CN1189838A - Diagnosis of, and vaccination against, a positive stranded RNA virus using an isolated, unprocessed polypeptide - Google Patents

Abstract

The unprocessed polyprotein originally translated from the positive-strand RNA viral genome has an epitope configuration that the processed protein does not. When processing splice sites between the genomic region encoding the core protein and the genomic region encoding core protein-adjacent proteins such as the envelope protein of HCV, especially the structural protein region loses its one-epitope configuration. The present invention relates to compositions, methods and assays for diagnosing and detecting the presence of positive-strand RNA viruses in a sample or the presence of antibodies to positive-strand RNA viruses. Compositions and methods for inducing an immune response in animals and for immunizing animals. Combination of unprocessed core region and non-structural proteins (eg NS5 from HCV or unprocessed NS3-NS4 fusion).

Description

Diagnosis and immunization against positive-strand RNA viruses using isolated unprocessed polypeptides

technical field

The present invention generally relates to highly specific, highly sensitive diagnostic methods and compositions for positive-strand RNA viruses. The methods and compositions are also suitable for stimulating an immune response in animals, and for immunizing animals against positive-strand RNA viruses.

Background of the invention

Acquired immune deficiency syndrome (AIDS) is caused by a group of retroviruses known as HIV (Barre-Sinoussi et al., Science 220:868-871, 1983; Gallo et al., Science 225:500-503, 1984; Coffin et al., Science 232:697, 1986). The first member of this group has been named HIV-1, the cause of the major cases of AIDS worldwide. It is distinct from the HIV-2 isolate found from WAf (Clavel et al., Science 233:343-346, 1986). Although HIV-2, like HIV-1, causes immunodeficiency syndrome in humans, it is also genetically distinct from HIV (Guyader et al., Nature 326:662-669, 1987).

Similar to other retroviruses, the genomes of HIV isolates include three basic genes: gag, pol and env (Weiss et al., Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1985). And the genome contains many other genes whose gene products play an important role 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).

Transmission of HIV-1 is typically through sexual contact, exposure to blood or certain blood products, or from an infected mother to her infant or child (Piot et al., Science 239:573-579, 1988). The first cases of transfusion-associated HIV-2 infection have been identified (Courouce et al., AIDS 2:261-265, 1988). Therefore, there is an urgent need for methods for sensitive and specific detection of HIV in blood.

EIAs have been developed to detect HIV based on whole virus or virus lysates. However, E1As and samples from non-HIV-infected individuals have been found to have unacceptable non-specific reactions, such as patients with autoimmune disease, history of multiple pregnancy, anti-HLA, EBV infection or blood hypergammaglobulinemia.

To avoid these non-specific reactions and attempt to detect anti-HIV-1 and anti-HIV-2 in samples, an ELISA using HIV-1 core and HIV-1 envelope and HIV-2 envelope proteins has been performed by HIV Developed and commercialized by Abbott Laboratories for Serological Diagnosis of Infection. However, this ELISA cannot yet provide the highly specific and sensitive detection required for superprotection of blood products or early diagnosis of HIV in patients.

Therefore, in order to provide super protection of blood products and super diagnosis of HIV in patients, a product and method capable of detecting HIV with high specificity and high sensitivity are needed. There is also a need for products and methods that stimulate an immune response to HIV, particularly an immune protective immune response to HIV. These needs and other related advantages are met by the present invention.

In addition to problems associated with HIV, other positive-strand RNA viruses are also important health risk factors worldwide. An example of such a positive-strand RNA virus is hepatitis C virus (HCV). HCV is distinct from other forms of known hepatitis viruses such as hepatitis A virus (HAV) and hepatitis B virus (HBV) that cause virus-associated liver disease. Like HIV, HCV is frequently transmitted through blood transfusions; post-transfusion hepatitis (PTH) accounts for approximately 10% of transfused patients, and HCV (ie, non-A, non-B hepatitis (NANBH)) accounts for more than 90% of these cases. The major problem arising from this disease is the frequent progression (25-55%) to chronic liver damage. Therefore, there is a great need for sensitive and specific methods for the detection of HCV in contaminated blood or blood products.

Hepatitis C virus (HCV) was first identified by Choo et al. (Science 244:359-362, 1989) by molecular cloning and determination of its RNA genome. A specific assay using an HCV antigen designated C100-3 was then generated by yeast recombinant DNA methods. This assay detects antibodies against HCV (Science 244:362-364). A detailed disclosure of the HCV genome and some of the cDNA sequences and polypeptides derived therefrom, as well as methodology for these materials, is provided in EP 0 318 216 A1 by Chiron Corporation. In particular, the disclosure provides a synthetic polypeptide C100-3 containing 363 virus-encoded amino acids that can be used to detect one type of HCV antibody. Currently, a kit for detecting HCV antibodies based on the C100-3 antigen has been commercialized by Abbott Laboratories.

HCV may be a flavivirus or flavivirus as described in EP 0 318 216 A1. In general morphology, flaviviruses contain a central nucleocapsid surrounded by a lipid bilayer. Hepatitis C virus is thought to consist of structural proteins including a nucleocapsid (core) protein (C), two glycosylated envelope proteins (E1, E2), and a number of nonstructural proteins (NS1-5). It has been confirmed that C100-3 discovered by Choo et al. is a protein encoded by the non-structural region 3-4 part of the HCV genome. It has been found that anti-C100-3 antibodies cannot be detected in all cases of post-transfusion NANBH. The failure to detect anti-C100-3 may be due to the hypermutation of the nucleotide sequence in the C100-3 region.

In addition to the work on the nonstructural C100-3 antigen, an enzyme-linked immunosorbent assay using the HCV core protein (p22) for the serological diagnosis of hepatitis C virus (HCV) infection has been developed. The core protein was synthesized using recombinant baculovirus as reported by Chiba et al. However, this core-based assay cannot detect a large number of HCV infection cases, even when the resulting sample volume is very large.

Therefore, like other positive-strand RNA viruses, products and methods capable of detecting HCV with high specificity and high sensitivity are required. Like other positive-strand RNA viruses, it is also desirable to be able to stimulate an immune response to HCV, especially an immunoprotective immune response to HCV. The present invention provides these needs and other advantages.

Summary of the invention

The present invention is concerned with the unprocessed complete polypeptide (such as polyprotein) of positive strand ((+) strand) RNA viral structural region or unprocessed particle polypeptide and the protein of nonstructural region can provide a superantigen and thus provide an improved Concept of detection and diagnostic methods for positive-strand RNA viruses in samples. The invention also provides improved methods of immune activation, including improved immune protective responses in animals.

Accordingly, in a first aspect the invention provides a positive-strand RNA virus-derived composition comprising an isolated, substantially intact, unprocessed polyprotein of a positive-strand RNA virus. In another aspect, the present invention provides positive-strand RNA virus-derived compositions comprising: a) an isolated polypeptide comprising a positive-strand RNA virus-like core antigen protein linked to an amino-terminal portion of an adjacent nucleic acid region of a positive-strand RNA virus , wherein the size of the amino-terminal portion of the adjacent nucleic acid region is such that the polypeptide has an epitope configuration specific to the unprocessed core-adjacent nucleic acid region of a positive-strand RNA virus (the polypeptide is sometimes referred to herein as a "core-like antigen-like antigen- Adjacent protein"; and b) a nonstructural protein of an isolated positive-strand RNA virus. As further described below, all clarifications herein regarding core antigen-like-adjacent proteins apply correspondingly to env proteins of positive-strand RNA viruses, which typically contain at least one unprocessed link to an adjacent protein.

In preferred embodiments relating to each aspect of the present invention, the positive-strand RNA virus is selected from the family Togaviridae, Coronaviridae, Retroviridae, Picornaviridae, Caliciviridae and Flaviviridae The group constituted is more preferably from the group consisting of hepatitis C virus, human immunodeficiency virus (HIV) and human T-cell leukemia virus (HTLV). Unless otherwise stated, all preferred embodiments relate to each aspect of the invention. In addition to HCV, the positive-strand RNA virus can be any positive-strand RNA virus. In other preferred embodiments, the composition is produced by a suitable prokaryotic host cell, in particular a bacterium, preferably E. coli BL21(DE3). Furthermore, the isolated polypeptide is produced by a suitable eukaryotic host cell that is incapable of processing the isolated polypeptide.

In another aspect, the present invention provides methods of preparing compositions comprising isolated, substantially intact, unprocessed polyproteins from positive-strand RNA viruses. This aspect also provides a method for preparing a plurality of polypeptides from a positive-strand RNA virus, the method comprising the steps of: a) expressing a positive-strand positive-strand RNA virus capable of expressing The first expression vector of the nucleic acid molecule of the polypeptide of the strand RNA virus-like core antigen protein is introduced in the first suitable host cell, wherein the size of the amino-terminal portion of the adjacent nucleic acid region makes the polypeptide have no specificity for the positive-strand RNA virus. processing class core-adjacent nucleic acid region-specific epitope configuration, b) incubating the first host cell under conditions suitable for expression vector production of the polypeptide, c) purifying the polypeptide to produce a purified polypeptide, and d) will be able to express introducing a second expression vector of a nucleic acid molecule encoding a nonstructural protein of an isolated positive-strand RNA virus into a second host cell, e) incubating the second host cell under conditions suitable for production of the nonstructural protein from the nucleic acid molecule , f) purifying the nonstructural protein to produce a purified nonstructural protein, followed by g) combining the purified polypeptide and the purified nonstructural protein in a composition.

In a preferred embodiment, the method further comprises a) converting a nucleic acid molecule capable of expressing a polypeptide that encodes an isolated positive-strand RNA virus-like core antigen protein that is linked to the amino-terminus of the adjacent nucleic acid region of the positive-strand RNA virus A first expression vector is introduced into a suitable host cell in which the amino-terminal portion of the adjacent nucleic acid region is of a size such that the polypeptide has an epitope configuration specific to the unprocessed core-adjacent nucleic acid region of a positive-strand RNA virus, b ) incubating the host cell under conditions suitable for the expression vector to produce the polypeptide and nonstructural protein, c) purifying the polypeptide and nonstructural protein to produce pure polypeptide and pure nonstructural protein. In another preferred embodiment, the method further comprises binding the polypeptide of the invention to a solid substrate.

In another aspect, the invention provides positive-strand RNA virus-derived compositions comprising an isolated substantially intact unprocessed polyprotein of a positive-strand RNA virus, wherein the polyprotein is bound to a solid substrate. Alternatively, the composition comprises a core-like antigen-adjacent protein bound to a solid matrix, preferably also non-structural proteins of a positive-strand RNA virus bound to a solid matrix.

In another preferred embodiment, the assay for detecting a positive-strand RNA virus in a sample comprises: a) providing an isolated polypeptide comprising a positive-strand RNA virus-like core antigen-adjacent protein, b) separating the polypeptide in Under appropriate conditions, contact with the sample for a sufficient time to ensure that the polypeptide binds to one or more positive-strand RNA virus-specific antibodies to produce an antibody-binding polypeptide, and c) detect the antibody-binding polypeptide, thereby determining that the sample contains a positive-strand RNA virus. In another embodiment, the method comprises a) providing an isolated polypeptide comprising an isolated, substantially intact unprocessed polyprotein of a positive-strand RNA virus, b) contacting the isolated polypeptide with a sample under suitable conditions sufficient time to ensure that the polypeptide binds to one or more positive-strand RNA virus-specific antibodies to produce antibody-binding polypeptides, and c) detect the antibody-binding polypeptides, thereby determining that the sample contains positive-strand RNA viruses.

In a preferred embodiment, the method further comprises a) in step a), providing a non-structural protein of a positive-strand RNA virus bound to a solid substrate, b) in step b), incorporating the non-structural protein in a suitable Conditions and sufficient time for sample contact to ensure that the nonstructural protein is combined with one or more positive-strand RNA virus specific antibodies to produce antibodies that bind to the positive-strand RNA virus nonstructural protein, and c) in step c), detection The antibody binds to the polypeptide, or the antibody binds to the nonstructural protein, thereby confirming that the sample contains a positive-strand RNA virus.

In another preferred embodiment, the assay further comprises the step of binding isolated polypeptides, nonstructural proteins, or polyproteins bound to a solid matrix. In another preferred embodiment, the sample is an unpurified sample, generally from an animal, preferably from a human. In other preferred embodiments, the assay is selected from the group consisting of countercurrent immunoelectrophoresis (CIEP) analysis, radioimmunoassay, radioimmunoprecipitation, enzyme-linked immunoabsorbent assay (ELISA), dot blot analysis, inhibition or competition assay, sandwich assay, immunoadhesion Assay (dip-stick), simultaneous assay, immunochromatography assay, immunofiltration assay, latex bead agglutination assay, immunofluorescence assay, biosensor assay, and low-light detection assay. The analysis is more preferably a non-Western blot analysis.

In yet another aspect, the present invention provides a method of producing an antibody comprising the steps of: a) administering to an animal an isolated positive-strand RNA virus-like core antigen comprising an amino-terminus of an adjacent nucleic acid region of a positive-strand RNA virus A polypeptide of protein, wherein the size of the amino-terminal part of the adjacent nucleic acid region makes the polypeptide have an epitope configuration specific to the unprocessed class core-adjacent nucleic acid region of a positive-strand RNA virus, and b) isolating antibodies against the polypeptide. Alternatively, the invention provides a method of producing an antibody comprising the steps of: a) administering to an animal an isolated polypeptide comprising an isolated, substantially intact, unprocessed polyprotein of a positive-strand RNA virus, b) isolating a polypeptide directed against the polyprotein antibodies.

The present invention also provides antibodies prepared according to one of the above methods, as well as antibodies to other proteins described herein (such as non-structural proteins). Antibodies are preferably bound to a solid substrate.

On the other hand, this method provides an analysis method for detecting positive-strand RNA viruses in a sample, which includes: a) contacting the sample with the above-mentioned one or more antibodies under appropriate conditions for a sufficient time to ensure that the given antibody binding the antigenic protein to generate binding antibody, and b) detecting the binding antibody, thereby confirming that the sample contains positive-strand RNA virus.

In a preferred embodiment, the sample is an unpurified sample, generally from an animal, preferably from a human. In other preferred embodiments, the analytical method is selected from the group consisting of countercurrent immunoelectrophoresis (CIEP) analysis, radioimmunoassay, radioimmunoprecipitation, enzyme-linked immunoabsorbent assay (ELISA), dot blot analysis, inhibition or competition assay, sandwich assay, immunoassay Adhesion assay (dip-stick), simultaneous assay, immunochromatographic assay, immunofiltration assay, latex bead agglutination assay, immunofluorescence assay, biosensor assay, and low-light detection assay. The analysis is more preferably a non-Western blot analysis.

On the other hand, the present invention also provides a composition capable of stimulating immune response in animals, which comprises isolated positive-strand RNA virus-like core antigen-adjacent protein-containing polypeptide in combination with a pharmaceutically acceptable carrier or diluent. The composition also preferably contains nonstructural proteins of positive-strand RNA viruses. In addition, the present invention also provides a composition capable of stimulating immune response in animals, which contains the isolated, substantially complete unprocessed polyprotein of positive-strand RNA virus, and a pharmaceutically acceptable carrier or diluent in combination.

For each aspect of the immunocompetence (and other aspects) of the invention, the animal is preferably a human.

Another aspect of the present invention also provides a vaccine against positive-strand RNA viruses, which contains isolated polypeptides containing positive-strand RNA virus-like core antigen-adjacent proteins, and is combined with a pharmaceutically acceptable carrier or diluent. The composition also preferably contains nonstructural proteins of positive-strand RNA viruses. In addition, the present invention also provides a vaccine against positive-strand RNA viruses, which comprises isolated, substantially intact, unprocessed polyproteins of positive-strand RNA viruses, combined with a pharmaceutically acceptable carrier or diluent.

The present invention also provides a method for inducing an immune response in an animal, the method comprising administering to the animal an isolated polypeptide containing positive-strand RNA virus-like core antigen-adjacent protein in combination with a pharmaceutically acceptable carrier or diluent. The method also preferably includes administering a nonstructural protein of a positive-strand RNA virus. Alternatively, the method comprises administering to the animal an isolated, substantially intact unprocessed polyprotein of a positive-strand RNA virus in combination with a pharmaceutically acceptable carrier or diluent.

The present invention also provides a method of immunizing an animal, the method comprising administering to the animal an isolated positive-strand RNA virus-like core antigen-adjacent protein-containing polypeptide in combination with a pharmaceutically acceptable carrier or diluent. The method preferably also includes administering a nonstructural protein of a positive-strand RNA virus. Alternatively, the method comprises administering to the animal an isolated substantially intact unprocessed polyprotein from a positive-strand RNA virus in combination with a pharmaceutically acceptable carrier or diluent.

Another aspect of the present invention also provides a test kit for detecting positive-strand RNA viruses, the kit comprising: a) an isolated amino group that is connected to a solid substrate and contains an adjacent nucleic acid region connected to a positive-strand RNA virus a polypeptide of the terminal positive-strand RNA virus-like core antigen protein, wherein the amino-terminal portion of the adjacent nucleic acid region is sized such that the polypeptide has an epitope configuration specific to the unprocessed core-like-adjacent nucleic acid region of a positive-strand RNA virus, and b) means for detecting the isolated polypeptide. The kit preferably contains nonstructural proteins of positive-strand RNA viruses and tools for detecting the nonstructural proteins. Alternatively, the kit for detecting a positive-strand RNA virus comprises a) an isolated, substantially intact, unprocessed polyprotein from a positive-strand RNA virus attached to a solid substrate, and b)) a method for detecting the isolated polyprotein tool.

Another aspect of the present invention also provides a kit for detecting positive-strand RNA viruses, the kit comprising: a) one or more of the above-mentioned antibodies, and b) a tool for detecting the antibodies.

The kit may also contain a) a composition, or vaccine, capable of stimulating an immune response, and b) means for administering the composition or vaccine to an animal.

