WO1996037606A1 - Procedes et compositions de diagnostic d'infections par le virus de l'hepatite c et de vaccination contre le virus de l'hepatite c - Google Patents
Procedes et compositions de diagnostic d'infections par le virus de l'hepatite c et de vaccination contre le virus de l'hepatite c Download PDFInfo
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- WO1996037606A1 WO1996037606A1 PCT/US1996/007378 US9607378W WO9637606A1 WO 1996037606 A1 WO1996037606 A1 WO 1996037606A1 US 9607378 W US9607378 W US 9607378W WO 9637606 A1 WO9637606 A1 WO 9637606A1
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
- C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
- C07K16/1081—Togaviridae, e.g. flavivirus, rubella virus, hog cholera virus
- C07K16/109—Hepatitis C virus; Hepatitis G virus
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
- C12N2770/00011—Details
- C12N2770/24011—Flaviviridae
- C12N2770/24211—Hepacivirus, e.g. hepatitis C virus, hepatitis G virus
- C12N2770/24222—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
Definitions
- the present invention relates generally to methods and compositions for the highly specific, highly sensitive diagnosis of Hepatitis C virus (HCV).
- HCV Hepatitis C virus
- the methods and compositions are also suitable for the elicitation of an immune response in an animal, and for the vaccination of an animal, against HCV.
- hepatitis arising from blood transfusion are induced virally, and are distinguishable from other forms of viral-associated liver diseases caused by known hepatitis viruses such as hepatitis A virus (HAV) and hepatitis B virus (HBV).
- HAV hepatitis A virus
- HBV hepatitis B virus
- the etiological agent(s) of such Non-A, Non-B hepatitis (NANBH) has long been sought by many research groups and is presently believed to be the hepatitis C virus (HCV).
- Post-transfusion hepatitis (PTH) occurs in approximately 10% of transfusion patients, and NANBH accounts for up to 90% of these cases.
- a major problem arising from this disease is the frequent progression to chronic liver damage (25-55%). Therefore, the demand for sensitive, specific methods for detecting HCV in contaminated blood or blood products is significant.
- HCV hepatitis C virus
- C nucleocapsid
- El glycosylated envelope proteins
- NS1-5 nonstructural proteins
- EP 0 318 216 Al discloses a synthesized polypeptide, C 100-3, which contains 363 virally-encoded amino acids that can be used for the detection of one type of HCV antibody.
- a specific assay using such HCV antigen designated C 100-3 has been created, using recombinant DNA methods in yeast (Science 244:362-364).
- kits for detecting HCV antibodies on the basis of the C 100-3 antigen have been commercialized by Abbott Laboratories. It has been confirmed that C 100-3 is a protein encoded by part of the nonstructural regions 3-4 of the HCV genome.
- anti-C 100-3 antibody is not detected in all post- transfusion NANBH cases. The failure to detect the anti-C 100-3 antibody is possibly due to hypermutation of the nucleotide sequence in the C 100-3 antigen.
- an enzyme- linked immunosorbent assay has been developed for serological diagnosis of hepatitis C virus (HCV) infection using the HCV core protein (p22).
- the core protein was synthesized by a recombinant baculovirus, as reported in Chiba et al. (Proc. Natl. Acad Sci. USA 55:4641-4645, 1991).
- the assay of Chiba, et al. used a nonglycosylated 22-kDa nucleocapsid (core) protein, in an effort to establish an antibody-based, specific, sensitive method for diagnosing HCV infection.
- this core protein-based assay still failed to detect a significant number of cases of HCV infection, even when relatively large sample volumes were available.
- compositions and methods capable of highly specific, highly sensitive detection of HCV there has gone unmet a need for compositions and methods capable of eliciting an immune response to HCV, especially an immunoprotective immune response to HCV.
- the present invention provides these and other related advantages.
- the present invention is directed to the discovery that there are significant advantages in antigenicity and epitopic configuration in unprocessed polypeptides derived from HCV. These advantages are particularly useful for the detection and diagnosis of HCV, and also provide significantly enhanced compositions and methods for the induction of immune responses in an animal, and are expected to provide significantly enhanced vaccination of such an animal.
- the present invention features an isolated polypeptide comprising an HCV core antigen protein joined to an amino-terminal portion of an HCV envelope region in unprocessed form, wherein the amino-terminal portion of the HCV envelope region is sized such that the polypeptide has an epitopic configuration specific to an unprocessed core-envelope region of the HCV (this polypeptide is also referred to herein as an "unprocessed core antigen-envelope protein").
- This polypeptide is also referred to herein as an "unprocessed core antigen-envelope protein").
- the present invention provides the first discovery that the epitopic configurations occur specifically in the unprocessed core-envelope region, and surprisingly finds that these epitopic configurations can be found in an isolated protein.
- the -present invention provides an HCV-derived composition
- an HCV-derived composition comprising, a) an isolated polypeptide comprising an HCV core antigen protein joined to an amino-terminal portion of an HCV envelope region in unprocessed form, wherein the amino-terminal portion of the HCV envelope region is sized such that the polypeptide has an epitopic configuration specific to an unprocessed core-envelope region of the HCV; and b) an isolated HCV nonstructural protein.
- the nucleic acid molecule is derived from a flavivirus or flavi-like virus other than HCV (other aspects of the invention disclosed herein are also suitable for use with such flavivirus or flavi-like virus).
- the unprocessed core antigen-envelope protein is encoded by a nucleic acid molecule as set forth in SEQ ID No. 7.
- the isolated HCV nonstructural protein comprises an NS5 nonstructural protein or an NS3-NS4 unprocessed nonstructural protein.
- the NS5 nonstructural protein is encoded by a nucleic acid molecule as set forth in SEQ ID No. 9, and the NS3-NS4 unprocessed nonstructural protein is encoded by a nucleic acid molecule as described below.
- the unprocessed core antigen-envelope protein and/or nonstructural protein is produced by a suitable prokaryotic host cell, preferably a bacterium, and further preferably an E. coli BL21 (DE3).
- a suitable prokaryotic host cell preferably a bacterium, and further preferably an E. coli BL21 (DE3).
- the unprocessed core antigen-envelope protein and/or nonstructural protein is produced by a suitable eukaryotic host cell that is unable to process the isolated polypeptide.
- the present invention provides a method of making a composition comprising multiple polypeptides obtained from an HCV, comprising the following steps: a) introducing into a first host cell a first expression vector capable of expressing a nucleic acid molecule encoding an isolated polypeptide comprising an unprocessed HCV core antigen protein and an amino-terminal portion of an HCV envelope region, wherein the amino-terminal portion of the HCV envelope region is sized such that the polypeptide has an epitopic configuration specific to an unprocessed core-envelope region of the HCV, b) incubating the first host cell under conditions suitable for the expression vector to produce the polypeptide, c) isolating the fusion polypeptide to provide an isolated fusion polypeptide, and also d) introducing into a second host cell a second expression vector capable of expressing a nucleic acid molecule encoding an isolated HCV nonstructural protein, e)incubating the second host cell under conditions suitable for the nucleic acid
- the present invention provides a method of making a composition comprising multiple polypeptides obtained from an HCV, comprising the following steps: a) introducing into a suitable host cell an expression vector capable of expressing a first nucleic acid molecule encoding an isolated polypeptide comprising an HCV core antigen protein joined to an amino-terminal portion of an HCV envelope region in unprocessed form, wherein the amino-terminal portion of the HCV envelope region is sized such that the polypeptide has an epitopic configuration specific to an unprocessed core-envelope region of the HCV, the expression vector further capable of expressing a second nucleic acid molecule encoding an HCV nonstructural protein, b) incubating the first host cell under conditions suitable for the expression vector to produce the polypeptide, and c) isolating the polypeptide and the HCV nonstructural protein.
- the unprocessed core antigen-envelope protein and/or the nonstructural protein are bound to a solid substrate. Further preferably, the protein(s) are covalently bound to the solid substrate.
- the present invention provides an assay for the detection of HCV in a sample, comprising: a) providing an isolated polypeptide comprising an unprocessed HCV core antigen protein and an amino-terminal portion of an HCV envelope region, wherein the amino-terminal portion of the HCV envelope region is sized such that the polypeptide has an epitopic configuration specific to an unprocessed core-envelope region of the HCV, b) contacting the isolated polypeptide with the sample under conditions suitable and for a time sufficient for the polypeptide to bind to one or more antibodies present in the sample, to provide an antibody-bound polypeptide, and c) detecting the antibody-bound polypeptide, and therefrom determining that the sample contains HCV.
- the assay further comprises: a) in step a), providing an HCV nonstructural protein bound to the solid substrate, b) in step b), contacting the HCV nonstructural protein with the sample under conditions suitable and for a time sufficient for the HCV nonstructural protein to bind to one or more antibodies present in the sample, to provide an antibody-bound HCV nonstructural protein, and c) in step c), detecting one or both of the antibody-bound polypeptide or the antibody- bound HCV nonstructural protein, and therefrom determining that the sample contains HCV.
- the sample in the assay is an unpurified sample, preferably from an animal, and further preferably from a human being.
- the assay is selected from the group consisting of a countercurrent immuno-electrophoresis (CIEP) assay, a radioimmunoassay, a radioimmunoprecipitation, an enzyme-linked immunosorbent assay (ELISA), a dot blot assay, an inhibition or competition assay, a sandwich assay, an immunostick (dip-stick) assays, a simultaneous assay, an immunochromatographic assay, an immunofiltration assay, a latex bead agglutination assay, an immunofluorescent assay, a biosensor assay, and a low-light detection assay. Still further, the assay is preferably not a western blot assay.
