus of positive filament in an unprocessed form, wherein said amino-terminal portion of said adjacent protein is sized such that said polypeptide has an epitopic configuration corresponding to an adjacent protein-antigen similar to the unprocessed nucleus of said filament RNA virus positive; and b) a non-structural protein isolated from said positive strand RNA virus. The composition of claim 1 wherein said positive filament RNA virus is selected from the group consisting of Togaviridae, Coronaviridae, Retroviridae, Picornaviridae, Caliciviridae and Flaviviridae. The composition of claim 2 wherein said positive filament RNA virus is selected from the group consisting of human immunodeficiency virus (HIV) and human T cell leukemia virus (HTLV). The composition of claim 1 wherein said isolated polypeptide is produced by a suitable prokaryotic host cell. The composition of claim 1 wherein said polypeptide is produced by a eukaryotic host cell that is incapable of processing said isolated polypeptide. A method for making a composition comprising multiple polypeptides obtained from a positive strand RNA virus, comprising the following steps: a) introducing into a first host a first expression vector capable of expressing a nucleic acid molecule encoding a polypeptide isolated comprising an antigen protein similar to the nucleus of positive strand RNA virus bound to an amino-terminal portion of a protein adjacent to said positive strand RNA virus in unprocessed form, wherein said amino-terminal portion of said adjacent protein is sized such that said polypeptide has an epitopic configuration corresponding to an adjacent protein-antigen similar to the nucleus of said positive filament RNA virus, b) incubating said first host cell under conditions suitable for said expression vector to produce said polypeptide, c) purifying said polypeptide to provide a purified polypeptide, and d) introducing into a second host cell a second expression vector capable of expressing a nucleic acid molecule encoding a nonstructural protein isolated from said positive filament RNA virus, e) incubating said second host cell under suitable for said nucleic acid molecule to produce said non-structural protein, f) purifying said non-structural protein to provide a purified non-structural protein, and then g) combining said purified polypeptide and said purified non-structural protein to form said composition. A method for making a composition comprising multiple polypeptides obtained from a positive filament RNA virus, comprising the following steps: a) introducing into an host cell an expression vector capable of expressing a first nucleic acid molecule encoding a polypeptide isolated comprising an antigen protein similar to the nucleus of positive strand RNA virus bound to an amino-terminal portion of an adjacent protein of said positive strand RNA virus in unprocessed form, wherein said amino-terminal portion of said adjacent protein is sized such that said polypeptide has an epitopic configuration corresponding to an adjacent protein-antigen similar to the unprocessed nucleus of said positive strand RNA virus, said expression vector is also capable of expressing a second protein comprising a non-structural protein derived of said filament RNA virus positive, b) incubating said host cell under conditions suitable for said expression vector to produce said polypeptide and said non-structural protein, and c) purifying said polypeptide and said non-structural protein to provide a purified polypeptide and a purified nonstructural protein. The method of claim 6 or 7 wherein said positive filament RNA virus is selected from the group consisting of Togaviridae, Coronaviridae, Retroviridae, Picornaviridae, Caliciviridae and Flaviviridae. A composition comprising a substantially complete, unprocessed polyprotein isolated from a positive strand RNA virus linked to a solid substrate. A composition comprising an isolated polypeptide comprising an antigen protein similar to the virus core of RNA of positive filament attached to an amino-terminal portion of an adjacent protein of said positive filament RNA virus in unprocessed form, wherein said amino-termination portion of said adjacent protein is sized such that said polypeptide has an epitopic configuration corresponding to an adjacent protein-antigen similar to the unprocessed nucleus of said positive filament RNA virus, bound to a solid substrate. The composition of claim 10 further comprising a non-structural protein of said positive filament RNA virus linked to said solid substrate. An assay for the detection of a positive filament RNA virus in a sample, comprising: a) providing an isolated polypeptide comprising an antigen protein similar to the nucleus of positive filament RNA virus bound to an amino-terminal portion of a protein adjacent said positive filament RNA virus in unprocessed form, wherein said amino-terminal portion of said adjacent protein is sized such that said polypeptide has an epitopic configuration corresponding to an adjacent protein-antigen similar to the unprocessed core of said RNA virus of positive filament. b) Contacting said isolated polypeptide with said sample under suitable conditions and for a sufficient time for said polypeptide to bind to one or more antibodies