Turning to another aspect, the present invention provides a positive-strand RNA virus-derived composition comprising: a) an isolated positive-strand RNA virus-like core antigen comprising the amino terminus of an adjacent nucleic acid region of a positive-strand RNA virus The polypeptide of albumen, wherein the size of the amino-terminal part of adjacent nucleic acid region makes polypeptide have the unprocessed class core-adjacent nucleic acid region specific epitope configuration to positive strand RNA virus, and b) can be connected to positive strand RNA The positive-strand RNA virus-like core antigen protein of the amino terminal of the adjacent nucleic acid region of the virus cooperates to improve the antigenicity of the positive-strand RNA virus-like core antigen protein connected to the amino-terminal of the adjacent nucleic acid region of the positive-strand RNA virus second protein. The present invention also provides the preparation of such a composition comprising a plurality of polypeptides including one or two of the above-mentioned polypeptides; these proteins may be from the same or different positive-strand RNA viruses.

The invention also provides a protein comprising a first protein isolated from a positive-strand RNA virus and a second protein isolated from a positive-strand RNA virus (preferably from the same positive-strand RNA virus), wherein according to other embodiments of the invention hereinafter described According to the method described above, the first and second proteins are selected so that the first and second proteins have a synergistic effect in the detection of positive-strand RNA viruses and/or the immune enhancement of animals against positive-strand RNA viruses.

The present invention also provides an isolated polypeptide comprising a positive-strand RNA virus-like core antigen protein linked to the amino terminus of an adjacent nucleic acid region of a positive-strand RNA virus linked to a solid substrate, wherein the amino-terminal portion of the adjacent nucleic acid region is The size of the polypeptide has an epitope configuration specific to the unprocessed core-adjacent nucleic acid region of a positive-strand RNA virus, which can be alone or linked to a solid substrate that can be linked to a positive-strand RNA virus. The positive-strand RNA virus-like core antigen protein of the amino terminal of the adjacent nucleic acid region cooperates to improve the second antigenicity of the positive-strand RNA virus-like core antigen protein connected to the amino-terminal of the adjacent nucleic acid region of the positive-strand RNA virus protein combination.

Another aspect of the present invention also provides an analytical method for detecting a positive-strand RNA virus in a sample, the analytical method comprising: a) providing an isolated positive-strand RNA containing an amino-terminus connected to an adjacent nucleic acid region of the positive-strand RNA virus A polypeptide of the virus-like core antigen protein, wherein a small portion of the amino-terminal portion of the adjacent nucleic acid region makes the polypeptide have an epitope configuration specific to the unprocessed class core-adjacent nucleic acid region of a positive-strand RNA virus, and b) will isolate The polypeptide is contacted with the sample under appropriate conditions for a sufficient time to ensure that the polypeptide binds to one or more positive-strand RNA virus-specific antibodies to produce antibody-binding polypeptides, and c) detect antibody-binding polypeptides, thereby determining the Contains positive-strand RNA viruses. The method may also include a) in step a), providing a kind of positive-strand RNA virus-like core antigen protein that is bound to the solid substrate and can cooperate with the amino-terminus of the adjacent nucleic acid region of the positive-strand RNA virus, to The second protein that improves the antigenicity of the positive-strand RNA virus-like core antigen protein connected to the amino terminus of the adjacent nucleic acid region of the positive-strand RNA virus, b) in step b), the second protein is placed under suitable conditions contact with the sample for sufficient time to ensure synergy with the positive-strand RNA virus-like core antigen protein connected to the amino-terminus of the adjacent nucleic acid region of the positive-strand RNA virus, and c) in step c), detect the bound antibody, according to This confirms that the sample contains positive-strand RNA viruses.

In a preferred embodiment, the assay further comprises the step of binding the isolated polypeptide or a second protein to a solid matrix. The assay is preferably selected from the group consisting of countercurrent immunoelectrophoresis (CIEP) analysis, radioimmunoassay, radioimmunoprecipitation, enzyme-linked immunoabsorbent assay (ELISA), dot blot analysis, inhibition or competition assay, sandwich assay, immunoadhesion assay (dip-stick assay). ), simultaneous assays, immunochromatographic assays, immunofiltration assays, latex bead agglutination assays, immunofluorescence assays, biosensor assays, and low-light detection assays. More preferably, the analysis is a non-Western blot analysis.

The present invention also provides a method for producing antibodies, the method comprising a) administering to an animal an isolated polypeptide comprising a positive-strand RNA virus-like core antigen protein linked to the amino terminus of an adjacent nucleic acid region of a positive-strand RNA virus, wherein the adjacent The amino-terminal portion of the nucleic acid region is sized such that the polypeptide has an epitope configuration specific to the raw-like core-adjacent nucleic acid region of a positive-strand RNA virus, b) isolating antibodies against the polypeptide. The method may further comprise administering to the animal a synergistic effect with the positive-strand RNA virus-like core antigen protein linked to the amino terminus of the adjacent nucleic acid region of the positive-strand RNA virus, to increase the binding to the adjacent nucleic acid region of the positive-strand RNA virus. Antigenic second protein of the amino-terminal positive-strand RNA virus-like core antigen protein. The invention features antibodies prepared as above, which antibodies can be bound to a solid substrate. These antibodies can also be used in assays as described above.

Another aspect of the present invention also provides a composition capable of stimulating an immune response in an animal, which contains an isolated polypeptide containing an amino-terminal positive-strand RNA virus-like core antigen protein connected to an adjacent nucleic acid region of a positive-strand RNA virus (wherein The size of the amino-terminal part of the adjacent nucleic acid region makes the polypeptide have an epitope configuration specific to the unprocessed core-adjacent nucleic acid region of a positive-strand RNA virus), and is combined with a pharmaceutically acceptable carrier or diluent. The composition may also contain a positive-strand RNA virus-like core antigen protein that can cooperate with the amino-terminus of the adjacent nucleic acid region of the positive-strand RNA virus to increase the amino-terminus of the adjacent nucleic acid region of the positive-strand RNA virus. Antigenic second protein of positive-strand RNA virus-like core antigen protein. The composition is preferably a vaccine.

The present invention also provides a method for inducing an immune response in an animal, the method comprising administering to the animal an isolated amino-terminal DNA containing an adjacent nucleic acid region linked to a positive-strand RNA virus in combination with a pharmaceutically acceptable carrier or diluent. A polypeptide of the positive-strand RNA virus-like core antigen protein, wherein the size of the amino-terminal part of the adjacent nucleic acid region makes the polypeptide have an epitope configuration specific to the unprocessed core-like adjacent nucleic acid region of the positive-strand RNA virus. The method preferably comprises administering to the animal a positive-strand RNA virus-like core antigen protein capable of synergizing with the amino-terminus of an adjacent nucleic acid region of a positive-strand RNA virus to increase the amino acid concentration of the adjacent nucleic acid region of a positive-strand RNA virus. Antigenic second protein at the end of the positive-strand RNA virus-like core antigen protein. More preferably the method comprises vaccination.

Another aspect of the present invention also provides a kit for detecting positive-strand RNA viruses, the kit comprising: a) an isolated amino group that is bound to a solid substrate and contains an adjacent nucleic acid region connected to a positive-strand RNA virus a polypeptide of the terminal positive-strand RNA virus-like core antigen protein, wherein the amino-terminal portion of the adjacent nucleic acid region is sized such that the polypeptide has an epitope configuration specific to the unprocessed-like core-like adjacent nucleic acid region of a positive-strand RNA virus, and b ) means for detecting the isolated polypeptide. The kit preferably also contains a positive-strand RNA virus-like core antigen protein that can cooperate with the amino-terminus of the adjacent nucleic acid region of the positive-strand RNA virus to increase the amino-terminus of the adjacent nucleic acid region of the positive-strand RNA virus. Antigenicity of the second protein of the positive-strand RNA virus-like core antigen protein and a tool for detecting the second protein.

Alternatively, a kit for detecting a positive-strand RNA virus may contain: a) an antibody prepared as described above, and b) means for detecting the antibody.

These and other aspects of the invention will become apparent upon reference to the following detailed description and accompanying drawings. Also, as indicated above, various references describing in greater detail certain steps and components (eg, plasmids, etc.) will be placed throughout this patent specification; these references are fully incorporated herein.

A brief description of the legend

Figure 1A shows the nucleotide sequence of a nucleic acid molecule encoding a polypeptide comprising the HCV core antigen protein linked to the amino-terminal portion of the HCV envelope region.

Figure 1B shows the amino acid sequence encoded by the nucleotide sequence shown in Figure 1A.

Fig. 2 shows the structure of expression vector pEN-2 constructed by inserting cDNA encoding HCV core antigen protein linked to the amino terminal portion of the HCV envelope region into a plasmid. The figure also shows a restriction map showing some important features of the vector pEN-2.

Figure 3A shows the nucleotide sequence of a nucleic acid molecule encoding a polypeptide containing the NS5 nonstructural region.

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 the cDNA encoding the non-structural region of NS5 into the plasmid. The figure also shows a restriction map showing some important features of the vector pEN-2.

Detailed description of the invention

The present invention is based on the discovery that the unprocessed polyprotein originally translated from the genome of a positive-strand RNA virus contains an epitope configuration that is not present in the processed protein. In particular, when a splice site is processed between a genomic region encoding a core protein (such as a gene) and a genomic region encoding a protein adjacent to the amino terminus of the core protein (such as the envelope protein of HCV), the core protein region (or other virus-encoded equivalent of the "core" protein) will lose one epitope configuration. As described in the Examples section set forth below, the unprocessed epitope configuration of the core region has a surprisingly enhanced ability to detect positive-strand RNA viruses, or antibodies against positive-strand RNA viruses, in samples, including unpurified samples or very small volumes (This is especially useful when testing samples from infants or people with little blood (or other suitable material) available for testing).

Even more surprisingly, combining the unprocessed core region with a non-structural protein, such as the NS5 protein from HCV or an unprocessed NS4-NS5 fusion protein, resulted in a significantly enhanced The resulting synergistic effect of increased sensitivity and specificity.

The important advantages of these antigenic and epitope configurations also provide compositions and methods for significantly enhancing the induction of immune responses in animals, and enhanced animal immunization.

Accordingly, the invention features compositions and methods employing isolated, substantially intact, unprocessed polyproteins from positive-strand RNA viruses.

The invention also features compositions and methods using an isolated polypeptide comprising a positive-strand RNA virus-like core antigen protein linked to the amino terminus of an adjacent protein of a positive-strand RNA virus, wherein the positive-strand RNA virus coat region The size of the amino-terminal portion of the polypeptide has an epitope configuration specific to the unprocessed core-adjacent protein region of positive-strand RNA viruses. The invention also features the combination of this unprocessed core-adjacent protein region and nonstructural proteins in compositions and methods that result in surprisingly sensitive and specific responses to a given positive-strand RNA virus.

The present invention provides for the first time the discovery that intact polyproteins have a unique configuration and that this configuration leads to important differences in antigenicity. The present invention also provides for the first time the discovery that there is a missing epitope configuration in the core protein-adjacent protein region.

An "isolated, substantially intact unprocessed polyprotein" from a positive-strand RNA virus is a polyprotein originally translated from the genome of a positive-strand RNA virus. This polyprotein is not processed, so the processing sites between the proteins of the polyprotein are not cleaved. A polyprotein is also isolated, meaning that the polyprotein has been separated from the genome it encodes. When the polyprotein retains the functional elements necessary to produce the immunologically active features of the invention, particularly an epitope configuration present only in the polyprotein and not in processed proteins or subunits derived from the polyprotein, then multiple The protein is substantially complete. However, for the protein of the present invention or other proteins, those skilled in the art can make conservative amino acid substitutions or minor amino acid insertions, modifications or deletions, which can change the amino acid sequence of the protein without significantly changing the function of the protein (i.e., still retain the unprocessed epitope configuration). And when desired, this modification can delete protein processing signals and/or sites. These modifications are discussed further below. For example, the integrity of a polyprotein can be determined by SDS-PAGE followed by amino acid sequencing. Integrity can also be determined by using the polyprotein in one or more of the assays discussed below and detecting the effect of the unprocessed state-specific epitope configuration.

"Core-like" proteins are structural proteins that serve the same type of function as the HCV core protein. Examples of "core-like" proteins from other viruses are the Japanese encephalitis virus core protein and the HIV gag protein. "Core-like antigenic protein" refers to a structural "core-like" protein that contains a core-like portion that exhibits core-like antigenicity. Although changes in epitope configuration upon processing are not known in the art, core-like proteins and core-like regions that are important for antigenicity are generally well known in the art (see, e.g., Okamoto, et al., J. Virol. 188 : 331, 1992; Wang, US Patent No. 5,106,726). As is well known in the art, the core-like antigen protein of a positive-strand RNA virus of interest can be determined by conventional core-type antigen reactivity ELISA or Western blot or both. Core-like antigen proteins can also be identified by SDS-PAGE followed by amino acid sequencing.

Generally, the core-like antigen protein is linked to the amino-terminal portion of an adjacent protein or peptide of a positive-strand RNA virus, forming an unprocessed "core-like antigen-adjacent protein" of the present invention. However, in some embodiments, particularly when the core-like protein is not the first protein domain of the polyprotein, the unprocessed form of the core-like protein is linked to the carboxy-terminal portion of an adjacent protein of a positive-strand RNA virus, forming the present Invented unprocessed class of core antigen-adjacent proteins. By unprocessed form it is meant that the core-like region and adjacent regions generally and preferably retain their linkage (ie encoding) exactly as they do in native positive-strand RNA viruses. As with the polyproteins and other proteins herein, the core-like protein can be slightly modified without changing the function of the core-like protein of the invention.

The portion of the "adjacent protein" adjacent to the core-like antigen protein is of a size such that the fusion protein has an epitope configuration specific to the unprocessed core-like adjacent protein of a positive-strand RNA virus. Therefore, the amino-terminal portion of the adjacent protein domain must generally be long enough to ensure that the fusion protein exhibits a transient epitope configuration specific for the unprocessed core-like domain.

In addition to traditional core-like proteins, the env proteins of positive-strand RNA viruses can also generate the surprisingly enhanced antigenic conformation and action exhibited by the core-like antigen-adjacent proteins described herein. This is especially true when the env protein is also used in combination with a second protein as described in the text. The env protein preferably includes an unprocessed linkage to an adjacent protein similar to that found in the core antigen-like-adjacent proteins disclosed herein (which itself could be an adjacent env protein, eg gp120 and gp41 in HIV). Furthermore, the second protein may be a core-like protein, such as the gag protein of HIV. The enhanced detection and immune induction due to the env region is similar to that shown for the core antigen-like-adjacent proteins of the present invention. So unless otherwise stated or clear from the text, references to the class of core antigen-adjacent proteins in the text apply equally to env and/or env-adjacent proteins. Determining whether a given env or env-adjacent protein has this enhanced detection and immunity-inducing effect can be performed by the analysis of the class of core antigen-adjacent proteins as described below.

The determination of whether a given polypeptide has the core antigen-like epitope configuration of the invention can be performed as follows. Unidentified core-like antigen-adjacent proteins can be included in a set of core-like antigen-adjacent proteins that contain established core-like antigen-adjacent proteins (eg, EN-80-2). The series of test subjects are placed in a series of wells of a microplate. The panel may also include other core antigen-like-adjacent proteins with adjacent proteins of different lengths. Additional wells are filled with a nonstructural protein or other protein identified to act synergistically with a core-like antigen-adjacent protein such as EN-80-1. For the identified core-like antigen-adjacent proteins, select sera that react weakly with the core-like antigen-adjacent proteins and have no reaction with the identified non-structural proteins. The rationale for selection was that the antisera would react as expected with individual proteins, but would be more reactive when the appropriate class of core antigen-adjacent proteins and identified nonstructural proteins were both present in the sample. Several examples of such antisera are shown in the Examples below, such as G614 (diluted 8 times), G614 (diluted 16 times), G615 (diluted 8 times), G615 (diluted 16 times) and 8-5. The antiserum is added to the sample protein under conditions suitable to elicit and detect a reaction between the antiserum and the given protein, and the reaction is detected and measured. The identified nonstructural proteins and other samples of each member of the class core antigen-adjacent protein series were then combined. In a next step, the antiserum is introduced into the combined protein under conditions suitable to elicit and detect a reaction between the antiserum and the protein, and this reaction is detected and measured. Those class core antigen-adjacent proteins that can produce synergistic effects are suitable for use in the present invention. The reactivity of the antiserum with the combined proteins is preferably at least 1.25 or 1.5 times stronger when compared to the sum of the reactivity of the antiserum with each protein alone. More preferably, the response of the antiserum is at least about two-fold stronger. Each of the steps described above is conventional in the art in light of the present specification.

The core-like antigen-adjacent protein is preferably isolated, which means that the core-like antigen-adjacent protein is separated from the rest of the polyprotein originally translated from the positive strand RNA viral genome. The core-like antigen-adjacent protein is also preferably isolated from its encoding nucleic acid molecule.

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

In a preferred embodiment, the second protein is a nonstructural protein. As is well known in the art, other names may be used for non-structural proteins of other positive-strand RNA viruses other than HCV. According to this patent specification, all such non-structurally similar proteins are referred to herein as "non-structural proteins". As indicated above, the non-structural coding partitions of positive-strand RNA viruses are well known in the art.

Determination of an appropriate second protein suitable for use with a core-like antigen-adjacent protein, which may include more than one (or shorter than an entire nonstructural protein) nonstructural protein The nonstructural coding region portion of a structural protein.

The unidentified second protein can be included in a second protein panel containing the identified second protein (eg EN-80-1). The series of test subjects are placed in a series of wells of a microplate. The series may also include other second proteins with adjacent proteins of different lengths. An identified core antigen-like-adjacent protein, such as EN-80-1, that synergizes with the second protein is added to additional wells. For the identified second protein, select sera that react weakly with the second protein and that do not react with the identified class of core antigen-adjacent proteins. The rationale for selection is that the antisera will react as expected with the protein alone, but will be more reactive when an appropriate second protein is present in the sample along with the identified class of core antigen-adjacent proteins. Several examples of such antisera are shown in the Examples below. The antiserum is added to the sample protein under conditions suitable to elicit and detect a reaction between the antiserum and the given protein, and the reaction is detected and measured. The identified classes of core antigen-adjacent proteins were then combined with other samples of each member of the second protein family. In a next step, the antiserum is introduced into the combined protein under conditions suitable to elicit and detect a reaction between the antiserum and the protein, and this reaction is detected and measured. Those second proteins that produce a synergistic effect are suitable for use in the present invention. Each of the steps described above is conventional in the art in light of this patent specification.