- CIEP countercurrent immuno-electrophoresis
- a radioimmunoassay a radioimmunoprecipitation
- ELISA enzyme-linked immunosorbent assay
- a dot blot assay an inhibition or competition assay
- a sandwich assay an
- the present invention provides a method of producing an antibody, comprising the following steps: a) administering to an animal an isolated polypeptide comprising an unprocessed HCV core antigen protein and an arnino- terminal portion of an HCV envelope region, wherein the amino-terminal portion of the HCV envelope region is sized such that the polypeptide has an epitopic configuration specific to an unprocessed core-envelope region of the HCV under conditions suitable and for a time sufficient to induce an immune response in the animal to the polypeptide, thereby providing antibodies to the polypeptide, and b) isolating the antibodies to the polypeptide.
- the present invention also provides antibodies produced according to this method. Preferably, the antibodies are bound to a solid substrate.
- the present invention provides an assay for the detection of HCV in a sample, comprising: a) contacting the sample with an antibody specific for an isolated polypeptide comprising an unprocessed HCV core antigen protein and an amino-terminal portion of an HCV envelope region, wherein the amino- terminal portion of the HCV envelope region is sized such that the polypeptide has an epitopic configuration specific to an unprocessed core-envelope region of the HCV, under conditions suitable and for a time sufficient for the antibody to bind the unprocessed HCV core antigen protein, to provide a bound antibody, and b)detecting the bound antibody, and therefrom determining that the sample contains HCV.
- the assay additionally includes a) in step a), contacting the sample with a further antibody specific for an HCV nonstructural protein under conditions suitable and for a time sufficient for the further antibody to bind the HCV nonstructural protein, to provide a bound further antibody, and b) in step b), detecting one or both of the bound antibody or the bound further antibody, and therefrom determining that the sample contains HCV.
- the assay is selected from the group consisting of a countercurrent immuno-electrophoresis (CIEP) assay, a radioimmunoassay, a radioimmunoprecipitation, an enzyme-linked immunosorbent assay (ELISA), a dot blot assay, an inhibition or competition assay, a sandwich assay, an immunostick (dip-stick) assays, a simultaneous assay, an immunochromatographic assay, an immunofiltration assay, a latex bead agglutination assay, an immunofluor escent assay, a biosensor assay, and a low-light detection assay. Even further preferably, the assay is not a western blot assay.
- CIEP countercurrent immuno-electrophoresis
- a radioimmunoassay a radioimmunoprecipitation
- ELISA enzyme-linked immunosorbent assay
- a dot blot assay an inhibition or competition assay
- a sandwich assay
- the present invention provides a composition capable of eliciting an immune response in an animal comprising an isolated polypeptide comprising an unprocessed HCV core antigen protein and an amino-terminal portion of an HCV envelope region, wherein the amino-terminal portion of the HCV envelope region is sized such that the polypeptide has an epitopic configuration specific to an unprocessed core-envelope region of the HCV, in combination with a pharmaceutically acceptable carrier or diluent.
- the composition further comprises an HCV nonstructural protein.
- the present invention provides a vaccine against HCV comprising an immunoprotective amount of an isolated polypeptide comprising an unprocessed HCV core antigen protein and an amino-terminal portion of an HCV envelope region, wherein the amino-terminal portion of the HCV envelope region is sized such that the polypeptide has an epitopic configuration specific to an unprocessed core-envelope region of the HCV, in combination with a pharmaceutically acceptable carrier or diluent.
- the vaccine further comprises an HCV nonstructural protein.
- the present invention provides a method of inducing an immune response in an animal comprising administering to the animal a composition comprising an isolated polypeptide comprising an unprocessed HCV core antigen protein and an amino-terminal portion of an HCV envelope region, wherein the amino- terminal portion of the HCV envelope region is sized such that the polypeptide has an epitopic configuration specific to an unprocessed core-envelope region of the HCV, in combination with a pharmaceutically acceptable carrier or diluent, under conditions suitable and for a time sufficient to induce the immune response.
- the composition further comprises an HCV nonstructural protein.
- the present invention provides a method of vaccinating an animal comprising administering to the animal a composition comprising an immunoprotective amount of an isolated polypeptide comprising an unprocessed HCV core antigen protein and an amino-terminal portion of an HCV envelope region, wherein the amino-terminal portion of the HCV envelope region is sized such that the polypeptide has an epitopic configuration specific to an unprocessed core-envelope region of the HCV, in combination with a pharmaceutically acceptable carrier or diluent, under conditions suitable and for a time sufficient to induce an immunoprotective immune response.
- the vaccine further comprises an HCV nonstructural protein.
- the present invention provides a kit for the detection of HCV comprising: a) an isolated polypeptide comprising an HCV core antigen protein joined to an amino-terminal portion of an envelope region of the HCV in unprocessed form, wherein the amino-terminal portion of the envelope region is sized such that the polypeptide has an epitopic configuration specific to an unprocessed core-envelope region of the HCV, covalently bound to a solid substrate, and b)means for detecting the isolated polypeptide, particularly when the isolated polypeptide is bound to an antibody specific for HCV.
- the kit further comprises an HCV nonstructural protein, and/or means for detecting the HCV nonstructural protein (particularly when bound to an antibody specific for HCV).
- the present invention provides a kit for the detection of HCV comprising: a) an antibody specific for an isolated polypeptide comprising an unprocessed HCV core antigen protein and an amino-terminal portion of an HCV envelope region, wherein the ammo-terminal portion of the HCV envelope region is sized such that the polypeptide has an epitopic configuration specific to an unprocessed core-envelope region of the HCV, under conditions suitable and for a time sufficient for the antibody to bind the unprocessed HCV core antigen protein, to provide a bound antibody, and b) means for detecting the antibody, particularly when bound to an antigen specific for HCV.
- the kit further comprises a further antibody specific for an HCV nonstructural protein and means for detecting the further antibody (particularly when bound to an antigen specific for HCV).
- the present invention provides the compositions and vaccines described herein for use as a medicament to inhibit, prevent and/or treat HCV infection in an animal.
- the present invention provides such a composition for use in the manufacture of a medicament to inhibit, prevent or treat HCV infection in an animal.
- the composition further comprises an HCV nonstructural protein, and/or is for use with a human being.
- Fig. 1A depicts the nucleotide sequence of a nucleic acid molecule encoding a polypeptide comprising an HCV core antigen protein joined to an amino- terminal portion of an HCV envelope region in unprocessed form.
- Fig. IB depicts the amino acid sequence encoded by the nucleotide sequence depicted in Fig. 1 A.
- Fig. 2 shows the structure of the expression vector pEN-2, which was constructed by inserting a cDNA encoding an HCV core antigen protein joined to an amino-terminal portion of an HCV envelope region in unprocessed form into a plasmid.
- the figure also shows a restriction map illustrating certain significant features of the vector pEN-2.
- Fig. 3 A depicts the nucleotide sequence of a nucleic acid molecule encoding a polypeptide comprising an NS5 nonstructural region.
- Fig. 3B depicts the amino acid sequence encoded by the nucleotide sequence depicted in Fig. 3 A.
- Fig. 4 shows the structure of the expression vector pEN-1, which was constructed by inserting a cDNA encoding an NS5 nonstructural region into a plasmid.
- the figure also shows a restriction map illustrating certain significant features of the vector pEN-1.
- the present invention is based on the discovery that the unprocessed core protein region as translated from the genome of HCV contains epitopic configurations that are not retained in the processed proteins.
- the core protein loses epitopic configuration(s) upon processing at the cleavage site between the genomic region (e.g., gene) encoding the core protein and the genomic region encoding the adjacent envelope protein.
- the unprocessed epitopic configuration of the core region provides a surprisingly improved ability to detect the presence of HCV, or antibodies to HCV, in a sample, including an unpurified sample or a sample of very small volume (which can be particularly helpful when testing a sample from an infant or other person having very little blood (or other suitable material) available for testing).
- the present invention features an isolated polypeptide comprising an HCV core antigen protein joined to an amino-terminal portion of an HCV envelope region in unprocessed form, wherein the amino-terminal portion of the HCV envelope region is sized such that the polypeptide has an epitopic configuration specific to an unprocessed core-envelope region of the HCV.
- the present invention provides the first discovery that the lost epitopic configuration occurs in the core-envelope region, and surprisingly finds that the epitopic configuration can be found in an isolated protein.
- core antigen protein it is meant a polypeptide comprising the portion of the core protein that displays the antigenicity of the core protein.
- core protein generally, and regions of the core protein that can be important to antigenicity, is well known in the art (see, e.g., Okamoto et al., J. Virol. 188:331, 1992; Wang, U.S. Patent No. 5,106,726; Sallberg et al., Immunology Letters 33:27-34, 1992; Clemens, J.M. et al., Blood 79:1, 169-72, 1992; Houghton et al.
- a core antigen protein may be determined by SDS-PAGE and amino acid sequence analysis in light of the above references, and may also be determined by use in established HCV assays in light of the above references.
- the present invention features this core antigen protein joined to an amino-terminal portion of an HCV envelope region in unprocessed form to supply the inventive "core-envelope fusion protein.”
- unprocessed form means that the core region and the envelope region are typically, and preferably, maintained precisely as they are joined (i.e., encoded) in a native HCV.