specific for said positive filament RNA virus present in said sample, to provide an antibody-bound polypeptide , and c) Detecting said antibody-bound polypeptide, and from there determining that said sample contains RNA virus of positive filament. The assay of claim 12 further comprising, a) in step a), providing a non-structural protein of said positive filament RNA virus linked to said solid substrate, b) in step b), contacting said non-target protein. structural with said sample under suitable conditions and for a sufficient time for said non-structural protein to bind to one or more antibodies specific for said positive filament RNA virus present in said sample, to provide a non-structural protein of strand RNA virus positive bound to antibody, and c) in step c); detecting one or both of said antibody-bound polypeptide or said non-structural protein bound to antibody, and from there determining that said sample contains said positive filament RNA virus. An assay for the detection of a positive strand RNA virus in a sample, comprising: a) providing an isolated polypeptide comprising a substantially complete unprocessed polyprotein isolated from a positive strand RNA virus, b) contacting said polypeptide isolated with said sample under suitable conditions and for a sufficient time for said polypeptide to bind to one or more antibodies specific for said positive filament RNA virus present in said sample, to provide an antibody-bound polypeptide, and c) detect said polypeptide bound to antibody, and hence determine that said sample contains said RNA virus of positive filament. The assay of claim 12, 13 or 14 further comprising the step of ligating said isolated polypeptide, said non-structural protein or said polyprotein to a solid substrate. The test of claim 12, 13 or 14 wherein said sample is an unpurified sample. The test of claim 12, 13 or 14 further comprising, prior to said contact, the step of obtaining said sample from an animal. The test of claim 17 wherein said animal is a human being. The assay of claim 12, 13 or 14 wherein said assay is selected from the group consisting of a countercurrent immunoelectrophoresis (CIEP) assay, a radioimmunoassay, a radioimmunoprecipitation, an enzyme-linked immunosorbent assay (ELISA), a dot blot assay, an inhibition or competition assay, a sandwich assay, an immunoadhesion assay (dipstick), 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. The assay of claim 12, 13 or 14 wherein said assay is not a western blot assay. A method for producing an antibody, comprising the following steps: a) administering to an animal an isolated polypeptide comprising an antigen protein similar to the nucleus of positive strand RNA virus bound to an amino-terminal portion of an adjacent protein of said virus of RNA of positive filament in unprocessed form, wherein said amino-terminal portion of said adjacent protein is sized such that said polypeptide has an epitopic configuration corresponding to an adjacent protein-antigen similar to the unprocessed core of said RNA virus. positive filament, and b) isolating said antibodies for said polypeptide. 22. An antibody produced according to claim 21. 23. A method for producing an antibody, comprising the following steps: a) administering to an animal an isolated polypeptide comprising a substantially complete, unprocessed polyprotein isolated from an RNA virus of positive filament, and b) isolating said antibodies for said polyprotein. 24. An antibody produced according to claim 23. 25. The antibodies of claim 22 or claim 24 wherein said antibodies are bound to a solid substrate. 26. An assay for the detection of a positive filament RNA virus in a sample, comprising: a) contacting said sample with the antibody of claim 22 under the appropriate conditions and for a sufficient time for said antibody to bind said antigen protein similar to the nucleus of positive filament RNA virus, to provide a bound antibody, and b) detect said bound antibody, and hence determine that said sample contains RNA virus of positive filament. The assay of claim 26 further comprising, a) in step a), contacting said sample with an additional antibody specific for a non-structural protein of RNA virus of positive filament under suitable conditions and for a sufficient time to said additional antibody for binding said non-structural protein of positive strand RNA virus, to provide an additional bound antibody, and b) in step b), detecting one or both of said bound antibody or said additional bound antibody, and thereby determining that said sample contains RNA virus of positive filament. 28. An assay for the detection of a positive filament RNA virus in a sample, comprising: a) contacting said sample with the antibody of claim 24 under suitable conditions and for a sufficient time for said antibody to bind an antigen specific for said positive filament RNA virus, to provide a bound antibody, and b) detect said bound antibody, and hence determine that said sample contains RNA virus of positive filament. 