The invention also provides antibodies, preferably monoclonal antibodies, directed against substantially intact polyproteins, core-like antigen-adjacent proteins, and/or nonstructural proteins of the invention and other proteins of the invention. Antibodies are preferably used in combination to yield a particularly sensitive and specific detection of positive-strand RNA viruses in a sample.

The invention also provides compositions and methods for activating an immune response (humoral or cellular or both) in an animal. The compositions and methods are even capable of immunizing animals against positive-strand RNA viruses.

The methods and compositions of the invention, including those of detection, immune response activation and immunization, are preferably applied to humans.

An example of the invention is hepatitis C virus (HCV). The discussion that follows generally focuses on HCV, and even more on the HCV core antigen protein linked to the amino-terminal portion of the HCV envelope region. The discussion also focuses on the combination of this class of core antigen-adjacent proteins and HCV nonstructural proteins (especially HCV NS5 and NS3-NS4 nonstructural proteins), or with a second protein of another positive-strand RNA virus (especially is a combination of HIV envelope protein and HTLV-1 envelope protein). As indicated above, the present discussion is generally the expected result obtained from positive-strand RNA virus-like core antigen-adjacent proteins. This statement is also generally expected from the substantially intact polyproteins of positive-strand RNA viruses, particularly HCV. Nucleic Acid Molecules Encoding Raw Polypeptides and Other Polypeptides of the Invention

As indicated above, the present invention includes nucleic acid molecules encoding polypeptides comprising substantially intact positive strand RNA viral polyproteins. The invention also provides nucleic acid molecules encoding polypeptides comprising a core-like protein, such as the HCV core antigen protein linked to the amino-terminal portion of the HCV envelope region. The present invention also provides nucleic acid molecules encoding polypeptides containing nonstructural proteins of such positive-strand RNA viruses. In preferred embodiments, 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 isolated from nucleic acid sequences present in the plasma of HCV-infected patients. As further described below, the nucleic acid isolation step includes isolating viral particles from the patient's plasma, extracting and purifying the viral nucleic acid sequence, and then cloning the target DNA molecule by polymerase chain reaction (PCR). Primers used for cloning are as follows:

(i) 5'-GGATCCATGAGCACAAATCCTAAACCT-3' (SEQ ID No: 1) and

(ii) 5'-GAATTCGGTGTGCATGATCATGTCCGC-3' (SEQ ID No: 2) To identify it, the cloned DNA molecule was sequenced. The resulting molecule was designated EN-80-2. The DNA sequence of molecule EN-80-2 is given in Figure 1A (SEQ ID No. 7) and has 699 bp. The amino acid sequence of molecule EN-80-2 is given in Figure 1B (SEQ ID No. 8) and has 233 residues. The molecule EN-80-2 in Escherichia coli BL12(DE3) was deposited with the American Type Culture Collection (ATCC) Rockville Maryland 20852 on July 14, 1993 and has been registered as ATCC accession number 55451. The culture was deposited under the conditions of the Budapest Treaty.

In another preferred embodiment, the nucleic acid molecule is a DNA molecule encoding the HCV NS5 nonstructural protein, which is isolated from a nucleic acid sequence present in the plasma of HCV-infected patients. As described above for the isolation of unprocessed core antigen-envelope protein (although a different patient was used), the isolation step involved isolating viral particles from the patient's plasma, extracting and purifying the viral nucleic acid sequence, and then performing a polymerase chain reaction ( PCR) technology to clone target DNA molecules. Primers used for PCR are as follows:

(i) 5'-GGATCCCGGTGGAGGATGAGAGGGAAATATCCG-3' (SEQ ID No: 3) and

(ii) 5'-GAATTCCCGGACGTCCTTCGCCCCGTAGCCAAATTT-3' (SEQ ID No: 4)

To identify it, the cloned DNA molecule was sequenced. The resulting molecule was designated EN-80-1. The DNA sequence of molecule EN-80-1 is given in Figure 3A (SEQ ID No. 9) and has 803 bp. The amino acid sequence of molecule EN-80-1 is given in Figure 3B (SEQ ID No. 10) and has 267 residues. The molecule EN-80-1 in E. coli BL12(DE3) was deposited with the American Type Culture Collection (ATCC) Rockville Maryland 20852 on July 14, 1993 and has been registered as ATCC accession number 55450. The culture was deposited under the conditions of the Budapest Treaty.

Figure 2 shows an expression plasmid pNE-2 containing a DNA molecule encoding the unprocessed core antigen-envelop protein isolated with the above primers SEQ ID No: 1 and 2. Fig. 4 shows an expression plasmid PNE-1 containing the DNA molecule encoding NS5 nonstructural protein isolated with the above primers SEQ ID No: 1 and 2.

This general method has been used to isolate representative nucleic acid molecules from the NS3-NS4 nonstructural region of HCV. See also Simmonds, Lancet 336:1469-1472, 1990. The primers 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 resulting molecule was designated EN-80-4. The molecular weight of the polypeptide encoded by the isolated molecule was determined by SDS-PAGE electrophoresis to be about 20,000 Daltons.

Other examples of polypeptides useful as the second protein include HIV envelope protein (molecular weight about 18,000 Daltons) and HTLV-1 envelope protein (molecular weight about 18,000 Daltons).

The present invention also provides manipulation and expression of the above-mentioned nucleic acid molecules by culturing host cells containing constructs capable of expressing the above-mentioned genes.

Various support structures suitable for the nucleic acid molecules of the present invention are available as convenient materials. In the present invention, a vector structure should generally be understood as referring to a DNA molecule or a clone (single-stranded or double-stranded) of such a molecule, which has been artificially modified to contain fragments formed by combining and juxtaposing DNA (as a whole, its not naturally occurring). The vector constructs of the invention contain a first DNA segment encoding one or more raw-like core antigen-adjacent proteins suitably linked to other DNA segments required for expression of the first DNA segment. In the present invention, other DNA fragments include promoters, generally include transcription terminators, and may include enhancers and other elements. See WO 94/25597 and WO/25598.

Mutagenesis of the nucleotide sequence expressing the protein of the invention preferably preserves the reading frame of the coding sequence. Furthermore, mutations preferably do not create complementary regions that can hybridize to form secondary mRNA structures, such as stem-loops and hairpins, which could adversely affect translation of the mRNA. While the site of mutation may be predetermined, the nature of the mutation itself need not be predetermined. For example, to screen for optimal mutations at a given position, random mutagenesis can be performed at the target codon and the expressed mutants screened for exhibited biological activity.

Mutations can be introduced at specific locations by synthesizing oligonucleotides containing the mutated sequence flanked by restriction enzyme sites that enable ligation to fragments of the native sequence. After ligation, the resulting reconstructed sequence encodes a derivative with the desired amino acid insertion, substitution or deletion.

Alternatively, oligonucleotide-directed site-directed mutagenesis can be used to generate genes with specific codon alterations as required for substitutions, deletions, and insertions. (Gene 42:133, 1986); Bauer et al. (Gene 37:73, 1985) Craik (Biotechnology, January 1985, 12-19); Smith et al. (Genetic Engineering: Principles and Methods, Plenum Publishing, 1981); and Sambrook et al. (supra).

The primary amino acid structure of the above-mentioned proteins can be modified by forming covalent or cohesive conjugates with other chemical groups, such as sugar groups, lipids, phosphates, acetyl groups, or with other proteins or polypeptides, as long as these modifications do not affect the protein. antigenicity. (See US Patent No. 4,851,341; see also Hopp et al., Biotechnology 6:1204, 1988). For example, these modifications should not affect the epitope configuration (including epitope proximity and other antigenic considerations) specific for the unprocessed core-like antigen-adjacent protein.

A preferred type of vector structure is known as an expression vector. As indicated above, plasmids pEN-1 and pEN-2 are examples of such expression vectors containing nucleic acid molecules encoding HCV nonstructural region and unprocessed HCV core antigen-envelope protein, respectively.

For expression, the nucleic acid molecule (typically DNA) is inserted into a suitable vector construct as described above, which in turn can be used to transform or transfect a suitable host cell for expression. The host cell used to express the gene sequence of the present invention is preferably a prokaryotic host cell, more preferably a bacterium, such as Escherichia coli. Other suitable host cells include Salmonella, Bacillus, Shigella, Pseudomonas, Streptomyces, and other genera known in the art. In a particularly preferred embodiment, the host cell is Escherichia coli containing DE3 lysogen or T7 RNA polymerase, such as BL21(DE3), JM109(DE3) or BL21(DE3)pLysS.

Vectors for expression of cloned DNA sequences in bacterial hosts generally contain selectable markers, such as antibiotic resistance genes, and promoters that are functional in the host cell. Suitable promoters include trp (Nichols and Yanofsky, Methods in Enzymology 101:155-164, 1983), lac (Casadaban et al., J. Bacteriology 143:971-980, 1980) and phage lambda (Queen, J. Molecular Applied Genetics , 2:1-10, 1983) promoter system. An expression unit may also include a transcription terminator. Plasmids used to transform bacteria include the pUC plasmid (Messing, Methods in Enzymology 101: 20-78, 1983; Vieira and Messing, Gene 19: 259-268, 1982), pBR322 (Bolibvar et al., Gene 2: 95-113, 1977 ), pCQV2 (Queen, ibid) and its derivative plasmids. Plasmids can contain viral and bacterial elements.

In another embodiment, if the host cell is engineered so that it cannot process unprocessed core antigen-like-adjacent proteins or unprocessed non-structural regions (such as NS3-NS4 non-structural proteins), or engineer processing in unprocessed polypeptides The signal and/or processing site renders it insensitive to processing (this modification cannot affect the antigenicity of the unprocessed protein), and the host cell can be a eukaryotic cell. Eukaryotic host cells suitable for use in the present invention include mammalian, avian, plant, insect and fungal cells. Preferred eukaryotic cells include cultured mammalian cell lines (e.g., rodent and human cell lines), insect cell lines (e.g., Sf-9) and various yeasts (e.g., Saccharomyces strains, especially S. Yeast, Schizosaccharomyces strains, or Kluyveromyces strains) or fungal cells of filamentous fungi (eg, Aspergillus strains, Neurospora strains).

Techniques for transforming these host cells and methods for expressing foreign DNA sequences cloned therein are well known in the art (see, e.g., Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, 1982; Sambrook et al., supra, " Gene Expression Technology", Methods in Enzymology, Volume 185, Goeddd (eds.) Academic Publishing, San Diego, Calif., 1990; "Introduction to Yeast Genetics and Molecular Biology", Methods in Enzymology, Guthrie and Fink (eds.), Academic Publishing Society, San Diego, Calif., 1991; Hizeman et al., Journal of Biochemistry 255: 12073-12080, 1980; Alber and Kawasaki, Journal of Molecular Applied Genetics 1: 419-434, 1982; Young et al., In Chemical Microbial Genetic Engineering, Hollaender et al. (eds.), p355, Plenum, New York, 1982; Ammerer, Methods in Enzymology 101:192-201, 1983; McKnight et al., US Patent No. 4,935,349).

In general, host cells are selected based on their ability to produce high levels of the protein of interest. In this way, the number of cloned DNA sequences introduced into the host cell is minimized while maximizing the total production of biologically active protein. Promoters, terminators, and methods for introducing expression vectors encoding proteins of the present invention into desired host cells will be understood by those skilled in the art based on the teachings herein.

Host cells containing the vector constructs of the present invention are then cultured to express the DNA molecules described above. Cells are cultured in a medium containing the nutrients required for growth of the selected host cells according to standard procedures. Various appropriate media are well known in the art, and generally include carbon sources, nitrogen sources, essential amino acids, vitamins and minerals, and other components required by specific host cells, such as growth factors or serum. The growth medium generally selects for cells containing the DNA construct, eg, by drug selection or lack of essential nutrients, the latter being complemented by a selectable marker on the DNA construct or its transfected DNA construct. Polypeptides comprising unprocessed polypeptides of the present invention

As indicated above, the present invention provides polypeptides comprising unprocessed, substantially intact polyproteins from positive-strand RNA viruses. The invention also provides a core-like antigen protein (eg HCV core protein) comprising an amino-terminal portion (eg HCV envelope region) that binds to a neighboring protein. The invention also provides certain nonstructural proteins. In a preferred embodiment, the amino acid sequence of the core antigen-like protein is shown in Figure 1B (SEQ ID No.8). In this preferred embodiment, the polypeptide has a molecular weight of about 25,000 Daltons as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis, and has been estimated to contain about 223 amino acids.

The unprocessed polypeptides of positive-strand RNA viruses bind antibodies specific for positive-strand RNA viruses. This has been confirmed by Western blot and enzyme-linked immunosorbent absorber (ELISA) on HCV. Unprocessed core antigen-envelope protein has been found to react specifically with HCV patient sera, but not with HCV-free human sera. The unprocessed polypeptides of positive-strand RNA viruses can detect the presence of positive-strand RNA virus-specific antibodies in samples, and thus can be used for the detection and diagnosis of positive-strand RNA viruses in patients, especially humans.

The present invention also provides polypeptides incorporating nonstructural proteins of positive-strand RNA viruses. In a preferred embodiment, the amino acid sequence of the core antigen-like protein is shown in Figure 3B (SEQ ID No.10). The polypeptide in FIG. 3B has a molecular weight of about 29,000 Daltons as determined by sodium dodecylsulfonate polyacrylamide gel (SDS-PAGE) electrophoresis, and has been estimated to contain about 267 amino acids.

Nonstructural proteins of positive-strand RNA viruses can bind antibodies specific for positive-strand RNA viruses. This has been demonstrated on HCV by Western blot and enzyme-linked immunosorbent absorbance (ELISA) for NS5 and NS3-NS4 nonstructural proteins shown in the text. The nonstructural proteins of the present invention have been found to react specifically with HCV patient sera, but not with HCV-free human sera. The non-structural protein can also detect the presence of antibodies specific to positive-strand RNA viruses under the conditions of the Budapest Treaty in a sample and can thus be used for the detection and diagnosis of positive-strand RNA viruses in patients, especially humans.

When the protein of the present invention is encoded by a part of a natural gene, a derivative of a natural gene, or has been modified in other ways, the protein basically maintains the same biological activity as the natural protein. For example, protein structures corresponding to unprocessed, substantially complete polyproteins, core antigen-adjacent proteins, or nonstructural proteins of positive-strand RNA viruses can be obtained, for example, using P/C Gene or Intelligenetics Suite (Intelligenetics, Mountain View, Calif. .) or predicted from its original translation product according to the method described by Kyte and Doolittle (J. Mol. Biol. 157: 105-132, 1982).

In a preferred embodiment, the invention provides isolated proteins. Proteins can be isolated by various methods including culturing appropriate host and vector systems to produce recombinant translation products of the invention. The protein may not be secreted or secreted into the supernatant. In order to isolate the target protein, the supernatant or protein inclusion body or whole cells of the cell line can be treated with various purification methods. For example, the supernatant can first be concentrated using commercially available filters, such as Amicon or Millipore Pellicon ultrafiltration units. After concentration, the concentrate is applied to a suitable purification matrix, such as an anti-protein antibody bound to a suitable support. Alternatively, anion or cation exchange resins can be used to purify proteins. Alternatively, one or more steps of reverse-phase high-performance liquid chromatography (RT-HPLC) can be applied to further purify the protein. Other methods of isolating the proteins of the invention are known to those skilled in the art. See W094/25597 and WO/25598.

A protein is considered "isolated" in the present invention if no other (non-interest) protein is detectable according to SDS-PAGE analysis followed by Coomassie Brilliant Blue staining. In other embodiments, the protein of interest can be separated such that no other proteins, preferably lipopolysaccharide (LPS), are detectable by SDS-PAGE analysis followed by silver staining. In other embodiments, a protein is isolated if no other protein with significant antigenic properties is present in the protein that would significantly interfere with detection assays and immunological events. Binding Mode of Unprocessed Polypeptides of the Invention

The present invention also provides monoclonal and polyclonal antibodies against unprocessed positive-strand RNA virus polyproteins, class core antigen-adjacent proteins of positive-strand RNA viruses, or non-structural proteins of the positive-strand RNA viruses, or other proteins of the invention . Using the polypeptide of the present invention as an immunogen, antibodies are prepared by standard hybridoma preparation procedures and/or other methods. The antibodies formed are particularly suitable for detecting positive-strand RNA viruses in a sample, preferably a human sample. See WO94/25597 and WO/25598.

Polyclonal antibodies can be readily prepared by one of ordinary skill in the art from various warm-blooded animals such as horses, cows, goats, sheep, dogs, chickens, donkeys, rabbits, mice or rats. Briefly, the protein or polypeptide of interest is used to immunize animals, usually by intraperitoneal, intramuscular, intraocular or intravenous injection. The use of adjuvants, such as Freund's complete or incomplete adjuvants, can improve the immunogenicity of proteins or peptides of interest. After multiple booster immunizations, collect a small amount of blood samples to detect the reactivity of multi-purpose proteins or peptides.

Once the animal's titer reaches a plateau of reactivity with the protein, large quantities of polyclonal antisera can be obtained by weekly bleeding or exsanguination of the animal.

Monoclonal antibodies are also readily available using known techniques (see U.S. Patent Nos: RE32,011, 4,902,614, 4,543,439, and 4,411,993; see also Monoclonal Antibodies, Hybridomas: New Approaches to Biological Analysis, plenum Publishing, Kennett, McKearn, and Bechtol (eds.), 1980, and Antibodies: A Laboratory Manual, supra). Briefly, in one embodiment a test animal such as a rat or mouse is injected with the protein or peptide of interest. Various techniques can be used to enhance the immune response generated by the protein and generate a higher antibody response, if desired. For example, the protein or peptide of interest can be conjugated to other proteins, such as ovalbumin or keyhole limpet hemocyanin (KLH), or by using Freund's complete or incomplete adjuvant. The initial immune response activation can be given intraperitoneally, intramuscularly, intraocularly or intravenously.