- conservative amino acid substitutions, or insignificant amino acid additions, modifications or deletions that may change the amino acid sequence of the core- envelope fusion protein but do not significantly alter the functioning of the protein (i.e., the unprocessed epitopic configuration is retained). Such modifications may, however, when desired, delete the processing signal and/or site of the protein.
- the amino-terminal portion of the HCV envelope region is sized such that the fusion protein has an epitopic configuration specific to an unprocessed core- envelope " region of said HCV.
- the amino-terminal portion of the HCV envelope region must be of sufficient length to permit the fusion protein to display the transient epitopic configuration specific to the unprocessed core region.
- a core-envelope protein in question may be included in a panel of core-envelope proteins that includes an established core-envelope fusion protein such as EN-80-2.
- the panel may be placed in a series of wells on a microtiter plate.
- the panel may also include other core-envelope proteins having different lengths of envelope protein.
- an established nonstructural protein capable of synergistic cooperation with the core-envelope fusion protein, such as EN-80-1.
- an antiserum is then selected for the established core-envelope fusion protein that reacts weakly with the established core-envelope fusion protein and that also is nonreactive with the established nonstructural protein.
- the basis for selection is that the antiserum will react with the separated proteins as expected, but the antiserum will react much more strongly when both a suitable core-envelope fusion protein and the established nonstructural protein are present in the sample.
- the antiserum will react at least about 1.25 or 1.5 times as strongly as with the combined proteins when compared to the additive reaction of the antiserum with each protein, alone. Further preferably, the antiserum will react at least about twice as strongly.
- antiserum such as G614 (diluted 8-fold), G614 (diluted 16-fold), G615 (diluted 8-fold), G615 (diluted 16-fold), and 8-5
- the antiserum is introduced to the sample proteins under conditions suitable for elicitation and detection of an immune response between the antiserum and the given protein, and then such response is detected and measured.
- the established nonstructural protein is combined with a further sample of each member of the core-envelope fusion protein panel.
- the antiserum is introduced to the combined proteins under conditions suitable for elicitation and detection of an immune response between the antiserum and the proteins, and such response is detected and measures.
- Those core-envelope proteins that provide a cooperative effect are suitable for use in the present invention.
- the core antigen-envelope fusion protein is preferably isolated, which means that the core antigen-envelope fusion protein is separated from the remainder of the polyprotein originally translated from the genome of HCV.
- the core antigen- envelope fusion protein of the present invention is used in combination with a nonstructural protein from HCV.
- the nonstructural coding region of HCV is well known in the art. See, e.g., EP 0 318 216 Al. The decision of which nonstructural protein, including portions of the nonstructural coding region that may include more than one nonstructural protein (or less than all of one nonstructural protein), can be made by selecting a nonstructural protein as follows.
- a nonstructural protein in question may be included in a panel of nonstructural proteins that includes an established nonstructural protein such as EN-80- 1.
- the panel may be placed in a series of wells on a microtiter plate.
- the panel may also include other nonstructural proteins.
- an established core-envelope fusion protein capable of synergistic cooperation with the nonstructural protein, such as EN-80-2.
- An antiserum is selected for the established core-envelope fusion protein that reacts weakly with the established core-envelope fusion protein and that also is nonreactive with the established nonstructural protein.
- the basis for selection is that the antiserum will react with the separated proteins as expected, but the antiserum will react much more strongly when both the established core-envelope fusion protein and a suitable nonstructural protein are present in the sample.
- an antiserum is introduced to the separated sample proteins under conditions suitable for elicitation and detection of an immune response between the antiserum and the given protein, and then such response is detected and measured.
- the established core- envelope protein is combined with each member of the nonstructural protein panel.
- the antiserum is introduced to the combined proteins under conditions suitable for elicitation and detection of an immune response between the antiserum and the proteins, and then such response is detected and measured.
- Those nonstructural proteins that provide a cooperative effect are suitable for use in the present invention.
- the present invention also provides antibodies, preferably monoclonal antibodies, to the core-envelope fusion protein and/or the nonstructural protein, as well as other proteins of the present invention.
- the antibodies are preferably used in combination to provide particularly sensitive and specific detection of HCV in a sample.
- the present invention provides compositions and methods for the elicitation on an immune response in an animal (either humoral, cellular, or both). Even further, the compositions and methods can vaccinate an animal against HCV. Preferably, the methods and compositions of the present invention, including those for detection, immune response elicitation and vaccination, are applied to a human being.
- the present invention provides a nucleic acid molecule encoding a polypeptide comprising an HCV core antigen protein joined to an amino- terminal portion of an HCV envelope region in unprocessed form.
- the present invention also provides a nucleic acid molecule encoding a polypeptide comprising a nonstructural protein of HCV.
- the nucleic acid molecule is DNA
- the nucleic acid molecule is a DNA molecule that encodes an unprocessed core antigen-envelope protein that was isolated from nucleic acid sequences present in the plasma of an HCV infected patient.
- the isolation of the DNA included the steps of isolating viral particles from the patient's plasma, extracting and purifying the viral nucleic acid sequences, and then cloning the desired DNA molecule via a Polymerase Chain Reaction (PCR) technique.
- PCR Polymerase Chain Reaction
- the cloned DNA molecule was sequenced in order to confirm its identity.
- the molecule thus obtained was designated EN-80-2.
- the DNA sequence of the molecule EN-80-2 is given in Fig. 1 A (SEQ ID No. 7), and has 669 bp.
- the amino acid sequence of the molecule EN-80-2 is given in Fig. IB (SEQ ID No. 8), and has 223 residues.
- the molecule EN-80-2, in E. coli strain BL21(DE3) was deposited with the American Type Culture Collection (ATCC) Rockville Maryland 20852, on July 14, 1993, and has been accorded ATCC Designation 55451. The culture has been deposited under the conditions of the Budapest Treaty.
- the nucleic acid molecule is a DNA molecule encoding an HCV NS5 nonstructural protein that was isolated from nucleic acid sequences present in the plasma of an HCV infected patient.
- the isolation included the steps of isolating viral particles from the patient's plasma, extracting and purifying the viral nucleic acid sequences, and then cloning the desired DNA molecule via a Polymerase Chain Reaction (PCR) technique.
- PCR Polymerase Chain Reaction
- the isolated DNA molecule was subjected to sequence analysis in order to confirm its identity.
- the molecule thus obtained was designated EN-80-1.
- the DNA sequence of the molecule EN-80-1 is given in Fig. 3A (SEQ ID No. 9) and has 803 bp.
- the amino acid sequence of the molecule EN-80-1 is given in Fig. 3B (SEQ ID No. 10), and has 267 residues.
- the molecule EN-80-1, in E. coli strain BL21(DE3) was deposited with the American Type Culture Collection (ATCC) Rockville Maryland 20852, on July 14, 1993, and has been accorded ATCC Designation 55450. The culture has been deposited under the conditions of the Budapest Treaty.
- the molecule thus obtained was designated EN-80-4.
- the polypeptide encoded by the isolated molecule has a molecular weight of about 20,000 Daltons as measure by electrophoresis through SDS-PAGE.
- Figure 2 depicts an expression plasmid, pEN-2, that contains the DNA molecule encoding the unprocessed core antigen-envelope protein isolated using the primers SEQ
- Figure 4 depicts an expression plasmid, pEN-1, that contains the DNA molecule encoding the NS5 nonstructural protein isolated using the primers SEQ ID No. 3 and 4, discussed above.
- the present invention provides for the manipulation and expression of the above described nucleic acid molecules by culturing host cells containing a construct capable of expressing the above-described genes.
- vector constructs suitable for use with the nucleic acid molecules of the present invention can be prepared as a matter of convenience.
- a vector construct is understood to typically refer to a DNA molecule, or a clone of such a molecule (either single-stranded or double- stranded), that has been modified through human intervention to contain segments of DNA combined and juxtaposed in a manner that as a whole would not otherwise exist in nature.
- Vector constructs of the present invention comprise a first DNA segment encoding one or more of an unprocessed core antigen-envelope protein and a nonstructural protein of HCV operably linked to additional DNA segments required for the expression of the first DNA segment.
- additional DNA segments will typically include a promoter and will generally include transcription terminators, and may further include enhancers and other elements. See WO 94/25597 and WO/25598.
- Mutations in nucleotide sequences constructed for expression of the inventive proteins preferably preserve the reading frame of the encoding sequences. Furthermore, the mutations will preferably not create complementary regions that could hybridize to produce secondary rnRNA structures, such as loops or hairpins, that would adversely affect translation of the rnRNA.
- a mutation site may be predetermined, it is not necessary that the nature of the mutation per se be predetermined. For example, in order to select for optimum characteristics of mutants at a given site, random mutagenesis may be conducted at the target codon and the expressed mutants screened for indicative biological activity.
- Mutations may be introduced at particular loci by synthesizing oligonucleotides containing a mutant sequence, flanked by restriction sites enabling ligation to fragments of the native sequence. Following ligation, the resulting reconstructed sequence encodes a derivative having the desired amino acid insertion, substitution or deletion.
- oligonucleotide-directed, site-specific mutagenesis procedures may be employed to provide an altered gene having particular codons altered according to the substitution, deletion, or insertion required.
- Exemplary methods of making the alterations set forth above are disclosed by Walder et al. (Gene 42:133, 1986); Bauer et al. (Gene 37:13, 1985); Craik (BioTechniques, January 1985, 12-19); Smith et al. (Genetic Engineering: Principles and Methods, Plenum Press, 1981); and Sambrook et al. (supra).