29. A composition capable of extracting an immune response in an animal comprising an isolated polypeptide comprising an antigen protein similar to the nucleus of positive strand RNA virus linked to an amino-terminal portion of an adjacent protein of said strand RNA virus. positive in unprocessed form, wherein said amino-terminal portion of said adjacent protein is sized such that said polypeptide has an epitopic configuration corresponding to an adjacent protein-antigen similar to the unprocessed core of said positive filament RNA virus, in combination with a pharmaceutically acceptable diluent or carrier. 30. The composition of claim 29 further comprising a non-structural protein of said positive strand RNA virus. 31. A composition capable of extracting an immune response in an animal comprising a substantially complete, unprocessed polyprotein isolated from a positive filament RNA virus, in combination with a pharmaceutically acceptable diluent or carrier. 32. The composition of claim 29, 30 or 31 wherein said animal is a human being. 33. A vaccine against a positive filament RNA virus comprising an isolated polypeptide comprising an antigen protein similar to the nucleus of positive strand RNA virus linked to an amino-terminal portion of an adjacent protein of said virus. RNA of positive filament in unprocessed form, wherein said amino-terminal portion of said adjacent protein is sized such that said polypeptide has an epitopic configuration corresponding to an adjacent protein-antigen similar to the unprocessed nucleus of said filament RNA virus positive, in combination with a pharmaceutically acceptable diluent or carrier. 34. A vaccine against a positive strand RNA virus comprising a substantially complete unprocessed polyprotein isolated from a positive strand RNA virus in combination with a pharmaceutically acceptable diluent. 35. The vaccine of claim 33 or 34 further comprising a non-structural protein of said positive strand RNA virus. 36. A set for the detection of a positive filament RNA virus comprising: a) an isolated polypeptide comprising an antigen protein similar to the nucleus of positive filament RNA virus bound to an amino-terminal portion of an adjacent protein of said virus. RNA of positive filament in unprocessed form, wherein said amino-terminal portion of said adjacent protein is sized such that said polypeptide has an epitopic configuration corresponding to an adjacent protein-antigen similar to the unprocessed nucleus of said strand RNA virus positive, linked to a solid substrate; and b) one or both of a reagent or a device for detecting said isolated polypeptide. 37. The assembly of claim 36 further comprising a non-structural protein of said positive filament RNA virus and one or both of a reagent or a device for detecting said non-structural protein. 38. A set for the detection of a positive strand RNA virus comprising: a) a substantially complete, unprocessed polyprotein isolated from a positive strand RNA virus, bound to a solid substrate, and b) one or both of a reagent or a device for detecting said isolated polyprotein. 39. A set for the detection of a positive filament RNA virus comprising: a) the antibody of claim 22, and b) one or both of a reagent or a device for detecting said antibody. 40. The assembly of claim 39 further comprising an additional antibody specific for a non-structural HCV protein and one or both of a reagent or a device for detecting said additional antibody. 41. A kit for the detection of a positive filament RNA virus comprising: a) the antibody of claim 24, and b) one or both of a reagent or a device for detecting said antibody. 42. A composition derived from positive filament RNA virus comprising the following: a) an isolated polypeptide comprising an antigen protein similar to the nucleus of positive strand RNA virus linked to an adjacent protein of said positive strand RNA virus in unprocessed form, wherein said adjacent protein is sized such that said polypeptide has an epitopic configuration corresponding to an adjacent protein-antigen similar to the unprocessed nucleus of said positive filament RNA virus; and b) a second protein capable of interacting cooperatively with said isolated polypeptide to increase the antigenicity of said isolated polypeptide. A method for making a composition comprising multiple polypeptides, comprising the following steps: a) introducing into a first host cell a first expression vector capable of expressing a nucleic acid molecule encoding an isolated polypeptide comprising an antigen protein similar to the nucleus of positive filament RNA virus linked to an adjacent protein of said positive filament RNA virus in unprocessed form, wherein said adjacent protein is sized such that said polypeptide has a corresponding epitopic configuration for an adjacent protein-antigen similar to the nucleus unprocessed said positive strand RNA virus, b) incubate said first host cell under conditions suitable for said expression vector to produce said polypeptide, c) purify said polypeptide to provide a purified polypeptide, and d) enter into a second host cell a second v expression molecule capable of expressing a nucleic acid molecule encoding a second isolated protein capable of interacting cooperatively with said isolated polypeptide to increase the antigenicity of said isolated polypeptide, e) incubating said second host cell under conditions suitable for said nucleic acid to produce said second protein, f) purifying said second protein to provide a second purified protein and