One to two weeks after the initial immunization, animals are reimmunized with a booster immunization. Animals are then experimentally bled and serum is tested for binding of unprocessed polypeptide using the assay described above. Additional immunizations can also be performed until the animal reaches a plateau of its reactivity with the protein or peptide of interest. Animals are then boosted with a final protein and peptide of interest and sacrificed 3 to 4 days later. This time the spleen and lymph nodes were collected and made into a single cell suspension by sieving the organ or disrupting the membrane of the spleen or lymph nodes surrounding the cells. In one embodiment, the erythrocytes are completely lysed by the addition of a hypotonic solution and immediately returned to isotonic state.

In another embodiment, cells suitable for producing monoclonal antibodies are obtained by in vitro immunization techniques. Briefly, animals were sacrificed and spleen and lymph node cells were removed as described above. A single cell suspension is prepared and the cells are placed in culture medium containing the protein and polypeptide of interest in a form suitable for raising an immune response as described above. Next, lymphocytes were harvested and fused as described above.

Cells from the immunized animal can be immortalized by virus transfection such as Epstein-Bar virus (EBV) using in vitro immunization as described above. (See Glasky and Reading, Hybridoma 8(4):377-389, 1989) Alternatively, in a preferred embodiment, the harvested spleen and/or lymph node cell suspensions are fused with appropriate myeloma cells to produce secretory "Hybridomas" of monoclonal antibodies. Appropriate myeloma cell lines are preferably deficient in antibody construction and expression, with additional synergy with cells from the immunized animal. Many such cell lines are well known in the art and can be obtained from sources such as the American Type Culture Collection (ATCC), Rockville, Maryland (see, Catalog of Hybridomas and Cell Lines, 6th Edition, ATCC, 1988). Representative myeloma cell lines include: human, UC729-6 (ATCC No. CRL8061), MC/CAR-Z2 (ATCC No. CRL8147), and SK0-007 (ATCC No. CRL8033); mouse, SP2 /0-Ag14 (ATCC No. CRL1581), and P3X63Ag8 (ATCC No. TIB9); and rat, Y3-Ag1.2.3 (ATCC No. CRL1631), and YB2/0 (ATCC No. CRL1662). Particularly preferred fusion lines include NS-1 (ATCC No. TIB18) and P3X63-Ag8.653 (ATCC No. CRL1580), which can be used to fuse mouse, rat or human cell lines. Fusion between myeloma cell lines and cells from immunized animals can be accomplished by various methods. These methods include the use of polyethylene glycol (PEG) (see Antibodies: A Laboratory Manual, supra) or electrofusion (see Zimmerman and Vienken, J. Membrane Biology 67:165-182, 1982).

After fusion, the cells are placed in a Petri dish containing an appropriate medium such as RPMI1640 or DMEM (Dulbecco's Modified Eagles Medium, JRH Biosciences, Lenexa, Kan.). The medium may also contain additional components such as fetal bovine serum (FBS, e.g., from Hyclone, Logan, Uth, or JRH Biosciences), thymocytes harvested from young animals of the same species as the animal used for immunization. or fixed medium agar. In addition, the medium should contain agents that selectively permit the growth of fused spleen and myeloma cells. It is particularly preferable to use a HAT medium (hypoxanthine, aminopterin, thymine) (Sigma Chemical Co., St. Louis, Mo.). After about 7 days, the resulting fusion cells or hybridoma cells can be screened for the presence of antibodies recognizing the core envelope region of the HCV or HCV nonstructural proteins. After dilution and reanalysis of many clones, hybridomas producing antibodies that bind the protein of interest can be isolated.

Other techniques can also be used to prepare monoclonal antibodies. (See Huse et al., "Generation of a large combinatorial library of immunoglobulin repertoires from bacteriophage lambda", Science 246:1275-1281, 1989; see also Sastry et al., "Immunoglobulin production of monoclonal catalytic antibodies in Escherichia coli. Cloning of repertoires: construction of heavy chain variable region-specific cDNA libraries", Proc. Natl. Acad. Sci. USA 86:5728-5732, 1989; see also Aliting-Mess et al., "Monoclonal Antibody Expression Libraries: A Rapid Alternative to Hybridomas." Methods in Molecular Biology 3:1-9, 1990; these references describe a commercial system from Stratacyte, La Jolla, California, which can produce antibodies by recombinant techniques.) Briefly, from B cell mRNA was isolated from the population and used to generate heavy and light chain immunoglobulin cDNA expression libraries in λIMMUNOZAP (H) and λIMMUNOZAP (L). These vectors can be selected individually or co-expressed to form Fabs or antibodies (see Huse et al., supra; see also Sastry et al., supra). Positive phages are then transformed into non-lytic plasmids that express high-level monoclonal antibodies in E. coli.

Recombinant DNA technology can also be used similarly to introduce variable regions encoding specific binding antibody genes to construct binding partners. The construction of these binding partners can be readily accomplished by one of ordinary skill in the art based on the teachings 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., "For Therapeutic Reconstruction of human antibodies", Nature, 332:323-327, 1988; Roberts et al., "Protein Engineering Generates Antibodies with Enhanced Affinity and Specificity for Antigens", Nature, 328:731-734, 1987; Verhoeyen et al., "Reconstructing Human Antibodies: Altering Anti-Lysozyme Activity", Science, 239:1534-1536, 1988: Chaudhary et al., "A recombinant immunotoxin consisting of two antibody variable regions fused to a pseudomonas exotoxin" Nature 339:394-397, 1989; see also US Patent No. 5,132,405, entitled "Biosynthetic Antibody Binding Sites"). Briefly, in one embodiment, a DNA segment encoding a specific antigen-binding domain of a protein or peptide of interest can be amplified from a hybridoma that produces a specifically binding monoclonal antibody and inserted directly into a human antibody-producing in the genome of the cell. (See Verhoeyen et al., supra; see also Reichmann et al., supra.). This technique transfers the antigen-binding site of a specifically binding mouse or rat monoclonal antibody into a human antibody. Such antibodies are preferred for human therapy because they do not have the antigenicity of rat or mouse antibodies.

In another embodiment, the gene encoding the variable region from a hybridoma producing a monoclonal antibody of interest can be amplified using primers directed to the variable region. These primers can be synthesized by one of ordinary skill in the art, or purchased from commercially available sources. For example, primers for mouse and human variable regions include various primers for V _Ha , V _Hb , V _Hc , V _Hd , _CH1 , V _L , and _CL regions, available from Stratacyte (La, Jolla, Calif.) get. These primers can be used to amplify the heavy chain or light chain variable regions, respectively, and then inserted into vectors, such as λIMMUNOZAP(H) and λIMMUNOZAP(L) (Stratacyte). These vectors can then be introduced into E. coli for expression. Using these techniques, large quantities of single-chain proteins containing fused _VH and _VL domains can be produced (see Bird et al., Science 242:423-426, 1988).

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

Once appropriate antibodies and binding partners are available, they can be isolated and purified by a number of techniques well known to those of ordinary skill in the art (see Antibodies: A Laboratory Manual, Harlow and Lane (eds.), Published by Cold Spring Harbor Laboratory, 1988 ; US Patent No. 4,736,110; and US Patent No. 4,486,530). Suitable separation techniques include affinity columns for peptides and proteins, HPLC or RP-HPLC, protein A and protein G purification columns, or any combination of these techniques. The term "isolated" used to define antibodies and binding partners in the present invention means "substantially free from other blood components".

The antibodies and binding partners of the invention have many uses. As discussed further below, the antibodies and binding partners of the invention are particularly useful in the detection and diagnosis of positive-strand RNA viruses. Other applications include, for example, flow cytometric sorting of cells with multiple proteins of the invention. Briefly, to detect proteins and peptides of interest on cells, cells are incubated with labeled monoclonal antibodies that specifically bind the protein of interest. The presence of bound antibody is then detected. These steps may also include additional steps of washing to remove unbound antibody. Labels suitable for use in the present invention are well known in the art and include various fluorescein isothiocyanate (FITC), phycoerythrin (PE), horseradish peroxidase (HRP) and colloidal gold. Particularly preferred for use in flow cytometry is FITC, which can be used according to the method in Kelkamp's "Coupling of fluorescein isothiocyanate and antibodies, I Experiment with coupling conditions" Immunology 18:865-873,1970, Conjugated to purified antibody. (See also Kelkamp, "Coupling of fluorescein isothiocyanate and antibodies, II. A reproducible method" Immunology 18:875-881, 1970; Goding, "Coupling of antibodies and fluorescein isothiocyanate: Improvements to standard methods", Methods in Immunology 13:215-226, 1970). Analysis of Positive Strand RNA Viruses in Detection Samples

As indicated above, the present invention provides polypeptides comprising unprocessed, substantially intact polyproteins from positive-strand RNA viruses. The invention also provides polypeptides comprising core-like antigen-adjacent proteins and nonstructural proteins. The invention also provides methods for detecting such polypeptides in a sample. As is known in the art, assays are generally based on the detection of antigens expressed by positive-strand RNA viruses or antibodies raised against positive-strand RNA viruses, but may also include nucleic acid-based assays (generally based on hybridization). The method is characterized in that the polypeptide of the present invention can be combined by an anti-positive-strand RNA virus antibody, and the prepared anti-positive-strand RNA virus antibody can bind to the antigen of the positive-strand RNA virus in the sample.

Surprisingly, the raw polypeptides of the present invention provide a significantly improved more sensitive detection of positive-strand RNA viruses. For example, using HCV as a reference, unprocessed core antibody-envelope protein provides significantly better detection of HCV in a sample than processed core protein (sometimes referred to as p22) or fragments of core protein alone. It is also surprising that the simultaneous application of the unprocessed core antigen-adjacent protein and a non-structural protein of positive-strand RNA viruses in the assay can produce a synergistic effect, making the detection of positive-strand RNA viruses more effective than using the unprocessed core alone. Antigen-adjacent or nonstructural proteins are significantly more sensitive.

A preferred assay for detecting positive-strand RNA viruses is a sandwich assay, such as an enzyme-linked immunosorbent assay (ELISA). In a preferred embodiment, the ELISA comprises the following steps: (1) coating the core antigen envelope protein of the present invention on a solid phase, (2) under appropriate conditions, combining samples suspected of containing HCV antibodies with the coating Incubated by the polypeptide on the solid phase, allowing the formation of the antigen-antibody complex, (3) adding a labeled anti-antibody (such as anti-IgG), which is formed by the antibody-antigen bound to the solid phase The complex is captured, and (4) the captured label is determined to determine whether there are HCV antibodies in the sample.

While a preferred assay is set forth above, various assays can be used to detect antibodies that specifically bind to a protein of interest in a sample, or to detect proteins of interest that bind to one or more antibodies in a sample. Exemplary analytical methods are described in detail in Antibodies: A Laboratory Manual, Harlow and Lane (eds.), published by Cold Spring Harbor Laboratory, 1988. Representative examples of such assays include: countercurrent immunoelectrophoresis (CIEP), radioimmunoassay, radioimmunoprecipitation, enzyme-linked immunosorbent assay (ELISA), dot blot analysis, inhibition or competition assay, sandwich assay, immunoadhesion assay ( dip-stick), simultaneous assays, immunochromatography assays, immunofiltration assays, latex bead agglutination assays, immunofluorescence assays, biosensor assays, and low-light detection assays (see U.S. Patent Nos: 4,376,110 and 4,486,530 ; WO94/25597; WO/25598; see also Antibodies: A Laboratory Manual, supra).

The fluorescently labeled antibody test (FA-test) uses a fluorescently labeled antibody that binds to a protein of the present invention. For detection, visual inspection is performed by a technician using a fluorescent microscope, which yields a definitive result. In one embodiment, the analysis is used to examine histologically sectioned tissue samples.

In latex bead agglutination assays, antibodies to one or more proteins of the invention are coupled to latex beads. Antibodies coupled to latex beads are then contacted with the sample under conditions that allow the antibodies to bind the protein of interest in the sample. The results are then visually inspected, yielding definite results. In one embodiment, the method can be used in localization experiments.

Enzyme immunoassays (E1A) include various assays to which the antibodies provided by the present invention can be used. For example, heterologous indirect E1A uses a solid phase conjugated antibody of the invention and an affinity purified anti-IgG immunoglobulin preparation. The solid phase is preferably a polystyrene microwell plate. The antibody and immunoglobulin preparations are then contacted with the sample under conditions that permit binding of the antibodies, conditions well known in the art. The results of this assay can be measured visually, but preferably read with a spectrophotometer, such as an ELISA plate reader, to yield quantitative results. Another solid-phase EIA method involves plastic-coated iron beads that can be removed using a magnet during the detection step. Yet another method is the low light detection immunoassay. In this highly sensitive method, light emission from appropriately labeled bound antibodies is automatically quantified. The reaction is preferably performed in a microwell plate.

In another embodiment, the enzyme-mediated detection in the EIA is replaced by a radioactive tracer to form a radioimmunoassay (RIA).

In a capture-antibody sandwich assay, the protein of interest is bound between an antibody attached to a solid phase, preferably a polystyrene microwell plate, and a labeled antibody. Results are preferably measured with a spectrophotometer, such as an ELISA plate reader. This analysis is a preferred embodiment of the invention.

In sequential assay methods, reagents and capture antibodies are allowed to incubate step by step. The first sample tested is incubated with capture antibody. After washing steps, incubation with labeled antibody. In a simultaneous analysis, the two incubation phases described in the sequential analysis are combined. This eliminates an incubation stage plus a wash step.

The dipstick/immunoadhesion method is basically an immunoassay in which polystyrene paddles or dipsticks are used as the solid phase instead of polystyrene microwell plates. The reagents are the same and the methods can be simultaneous or sequential.

In the chromatographic band detection method, a capture antibody and a labeled antibody are dried on a chromatographic band, typically nitrocellulose or high-porosity nylon bound to cellulose acetate. The capture antibody is often sprayed on one end of the strip and dries into a line. At this end there is adsorbent material in contact with the belt. The labeled antibody is located at the other end of the band, preventing absorption into the membrane. Labels attached to antibodies are typically latex beads or colloidal gold. The reaction was initiated by adding the sample immediately before the labeled antibody.

Immunofiltration and immunoconcentration methods combine a large solid surface with a sample/reagent stream that concentrates and accelerates antigen and antibody binding. In a preferred method, the sample to be tested is pre-incubated with labeled antibody and then placed on a solid phase, such as a fiber filter or nitrocellulose membrane or the like. The solid phase can be pre-coated with latex or glass beads coated with capture antibodies. Analytical assays are the same as standard immunoassays. Sample/reagent flow rate can be adjusted by vacuum or wicking action of adsorbent material placed on the bottom.

Threshold value biosensor assays are sensitive device-assisted assays that can screen large numbers of samples at low cost. In one embodiment, such assays include the use of a light-accepting potentiometric sensor, where the response involves detecting a change in pH resulting from the binding of the protein of interest to the capture antibody, bridging antibody, and urease-conjugated antibody. The effect of the pH change caused by binding is measured as conversion to electrical energy (microvolts). This analysis is generally performed in very small volumes and is very sensitive. Furthermore, the detection limit of the assay was reported to be 1000 molecules of urease per minute. Compositions and methods for stimulating an immune response to HCV

The present invention also provides compositions and methods for stimulating a humoral, cellular, or both immune response to positive-strand RNA viruses, the immune response preferably being induced by a vaccine against positive-strand RNA viruses, and thus being an immunoprotective immune response. These compositions and methods generally include an immunogen comprising a raw polypeptide of the invention in combination with a pharmaceutically acceptable carrier or diluent. In a preferred embodiment, the composition and method contain the unprocessed core antigen of HCV - membrane protein and non-structural protein of HCV, more preferably NS5 non-structural protein or NS3-N84 non-structural protein. The compositions and methods also include inactivated and attenuated preparations comprising the proteins of the invention.

Accordingly, another aspect of the present invention provides an isolated antigen capable of stimulating an immune response, preferably an immunogen capable of immunizing an animal. In a preferred embodiment, it comprises the amino acid sequence or molecule shown in or derived from the sequences shown in Figures 1A, 1B, 3A or 3B or an actual equivalent thereof. Those of ordinary skill in the art can understand that slight modifications can be made to the amino acid sequence of the polypeptide of the present invention without affecting the immunogenicity of the immunogen. Actual equivalents of the above proteins include conservative amino acid substitutions that fully retain the same charge and hydrophobicity as the original amino acid. Conservative substitutions include substitution of isoleucine and leucine for valine, glutamic acid for aspartic acid, and other substitutions of equivalent nature (see, Dayhoff et al. (eds.), "Atlas of Protein Sequence and Structure", Natl . Biomed. Res. Fdn., 1978)

As will be appreciated by those of ordinary skill in the art, the above-mentioned immunogens (including their actual equivalents) may stimulate different levels of immune responses in different animals. The above-mentioned immunogens (including their actual equivalents) can be tested for their efficacy as vaccines. These tests include T-cell proliferation assays, post-stimulation assays for lymphokine production, and immune protection assays. Briefly, T cell proliferation assays can be used as indicators of cell-mediated immunity competence. In addition, the production of lymphokines following stimulation by the immunogen can be used to determine the ability of the immunogen to confer protection.

In summary, to determine protection in animals, precise immunoprotection assays were performed as described below. But for humans, the first choice is to screen peripheral blood lymphocytes (PBL) of patients infected with HCV as follows rather than immune protection testing. Briefly, PBLs were isolated from diluted whole blood using Ficoll density gradient centrifugation, and cell proliferation studies were performed with [ ^3H ]-thymidine as described below. Positive peptides were then screened and used in primate trials.

Many other techniques well known in the art can be used to prepare the immunogens or polypeptides of the invention (see Sambrook et al., supra, A Laboratory Manual of Molecular Cloning, Cold Spring Harbor Laboratory Press, 1989).

The immunogen containing the polypeptide of the present invention can be combined with a pharmaceutically acceptable carrier or diluent, and administered to patients according to various methods known in the art (see WO94/25597 and WO/25598).

According to the present invention, warm-blooded animals include humans and primates, among other species.

Many suitable carriers and diluents that can be used in the present invention include various saline solutions, buffered saline solutions and saline solutions mixed with non-specific serum albumin. Such pharmaceutical compositions may also contain other excipient components including adjuvants, buffers, antioxidants, sugars (such as glucose, sucrose or dextran) and complexing agents such as EDTA. In a particularly preferred embodiment of the invention, an adjuvant is used together with the immunogen. Examples of such adjuvants include alum and aluminum hydroxide for humans.