- the primary amino acid structure of the above described proteins may also be modified by forming covalent or aggregative conjugates with other chemical moieties, such as glycosyl groups, lipids, phosphate, acetyl groups, or with other proteins or polypeptides, provided that such modifications should not interfere with the antigenicity of the proteins.
- modifications should not interfere with the epitopic configuration (including access to the epitope and other antigenic considerations) specific to the unprocessed core antigen-envelope protein.
- a preferred type of vector construct is known as an expression vector.
- the plasmids pEN-1 and pEN-2 are examples of such expression vectors, and contain nucleic acid molecules encoding the NS5 nonstructural region and the unprocessed core antigen-envelope protein, respectively.
- a DNA molecule as described above is inserted into a suitable vector construct, which in turn is used to transform or transfect appropriate host cells for expression.
- the host cell for use in expressing the gene sequences of the present invention is a prokaryotic host cell, further preferably a bacterium such as E. coli.
- host cells include Salmonella, Bacillus, Shigella, Pseudomonas, Streptomyces and other genera known in the art.
- the host cell is an E. coli containing a DE3 lysogen or T7 RNA polymerase, such as BL21(DE3), JM109(DE3) or BL21(DE3) pLysS.
- Vectors used for expressing cloned DNA sequences in bacterial hosts will generally contain a selectable marker, such as a gene for antibiotic resistance, and will contain a promoter that functions in the host cell.
- Appropriate promoters include the trp (Nichols and Yanofsky, Meth. Enzymol. 707:155-164, 1983), lac (Casadaban et al., J. Bacteriol. 743:971-980, 1980), and phage ⁇ (Queen, J. Mol. Appl. Genet. 2:1- 10, 1983) promoter systems.
- the expression units may also include a transcriptional terminator.
- Plasmids useful for transforming bacteria include the pUC plasmids (Messing, Meth.
- Plasmids may contain both viral and bacterial elements.
- the host cell may be a eukaryotic cell, provided that either the host cell has been modified such that the host cell cannot process, for example, the unprocessed core antigen-envelope protein or unprocessed nonstructural region (such as the NS3-NS4 nonstructural protein), or the processing signals and/or processing sites in the unprocessed protein have been modified such that the protein is no longer susceptible to processing (such modifications should not affect the antigenicity of the unprocessed protein).
- Eukaryotic host cells suitable for use in practicing the present invention include mammalian, avian, plant, insect and fungal cells.
- Preferred eukaryotic cells include cultured mammalian cell lines (e.g., rodent or human cell lines), insect cell line (e.g., Sf-9) and fungal cells, including species of yeast (e.g., Saccharomyces spp., particularly S. cerevisiae, Schizosaccharomyces spp., or Kluyveromyces spp.) or filamentous fungi (e.g., Aspergillus spp., Neurospora spp.).
- yeast e.g., Saccharomyces spp., particularly S. cerevisiae, Schizosaccharomyces spp., or Kluyveromyces spp.
- filamentous fungi e.g., Aspergillus spp., Neurospora spp.
- a host cell will be selected on the basis of its ability to produce the protein of interest at a high level. In this way, the number of cloned DNA sequences that must be introduced into the host cell can be minimized and overall yield of biologically active protein can be maximized.
- promoters, terminators and methods for introducing such expression vectors encoding the proteins of the present invention into desired host cells would be evident to those of skill in the art.
- Host cells containing vector constructs of the present invention are then cultured to express a DNA molecule as described above.
- the cells are cultured according to standard methods in a culture medium containing nutrients required for growth of the chosen host cells.
- suitable media are known in the art and generally include a carbon source, a nitrogen source, essential amino acids, vitamins and minerals, as well as other components, e.g., growth factors or serum, that may be required by the particular host cells.
- the growth medium will generally select for cells containing the DNA construct(s) by, for example, drug selection or deficiency in an essential nutrient which is complemented by the selectable marker on the DNA construct or co-transfected with the DNA construct.
- Polypeptides Comprising An Unprocessed Core Antigen-Envelope Protein And/Or A Nonstructural Protein Of The Invention
- the invention provides a polypeptide comprising the HCV core antigen protein joined to an amino-terminal portion of an HCV envelope region in unprocessed form.
- the amino acid sequence of the polypeptide is that given in Fig. IB (Seq. ID. No. 8).
- the polypeptide has a molecular weight of about 25,000 daltons as measured by electrophoresis through a sodium dodecyl sulfate-polyacrylamide gel and has been deduced to have about 223 amino acids.
- the unprocessed core antigen-envelope protein is capable of binding
- HCV antibodies which has been confirmed by Western Blotting and by enzyme-linked immunosorbent assay (ELISA).
- ELISA enzyme-linked immunosorbent assay
- the unprocessed core antigen-envelope protein is specifically reactive with the sera of patients with HCV, and therefore is not significantly reactive with the sera of persons without HCV.
- the unprocessed core antigen-envelope protein is also capable of detecting the presence of HCV antibodies in samples, and therefore is useful for diagnosis of HCV in a patient, particularly a human being.
- the present invention also provides a polypeptide comprising an HCV nonstructural protein.
- the polypeptide has the amino acid sequence of the polypeptide depicted in Fig. 3B (SEQ ID No. 10).
- the polypeptide of Figure 3B (SEQ ID No. 10) has a molecular weight of about 29,000 daltons as measured by electrophoresis through a sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) and has been deduced to have about 267 amino acids.
- the nonstructural protein of the present invention is capable of binding HCV antibodies, which has been confirmed by Western Blotting and by enzyme-linked immunosorbent assay (ELISA) for both the NS5 and the NS3-NS4 nonstructural proteins disclosed herein.
- the nonstructural protein of the present invention is specifically reactive with the sera of patients with HCV, and therefore is not reactive with the sera of persons without HCV.
- the nonstructural protein is also capable of detecting the presence of HCV antibodies in samples, and therefore is useful for diagnosis of HCV in a patient, particularly a human being.
- the protein of the present invention is encoded by a portion of a native gene, a derivative of a native gene, or has been otherwise modified, the protein maintains substantially the same biological activity of the native protein.
- the structure of proteins corresponding to the unprocessed core antigen-envelope protein or the nonstructural protein can be predicted from the primary translation products thereof using the hydrophobicity plot function of, for example, P/C Gene or Intelligenetics Suite (Intelligenetics, Mountain View, Calif), or according to the methods described by Kyte and Doolittle (J. Mol. Biol. 757:105-132, 1982).
- the present invention provides isolated proteins. Proteins can be isolated by, among other methods, culturing suitable host and vector systems to produce the recombinaut translation products of the present invention. Supernatants from such cell lines, or protein inclusions or whole cells where the protein is not excreted or secreted into the supernatant, can then be treated by a variety of purification procedures in order to isolate the desired proteins. For example, the supernatant may be first concentrated using commercially available protein concentration filters, such as an Amicon or Millipore Pellicon ultrafiltration unit. Following concentration, the concentrate may be applied to a suitable purification matrix such as, for example, an anti-protein antibody bound to a suitable support.
- a suitable purification matrix such as, for example, an anti-protein antibody bound to a suitable support.
- anion or cation exchange resins may be employed in order to purify the protein.
- one or more reverse-phase high performance liquid chromatography (RP-HPLC) steps may be employed to further purify the protein.
- RP-HPLC reverse-phase high performance liquid chromatography
- a protein is deemed to be "isolated" within the context of the present invention if the protein accounts for at least about 90% of the protein, by weight, in a mixture.
- the desired protein can be isolated such that no other (undesired) protein, and preferably no lipopolysaccharide (LPS), is detected by SDS-PAGE analysis followed by coomassie blue staining, further preferably by SDS- PAGE analysis followed by silver staining.
- the protein is isolated if no other protein having significant antigenic activity that significantly interferes with detection assays or immunological events is included with the protein.
- the present invention also provides monoclonal and polyclonal antibodies directed against the unprocessed core antigen-envelope protein or the nonstructural protein of HCV.
- the antibodies are produced by using the polypeptide of the invention as an immunogen through standard procedures for preparing a hybridoma, and/or other methods.
- the resulting antibodies are particularly useful for detecting HCV in a sample, preferably a sample from a human being.
- Polyclonal antibodies can be readily generated by one of ordinary skill in the art from a variety of warm-blooded animals such as horses, cows, goats, sheep, dogs, chickens, turkeys, rabbits, mice, or rats.
- the desired protein or peptide is utilized to immunize the animal, typically through intraperitoneal, intramuscular, intraocular, or subcutaneous injections.
- the immunogenicity of the protein or peptide of interest may be increased through the use of an adjuvant such as Freund's complete or incomplete adjuvant.
- an adjuvant such as Freund's complete or incomplete adjuvant.
- small samples of serum are collected and tested for reactivity to the desired protein or peptide.
- polyclonal antisera may be readily obtained either by weekly bleedings, or by exsanguinating the animal.
- Monoclonal antibodies can also be readily generated using well-known techniques (see U.S. Patent Nos. RE 32,011, 4,902,614, 4,543,439, and 4,411,993; see also Monoclonal Antibodies, Hybridomas: A New Dimension in Biological Analyses, Plenum Press, Kennett, McKearn, and Bechtol (eds.), 1980, and Antibodies: A Laboratory Manual, supra). Briefly, in one embodiment, a subject animal such as a rat or mouse is injected with a desired protein or peptide. If desired, various techniques may be utilized in order to increase the resultant immune response generated by the protein, in order to develop greater antibody reactivity.