then, g) combining said purified polypeptide and said second purified protein to form said composition. A method for making a composition comprising multiple polypeptides, at least one of which is obtained from a positive filament RNA virus, comprising the following steps: a) introducing into an host cell an expression vector capable of expressing a first molecule of nucleic acid encoding an isolated polypeptide comprising an antigen protein similar to the nucleus of positive strand RNA virus linked to an adjacent protein of said positive strand RNA virus in unprocessed form, wherein said adjacent protein is dimensioned in a manner that said polypeptide has an epitopic configuration corresponding to an adjacent protein-antigen similar to the unprocessed nucleus of said positive filament RNA virus, said expression vector also capable of expressing a second protein capable of interacting cooperatively with said isolated polypeptide for increase the antigenicity of said pol isolated peptide, b) incubating said host cell under conditions suitable for said expression vector to produce said polypeptide and said second protein, and c) purifying said polypeptide and said second protein to provide a composition comprising a purified polypeptide and a second purified protein. The method of claim 43 or 44 wherein said second protein is derived from a positive filament RNA virus. A composition comprising an isolated polypeptide comprising an antigen protein similar to the core of positive strand RNA virus linked to an adjacent protein of said positive strand RNA virus in unprocessed form, wherein said adjacent protein is sized so that said polypeptide has an epitopic configuration corresponding to an adjacent protein-antigen similar to the unprocessed nucleus of said virus. RNA of positive filament, linked to a solid substrate. The composition of claim 46 further comprising a second protein capable of interacting cooperatively with said isolated polypeptide to increase the antigenicity of said isolated polypeptide. An assay for the detection of a positive strand RNA virus in a sample, comprising: a) providing an isolated polypeptide comprising an antigen protein similar to the nucleus of positive strand RNA virus bound to an adjacent protein of said RNA virus of positive filament in unprocessed form, wherein said adjacent protein is sized such that said polypeptide has an epitopic configuration corresponding to an adjacent protein-antigen similar to the unprocessed nucleus of said positive filament RNA virus, b) contacting said polypeptide with said sample under suitable conditions and for a sufficient time for said polypeptide to bind to one or more antibodies specific for said positive filament RNA virus present in said sample, to provide an antibody-bound polypeptide, and c) detect said polypeptide bound to antibody, and from there determine that said sample c contains said positive filament RNA virus. The assay of claim 48 further comprising, a) in step a), providing a second protein capable of interacting cooperatively with said isolated polypeptide to increase the antigenicity of said isolated polypeptide, b) in step b), contact said second protein with said sample under suitable conditions and for a sufficient time for said second protein to interact cooperatively with said isolated polypeptide, and c) in step c), detect bound antibodies, and from there determine that said sample contains said RNA virus of positive filament. 50. The assay of claim 48 or 49 further comprising the step of ligating said isolated polypeptide or said second protein to a solid substrate. 51. A method for producing an antibody, comprising the following steps: a) administering to an animal an isolated polypeptide comprising an antigen protein similar to the nucleus of positive strand RNA virus linked to an adjacent protein of said strand RNA virus positive in unprocessed form, wherein said adjacent protein is sized such that said polypeptide has an epitopic configuration corresponding to an adjacent protein-antigen similar to the unprocessed core of said positive filament RNA virus, and b) isolating said antibodies for said polypeptide. 52. The method of claim 51 further comprising administering to said animal a second protein capable of interacting cooperatively with said isolated polypeptide to increase the antigenicity of said isolated polypeptide. 53. An antibody produced according to claim 51 or 52. The antibodies produced according to claim 51 or 52 wherein said antibodies are bound to a solid substrate. 55. An assay for the detection of a positive filament RNA virus in a sample, comprising: a) contacting said sample with an antibody produced according to claim 51 or 52 under suitable conditions and for a sufficient time for said antibody for ligating said antigen protein similar to the nucleus of unprocessed positive filament RNA virus, to provide a bound antibody, and b) detecting said bound antibody, and hence determining that said sample contains positive filament RNA virus. 