Dosage and frequency of administration will be determined in clinical trials, depending on such factors as the positive-sense RNA virus species for which protection is sought, the particular antigen, the degree of protection desired and many other factors. In a preferred embodiment, immunization is administered orally. In addition, vaccines may be administered parenterally by intravenous or other routes. Depending on the application, the amount of immunogen in the injected adjuvant vehicle varies from 50 μg to several milligrams, preferably about 100 μg to 1 mg, in combination with physiologically acceptable carriers and diluents. A booster immunization is given 4-6 weeks later.

The present invention also includes administering to animals a nucleic acid carrier capable of expressing HCV unprocessed core antigen-envelope protein or nonstructural protein (or both), wherein the nucleic acid molecule can activate an immune response, preferably the protein expressed by the immunized animal against the nucleic acid molecule Anti-HCV. In one embodiment of the method, naked DNA can be introduced into appropriate cells, such as muscle cells, where the DNA can produce proteins that are then displayed on the cell surface, thereby stimulating a host cytotoxic T lymphocyte response (CTL). This is an advantage over traditional immunogens where the activated response includes specific antibodies. Specific antibodies are generally strain-specific and do not recognize corresponding antigens on different strains. In other respects, CTLs are specific for conserved antigens and can respond to corresponding antigens expressed by different strains (Ulmer et al., "Heterologous protection against influenza by injection of DNA encoding viral proteins" Science 259:1754-1759, 1993; Lin et al., "Expression of recombinant genes in myocardium in vivo following direct DNA injection," Circulation 82: 2217-21, 1990; Wolff et al., "Long-term persistence of plasmid DNA and expression of exogenous genes in mouse muscle." Human Molecular Genetics 1: 363-69, 1992).

The naked carrier structure is introduced into animal cells, and then the structure can express the nucleic acid molecule (generally a gene) it carries, and the gene preferably contains one (or more) unprocessed core antigens of HCV-envelope protein or non-structural protein. Thus, expression of the peptide of interest activates the immune response in the host animal. The immune response preferably includes CD8 ⁺ CTLs that respond to different strains presenting the protein of interest. Kits for practicing various aspects of the invention

The present invention also provides kits for the analysis of antigens and antibodies to positive-strand RNA viruses present in a sample. The kit contains the polypeptide or antibody of the present invention and an appropriate solid phase. The polypeptide is preferably bound to a solid phase. The kit can also provide one or more reagents and/or devices for detecting HCV antigens or antibodies. Various operating procedures, reagents and devices included in the kit, including tools for detecting antigens or antibodies, are as described in the text.

The invention also provides kits for inducing an immune response. The kit contains the composition of the polypeptide of the present invention combined with a pharmaceutically suitable carrier or diluent, and also provides a device for administering or assisting administering.

Also provided are other kits suitable for use of the features of the invention.

The following examples are provided by way of illustration and not limitation. Example

The following examples are divided into three groups. The first is an example relating to the isolation and preparation of a suitable quasi-core antigen-adjacent protein, the HCV unprocessed core antigen-envelope fusion protein, and its use in the absence of nonstructural proteins. The second group concerns the isolation and preparation of an appropriate second protein for use with the class of core antigen-adjacent proteins, i.e. HCV non-structural proteins and their use in the absence of the HCV core antigen-envelope fusion protein the embodiment. The third group is examples related to the combination and application of core-like antigen-adjacent proteins and second proteins such as HCV NS5 protein, HCV NS3-NS4 protein, HIV envelope protein and HTLV-1 envelope protein. The fourth is an example of preparing a monoclonal antibody of the core antigen-adjacent protein. The fifth is an example involving the use of an appropriate quasi-core antigen-adjacent protein, HCV unprocessed core antigen-envelope fusion protein, to induce immune responses in animals. Isolation and preparation of core-like antigen-adjacent proteins 1. Cloning of HCV cDNA

Plasma from patients infected with hepatitis C virus was collected and ultracentrifuged at 4°C to obtain virus particles. Viral nucleic acid (RNA) is then extracted and purified from viral particles using guanidinium isothiocyanate and acid phenol (Chomczynski et al., Annals Biochem. 162: 156-159, 1987).

The following oligonucleotide sequences:

(i) 5'-GGATCCATGAGCACAAATCCTAAACCT-3' (SEQ ID No: 1) and

(ii) 5'-GAATTCGGTGTGCATGATCATGTCCGC-3' (SEQ ID No: 2) was used as a primer for cloning cDNA. Single-stranded DNA molecules are prepared using random primers, reverse transcriptase, and an RNA template. Using Taq polymerase and primers (i) and (ii), a double-stranded DNA molecule containing the sequence of the HCV core-envelope region is amplified by PCR.

The cloned DNA molecules were identified by sequence analysis. The obtained molecule is called EN-80-2. The DNA sequence of molecule EN-80-2 is given in Figure 1A (SEQ ID No: 7). This DNA molecule is derived from HCV core and envelope regions and has 669 bp. 2. Construction of plasmid containing HCV cDNA

The molecule EN-80-2 was treated with restriction endonucleases BamHI and EcoRI to obtain a DNA fragment containing the desired HCV cDNA. The resulting fragment was inserted into the vector plasmid previously cut with restriction enzymes BamHI and EcoRI to obtain an expression plasmid called pEN-2. The expression of HCV cDNA is under the regulation of T7 promoter. The structure and restriction enzyme map of expression plasmid pEN-2 are shown in FIG. 2 . 3. Transformation of E. coli

The expression plasmid was transformed into Escherichia coli BL21(DE3), spread on an ampicillin plate and placed in a 37°C incubator overnight. E. coli colonies producing HCV cDNA antigenic proteins were selected by screening their expression products by SDS-PAGE and Western blotting. 4. Preparation of Raw Core Antigen-Envelope Protein

Transformed E. coli colonies were incubated in conditioned medium. Colonies were centrifuged and lysed by repeated freeze-thaw and lysozyme digestion. The unprocessed core antigen-envelope protein product is released from lysed cells and purified by column chromatography. The purity of the polypeptide is more than 90%.

The unprocessed core antigen-envelope protein has a molecular weight of approximately 25,000 Daltons as determined by sodium dodecyl sulfate polyacrylamide electrophoresis. 5. Immunoreactivity of HCV Core Antigen with HCV Antibody in Western Blot

The purified unprocessed core antigen-envelope protein was subjected to SDS-PAGE electrophoresis using standard methods. The SDS-PAGE gel was washed with deionized water at 4°C for 15 minutes and with blotting buffer (0.15M sodium phosphate buffer, pH 6.7) at 4°C for 20 minutes. The peptides on the gel were then electroblotted onto nitrocellulose membranes at 1.3 A ₁ -1.5 hours in blotting buffer. Wash the membrane with washing buffer (PBS-Tween 20, pH7.4) and block buffer (0.1M NaCl, 5mM EDTA, 50mM Tris, pH7.2-7.4, 0.2% bovine serum albumin, 0.05% NP40, 1M urea) blocked overnight.

The membrane was reacted with human serum infected/uninfected with hepatitis C virus at 40° C. for 2 hours, and the serum was diluted 10× with 40% fetal bovine serum/Tris-HCl (pH 7.4) in advance. After the reaction, the membrane was washed three times with washing buffer. Membranes were reacted with anti-hlgG:HRPO conjugate (prepared as described below) for 2 hours at 40°C. After the reaction, the membrane was washed three times with washing buffer, and then mixed with the substrate solution (0.01% 4-chloro-1-naphthol, 18% methanol, 0.04M Tris, pH7.2-7.4, 0.1M NaCl and 0.01% H ₂ O ₂ ) for 20 minutes. The unprocessed core antigen-envelope protein of the present invention reacts with the sera of HCV patients but not with the sera of normal persons. 6. ELISA of HCV antibody

(A) Handling of microplates

The microwell plate is coated with the purified unprocessed core antigen-envelope protein of the present invention at an appropriate concentration, and blocked with a buffer containing bovine serum albumin. Store treated microplates at 2-8°C.

(B) Preparation of anti-higG:HRPO conjugate

Pure anti-human immunoglobulin G (anti-hlgG) was conjugated with horseradish peroxidase (HRPO) using _NaIO4 to give an 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:HRPO conjugate solution: Anti-hlgG:HRPO conjugate prepared as above was dissolved in Tris buffer containing protein stabilizers and preservatives.

(c) Sample diluent: Tris buffer containing protein stabilizers and preservatives

(d) OPD substrate solution: o-phenylenediamine (OPD) was dissolved in citric acid-phosphate buffer containing H ₂ O ₂ . (If the solution turns orange, the solution is contaminated and can no longer be used)

(e) Stop solution: 2N H ₂ SO ₄ solution

(f) Positive/negative control: human serum samples infected/uninfected with hepatitis C virus were diluted with phosphate buffer containing protein stabilizers and preservatives at appropriate concentrations.

(D) step:

(a) 100 and 50 microliters ([mu]l) of test samples were diluted (1:10) with sample diluent, and positive/negative controls were added to treated microwell plates. Keep some wells as substrate blanks.

(b) Mix plate gently by shaking and incubate at 37-40°C for 1 hour.

(c) Wash the plate three times with 0.3 μl of wash solution per well.

(d) Add 100 [mu]l anti-hlgG:HRPO conjugate solution per well.

(e) Mix plate gently by shaking and incubate at 37-40°C for 30 minutes.

(f) Wash the plate 5 times.

(g) Add 100 μl OPD substrate solution to each well and incubate the plate at 15-30°C for 30 minutes in the dark.

(h) Add 100 μl of stop solution per well and mix gently to stop the reaction.

(i) Measure the OD value of each well at 492 nm on a spectrophotometer.

(E) to determine:

The average reading of the blank (background) was subtracted from the _OD492nm value of each well. The difference (PCx-NCx) of the mean values of the positive control (PCx) and the negative control (NCx) should be equal to or greater than 0.5.

The cut-off value (CO) was calculated according to the following formula:

CO=PCx×0.15+Ncx

When the reading of the test sample was less than the CO value, the sample was considered negative (ie, no HCV antibody could be detected in the sample).

A sample is considered positive when the reading for the test sample is equal to or greater than the CO value; however, it is best if the sample is analyzed in multiple replicates. A sample was considered negative if any of the duplicate sample readings were less than the CO value. A sample was considered positive if all replicates were greater than or equal to the cutoff value.

When a test sample has a reading greater than NCx but less than 20% of the CO value, that sample should be considered suspect and those samples must be analyzed repeatedly.

27 samples were tested by ELISA according to the present invention. Simultaneously, samples were also assayed with HCV Antibody Analysis Abbott Kit (II), which contains structural and nonstructural proteins (ie, core (amino acids: 1-151), NS-3 and NS-4). A comparison of the analytical results of the Abbott kit (II) and the present invention is given in Table 1. It should be noted that sample G229 was negative according to the Abbott kit (II), but positive according to the analysis of the present invention. Sample G229 was confirmed to be HCV positive.

Table 1 Sample number OD _492nm result Abbott kit (II) refer to TSGH 56 >2.0 positive positive TSGH 57 >2.0 positive positive G 23 1.469 positive positive G 30 >2.0 positive positive G 32 >2.0 positive positive G 49 >2.0 positive positive G 56 >2.0 positive positive G 58 >2.0 positive positive G 114 1.559 positive positive G 128 >2.0 positive positive G 186 >2.0 positive positive G 208 >2.0 positive positive G 214 >2.0 positive positive G 231 >2.0 positive positive G 250 >2.0 Positive 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 0.952 Positive Positive USB 27 0.753 Positive Positive G 11 0.147 Negative Negative G 12 0.077 Negative Negative G 13 0.061 Negative Negative G 14 0.116 Negative Negative G 15 0.139 Negative Negative G 229 0.517 Positive Negative Applicable Second Protein HCV Nonstructural Protein Isolation and Preparation 7. Cloning of HCV cDNA Encoding NS5 Nonstructural Protein

Plasma from patients infected with hepatitis C virus was collected and ultracentrifuged at 4°C to obtain virus particles. Viral nucleic acid (RNA) is then extracted and purified from viral particles using guanidinium isothiocyanate and acid phenol (Chomczynski et al., Annals Biochem. 162: 156-159, 1987).

The following oligonucleotide sequences:

(i) 5'-GGATCCCGGTGGAGGATGAGAGGGAAATATCCG-3' (SEQ ID No: 3) and

(ii) 5'-GAATTCCCGGACGTCCTTCGCCCCGTAGCCAAATTT-3' (SEQ ID No: 4) was used as a primer for cloning cDNA. Single-stranded DNA molecules are prepared using random primers, reverse transcriptase, and an RNA template. Using Taq polymerase and primers (i) and (ii), a double-stranded DNA molecule containing the NS-5 sequence was amplified by PCR method.

The cloned DNA molecules were identified by sequence analysis. The obtained molecule was called EN-80-1. The DNA sequence of molecule EN-80-1 is given in Figure 3A and the amino acid sequence encoded by this molecule is given in Figure 3B. The DNA molecule was derived from the non-coding region 5 of the HCV genome and had 803 bp (SEQ ID No: 9). The amino acid sequence of molecule EN-80-1 is given in Figure 3B (SEQ ID No: 10) and has 267 residues. 8. Construction of plasmid containing HCV cDNA

The molecule EN-80-1 was treated with restriction endonucleases BamHI and EcoRI to obtain a DNA fragment containing the desired HCV cDNA. The obtained fragment was inserted into the vector plasmid previously cut with restriction endonucleases BamHI and EcoRI to obtain an expression plasmid called pEN-1. The expression of HCV cDNA is under the regulation of T7 promoter. The structure and restriction enzyme map of the expression plasmid pEN-1 are shown in FIG. 4 . 9. Transformation of E. coli

The expression plasmid was transformed into Escherichia coli BL21(DE3), spread on an ampicillin plate and placed in a 37°C incubator overnight. E. coli colonies producing HCV nonstructural proteins were selected by screening their expression products by SDS-PAGE and Western blot. 10. Preparation of NS5 nonstructural protein

Transformed E. coli colonies were incubated in conditioned medium. Colonies were centrifuged and lysed by repeated freeze-thaw and lysozyme digestion. The protein product is released from lysed cells and purified by column chromatography. The purity of the polypeptide is more than 90%.

The polypeptide has a molecular weight of about 29,000 Daltons as determined by sodium dodecyl sulfate polyacrylamide electrophoresis. 11. Immunoreactivity of NS5 Nonstructural Proteins with HCV Antibody in Western Blot

Purified peptides were subjected to SDS-PAGE electrophoresis using standard methods. The SDS-PAGE gel was washed with deionized water at 4°C for 15 minutes and with blotting buffer (0.15M sodium phosphate buffer, pH 6.7) at 4°C for 20 minutes. The peptides on the gel were then electroblotted onto nitrocellulose membranes in blotting buffer for 1.3A1-1.5 hours. Wash the membrane with washing buffer (PBS-Tween 20, pH7.4) and block buffer (0.1M NaCl, 5mM EDTA, 50mM Tris, pH7.2-7.4, 0.2% bovine serum albumin, 0.05% NP40, 1M urea ) closed overnight.

The membrane was reacted with HCV-infected/uninfected human serum at 40°C for 2 hours, and the serum was pre-diluted with 40% fetal bovine serum/Tris-HCl (pH 7.4), 10×. After the reaction, the membrane was washed three times with washing buffer. Membranes were reacted with anti-hlgG:HRPO conjugate (prepared as described below) for 2 hours at 40°C. After the reaction, the membrane was washed three times with washing buffer, and then mixed with a substrate solution (0.01% 4-chloro-1-naphthol, 18% methanol, 0.04M Tris, pH7.2-7.4, 0.1M NaCl and 0.01% H ₂ O ₂ ) react for 20 minutes. The unprocessed core antigen-envelope protein of the present invention reacts with the sera of HCV patients but not with the sera of normal persons. 12. ELISA of HCV antibody

(A) Handling of microplates

The microwell plate is coated with the NS5 nonstructural protein of the present invention at an appropriate concentration, and blocked with a buffer solution containing bovine serum albumin. Store treated microplates at 2-8°C.

(B) Preparation of anti-higG:HRPO conjugate

Pure anti-human immunoglobulin G (anti-hlgG) was conjugated with horseradish peroxidase (HRPO) using _NaIO4 to obtain an 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:HRPO conjugate solution: Anti-hlgG:HRPO conjugate prepared as above was dissolved in Tris buffer containing protein stabilizers and preservatives.

(c) Sample diluent: Tris buffer containing protein stabilizers and preservatives

(d) OPD substrate solution: o-phenylenediamine (OPD) was dissolved in citric acid-phosphate buffer containing H ₂ O ₂ . (If the solution turns orange, the solution is contaminated and can no longer be used)

(e) Stop solution: 2N H ₂ SO ₄ solution

(f) Positive/negative control: human serum samples infected/uninfected with hepatitis C virus were diluted with phosphate buffer containing protein stabilizers and preservatives at appropriate concentrations.

(D) step:

(a) 100 and 50 microliters ([mu]l) of test samples were diluted (1:10) with sample diluent, and positive/negative controls were added to treated microwell plates. Keep some wells as substrate blanks.

(b) Mix plate gently by shaking and incubate at 37-40°C for 1 hour.

(c) Wash the plate three times with 0.3 μl of wash solution per well.

(d) Add 100 [mu]l anti-hlgG:HRPO conjugate solution per well.

(e) Mix plate gently by shaking and incubate at 37-40°C for 30 minutes.

(f) Wash the plate 5 times.

(g) Add 100 μl OPD substrate solution to each well and incubate the plate at 15-30°C for 30 minutes in the dark.

(h) Add 100 μl of stop solution per well and mix gently to stop the reaction.

(i) Measure the OD value of each well at 492 nm on a spectrophotometer.

(E) to determine:

The average reading of the blank (background) was subtracted from the _OD492nm value of each well. The difference (PCx-NCx) of the mean values of the positive control (PCx) and the negative control (NCx) should be equal to or greater than 0.5.