- the desired protein or peptide may be coupled to another protein such as ovalbumin or keyhole limpet hemocyanin (KLH), or through the use of adjuvants such as Freund's complete or incomplete adjuvants.
- KLH keyhole limpet hemocyanin
- the initial elicitation of an immune response may be through intraperitoneal, intramuscular, intraocular, or subcutaneous routes.
- the animal may be reimmunized with booster immunization.
- the animal may then be test bled and the serum tested for binding to the unprocessed core antigen-envelope region, or to an HCV nonstructural protein using assays as described above. Additional immunizations may also be accomplished until the animal has reached a plateau in its reactivity to the desired protein or peptide.
- the animal may then be given a final boost of the desired protein or peptide, and three to four days later sacrificed.
- the spleen and lymph nodes may be harvested and disrupted into a single cell suspension by passing the organs through a mesh screen or by rupturing the spleen or lymph node membranes which encapsulate the cells.
- the red cells are subsequently lysed by the addition of a hypotonic solution, followed by immediate return to isotonicity.
- suitable cells for preparing monoclonal antibodies are obtained through the use of in vitro immunization techniques. Briefly, an animal is sacrificed, and the spleen and lymph node cells are removed as described above. A single cell suspension is prepared, and the cells are placed into a culture containing a form of the protein or peptide of interest that is suitable for generating an immune response as described above. Subsequently, the lymphocytes are harvested and fused as described below.
- Cells that are obtained through the use of in vitro immunization or from an immunized animal as described above may be immortalized by transfection with a virus such as the Epstein-Barr Virus (EBV). (See Glasky and Reading, Hybridoma 8(4):377-3S9, 1989.)
- EBV Epstein-Barr Virus
- the harvested spleen and/or lymph node cell suspensions are fused with a suitable myeloma cell in order to create a "hybridoma" which secretes monoclonal antibodies.
- Suitable myeloma lines are preferably defective in the construction or expression of antibodies, and are additionally syngeneic with the cells from the immunized animal.
- myeloma cell lines are well known in the art and may be obtained from sources such as the American Type Culture Collection (ATCC), Rockville, Maryland (see Catalogue of Cell Lines & Hybridomas, 6th ed., ATCC, 1988).
- Representative myeloma lines include: for humans, UC 729-6 (ATCC No. CRL 8061), MC/CAR-Z2 (ATCC No. CRL 8147), and SKO-007 (ATCC No. CRL 8033); for mice, SP2/0-Agl4 (ATCC No. CRL 1581), and P3X63Ag8 (ATCC No. TIB 9); and for rats, Y3-Agl.2.3 (ATCC No.
- fusion lines include NS-1 (ATCC No. ⁇ B 18) and P3X63 - Ag 8.653 (ATCC No. CRL 1580), which may be utilized for fusions with either mouse, rat, or human cell lines. Fusion between the myeloma cell line and the cells from the immunized animal can be accomplished by a variety of methods, including the use of polyethylene glycol (PEG) (see Antibodies: A Laboratory Manual, supra) or electrofusion (see Zimmerman and Vienken, J. Membrane Biol. 67:165-182, 1982).
- PEG polyethylene glycol
- the cells are placed into culture plates containing a suitable medium, such as RPMI 1640 or DMEM (Dulbecco's Modified Eagles Medium, JRH Biosciences, Lenexa, Kan.).
- a suitable medium such as RPMI 1640 or DMEM (Dulbecco's Modified Eagles Medium, JRH Biosciences, Lenexa, Kan.).
- the medium may also contain additional ingredients, such as Fetal Bovine Serum (FBS, e.g., from Hyclone, Logan, Utah, or JRH Biosciences), thymocytes that were harvested from a baby animal of the same species as was used for immunization, or agar to solidify the medium.
- FBS Fetal Bovine Serum
- the medium should contain a reagent which selectively allows for the growth of fused spleen and myeloma cells.
- HAT medium hyperxanthine, aminopterin, and thymidine
- HAT medium sodium xanthine, aminopterin, and thymidine
- the resulting fused cells or hybridomas may be screened in order to determine the presence of antibodies which recognizes the core-envelope region of said HCV or the HCV nonstructural protein.
- hybridoma producing antibodies that bind to the protein of interest can be isolated. Other techniques can also be utilized to construct monoclonal antibodies.
- rnRNA is isolated from a B cell population and utilized to create heavy and light chain immunoglobulin cDNA expression libraries in the ⁇ _MMUNOZAP(H) and ⁇ _MMUNOZAP(L) vectors.
- vectors may be screened individually or co-expressed to form Fab fragments or antibodies (see Huse et al., supra; see also Sastry et al., supra). Positive plaques can subsequently be converted to a non-lytic plasmid which allows high level expression of monoclonal antibody fragments from E. coli.
- binding partners can also be constructed utilizing recombinant DNA techniques to incorporate the variable regions of a gene that encodes a specifically binding antibody.
- the construction of these binding partners can be readily accomplished by one of ordinary skill in the art given the disclosure provided herein. (See Larrick et al., "Polymerase Chain Reaction Using Mixed Primers: Cloning of Human Monoclonal Antibody Variable Region Genes From Single Hybridoma Cells," Biotechnology 7:934-938, 1989; Riechmann et al., “Reshaping Human Antibodies for Therapy," Nature 332:323-327, 1988; Roberts et al., "Generation of an Antibody with Enhanced Affinity and Specificity for its Antigen by Protein Engineering," Nature 328:731-734, 1987; Verhoeyen et al., “Reshaping Human Antibodies: Grafting an Antilysozyme Activity," Science 239:1534-1536, 1988; Chaudhary et al., "A Re
- DNA segments encoding the desired protein or peptide interest-specific antigen binding domains are amplified from hybridomas that produce a specifically binding monoclonal antibody, and are inserted directly into the genome of a cell that produces human antibodies.
- This technique allows the antigen-binding site of a specifically binding mouse or rat monoclonal antibody to be transferred into a human antibody.
- Such antibodies are preferable for therapeutic use in humans because they are not as antigenic as rat or mouse antibodies.
- genes that encode the variable region from a hybridoma producing a monoclonal antibody of interest are amplified using oligonucleotide primers for the variable region.
- primers for mouse and human variable regions including, among others, primers for V H b, V Hc , V H( j, C H ⁇ , V L and C regions, are available from Stratacyte (La Jolla, Calif).
- These primers may be utilized to amplify heavy or light chain variable regions, which may then be inserted into vectors such as _MMUNOZAPTM(H) or IMMUNOZAPTM(L) (Stratacyte), respectively. These vectors may then be introduced into E. coli for expression. Utilizing these techniques, large amounts of a single-chain protein containing a fusion of the V H and V L domains may be produced (see Bird et al., Science 242:423-426, 1988). Monoclonal antibodies and binding partners can be produced in a number of host systems, including tissue cultures, bacteria, eukaryotic cells, plants and other host systems known in the art.
- suitable antibodies or binding partners may be isolated or purified by many techniques well known to those of ordinary skill in the art (see Antibodies: A Laboratory Manual, Harlow and Lane (eds.), Cold Spring Harbor Laboratory Press, 1988; U.S. Patent No. 4,736,110; and U.S. Patent No. 4,486,530).
- Suitable isolation techniques include peptide or protein affinity columns, HPLC or RP-HPLC, purification on protein A or protein G columns, or any combination of these techniques.
- the term "isolated" as used to define antibodies or binding partners means "substantially free of other blood components.”
- the antibodies and binding partners of the present invention have many uses. As discussed further below, the antibodies and binding partners of the present invention are particularly useful for the detection and diagnosis of HCV. Other uses include, for example, flow cytometry to sort cells displaying one more of the proteins of the present invention. Briefly, in order to detect the protein or peptide of interest on cells, the cells are incubated with a labeled monoclonal antibody which specifically binds to the protein of interest, followed by detection of the presence of bound antibody. These steps may also be accomplished with additional steps such as washings to remove unbound antibody.
- Labels suitable for use within the present invention are well known in the art including, among others, flourescein isothiocyanate (FITC), phycoerythrin (PE), horse radish peroxidase (HRP), and colloidal gold.
- FIT C flourescein isothiocyanate
- PE phycoerythrin
- HRP horse radish peroxidase
- colloidal gold particularly preferred for use in flow cytometry is FIT C, which may be conjugated to purified antibody according to the method of Keltkamp in "Conjugation of Fluorescein Isothiocyanate to Antibodies. I. Experiments on the Conditions of Conjugation," Immunology 75:865-873, 1970. (See also Keltkamp, "Conjugation of Fluorescein Isothiocyanate to Antibodies. 13. A Reproducible Method," Immunology 75:875-881, 1970; Goding, "Conjugation of Antibodies with Fluorochromes: Modification to the Standard
- the present invention provides methods for detecting HCV in a sample.
- the assays are typically based on the detection of antigens displayed by HCV or antibodies produced against HCV, but may also include nucleic acid based assays (typically based upon hybridization), as known in the art.
- the methods are characterized by the ability of the polypeptides of the present invention to be bound by anti-HCV antibodies, and the ability of antibodies produced against the proteins of the present invention to bind to HCV antigens in a sample.
- the unprocessed core antigen-envelope protein of HCV of the present invention provides significantly better and more sensitive detection of HCV in a sample than processed core protein (sometimes referred to as p22) or fragments of the core protein, alone.
- processed core protein sometimes referred to as p22
- the use of both unprocessed core antigen-envelope protein and a nonstructural protein of HCV in the assay provides a synergistic effect that permits significantly more sensitive detection of HCV than when either the unprocessed core antigen-envelope protein or nonstructural protein of HCV is utilized alone.