56. A composition capable of extracting an immune response in an animal comprising an isolated polypeptide comprising an antigen protein similar to the nucleus of positive filament RNA virus linked to an adjacent protein of said positive filament RNA virus in unprocessed form, wherein said adjacent protein is sized such that said polypeptide has an epitopic configuration corresponding to an adjacent protein-antigen similar to the unprocessed core of said positive filament RNA virus, in combination with a pharmaceutically acceptable diluent or carrier. 57. The composition of claim 56 further comprising a second protein capable of interacting cooperatively with said isolated polypeptide to increase the antigenicity of said isolated polypeptide. 58. A positive filament RNA virus vaccine comprising an isolated polypeptide comprising an antigen protein similar to the core of positive filament RNA virus linked to an adjacent protein of said positive filament RNA virus in unprocessed form, in wherein said adjacent protein is sized such that said polypeptide has an epitopic configuration corresponding to an adjacent protein-antigen similar to the unprocessed core of said positive filament RNA virus, in combination with a pharmaceutically acceptable diluent or carrier. 59. The vaccine of claim 58 further comprising a second protein capable of interacting cooperatively with said isolated polypeptide to increase the antigenicity of said isolated polypeptide. 60. The composition of any of claims 1-5, 9-11, 29-32, 42, 46, 47, 56 or 57 for use as an active therapeutic substance. 61. The vaccine of any of claims 33-35-58 or 59 for use as an active therapeutic substance. 62. The composition of any of claims 1-5, 9-11, 29-32, 42, 46, 47, 56 or 57 for use in the manufacture of a medicament for inhibiting, preventing or treating an HCV infection in a animal. 63. The vaccine of any of claims 33-35, 58 or 59 for use in the manufacture of a medicament for inhibiting, preventing or treating an HCV infection in an animal. 64. A set for the detection of a positive filament RNA virus comprising: a) an isolated polypeptide comprising an antigen protein similar to the nucleus of positive filament RNA virus linked to an adjacent protein of said positive filament RNA virus in unprocessed form, wherein said adjacent protein is sized such that said polypeptide has an epitopic configuration corresponding to an adjacent protein-antigen similar to the unprocessed core of said positive filament RNA virus, bound to a solid substrate, and b) one or both of a reagent or a device for detecting said isolated polypeptide. The assembly of claim 64 further comprising a second protein capable of interacting cooperatively with said isolated polypeptide and one or both of a reagent or a device for detecting said second protein. A set for the detection of a positive filament RNA virus comprising: a) an antibody produced according to claim 51 or 52, and b) one or both of a reagent or a device for detecting said antibody. The assay of claim 12, 13, 14, 48 or 49 wherein the step of providing comprises providing at least two isolated polypeptides of which are obtained from different positive filament RNA viruses from the group consisting of Hepatitis C virus (HCV), immunodeficiency virus (HIV) and human T-cell leukemia virus (HLTV), and wherein the step of contacting comprises contacting the isolated polypeptides with said sample under suitable conditions and for a sufficient time to each of said isolated polypeptides to bind to one or more antibodies specific therefor, thereby providing one or more polypeptides linked to antibodies. The assay of claim 12, 13, 14, 48, 49 wherein the step of providing comprises providing at least three isolated polypeptides which are from different positive filament RNA viruses from the group consisting of Hepatitis C virus (HCV) ), immunodeficiency virus (HIV) and human T cell leukemia virus (HLTV), and wherein the step of contacting comprises contacting the isolated polypeptides with said sample under suitable conditions and for a sufficient time for each of said isolated polypeptides to bind to one or more antibodies specific for them, thereby providing one or more polypeptides linked to antibodies. The set of claim 36, 37, 64 or 65 wherein said set comprises a) at least two of said isolated polypeptides of which at least two different positive filament RNA viruses selected from the group consisting of Hepatitis virus C (HCV), human immunodeficiency virus (HIV) and human T cell leukemia virus (HTLV) and b) means for detecting said at least two isolated polypeptides. The set of claim 36, 37, 64 or 65 wherein said set comprises a) at least three of said polypeptides isolated from each of Hepatitis C virus (HCV), human immunodeficiency virus (HIV) and human T cell leukemia virus (HTLC) and b) means for detecting said at least three isolated polypeptides. 71. The set of claim 38 wherein said set comprises a) at least two of said polyproteins isolated from at least two different positive strand RNA viruses selected from the group consisting of Hepatitis C virus (HCV), virus of human immunodeficiency (HIV) and human T cell leukemia virus (HTLV) and b) means for detecting said at least two isolated polyproteins. 72. The set of claim 38 wherein said set comprises a) at least three of said polyproteins isolated from each of the Hepatitis C virus (HCV), human immunodeficiency virus (HIV) and human T cell leukemia virus. (HTLV) and b) means for detecting said at least three isolated polyproteins.