The cut-off value (CO) was calculated according to the following formula:

CO=PCx×0.15+Ncx

When the reading of the test sample was less than the CO value, the sample was considered negative (ie, no HCV antibody could be detected in the sample). A sample is considered positive when the reading for the test sample is equal to or greater than the CO value; however, it is best if the sample is analyzed in multiple replicates. A sample was considered negative if any of the duplicate sample readings were less than the CO value. A sample was considered positive if all replicates were greater than or equal to the cutoff value.

When a test sample has a reading greater than NCx but less than 20% of the CO value, that sample should be considered suspect and those samples must be analyzed repeatedly.

Eighteen samples were tested by ELISA according to the invention. Simultaneously, samples were also analyzed with the Abbott kit (I) and HCV antibodies containing nonstructural protein C100-3 (i.e., core (amino acids: 1-151), NS-3 and NS-4). HCV antibody analysis Abbott kit (II) detection. A comparison of the analytical results of the Abbott kit and the present invention is given in Table 2. It should be noted that sample G30 and sample G128 were negative according to the Abbott kit (I), but positive according to the analysis of the present invention. These samples proved positive for HCV.

Table 2 Sample number OD _492nm result Abbott kit reference

(I) (ii) TSGH 56 ＞ 2.0 positive positive G 23 0.813 positive positive G 26 1.607 positive positive G 30> 2.0 positive negative G 32> 2.0 positive positive G 56> 2.0 positive positive G 128> 2.0 Positive-negative-positive G 186 ＞ 2.0 positive positive G208> 2.0 positive-positive G 214> 2.0 positive-positive G 231 ＞ 2.0 positive-positive Y 1> 2.0 positive USB 9> 2.0 positive USB 19 >0 positive -Host USB 20 ＞ 2.0 Positive-Positive G 201 0.062 negative-negative G 202 0.072 negative-negative G 211 0.059 negative use of core antigen-adjacent protein and second protein detection 13. Used non-processed core antigen- HCV ELISA of envelope protein and one NS5 nonstructural protein

A. Analytical comparison of core antigen-envelope protein and NS5 nonstructural protein with ABBOTTHCV assay

I. First Analysis

The method is similar to the above ELISA, except that the unprocessed core antigen-envelope protein is combined with NS5 non-structural protein (9:1) (known as EverNew anti-HCV EIA).

In the first analysis, 24 samples were tested as described above. At the same time, the samples were also detected with Abbott (II) kit. The results are given in Table 3. In this analysis, the result of the Abbott (II) kit was the same as that of the analysis using the antigen of the present invention.

Table 3 Sample number OD _492nm results Abbott kit (II) refer to 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 114 >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. The second analysis of the clinical test report of the EverNew anti-HCV EIA of blood donors is shown in Table 4: Hospital: Taipei Third Service General Hospital Sample source: Collect samples from blood banks Classification: Volunteer blood donors reference reagent Box Abbott kit (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 show that the two analytical methods produced the same detection results. III. The clinical detection report of the third analysis of the EverNew anti-HCV EIA of high-risk patients is shown in Table 5: Hospital: Taipei Veterans General Hospital Sample source: collected from the Department of Clinical Virology Classification: NANB, sporadic 20NANB, PHT 12HCC 15 liver Cirrhosis 9 Chronic hepatitis B and carriers 10 Biliary stones 4 Alcoholic liver disease 3 Fatty liver 2 Acute hepatitis, etiology? 2 liver blood absorption disease 1 liver cysts 1 bile duct disease-CA 1 non-liver and gallbladder disease 6 Unprecedented data 2 Total 88 reference kit: Abbott HCV E1A second-generation results:

table 5

ABBOTT

+ + - - Total

+ 54(61.36%) 0(0%) 54(61.36%)EverNew - 1(1.14%)@ 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 show that EverNew anti-HCV antibody detection is better than the second generation of ABBOTT HCV EIA licensed by the US FDA.

B. Analysis showing synergy of the core-like antigen-adjacent protein and different second proteins and comparison of HCV core antigen-envelope protein and HCV particle core protein

I. First Analysis

This analysis showed that the results of the ELISA were similar to those above and showed a synergistic relationship between the EN-80-2 and EN-80-1 proteins of HCV. The method of ELISA is as follows:

Coating buffer: 0.05M Tris-HCl/0.15N NaCl/6M urea pH: 7.4±0.2

Wash buffer: PBS with 0.05% Tween 20

Buffer after coating: PBS buffer with 1% BSA

Coating step: EN-80-1 and EN-80-2 proteins were added to the coating buffer (final concentration: about 1.5 μg/ml) and mixed at room temperature for 30 minutes. After mixing, the diluted EN-80-1 and EN-80-2 proteins were added to the wells of the microplate at 100 μl/well and incubated in a 40°C incubator for 24 hours. The wells of the microplate are then washed, and the post-coating buffer is added to the wells of the microplate. The wells of the microplate were then kept at 4°C overnight. After post-coating treatment, the wells of the coated microplate can be used for detection of anti-HCV antibodies.

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

Conjugation: Anti-human IgG monoclonal antibody was conjugated to HRPO using _NaIO4 . After conjugation, the anti-human IgG:HRPO conjugate was purified by S-200 gel filtration and diluted in sample diluent.

OPD tablets: purchased from Beckman

Substrate diluent: citrate-phosphate _buffer with _H2O2

Stop solution: 2N H ₂ SO ₄

Positive control: anti-HCV positive serum diluted in sample diluent

Negative control: recalcified human sera that did not respond to HBV markers, anti-HIV, anti-HTLV1 and anti-HCV.

Analysis steps:

100 μl of sample diluent was added to each well.

50 μl of sample, positive and negative controls were added to appropriate wells.

Sample incubation: incubate at 40±1°C for 30±2 minutes

Sample washing: wash the wells three times with washing buffer

100 μl of anti-human IgG:HRPO conjugate was added to each well.

Conjugate incubation: 30±2 minutes at 40±1°C

Washing of conjugates: wash wells 6 times with washing buffer

After washing, 100 µl of a substrate solution (a substrate solution prepared by dissolving a tablet of OPD in a substrate diluent) was added, and the mixture was kept at room temperature for 10 minutes. To protect from light, the microplate is covered with a black lid.

Add 100 μl of stop solution to each well and mix gently.

Measurement: Measure the OD value of each well on a spectrophotometer at 492 nm.

clarify:

Determination of cut-off value: cut-off value=PCx×0.25+Ncx

Absorbance equal to or greater than the cutoff value indicates a positive reaction, ie, an anti-HCV antibody response. An absorbance less than the cutoff value indicates a negative reaction, ie no anti-HCV antibody response.

The sources of the analytical samples listed in Table 6 are as follows:

Sample source I: G83, G191, G205 and G2 35 are GPT abnormal samples, which are anti-HCV antibody negative and collected from Taipei Blood Donation Center.

Sample source II: G614 and G615 were positive for anti-HCV antibody, purchased from the United States.

Sample source III: 8-5 were anti-HCV positive, collected from Taichun Blood Donation Center.

Sample source IV: N345 is patient serum.

Table 6 sample EN-80-1 EN-80-2 EN-80-1+EN-80-2 G83 0.027@ 0.047 0.055 G191 0.071 0.209 0.056 G205 0.027 0.034 0.039 G235 0.025 0.044 0.043 G614 8X# 0.066 0.831 1.894 G614 16X 0.059 0.348 0.848 G615 8X 0.048 0.495 1.592 G615 16X 0.053 0.209 0.740 8-5 0.059 0.352 0.690 N345$ 0.008 0.420 0.730 @: Absorbance at 492 nm #: Samples were diluted with recalcified human serum negative for HBV, HCV and HIV. $: The sample was found to be negative with the Abbott kit (II).

These data indicate that the absorbance at 492 nm of anti-HCV positive samples is synergistic rather than additive when EN-80-2 and EN-80-1 proteins are combined. Thus, a synergistic relationship between EN-80-2 and EN-80-1 proteins could be found. An advantage of this synergy is shown, for example, in sample N345, which was found to be HCV negative with the Abbott kit (II), but positive with the present invention due to the synergy. These data also suggest that this synergy is useful in screening samples for anti-HCV antibodies, especially in early diagnostic situations.

II. Second analysis

This assay was performed as described above for the first assay and included providing a single well of a core-envelope fusion protein of the invention in combination with the NS3-NS4 protein identified as EN-80-4. ELISA results are shown in Table 7.

Table 7 sample EN-80-2 EN-80-4 EN-80-2+EN-8 0-4 G83 0.047@ 0.032 0.049 G191 0.209 0.103 0.102 G205 0.034 0.045 0.046 G235 0.044 0.064 0.068 G58 21X# 0.561 0.041 1.729 G612 161X 1.298 0.218 >2.0 G613 40X 0.202 0.243 0.708 @: Absorbance at 492 nm #: Samples were diluted with calcified human serum negative for HBV, HCV and HIV.

The data in Table 7 show that when EN-80-2 and EN-80-4 proteins are combined, the absorbance at 492 nm of anti-HCV positive samples shows a synergistic effect, not just an additive effect. Thus, a synergistic relationship between the EN-80-2 and EN-80-4 proteins of HCV can be found.

III. THIRD ANALYSIS

This assay was performed as described above for the first assay and included providing a single well of a core-envelope fusion protein of the invention in combination with HIV envelope protein. ELISA results are shown in Table 8.

Table 8 sample EN-80-2 HIV env EN-80-2+HIV env recalcification into serum 0.030@ 0.056 0.093 G614 30.0X# 0.116 0.064 0.250 G614 15.0X 0.221 0.055 0.411 G614 9.9X 0.403 0.054 0.798 G614 7.5X 0.598 0.046 1.061 G614 6.0X 0.821 0.045 1.282 G614 5.0X 1.022 0.040 1.656 G614 4.3X 1.445 0.042 1.889 @: Absorbance at 492 nm #: Samples were diluted with calcified human serum negative for HBV, HCV and HIV.

The data in Table 8 show that when EN-80-2 of HCV (ie, the core-envelope fusion protein) and HIV envelope protein are combined, the absorbance at 492 nm of anti-HCV positive samples shows a synergistic rather than just additive effect. Thus, a synergistic relationship between EN-80-2 of HCV and HIV envelope protein can be found.

IV. Fourth Analysis

This assay was performed as described above for the first assay and included providing a single well of a core-envelope fusion protein of the invention in combination with the HTLV-I envelope protein. ELISA results are shown in Table 9.

Table 9 sample EN-80-2 HTLV-I env EN-80-2+HTLV-I env recalcified human serum 0.030@ 0.035 0.084 G614 30.0X# 0.116 0.031 0.375 G614 15.0X 0.221 0.027 0.561 G614 9.9X 0.403 0.034 1.017 G614 7.5X 0.598 0.033 1.303 G614 6.0X 0.821 0.025 1.502 G614 5.0X 1.022 0.017 >2.0 G614 4.3X 1.445 0.021 >2.0 @: Absorbance at 492 nm #: Samples were diluted with recalcified human serum negative for HBV, HCV and HIV.

The data in Table 9 show that when EN-80-2 of HCV (i.e. core-envelope fusion protein) and HTLV-1 envelope protein are combined, the 492nm absorbance of anti-HCV positive samples shows a synergistic effect, not just additive effect. Thus, a synergistic relationship between EN-80-2 of HCV and the envelope protein of HTLV-1 can be found.

V. Fifth Analysis

This assay was performed as described above for the first assay and included providing a single well of a core-envelope fusion protein of the invention in combination with the HTLV-1 pol protein. ELISA results are shown in Table 10.

Table 10 sample EN-80-2 HTLV-I pol& EN-80-2+HTLV-I pol recalcified human serum 0.027@ 0.039 0.073 G614 15.0X 0.167 0.057 0.379 G614 9.9X 0.288 0.047 0.543 G614 7.5X 0.418 0.060 0.805 G614 6.0X 0.600 0.053 1.188 G614 5.0X 0.706 0.040 1.568 G614 4.3X 0.867 0.047 1.644 8-5 0.436 0.052 0.779 &: HTLV-1 pol protein has a molecular weight of approximately 16,000 Daltons. @: Absorbance at 492nm. #: Samples were diluted with recalcified human serum negative for HBV, HCV and HIV.

The data in Table 10 show that when EN-80-2 (i.e. core-envelope fusion protein) of HCV and HTLV-1 pol protein are combined, the 492nm absorbance of anti-HCV positive samples shows a synergistic effect, rather than just an additive effect . Thus, a synergistic relationship between EN-80-2 of HCV and HTLV-1 pol protein could be found.

VI. Sixth Analysis

Table 11 shows that the results of the analysis are similar to the fifth analysis (V), and show that HBV antigens HBsAg and HBcAg have no synergistic relationship with HCV EN-80-1 protein.

HBsAg: Purified from HBsAg positive human serum.

HBcAg: derived from HBV cDNA fragment.

Sample source I: G30 and G49 are GPT abnormal samples, which are anti-HCV antibody positive and collected from Taipei Blood Donation Center.

Sample source II: G612, G613, G614 and G615 were positive for anti-HCV antibody and purchased from the United States.

Table 11 sample EN-80-1 HBsAg HBcAg EN-80-1+HBsAg EN-80-1+HBcAg G30 102X@ 0.088# 0.117 0.162 0.186 0.219 G49 42X 0.063 0.125 0.174 0.146 0.190 G612 804X 0.096 0.111 0.145 0.178 0.187 G613 52X 0.195 0.165 0.137 0.232 0.239 G614 16X 0.059 0.124 0.123 0.111 0.116 G615 16X 0.053 0.107 0.134 0.158 0.232 @: Samples were serially diluted with recalcified human sera negative for HBV, HCV and HIV. #: Absorbance at 492nm.

The data in Table 11 show that there is no synergistic effect on the absorbance at 492 nm of anti-HCV positive samples when HBsAg and HBcAg are coated with EN-80-1 (NS5) protein. No obvious relationship between HBsAg and EN-80-1 protein, or HBcAg and EN-80-1 protein was found.

VII. Seventh Analysis

Table 12 shows the comparison of EverNew anti-HCV EIA and Abbott kit (II) for detecting anti-HCV antibodies. Test samples were obtained from the following sources:

Sample source I: G23, G26, G30, G49, G58, G114, G128, G186, G231, G250 and G262 are GPT abnormal samples, which are anti-HCV antibody positive and collected from Taipei Blood Donation Center.

Sample source II: G612, G613, G614 and G615 were positive for anti-HCV antibody and purchased from the United States.

Sample source III: VGH7, VGH11, VGH12, VGH13, VGH16, VGH26, VGH27, VGH29, VGH30, VGH32, VGH33, VGH40, VGH43, VGH46, and VGH52 were positive for anti-HCV antibodies and collected from Taipei Veterans General Hospital.

Classification of samples from source III:

VGH7 IHD stones

VGH11 NANB, distributed

                                     

VGH13 NANB, PTH

VGH16 HCC

                                   

VGH27 NANB, distributed

VGH29 IDH stones

VGH30 Hepatic schistosomiasis

VGH32 NANB, Distributed

                                       

                                            

                                     

VGH46 HCC-induced liver cirrhosis

VGH52 NANB, Distributed

Control: recalcified human serum (non-reactive with HBV, HCV and HIV), this human serum can be used to dilute the above anti-HCV positive samples.

Kits tested:

EverNew anti-HCV E1A...microwells coated with EN-80-1 antigen

EverNew anti-HCV E1A...microwells coated with EN-80-2 antigen

EverNew anti-HCV E1A...microwells coated with EN-80-1 and EN-80-2 antigens. Refer to the kit Abbott kit (II). result:

Table 12 Sample Dilution Factor EN-80-1 EN-80-2 EN-80-1+ ABB0TT

EN-80-2 recalcified human serum n/a negative negative negative (control) G23 20X@ negative $ positive # positive positive

40X Negative Negative Positive Positive G26 8X Negative Positive Positive Positive

16X Negative Negative Positive Positive G30 51X Negative Negative Positive Positive

102X Negative Negative Positive Positive G32 51X Positive Negative Positive Positive

                                                                     

42X Negative Negative Positive Positive G58 16X Negative Positive Positive Positive

32X Negative Negative Positive Positive G114 10X Negative Positive Positive Positive

20X Negative Negative Negative Positive Positive G128 120X Negative Negative Positive Positive

240X Negative Negative Positive Negative G186 42X Negative Negative Positive Positive

84X Negative Negative Negative Negative Negative G231 336X Negative Negative Positive Positive

672X Negative Negative Negative Positive Negative G250 168X Negative Negative Positive Positive

                                                                                                                                           

                                                                                                             

804X Negative Negative Positive Negative G613 26X Negative Negative Positive Positive

                                                                                                                                         

                                                                                                                                                                         

                                                                                               

84X Negative Negative Positive Positive VGH11 126X Positive Negative Positive Positive

                                                                               

                                                                         

504X Negative Negative Positive Positive VGH16 252X Negative Negative Positive Positive

504X Negative Negative Positive Positive VGH26 84X Negative Negative Positive Positive

                                                                                                                   

84X Negative Negative Positive Negative VGH29 42X Negative Positive Positive

84X Negative Negative Positive Negative VGH30 42X Positive Negative Positive Positive

84X Negative Negative Positive Negative VGH32 504X Negative Negative Negative Positive Negative

        1008X   Negative Negative   Positive   Negative VGH33   84X   Negative  Positive  Positive  Negative

        168X   Negative Negative   Positive   Negative VGH40   9X   Negative Negative Negative Positive Negative

                                                                                     

                                                                       

                                                         

252X Negative Negative Negative Negative Negative Negative@: Serial dilutions of samples with calcified human sera negative for HBV, HCV and HIV. $: Negative...No reaction with anti-HCV antibody. #: Positive...Reactive with anti-HCV antibodies. &: N.D....not done.

Data in bold in Table 12 show an example of synergy between the HCV core antigen-envelope protein and the non-structural (NS5) region. The data in bold also shows an example where the detection provided by the present invention is superior to that of the reference Abbott kit (II) HCV detection kit. These data indicate that wells of microplates coated with EN-80-1 and EN-80-2 antigens are more detectable than wells of microplates coated with EN-80-1 and EN-80-80-2 antigens alone. The hole efficiency of the orifice plate is higher. Moreover, samples G128 240X, G231 672X, G612 804X, VGH27 42X, VGH27 84X, VGH29 84X, VGH30 84X, VGH32 504X, VGH32 1008X, VGH33 84X, VGH40 9X, VGH43 9X and VGH43 18X. EverNew anti-HCV EIA (microwells coated with EN-80-1 and EN-80-2 antigens), but not with Abbott kit (II).