- a preferred assay for the detection of HCV is a sandwich assay such as an enzyme-linked immunosorbent assay (ELISA).
- One preferred ELISA comprises the following steps: (1) coating a core antigen-envelope protein of the present invention onto a solid phase, (2) incubating a sample suspected of containing HCV antibodies with the polypeptide coated onto the solid phase under conditions that allow the formation of an antigen-antibody complex, (3) adding an anti-antibody (such as anti-IgG) conjugated with a label to be captured by the resulting antigen-antibody complex bound to the solid phase, and (4) measuring the captured label and determining therefrom whether the sample has HCV antibodies.
- an anti-antibody such as anti-IgG conjugated with a label to be captured by the resulting antigen-antibody complex bound to the solid phase
- Such assays include: countercurrent immuno-electrophoresis (CIEP), radioimmunoassays, radioimmunoprecipitations, enzyme-linked immunosorbent assays (ELISA), dot blot assays, inhibition or competition assays, sandwich assays, immunostick (dip-stick) assays, simultaneous assays, immunochromatographic assays, immunofiltration assays, latex bead agglutination assays, immunofluorescent assays, biosensor assays, and low-light detection assays (see U.S. Patent Nos. 4,376,110 and 4,486,530; see also Antibodies: A Laboratory Manual, supra).
- CIEP countercurrent immuno-electrophoresis
- ELISA enzyme-linked immunosorbent assays
- dot blot assays inhibition or competition assays
- sandwich assays sandwich assays
- immunostick (dip-stick) assays simultaneous assays
- immunochromatographic assays immunofiltration assays
- a fluorescent antibody test uses a fluorescently labeled antibody able to bind to one of the proteins of the invention. For detection, visual determinations are made by a technician using fluorescence microscopy, yielding a qualitative result. In one embodiment, this assay is used for the examination of tissue samples or histological sections.
- EIA enzyme immunoassays
- a heterogeneous indirect EIA uses a solid phase coupled with an antibody of the invention and an affinity purified, anti-IgG immunoglobulin preparation.
- the solid phase is a polystyrene microtiter plate.
- the antibodies and immunoglobulin preparation are then contacted with the sample under conditions permitting antibody binding, which conditions are well known in the art.
- the results of such an assay can be read visually, but are preferably read using a spectrophotometer, such as an ELISA plate reader, to yield a quantitative result.
- An alternative solid phase EIA format includes plastic-coated ferrous metal beads able to be moved during the procedures of the assay by means of a magnet.
- Yet another alternative is a low-light detection immunoassay format. In this highly sensitive format, the light emission produced by appropriately labeled bound antibodies are quantitated automatically.
- the reaction is performed using microtiter plates.
- a radioactive tracer is substituted for the enzyme mediated detection in an EIA to produce a radioimmunoassay (RIA).
- the desired protein is bound between an antibody attached to a solid phase, preferably a polystyrene microtiter plate, and a labeled antibody.
- a solid phase preferably a polystyrene microtiter plate
- the results are measured using a spectrophotometer, such as an ELISA plate reader.
- This assay is one preferred embodiment for the present invention.
- reagents are allowed to incubate with the capture antibody in a step wise fashion. ' The test sample is first incubated with the capture antibody. Following a wash step, an incubation with the labeled antibody occurs. In a simultaneous assay, the two incubation periods described in the sequential assay are combined. This eliminates one incubation period plus a wash step.
- a dipstick/immunostick format is essentially an immunoassay except that the solid phase, instead of being a polystyrene microtiter plate, is a polystyrene paddle or dipstick. Reagents are the same and the format can either be simultaneous or sequential.
- a capture antibody and a labeled antibody are dried onto a chromatographic strip, which is typically nitrocellulose or nylon of high porosity bonded to cellulose acetate.
- the capture antibody is usually spray dried as a line at one end of the strip. At this end there is an absorbent material that is in contact with the strip.
- the labeled antibody is deposited in a manner that prevents it from being absorbed into the membrane.
- the label attached to the antibody is a latex bead or colloidal gold.
- the assay may be initiated by applying the sample immediately in front of the labeled antibody.
- Immunofiltration/immunoconcentration formats combine a large solid phase surface with directional flow of sample/reagents, which concentrates and accelerates the binding of antigen to antibody.
- the test sample is preincubated with a labeled antibody then applied to a solid phase such as fiber filters or nitrocellulose membranes or the like.
- the solid phase can also be precoated with latex or glass beads coated with capture antibody. Detection of analyte is the same as standard immunoassay.
- the flow of sample/reagents can be modulated by either vacuum or the wicking action of an underlying absorbent material.
- a threshold biosensor assay is a sensitive, instrumented assay amenable to screening large numbers of samples at low cost.
- such an assay comprises the use of light addressable potentiometric sensors wherein the reaction involves the detection of a pH change due to binding of the desired protein by capture antibodies, bridging antibodies and urease-conjugated antibodies. Upon binding, a pH change is effected that is measurable by translation into electrical potential ( ⁇ volts).
- the assay typically occurs in a very small reaction volume, and is very sensitive. Moreover, the reported detection limit of the assay is 1,000 molecules of urease per minute.
- compositions and methods for the elicitation of an immune response to HCV which may be either humoral, cellular, or both.
- the immune response * is induced by a vaccine against HCV, and is therefore an immunoprotective immune response.
- These compositions and methods typically involve an immunogen comprising an unprocessed core antigen-envelope protein or nonstructural protein of HCV in combination with a pharmaceutically acceptable carrier or diluent.
- the compositions and methods comprise both an unprocessed core antigen-envelope protein and a nonstructural protein of HCV, further preferably an NS5 nonstructural protein or a NS3-NS4 nonstructural protein.
- the compositions and methods may also include an inactivated preparation or an attenuated preparation comprising the proteins of the invention.
- another aspect of the present invention provides isolated antigens capable of eliciting an immune response, preferably immunogens capable of immunizing an animal.
- the immunogens comprise amino acid sequences or molecules shown in or derived from the sequences shown in Figures 1A, IB, 3A or 3B or substantial equivalents thereof.
- slight deviations of the amino acid sequences can be made without affecting the immunogenicity of the immunogen.
- Substantial equivalents of the above proteins include conservative substitutions of amino acids that maintain substantially the same charge and hydrophobicity as the original amino acid.
- Conservative substitutions include replacement of valine for isoleucine or leucine, and aspartic acid for glutamic acid, as well as other substitutions of a similar nature (See Dayhoff et al. (ed.), "Atlas of Protein Sequence and Structure,” Natl. Biomed Res. Fan., 1978).
- the immunogens listed above, including their substantial equivalents may stimulate different levels of response in different animals.
- the immunogens listed above, including their substantial equivalents can be tested for effectiveness as a vaccine. These tests include T-cell proliferation assays, determination of lymphokine production after stimulation, and immunoprotection trials. Briefly, T-cell proliferation assays can be utilized as an indicator of potential for cell-mediated immunity. Additionally, evidence of lymphokine production after stimulation by an immunogen can be utilized to determine the potential for protection provided by an immunogen.
- PBLs peripheral blood lymphocytes
- the immunogens, or polypeptides, of the present invention can be readily produced utilizing many other techniques well known in the art (see Sambrook et al., supra, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1989).
- the immunogens comprising an unprocessed core antigen-envelope protein or nonstructural protein of HCV (or both) in combination with a pharmaceutically acceptable carrier or diluent can be administered to a patient in accordance with a number procedures known in the art.
- warm-blooded animals include, among others, humans and primates.
- Suitable carriers or diluents can be utilized in the present invention, including among others saline, buffered saline, and saline mixed with nonspecific serum albumin.
- the pharmaceutical composition may also contain other excipient ingredients, including adjuvants, buffers, antioxidants, carbohydrates such as glucose, sucrose, or dextrins, and chelating agents such as EDTA.
- an adjuvant is utilized along with the immunogen. Examples of such adjuvants include alum or aluminum hydroxide for humans.
- immunizations will involve oral administration.
- the vaccine can be parenterally administrated via the subcutaneous route, or via other routes.
- quantities of injected immunogen will vary from 50 ⁇ g to several milligrams in an adjuvant vehicle and preferably about 100 ⁇ g to 1 mg, in combination with a physiologically acceptable carrier or diluent.
- Booster immunizations can be given from 4-6 weeks later.
- the present invention also includes the administration of a nucleic acid vector capable of expressing the unprocessed core antigen-envelope protein or nonstructural protein of HCV (or both) into an animal, wherein the nucleic acid molecule can elicit an immune response in, and preferably immunize, an animal against the expressed protein expressed from the nucleic molecule, and therefore HCV.
- naked DNA is introduced into an appropriate cell, such as a muscle cell, where it produces protein that is then displayed on the surface of the cell, thereby eliciting a response from host cytotoxic T-lymphocytes (CTLs). This can provide .an advantage over traditional immunogens wherein the elicited response comprises specific antibodies.
- CTLs are specific for conserved antigens and can respond to different strains expressing a corresponding antigen (Ulmer et al., "Heterologous protection against influenza by injection of DNA encoding a viral protein," Science 259:1745-1749, 1993; Lin et al., “Expression of recombinant genes in myocardium in vivo after direct injection of DNA” Circulation 52:2217-21, 1990); Wolff et al., "Long-term persistence of plasma DNA and foreign gene expression in mouse muscle,” Human Mol. Gen. 7:363-69, 1992).
- the construct Upon introduction of the naked vector construct into the animal's cell, the construct is then able to express the nucleic acid molecule (typically a gene) that it carries, which gene preferably comprises one (or more) of the unprocessed core antigen- envelope protein or nonstructural protein of HCV. Accordingly, upon expression of the desired peptide, an immune response is elicited from the host animal.
- the immune response includes CD8 + CTLs able to respond to different strains that exhibit a form of the desired peptide.
- kits for analyzing samples for the presence of HCV antigens or antibodies comprise a polypeptide or antibody of the invention and an appropriate solid phase. Preferably, the polypeptide is bound to the solid phase.
- kits can also provide one or more reagents and/or devices for the detection of the polypeptides or antibodies. A variety of formats, reagents and devices for inclusion within the kits, including means for detecting the antigens or antibodies, are discussed herein.
- kits for the induction of an immune response comprise compositions comprising a polypeptide of the invention in combination with an pharmaceutically acceptable carrier or diluent, and can also provide devices for administering or assisting in the administration of the composition.
- Examples relating to the isolation and production of the HCV unprocessed core antigen- envelope fusion protein, and uses thereof without a nonstructural protein are presented.
- Second, Examples relating to the isolation and production of a nonstructural protein, and uses thereof without the HCV core antigen-envelope fusion protein are presented.
- Third, Examples relating to the combination and use of a nonstructural protein with the HCV core antigen-envelope fusion protein are presented.
- Examples relating to the use of an HCV unprocessed core antigen-envelope fusion protein to induce an immune response in an animal are presented.
- RNA Ribonucleic acid
- a single-stranded DNA molecule was produced using random primers, reverse transcriptase and the RNA template.
- a double- stranded DNA molecule containing the HCV core-envelope region sequence was amplified by the PCR method using Taq polymerase and primers (i) and (ii).
- the cloned DNA molecule was subjected to sequence analysis for identification.
- the obtained molecule was designated EN-80-2.
- the DNA sequence of the molecule EN-80-2 is given in Fig. 1A (SEQ ID No. 7).
- the DNA molecule was derived from the HCV core and envelope regions and has 669bp. 2. Construction of a Plasmid Containing an HCV cDNA
- the molecule EN-80-2 was treated with restriction endonucleases Bam HI and EcoRI to obtain a DNA fragment containing the desired HCV cDNA.
- the obtained DNA fragment was inserted into a vehicle plasmid which had been first cleaved with the restriction endonucleases Bam HI and EcoRI, to obtain an expression plasmid, designated pEN-2.
- the expression of the HCV cDNA is under the control of a T7 promoter.
- the structure of the expression plasmid pEN-2 and a restriction map are depicted in Fig. 2.
- the expression plasmid pEN-2 was transformed into E. coli BL21 (DE3), spread onto an ampicillin-agar plate and placed at 37°C overnight. E. coli colonies producing HCV core antigen protein were selected by screening their expression products by SDS-PAGE and Western Blotting.
- the transformed E. coli colonies were incubated in a conditioned culture medium. The colonies were centrifuged and lysed by freezing-thawing cycles and lysozyme-digestion. The unprocessed core antigen-envelope protein product was released by the lysed cells and purified by column chromatography. The polypeptide was more than 90% pure.
- the unprocessed core antigen-envelope protein has a molecular weight of about 25,000 daltons as measured by electrophoresis through a sodium dodecyl sulfate-polyacrylamide gel.
- the purified unprocessed core antigen-envelope protein was subjected to an SDS-PAGE electrophoresis using standard procedures.
- the SDS-PAGE gel was washed with deionized water at 4°C for 15 minutes and washed with Blotting Buffer (0.15M sodium phosphate buffer, pH 6.7) at 4°C for 20 minutes.
- Blotting Buffer (0.15M sodium phosphate buffer, pH 6.7) at 4°C for 20 minutes.
- the polypeptide on the gel was then electroblotted onto nitrocellulose membrane under the Blotting Buffer at 1.3A for 1-1.5 hours.
- the membrane was washed with Wash Buffer (PBS-Tween 20, pH 7.4) and blocked with Blocking Buffer (0.1M NaCl, 5mM EDTA 50mM Tris, pH 7.2-7.4, 0.2% bovine serum albumin, 0.05% Nonidet p-40, 1M urea) overnight.
- Wash Buffer PBS-Tween 20, pH 7.4
- Blocking Buffer 0.1M NaCl, 5mM EDTA 50mM Tris, pH 7.2-7.4, 0.2% bovine serum albumin, 0.05% Nonidet p-40, 1M urea
- the membrane was reacted with the sera of the persons infected with/without hepatitis C, which were first diluted with 40% new born bovine serum/Tris-HCl (pH 7.4), 10X, at 40°C for 2 hours. After the reaction, the membrane was washed with Wash Buffer three times. The membrane was reacted with an anti- hIgG:HRPO conjugate (which was prepared as described hereafter) at 40°C for 2 hours. After the reaction, the membrane was washed with Wash Buffer three times and then reacted with 10 ml Substrate Solution.
- the unprocessed core antigen-envelope protein of the present invention was reactive with the sera of HCV patients but not reactive with the sera of healthy persons.
- a microtiter plate was coated with the purified unprocessed core antigen- envelope protein of the invention at appropriate concentrations and blocked with a buffer containing bovine serum albumin.
- the treated microtiter plate was stored at 2- 8°C.
- anti-hlgG Purified anti-human Immunoglobulin G
- HRPO horse radish peroxidase
- the OD4 9 2 JUI1 value per well subtracts the mean of the readings of the blanks (backgrounds).
- the difference (PCx-NCx) between the mean of the readings of the positive controls (PCx) and that of the negative controls (NCx) is equal to or more than 0.5.
- the Cut-off value (CO) is calculated by the following formula:
- the samples were expected to be positive; however, it is preferred to repeat the assay for the samples in duplicate. If the readings of either of the duplicate samples were less than the CO value, the samples were considered to be negative. If the duplicate samples were both more than or equal to the Cut-off value, the samples were considered to be positive. When the readings of test samples are more than NCx but less than the
- RNA nucleic acid
- a single-stranded DNA molecule was produced using random primers, reverse transcriptase and the RNA template.
- a double- stranded DNA molecule containing the HCV NS5 sequence was amplified by the PCR method using Taq polymerase and primers (i) and (ii).
- the cloned DNA molecule was subjected to sequence analysis for identification.
- the obtained molecule was designated EN-80-1.
- the DNA sequence of the molecule EN-80-1 is given in Figure 3 A, and the amino acid sequence encoded by the molecule is given in Figure 3B.
- the DNA molecule was derived from the genome of HCV nonstructural region 5 and has 803 bp (SEQ ID No. 9).
- the amino acid sequence of the molecule EN-80-1 is given in Fig. 3B (SEQ ID No. 10), and has 267 residues.
- the molecule EN-80-1 was treated with restriction endonucleases Bam HI and EcoRI to obtain a DNA fragment containing said HCV cDNA.
- the resulting DNA fragment was inserted into a vehicle plasmid which had been first cleaved with restriction endonucleases Bam HI and EcoRI, to obtain an expression plasmid, designated pEN-1.
- the expression of the HCV cDNA is under the control of a T7 promoter.
- the structure of the expression plasmid pEN-1 and restriction map are given in Fig. 4.
- E. coli colonies producing the HCV nonstructural protein were selected by screening their expression products by SDS-PAGE and Western Blotting.
- the transformed __. coli colonies were incubated in a conditioned culture medium. The colonies were centrifuged and lysed by freezing-thawing cycles and lysozyme-digestion. The protein product was released by the lysed cells and purified by column chromatography. The resulting polypeptide was more than 90% pure.
- the polypeptide has a molecular weight of about 29,000 daltons as measured by electrophoresis through a sodium dodecyl sulfate-polyacrylamide gel.
- the membrane was reacted with the sera of the persons infected with/without hepatitis C, which were first diluted with 40% New Born Bovine Serum/Tris-HCl (pH 7.4), 10X, at 40°C for 2 hours. After the reaction, the membrane was washed with Wash Buffer three times. The membrane was then reacted with an anti-hIgG:HRPO conjugate (which is prepared as described hereafter) at 40°C for 2 hours.
- the membrane was washed with Wash Buffer three times and then reacted with 10 ml Substrate Solution (0.01% 4-chloro-l-Naphthol, 18% methanol, 0.04M Tris, pH 7.2-7.4, 0.1 M NaCl and 0.01% H 2 0 2 ) for 20 minutes.
- the polypeptide of the present invention was reactive with the sera of HCV patients but was not reactive with the sera of healthy persons.
- a microtiter plate was coated with the NS5 nonstructural protein of the invention at appropriate concentrations and blocked with a buffer containing bovine serum albumin. The treated microtiter plate was stored at 2-8°C.
- Anti-hIgG:HRPO Conjugate Solution the anti-MgG:HRPO conjugate prepared as described above dissolved in Tris Buffer containing a proteineous stabilizer and antiseptics.
- Sample Diluent Tris Buffer containing a proteineous stabilizer and antiseptics.
- OPD Substrate Solution o-phenylene diamine (OPD) dissolved in citrate-phosphate buffer containing H 2 0 2 . (If the solution becomes orange, it means that the solution has been contaminated and cannot be used any more.)
- Stopping Solution 2N H 2 SO 4 solution.
- the OD 49 2nm va l ue P er e N subtracts the mean of the readings of the blanks (backgrounds).