VIII. Eighth Analysis

This assay shows the results of an ELISA performed as described for the first assay above, in which a portion of the core protein was combined with the EN-80-1 (NS5) protein of HCV. The core protein is composed of amino acids 1 to 120, donated by Taiwan Biotechnology Development Center (DCB).

Sample source 1: G2 35 is a GPT abnormal sample, which is anti-HCV antibody negative and collected from Taipei Blood Donation Center.

Sample source II: G614 and G615 were positive for anti-HCV antibody, purchased from the United States.

Table 13 sample EN-80-1 part core EN-80-1+partial core G235 0.002@ 0.082 0.078 G614 0.004 1.142 1.243 G615 0.000 1.332 1.430 @: Absorbance at 492nm.

The data in Table 13 show that there is no synergistic effect on the absorbance at 492 nm of anti-HCV positive samples when part of the core (amino acids 1 to 120) is coated with EN-80-1 protein. No apparent relationship between partial core and NS5 protein of HCV was found.

IX. Ninth Analysis

Table 14 confirms the results given above and shows the partial core (EN-80-5 antigen, which is the HCV partial core with a molecular weight of about 15,000 Daltons as determined by SDS-polyacrylamide gel electrophoresis) Anti-HCV antigen), core antigen-envelope protein (EN-80-2 antigen) and/or HCV nonstructural protein (NS5; EN-80-1 antigen discussed above) by enzyme immunoassay detection of anti-HCV antibodies. The samples analyzed were anti-HCV positive samples N8, N81 , N89, N12 and N302 and anti-HCV negative samples N202, N203 and N302. Positive samples were diluted between 25X and 672X with 0.1M Tris-HCl pH 7.4 (+/-0.2) containing 40% fetal calf serum, 1% BSA and 2% mouse serum. Analysis of the samples was carried out in a solution of monoclonal anti-human IgG:HRPO conjugate in wells of a microplate, plus the following antigen or combination of antigens: a.) NS5; b.) core antigen-envelope protein; c .) part of core protein; d.) NS5 and core antigen-envelope protein e.) NS5 and part of core protein; f.) core antigen-envelope protein and part of core; and g.) NS5, core antigen-envelope Egg whites and part of the core.

The following results are obtained:

Table 14

Sample NS5 Core-Envelope Core NS5+ NS5+ Core+ NS5+

ID Core-Envelope Core Core-Envelope Core+

Core-Package 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 0.781N81 336X 0.018 1.572 1.778 0.0 9 ＞2.0

672X 0.019 0.697 0.633 0 742 0.344 0.912 0.982N89 336X 0.083 ＞2.0 ＞2.0 1 .0 8 ＞2.0 9 ＞2.0 ＞2.0 .0 1 .0 9 ＞2.0 .

672X 0.040 1.301 0.794 1.671 0.589 1.321 1.694N12 25X 0.019 1.848 ＞2.0 .0 .2 7 ＞2.0 ＞2.0 .0 .6 ＞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 0.435N302 168X 0.188 ＞2.0 ＞2.0 .2 0 .2 0 ＞2.0 ＞2

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    1.025N202          0.043      0.081    0.069    0.077      0.048    0.081    0.075N203          0.100      0.208    0.124    0.185      0.117    0.189    0.169N209          0.023      0.033    0.054    0.036      0.037    0.045    0.042样品稀释液#   0.018      0.028    0.018    0.021      0.025    0.028    0.027@：用样品稀释液稀释Anti-HCV positive serum*: Absorbance at 492 nm. #: Sample diluent: 0.1 M Tris-HCl pH: 7.4±0.2, containing 40% fetal bovine serum, 1% BSA and 2% mouse serum.

X. Tenth Analysis

The tenth assay was an enzyme immunoassay using HIV gag protein in combination with HIV env protein to detect the presence of anti-HIV-1 antibodies in human sera.

The antigens used for the assay were as follows: first, an amino-terminal fragment (377 amino acids) containing β-galactosidase fused to gag-17 (amino acids 15-132) followed by gag p24 (amino acids 133-363) and gag p15 ( Amino acid 364-437) recombinant fusion protein. The protein has a molecular weight of 92.8 kDa and 831 amino acids (including spacer amino acids), which is named EN-I-5 antigen. The protein used for the analysis was purified from E. coli with a purity of more than 90% and was non-glycosylated. Second, a recombinant fusion protein containing the amino-terminal fragment (311 amino acids) of β-galactosidase fused to env, ie, amino acids 474-863 of gp160. The protein has a molecular weight of 80.7 kDa and 705 amino acids (including spacer amino acids), which is named EN-I-6 antigen. According to Ratner et al., AIDS Research and Human Retroviruses 3(1):57-69, 1987, the envelope cleavage site in gp160 was found between amino acids 491 and 492. Thus the EN-I-6 antigen includes the carboxyl terminus of gp120 and the amino terminus of gp41. The protein used for the analysis was purified from E. coli with a purity of more than 90% and was non-glycosylated.

Positive samples analyzed were from clinically proven HIV-positive persons, and thus were confirmed anti-HIV-1 positive sera, and were numbered T1, T2, T3, T4, T5, T6, P1, P2 and P3. The control sample number is NC, which is anti-HIV-1 antibody negative serum. Analysis of the samples was carried out in the wells of a microplate with monoclonal anti-human IgG:HRPO conjugate solution, plus the following antigen or combination of antigens: a.) EN-1-5 antigen (1 μg/ml, 0.1 ml b.) EN-I-6 antigen (1 μg/ml, 0.1ml/hole); c.) EN-I-5 and EN-I-6 (each 1 μg/ml, antigen, 0.1ml/hole ).

The following results are obtained:

Table 15 Sample HIV _gag HIV _env HIV _gag+

HIV _env p1 0.043@ 0.942 1.586p2 0.031 0.698 1.142p3 0.019 0.342 0.468T1 24X# 0.007 0.957 1.520T1 72X 0.000 0.440 0.863T2 72X 0.000 0.407 0.644T3 8X 0.000 0.350 0.548T4 648X 0.001 0.3 19 0.488T5 72X 0.000 0.227 0.353T6 72X 0.005 0.560 0.799NC 0.019 0.028 0.030NC 4X 0.012 0.027 0.025@: 492nm Absorbance #: Sample diluted with Sample Diluent

Table 15 surprisingly shows that there is a synergy between the HIV-1 gag and env proteins.

XI. Eleventh Analysis

The eleventh assay was an enzyme immunoassay using the HIV env protein in combination with other secondary proteins to detect the presence of anti-HIV-1 antibodies in human serum.

The antigens used for the assay were HIV env protein (EN-1-6 antigen above), HCV NS5 protein (EN-80-1 antigen above), and HCV class core antigen-adjacent protein (EN-80-2 antigen above). The positive samples analyzed were anti-HIV-1 antibody positive sera T1, T2, T3, T4, T5 and T6; the negative controls were anti-HCV and anti-HIV-1 antibody negative sera N639, N626, N634, N6 32 and N637 .

Analysis of the samples was performed with a monoclonal anti-human IgG:HRPO conjugate solution in wells of a microplate, using the antigen or combination of antigens shown in Table 16 below. The results of the analysis are also shown in Table 16.

Table 16

HCV NS5& HCV核心_env样品： HCV NS5 HIV _env HIV _env &HIV _env HCV核心_env T1 24X@ 0.048 ^* 0.833 0.602 0.930 0.03872X 0.057 0.599 0.460 0.679 0.048216X 0.055 0.278 0.213 0.314 0.039N639 0.077 0.092 0.097 0.110 0.069T2 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 Sample 0.069 0.052 0.029 0.040 0.047 Dilute Liquid T3 4X 0.029 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 0.024N626 0.065 0.073 0.101 0.094T4 72X 0.031 1.4293 1.6666 0.08444

216X 0.051 1.065 1.008 1.259 0.109

648x 0.03 5 0.724 0.641 0.233 0.099N634 0.076 0.059 0.108 0.155T5 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 0.017N632 0.036 0.053 0.041 0.051T6 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 0.026n637 0.051 0.050 0.042 0.052 0.072@: Diluted with sample dilution (0.1 m Tris-HCL PH: 7.4 ± 0.2, containing 40 % fetal beef serum, 1 % BSA and 2 % mice). HIV-1 positive sample*: Absorbance at 492nm.

These results suggest that the HIVenv protein can synergize with a second protein, similar to the synergy shown above for the HCV core-env protein. 14. Preparation of anti-HCV antibody

Antibodies against raw core antigen-envelope protein and NS5 nonstructural protein were prepared according to standard methods for preparing monoclonal antibodies. Specifically, BALB/c mice were immunized with the purified protein described in Examples 2 and 10 above mixed with an adjuvant; then splenocytes and murine lymphocytes (F0 cell line) were fused with polyethylene glycol to form heterozygous cells. The target clone producing the target monoclonal antibody is obtained by screening the titer of the antibody produced by the prepared hybridoma clone. In a specific embodiment of the invention, the hybridoma clone is designated EN-80-1-99. Application of HCV-like core antigen-adjacent protein to induce immune response 15. Administration method of HCV-like core antigen-adjacent protein

Core antigen-envelope protein (EN-80-2) was injected intramuscularly into 6-8 week old ICR mice. The first administration method, booster and sampling arrangements are as follows: Negative control group: (ID no.0-1 and 0-2) Day 0: No immunity. Day 13: First blood collection. 28 days: the second blood collection. Test group 1: (ID no.1-1, 1-2, 1-3, 1-4, 1-5 and 1-6) Day 0: 50 μg/mouse EN-80-2 protein, using complete Freund's auxiliary agent (CFA) (Gaithersburg, USA, 20877). Day 13: First blood collection. 28 days: the second blood collection. 39 days: the third blood collection. Test group 2: (ID no. 2-1, 2-2, 2-3, 2-4, 2-5 and 2-6) Day 0: 50 μg/mouse EN-80-2 protein, using complete Freund's auxiliary (CFA), GIBCO 13 days: the first boost, 80 μg/mouse EN-80-2 protein, using incomplete Freund's adjuvant (IFA), GIBCO (Gaithersburg, USA, 20877). 28 days: the first blood collection. Day 39: Second blood collection. Test group 3: (ID no.3-1, 3-2, 3-3, 3-4, 3-5 and 3-6) Day 0: 50 μg/mouse EN-80-2 protein, using complete Freund's auxiliary agent (CFA), GIBCO. Day 13: First boost, 80 μg/mouse EN-80-2 protein, with Incomplete Freund's Adjuvant (IFA), GIBCO. Day 28: Second boost, 80 μg/mouse of EN-80-2 protein in PBS. Day 39: First blood collection. 16. Detection of immune response induced by core antigen-envelope protein treatment

Two enzyme immunoassays (EIA) similar to those described above were used to determine the presence or absence of an immune response in the experimental animals. In the first EIA, the rat anti-mouse:HRPO conjugate was added to microarrays that had been coated with core antigen-envelope protein (EN-80-2) and mouse anti-mouse:HRPO conjugate. in the wells of the orifice plate. The results of the first E1A are shown in Table 17 below.

Form 17 Sample ID Day 13 Day 28 Day 39

Negative control: 0-1 50X@ 0.141# 0.160 N.D.S

500X 0.058 0.060 N.D.

2500X 0.008 0.025 N.D.

    12500X 0.000 0.010 N.D.

62500X 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 ^* ND ND

2500X 0.018 N.D. N.D.

12500X 0.000 N.D. N.D.

62500X 0.000 ND ND1-2 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 0.0271-3 50X 0.213 ^* >2.0 ND

500X 0.042 0.424 ^* ND

2500X 0.000 0.058 N.D.

12500X 0.000 0.000 N.D.

62500X 0.000 0.000 ND1-4 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 0.0391-5 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 0.0211-6 50X 0.443 0.341 N.D.

500X 0.161 ^* 0.191 ^* ND

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 0.0222-2 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 0.0612-3 50X 0.544 N.D.

500X 0.121 ^* ND

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 0.0512-5 50X 1.860 ＞2.0

500X 0.379 ^* 0.836

2500X 0.071 0.155 ^*

12500X 0.018 0.030

62500X 0.010 0.0192-6 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 3:3-1 50X ＞2.0

500X ＞2.0

2500X ＞2.0

12500X 1.647

62500X 0.362 ^* 3-2 50X ＞2.0

500X ＞2.0

2500X 1.032

12500X 0.195 ^*

62500X 0.0533-3 50X ＞2.0

500X 1.814

2500X 0.312 ^*

12500X 0.060

62500X 0.0263-4 50X ＞2.0

500X ＞2.0

2500X 0.895

12500X 0.181 ^*

62500X 0.0483-5 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 ^* @: Rat serum diluted with 1% BSA 50×, 500×, 2500×, 12500× and 62500× #: Absorbance at 492nm *: Endpoint of detection capability $: ND not analyzed because no serum can be detected

In a second EIA, rat anti-mouse:HRPO conjugates were added to wells of microplates that had been coated with the following antigen or combination of antigens: a.) NS5 (EN-80-1 antigen); b.) core antigen-envelope protein (EN-80-2 antigen); c.) part of core protein (EN-80-5 antigen); d.) NS5 and core antigen-envelope protein; e.) NS5 and Part of core protein; f.) core antigen-envelope and part of core; and g.) NS5, core antigen-envelope and part of core. The samples used in the second analysis were as follows: 0-2 (50× dilution, taken on day 28); 0-2 (500× dilution, taken on day 28); 2-2 (2500× dilution, taken on day 28); from day 28); 3-1 (12500× dilution, taken from day 39); 3-4 (2500× dilution, taken from day 39); 3-5 (2500× dilution, taken from day 39) ; 3-6 (2500X dilution, taken from day 39); and 3-6 (12500X dilution, taken from day 39).

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

Table 18 Sample NS5 Core-Envelope Core NS5+ NS5+ Core + NS5+ID Core-Envelope Core

Core-Encades-negative control: 0-2 50X 0.018@0.024 0.025 0.026 0.020 0.027 0.0290-2 500x 0.010 0.011 0.014 0.014 0.022 0.019 group II: 2-2

0.004 0.398 0.007 0.489 0.009 0.313 0.3882500X Group III: 3-1

0.002 0.506 0.009 0.760 0.009 0.513 0.47212500x3-4 0.003 0.007 0.344 0.006 0.192 0.2272500x3-55

0.003 0.705 0.007 1.168 0.006 0.592 0.7472500x3-6 0.005 0.005 1.012 0.008 0.542 0.7042500x3-6 0.008 0.008 0.009 0.13412500x

@: Absorbance at 492nm.

Claims (66)

1. positive chain RNA virus deutero-composition, wherein contain:

A) a kind of polypeptide of undressed form of the isolating aminoterminal positive chain RNA virus class core antigen protein that contains the adjacent nucleic acid district that is connected to positive chain RNA virus, the size of the N-terminal part in wherein said adjacent nucleic acid district makes described polypeptide have the epi-position configuration special to the undressed class core-adjacent nucleic acid district of described positive chain RNA virus; With

B) a kind of isolating Nonstructural Protein of described positive chain RNA virus.

2. the composition of claim 2, wherein said positive chain RNA virus is selected from the group that Alphaherpesvirinae, coronaviridae, Retroviridae, Picornaviridae, Caliciviridae and flaviviridae constitute.

3. the composition of claim 2, wherein said positive chain RNA virus are selected from the group that human immunodeficiency virus (HIV) and human T-cell leukemia virus (HTLV) are constituted.

4. the composition of claim 1, wherein said isolated polypeptide is produced by suitable prokaryotic host cell.

5. the composition of claim 1, wherein said isolated polypeptide is produced by the eukaryotic host cell that can not process described isolated polypeptide.

6. preparation contains the method for compositions of the multiple polypeptide that obtains from positive chain RNA virus, comprising the following step:

A) first expression vector of nucleic acid molecule of polypeptide that will express the undressed form of the isolating aminoterminal positive chain RNA virus class core antigen protein that contains the adjacent nucleic acid district that is connected to positive chain RNA virus of coding imports in first host cell, the size of the N-terminal part in wherein said adjacent nucleic acid district makes described polypeptide have the epi-position configuration special to the undressed class core-adjacent nucleic acid district of described positive chain RNA virus

B) be suitable for hatching described first host cell under the condition that described expression vector produces described polypeptide,

C) the described polypeptide of purifying to be producing purified polypeptide, and

D) second expression vector of nucleic acid molecule that will express the isolating Nonstructural Protein of the described positive chain RNA virus of coding imports in second appropriate host cell,

E) be suitable for hatching described second host cell under the condition that described nucleic acid molecule produces described Nonstructural Protein,

F) the described Nonstructural Protein of purifying is to produce the Nonstructural Protein of purifying, then

G) polypeptide of described purifying and the Nonstructural Protein of described purifying are made up to form described composition.

7. preparation contains the method for compositions of the multiple polypeptide that obtains from positive chain RNA virus, comprising the following step:

A) expression vector of first kind of nucleic acid molecules of polypeptide that will express the undressed form of the aminoterminal positive chain RNA virus class core antigen protein that contains the adjacent nucleic acid district that is connected to positive chain RNA virus that coding separates imports in the host cell; The size of the amino terminal in wherein said adjacent nucleic acid district part is so that described polypeptide has undressed class core to described positive chain RNA virus-special epi-position configuration in adjacent nucleic acid district; Described expression vector also can be expressed the second albumen that contains from the non-structural protein of described positive chain RNA virus

B) be suitable for hatching under the condition that described expression vector produces described polypeptide and described Nonstructural Protein described host cell and

C) described polypeptide of purifying and described Nonstructural Protein are with the polypeptide of generation purifying and the Nonstructural Protein of purifying.

8. claim 6 or 7 method, wherein said positive chain RNA virus is selected from the group that Alphaherpesvirinae, coronaviridae, Retroviridae, Picornaviridae, Caliciviridae and flaviviridae constitute.

9. contain the composition that is combined on the solid-phase matrix from isolating, the complete substantially undressed polyprotein of positive chain RNA virus.

10. contain a kind of isolating composition undressed form and that be combined in the polypeptide on the solid-phase matrix that contains the aminoterminal positive chain RNA virus class core antigen protein in the adjacent nucleic acid district that is connected to positive chain RNA virus, the size of the N-terminal part in wherein said adjacent nucleic acid district makes described polypeptide have the epi-position configuration special to the undressed class core-adjacent nucleic acid district of described positive chain RNA virus.

11. the composition of claim 10, it also contains the Nonstructural Protein that is combined in the described positive chain RNA virus on the described solid-phase matrix.

12. the analytical procedure of positive chain RNA virus in the test sample, comprising:

A) provide a kind of polypeptide of undressed form of the isolating aminoterminal positive chain RNA virus class core antigen protein that contains the adjacent nucleic acid district that is connected to positive chain RNA virus, the size of the N-terminal part in wherein said adjacent nucleic acid district makes described polypeptide have the epi-position configuration special to the undressed class core-adjacent nucleic acid district of described positive chain RNA virus

B) with described isolated polypeptide under suitable condition with time of described sample full contact, guarantee the special antibodies of one or more described positive chain RNA virus in described polypeptide and the described sample, with produce the antibodies polypeptide and

C) detect described antibodies polypeptide, determine in view of the above to contain positive chain RNA virus in the described sample.

13. the analytical procedure of positive chain RNA virus in the test sample, comprising:

A) provide a kind of isolating polypeptide that contains isolating, the complete substantially undressed polyprotein of positive chain RNA virus,

B) with described isolated polypeptide under suitable condition with time of described sample full contact, guarantee the special antibodies of one or more described positive chain RNA virus in described polypeptide and the described sample, with produce the antibodies polypeptide and

C) detect described antibodies polypeptide, determine in view of the above to contain positive chain RNA virus in the described sample.

14. the analytical procedure of claim 12 or 13 wherein also comprises:

A) in step a), a kind of Nonstructural Protein that is attached to the described positive chain RNA virus on the described solid-phase matrix is provided,

B) in step b), with described Nonstructural Protein under suitable condition with time of described sample full contact, guarantee the special antibodies of one or more described positive chain RNA virus in described Nonstructural Protein and the described sample, with the positive chain RNA virus Nonstructural Protein that produces antibodies and

C) in step c), detect described antibodies polypeptide, or described antibodies Nonstructural Protein one or both of, determine in view of the above to contain positive chain RNA virus in the described sample.

15. the analytical procedure of claim 14, it also comprises described polypeptide, described Nonstructural Protein or described polyprotein is combined in step on the solid-phase matrix.

16. claim 12,13 or 14 analytical procedure, wherein said sample is unpurified sample.

17. claim 12,13 or 14 analytical procedure, it also is included in the step that obtains described sample before the described contact from animal.

18. the analytical procedure of claim 17, wherein said animal is the people.

19. claim 12,13 or 14 analytical procedure, wherein said analytical procedure are selected from counterimmunoelectrophoresis (CIEP) analysis, radioimmunoassay, radioimmunoprecipitation, enzyme linked immunosorbent assay (ELISA), Dot blot analysis, inhibition or competition analysis, sandwich assay, the group that analysis (dip-stick), analysis simultaneously, immunochromatographiassays assays, immunofiltration assay, latex bead aggegation analysis, immunofluorescence analysis, biosensor analysis and low light check and analysis are formed is sticked in immunity.

20. claim 12,13 or 14 analytical procedure, wherein said analytical procedure is not a western blot analysis.

21. prepare the method for antibody, comprising the following step:

A) use the polypeptide of the undressed form of the isolating aminoterminal positive chain RNA virus class core antigen protein that contains the adjacent nucleic acid district that is connected to positive chain RNA virus to animal, the size of the N-terminal in wherein said adjacent nucleic acid district part make described polypeptide have to the special epi-position configuration in the undressed class core-adjacent nucleic acid district of described positive chain RNA virus and

B) separation is at the described antibody of described polypeptide.

22. antibody according to claim 21 preparation.

23. prepare the method for antibody, comprising the following step:

A) use the isolating polypeptide that contains from isolating, the complete substantially undressed polyprotein of positive chain RNA virus to animal,

B) separation is at the described antibody of described polyprotein.

24. antibody according to claim 23 preparation.

25. the antibody of claim 22 or 24, wherein said antibodies is on solid-phase matrix.

26. the analytical procedure of positive chain RNA virus in the test sample, comprising:

A) with described sample under suitable condition with time of the antibody full contact of claim 22, guarantee the described undressed positive chain RNA virus class core antigen protein of described antibodies, with produce binding antibody and

B) detect described binding antibody, determine in view of the above to contain positive chain RNA virus in the described sample.

27. the analytical procedure of claim 26 wherein also comprises,

A) in step a), with described sample under suitable condition with another time to the special antibody full contact of positive chain RNA virus Nonstructural Protein, guarantee the described positive chain RNA virus Nonstructural Protein of described another antibodies, with produce another antibody of bonded and

B) in step b), detect described binding antibody or described another antibody of bonded one or both of, determine in view of the above to contain positive chain RNA virus in the described sample.

28. the analytical procedure of positive chain RNA virus in the test sample, comprising:

A) with described sample under suitable condition with time of the antibody full contact of claim 24, guarantee the described positive chain RNA virus specific antigens of described antibodies, with produce binding antibody and

B) detect described binding antibody, determine in view of the above to contain positive chain RNA virus in the described sample.

29. composition that can stimulate animal immune to reply, it contains a kind of polypeptide of undressed form of the isolating aminoterminal positive chain RNA virus class core antigen protein that contains the adjacent nucleic acid district that is connected to positive chain RNA virus, the size of the N-terminal part in wherein said adjacent nucleic acid district makes described polypeptide have the epi-position configuration special to the undressed class core-adjacent nucleic acid district of described positive chain RNA virus, and carrier or the thinner combination suitable with pharmacology.

30. the composition of claim 29 wherein also contains the Nonstructural Protein from described positive chain RNA virus.

31. the composition that can stimulate animal immune to reply, it contains isolating, the complete substantially undressed polyprotein from positive chain RNA virus, and carrier or the thinner combination suitable with pharmacology.

32. claim 29,30 or 31 composition, wherein said animal is the people.

33. the vaccine of an anti-positive chain RNA virus, it contains a kind of polypeptide of undressed form of the isolating aminoterminal positive chain RNA virus class core antigen protein that contains the adjacent nucleic acid district that is connected to positive chain RNA virus, a certain size of the N-terminal part in wherein said adjacent nucleic acid district makes described polypeptide have the epi-position configuration special to the undressed class core-adjacent nucleic acid district of described positive chain RNA virus, and carrier or the thinner combination suitable with pharmacology.

34. the vaccine of an anti-positive chain RNA virus, it contains isolating, the complete substantially undressed polyprotein from positive chain RNA virus, and carrier or the thinner combination suitable with pharmacology.

35. the vaccine of claim 33 or 34 wherein also contains the Nonstructural Protein from described positive chain RNA virus.

36. detect the test kit of positive chain RNA virus, comprising:

A) a kind of undressed form of the isolating aminoterminal positive chain RNA virus class core antigen protein that contains the adjacent nucleic acid district that is connected to positive chain RNA virus and be combined in polypeptide on the solid-phase matrix, the size of the N-terminal in wherein said adjacent nucleic acid district part make described polypeptide have to the special epi-position configuration in the undressed class core-adjacent nucleic acid district of described positive chain RNA virus and

B) be used to detect the reagent of described isolated polypeptide or device one or both of.

37. the test kit of claim 36, it also comprises from the Nonstructural Protein of described positive chain RNA virus and is used to detect the reagent of described Nonstructural Protein or device one or both of.

38. detect the test kit of positive chain RNA virus, comprising:

A) be combined on the solid-phase matrix from isolating, the complete substantially undressed polyprotein of positive chain RNA virus and

B) be used to detect the reagent of described isolating polyprotein or device one or both of.

39. detect the test kit of positive chain RNA virus, comprising:

A) antibody of claim 22 and

B) be used to detect the reagent of described antibody or device one or both of.

40. the test kit of claim 39, it also comprises special another antibody of HCV Nonstructural Protein and is used to detect the reagent of described another antibody or device one or both of.

41. detect the test kit of positive chain RNA virus, comprising:

A) antibody of claim 24 and

B) be used to detect the reagent of described antibody or device one or both of.

42. positive chain RNA virus deutero-composition wherein contains:

A) a kind of polypeptide of undressed form of the isolating positive chain RNA virus class core antigen protein that contains the adjacent nucleic acid district that is connected to described positive chain RNA virus, the size in wherein said adjacent nucleic acid district makes described polypeptide have the undressed nuclear class heart-special epi-position configuration in adjacent nucleic acid district to described positive chain RNA virus; With

B) can with the described positive chain RNA virus class core antigen protein synergy in the described described adjacent nucleic acid district that is connected to described positive chain RNA virus, be connected to antigenic second kind of albumen of described positive chain RNA virus class core antigen protein in the described adjacent nucleic acid district of described positive chain RNA virus with raising.

43. preparation contains the method for compositions of multiple polypeptides, comprising the following step:

A) first expression vector of nucleic acid molecule of polypeptide that will express the undressed form of a kind of isolating positive chain RNA virus class core antigen protein that contains the adjacent nucleic acid district that is connected to described positive chain RNA virus of coding imports in first host cell, the size in wherein said adjacent nucleic acid district makes described polypeptide have the undressed nuclear class heart-special epi-position configuration in adjacent nucleic acid district to described positive chain RNA virus

B) be suitable for hatching described first host cell under the condition that described expression vector produces described polypeptide,

C) the described polypeptide of purifying to be producing the polypeptide of purifying, and

D) will express coding can act synergistically with the described described positive chain RNA virus class core antigen protein that is connected to the described adjacent nucleic acid district of described positive chain RNA virus, second expression vector of antigenic second kind of proteic nucleic acid molecule of described positive chain RNA virus class core antigen protein that is connected to the described adjacent nucleic acid district of described positive chain RNA virus with raising imports in second appropriate host cell

E) produce and hatch described second host cell under described second kind of proteic condition being suitable for described nucleic acid molecule,

F) the described second kind of albumen of purifying is to produce second kind of albumen of purifying, then

G) polypeptide of described purifying and second kind of protein combination of described purifying are formed described composition.

44. preparation contains method for compositions multiple but wherein at least a polypeptide from positive chain RNA virus, comprising the following step:

A) expression vector of first kind of nucleic acid molecule of polypeptide that will express the undressed form of a kind of isolating positive chain RNA virus class core antigen protein that contains the adjacent nucleic acid district that is connected to described positive chain RNA virus of coding imports in the host cell, the size in wherein said adjacent nucleic acid district makes described polypeptide have the epi-position configuration special to the undressed class core-adjacent nucleic acid district of described positive chain RNA virus, described expression vector also can be expressed and can be acted synergistically with the described described positive chain RNA virus class core antigen protein that is connected to the described adjacent nucleic acid district of described positive chain RNA virus, be connected to antigenic second kind of albumen of described positive chain RNA virus class core antigen protein in the described adjacent nucleic acid district of described positive chain RNA virus with raising

B) be suitable for described expression vector produce hatch under described polypeptide and the described second kind of proteic condition described host cell and

C) the described polypeptide of purifying and described second kind of albumen contain the polypeptide of purifying and second kind of proteic composition of purifying with generation.

45. the method for claim 43 or 44, wherein said second kind of albumen is from positive chain RNA virus.

46. contain a kind of isolating composition undressed form and that be combined in the polypeptide on the solid-phase matrix that contains the positive chain RNA virus class core antigen protein in the adjacent nucleic acid district that is connected to described positive chain RNA virus, the size in wherein said adjacent nucleic acid district makes described polypeptide have the epi-position configuration special to the undressed class core-adjacent nucleic acid district of described positive chain RNA virus.

47. the composition of claim 46, wherein also contain can with the described positive chain RNA virus class core antigen protein synergy in the described described adjacent nucleic acid district that is connected to described positive chain RNA virus, be connected to raising described positive chain RNA virus described adjacent nucleic acid district described positive chain RNA virus class core antigen protein antigenic and be combined in second kind of albumen on the described solid-phase matrix.

48. the analytical procedure of positive chain RNA virus in the test sample, comprising:

A) provide a kind of polypeptide of undressed form of the isolating positive chain RNA virus class core antigen protein that contains the adjacent nucleic acid district that is connected to described positive chain RNA virus, the size in wherein said adjacent nucleic acid district makes described polypeptide have the epi-position configuration special to the undressed class core-adjacent nucleic acid district of described positive chain RNA virus

B) with described isolated polypeptide under suitable condition with time of described sample full contact, guarantee the special antibodies of one or more described positive chain RNA virus in described polypeptide and the described sample, with produce the antibodies polypeptide and

C) detect described antibodies polypeptide, determine in view of the above to contain positive chain RNA virus in the described sample.

49. the analytical procedure of claim 48, it also comprises:

A) in step a), a kind of can act synergistically with the described described positive chain RNA virus class core antigen protein that is connected to the described adjacent nucleic acid district of described positive chain RNA virus on the described solid-phase matrix that be attached to is provided, be connected to antigenic second kind of albumen of described positive chain RNA virus class core antigen protein in the described adjacent nucleic acid district of described positive chain RNA virus with raising

B) in step b), with described second kind of albumen under suitable condition with time of described sample full contact, guarantee that described second kind of albumen and the described positive chain RNA virus class core antigen protein that is connected to the described adjacent nucleic acid district of described positive chain RNA virus can act synergistically and

C) in step c), detect binding antibody, determine in view of the above to contain positive chain RNA virus in the described sample.

50. the analytical procedure of claim 49, it also comprises the step on solid-phase matrix with described isolated polypeptide or described second kind of protein binding.

51. prepare the method for antibody, comprising the following step:

A) use a kind of polypeptide of undressed form of the isolating positive chain RNA virus class core antigen protein that contains the adjacent nucleic acid district that is connected to positive chain RNA virus to animal, the size in wherein said adjacent nucleic acid district makes described polypeptide have the epi-position configuration special to the undressed class core-adjacent nucleic acid district of described positive chain RNA virus

B) separation is at the described antibody of described polypeptide.

52. the method for claim 51, its also comprise to described animal uses can with the described positive chain RNA virus class core antigen protein synergy in the described described adjacent nucleic acid district that is connected to described positive chain RNA virus, be connected to antigenic second kind of albumen of described positive chain RNA virus class core antigen protein in the described adjacent nucleic acid district of described positive chain RNA virus with raising.

53. antibody according to claim 51 or 52 preparations.

54. according to the antibody of claim 51 or 52 preparations, wherein said antibodies is on solid-phase matrix.

55. the analytical procedure of positive chain RNA virus in the test sample, comprising:

A) with described sample under suitable condition with time of antibody full contacts according to claim 51 or 52 preparations, guarantee the described undressed positive chain RNA virus class core antigen protein of described antibodies, with produce binding antibody and

B) detect described binding antibody, determine in view of the above to contain positive chain RNA virus in the described sample.

56. composition that can stimulate animal immune to reply, it contains a kind of polypeptide of undressed form of the isolating positive chain RNA virus class core antigen protein that contains the adjacent nucleic acid district that is connected to described positive chain RNA virus, the size in wherein said adjacent nucleic acid district makes described polypeptide have the epi-position configuration special to the undressed class core-adjacent nucleic acid district of described positive chain RNA virus, and carrier or the thinner combination suitable with pharmacology.

57. the composition of claim 56, its also contain can with the described positive chain RNA virus class core antigen protein synergy in the described described adjacent nucleic acid district that is connected to described positive chain RNA virus, be connected to antigenic second kind of albumen of described positive chain RNA virus class core antigen protein in the described adjacent nucleic acid district of described positive chain RNA virus with raising.

58. the vaccine of an anti-positive chain RNA virus, it contains a kind of polypeptide of undressed form of the isolating positive chain RNA virus class core antigen protein that contains the adjacent nucleic acid district that is connected to described positive chain RNA virus, the size in wherein said adjacent nucleic acid district makes described polypeptide have the epi-position configuration special to the undressed class core-adjacent nucleic acid district of described positive chain RNA virus, and carrier or the thinner combination suitable with pharmacology.

59. the vaccine of claim 58, its also contain can with the described positive chain RNA virus class core antigen protein synergy in the described described adjacent nucleic acid district that is connected to described positive chain RNA virus, be connected to antigenic second kind of albumen of described positive chain RNA virus class core antigen protein in the described adjacent nucleic acid district of described positive chain RNA virus with raising.

60. as each composition among claim 1-5, the 9-11 of active treatment material, the 29-32,42,46,47,56 or 57.

61. as each vaccine among the claim 33-35,58 or 59 of active treatment material.

62. be used for suppressing, prevent or treat claim 1-5,9-11,29-32,42,46,47,56 or 57 each the compositions of the drug manufacture that animal HCV infects.

63. be used for suppressing, prevent or treat claim 33-35, the 58 or 59 arbitrary vaccines of the drug manufacture that animal HCV infects.

64. detect the test kit of positive chain RNA virus, comprising:

A) a kind of undressed form of the isolating positive chain RNA virus class core antigen protein that contains the adjacent nucleic acid district that is connected to described positive chain RNA virus and be combined in polypeptide on the solid-phase matrix, the size in wherein said adjacent nucleic acid district make described polypeptide have to the special epi-position configuration in the undressed class core-adjacent nucleic acid district of described positive chain RNA virus and

B) be used to detect the reagent of described isolated polypeptide and install one or both of.

65. the test kit of claim 64, it also comprises and can act synergistically with the described described positive chain RNA virus class core antigen protein that is connected to the described adjacent nucleic acid district of described positive chain RNA virus, be connected to raising described positive chain RNA virus described adjacent nucleic acid district described positive chain RNA virus class core antigen protein antigenic second kind of albumen and be used to detect described second kind of proteic reagent and the device one of or both.

66. detect the test kit of positive chain RNA virus, comprising:

A) according to claim 51 or 52 the preparation antibody and

B) be used to detect the reagent of described antibody and install one or both of.

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