- the difference (PCx-NCx) between the mean of the readings of the positive controls (PCx) and that of the negative controls (NCx) is equal to or more than 0.5.
- the samples When the readings of test samples were less than the CO value, the samples were considered to be negative (i.e., HCV antibodies could not be detected in the samples). When the readings of test samples were equal to or more than the CO value, the samples were expected to be positive; however, it is preferred to repeat the assay for the samples in duplicate. If the readings of either of the duplicate samples were less than the CO value, the samples will be negative. If the duplicate samples were both more than or equal to the CO value, the samples were considered to be positive.
- the samples When the readings of the test samples are more than NCx but less than the CO value by 20%, the samples should be regarded as questionable samples and the assay has to be repeated for the samples.
- the method was analogous to the ELISAs described above, except that unprocessed core antigen-envelope protein was combined with an NS5 nonstructural protein (9:1) (known as the EverNew Anti-HCV EIA ).
- This assay shows the results of an ELISA similar to those set forth above, and shows cooperative interaction between EN-80-2 and EN-80-1 proteins of HCV.
- the protocol for the ELISA is as follows:
- Coating buffer 0.05 M Tris-HCl/ 0.15 N NaCl/6 M Urea pH: 7.4 ⁇ 0.2.
- Washing buffer PBS with 0.05% Tween 20.
- Postcoating buffer PBS buffer with 1% BSA
- Coating procedure EN-80-1 and EN-80-2 proteins were added into coating buffer (final concentration: about 1.5 ⁇ g ml) and mixed at room temperature for 30 minutes. After mixing, the diluted EN-80-1 and EN-80-2 proteins were added into microtiter wells, 100 ⁇ g/well, and incubated in a 40°C incubator for 24 hours. The microtiter wells were then washed, and postcoating buffer was added into the wells. The microtiter wells were then let stand at 4°C for overnight. After postcoating, the coated microtiter wells can be used for anti-HCV antibody detection.
- Sample diluent 0.1 M Tris-HCl pH: 7.4 ⁇ 0.2 with 40% NBBS, 1% BSA and 2% mouse serum.
- Conjugate anti-human IgG monoclonal antibody coupled with HRPO using NalO After coupling, the anti-human IgG:HRPO conjugates were purified by S-200 gel filtration and were diluted in sample diluent.
- OPD tablets purchased from Beckman.
- Substrate diluent citrate-phosphate buffer containing H 2 O 2 .
- Stopping solution 2N H 2 SO4- Positive control: anti-HCV positive serum diluted in sample diluent.
- Negative control recalcified human serum, which is non-reactive for HBV markers, anti-HTV, anti-HTLV I and anti-HCV.
- Sample incubation incubated at 40 ⁇ 1°C for 30 ⁇ 2 minutes.
- Sample wash the wells were washed 3 times using washing buffer. 100 ⁇ l anti-human IgG:HRPO conjugate was added into each well.
- Conjugate incubation incubated at 40 ⁇ 1°C for 30 ⁇ 2 minutes.
- Conjugate wash the wells were washed 6 times using washing buffer. After washing, 100 ⁇ l substrate solution was added (the substrate solution was prepared by dissolving one tablet OPD in 5 ml substrate diluent), then the mixture was allowed to stand at room temperature for 10 minutes. In order to prevent light, the microtiter wells were covered with a black cover.
- cutoff value PCx X 0.25 + NCx.
- An absorbance equal to or greater than cutoff value indicated that a reaction was considered to be positive, which means reactive for anti-HCV antibody.
- An absorbance less than the cutoff value was considered to be negative, which means non-reactive for anti-HCV antibody.
- Sample source I G83, G191, G205 and G235 were GPT abnormal samples that were anti-HCV antibody negative and were collected from Taipei blood donation center.
- Sample source II G614 and G615 were anti-HCV antibody positive and were purchased from the U.S.A.
- Sample source HI: 8-5 was anti-HCV antibody positive and was collected from the Taichung blood donation center.
- Sample source IV N345 was a patient serum.
- This assay shows the results of an ELISA performed according to the protocol set forth in the First Assay, above, wherein a partial core protein was combined with the EN-80-1 (NS5) protein of HCV.
- the partial core protein consisted of amino acids 1 through 120, and was a gift from the Development Center of Biotechnology (DCB) in Taiwan.
- Sample source I G235 was a GPT abnormal sample, which was anti- HCV antibody negative and was collected from the Taipei blood donation center.
- Sample source II G614 and G615 were anti-HCV positive samples and were purchased from the U.S.A. TABLE 8
- Table 9 depicts the results of an assay that was similar to that in the Fifth Assay (V), and shows that there were no cooperative interactions between the HBV antigens HBsAg and HBcAg and the EN-80-1 protein of HCV.
- HBsAg purified from HBsAg positive human plasma.
- HBcAg derived from HBV cDNA fragment.
- Sample source I G30 and G49 were GPT abnormal samples, which were anti-HCV antibody positive and were collected from the Taipei Blood Donation Center.
- Sample source II G612, G613, G614 and G615 were anti-HCV antibody positive and were purchased from the United States of America.
- Table 10 shows a comparison of the detection of anti-HCV antibodies between the EverNew Anti-HCV EIA and the Abbott's kit (II).
- the samples for the test were obtained from the following sources:
- Sample source I G23, G26, G30, G32, G49, G58, G114, G128, G186, G231, G250 and G262 were GPT abnormal samples, which were anti- HCV antibody positive and were collected from Taipei blood donation center.
- Sample source II G612, G613, G614 and G615 were anti-HCV antibody positive and were purchased from U.S.A.
- Sample source HI VGH7, VGH11, VGH12, VGH13, VGH16, VGH26, VGH27, VGH29, VGH30, VGH32, VGH33, VGH40, VGH43, VGH46 and VGH52 were anti-HCV antibody positive and were collected from Taipei Veteran General Hospital. Classification for the samples from source HI:
- Control Recalcified human serum (non-reactive with HBV, anti-HCV and HTV). This human serum was also used to dilute the above-mentioned anti-HCV positive samples.
- EverNew Anti-HCV EIA Microtiter wells coated with EN-80-2 antigen.
- EverNew Anti-HCV EIA Microtiter wells coated with EN-80-1 and EN-80-2 antigens. Reference Kit: Abbott's kit (II).
- anti-HCV antibody in samples G128 240X, G231 672X, G612 804X, VGH2742X, VGH27 84X, VGH29 84X, VGH30 84X, VGH32 504X, VGH32 1008X, VGH33 84X, VGH33 168X, VGH40 9X, VGH43 9X and VGH43 18X could be detected by using EverNew Anti-HCV EIA (microtiter wells coated with EN-80-1 and EN-80-2 antigens) but was not detected using the Abbott's kit (II).
- SIXTH ASSAY Table 11 confirms the above-presented results and shows an enzyme immunoassay comparison of the detection of anti-HCV antibodies using partial core (EN-80-5 antigen, which is an HCV partial core antigen having a molecular weight of about 15,000 daltons as measured by electrophoresis through a SDS-polyacrylamide gel), core antigen-envelope protein (EN-80-2 antigen) and/or an HCV nonstructural protein (NS5; the EN-80-1 antigen discussed above).
- the samples for the assay were anti-HCV positive samples nos. N8, N81, N89, N12 and N302, and anti-HCV negative samples nos. N202, N203 and N302.
- the positive samples were diluted between 25X and 672X with 0.1M Tris-HCl, pH 7.4 (+/- 0.2) with 40% new born bovine serum, 1% BSA and 2% mouse serum.
- the samples were assayed in microtiter wells with a monoclonal anti-human IgG:HRPO conjugate solution, in combination with the following antigens or combinations of antigens: a.) NS5; b.) core antigen-envelope protein; c.) partial core protein; d.) NS5 and core antigen-envelope protein; e.) core antigen-envelope protein and partial core; and, f.) NS5, core antigen-envelope protein, and partial core.
- the following results were obtained:
- NS5 nonstructural protein were produced according to a standard procedure for producing monoclonal antibodies.
- a BALB/c mouse was immunized with the purified proteins described above in Examples 2 and 10, mixed with an adjuvant; and then the spleen cells were fused with mouse myeloma cells (FO cell line) using polyethylene glycol to form hybridoma cells.
- the desired clones producing desired monoclonal antibodies was obtained by screening the titer of the antibodies produced by the hybridoma clones so prepared.
- a hybridoma clone was designated EN-80-1-99.
- a core antigen-envelope protein (EN-80-2) was administered intramuscularly to ICR mice at 6-8 weeks of age.
- the first administration, boost and sampling schedule was as follows:
- Negative control Group (ID nos. 0-1 and 0-2) Day 0: no immunization.
- Test Group 1 (ID nos. 1-1, 1-2, 1-3, 1-4, 1-5 and 1-6)
- Test Group 2 (ID nos. 2-1, 2-2, 2-3, 2-4, 2-5 and 2-6) Day 0: 50 ⁇ g/mouse of EN-80-2 protein using complete Freund's adjuvant (CFA), GIBCO.
- CFA complete Freund's adjuvant
- Test Group 3 (ID nos. 3-1, 3-2, 3-3, 3-4, 3-5 and 3-6) Day 0: 50 ⁇ g/mouse of EN-80-2 protein using complete Freund's adjuvant (CFA), GIBCO.
- CFA complete Freund's adjuvant
- Day 13 1st boost, with 80 ⁇ g/mouse of EN-80-2 protein using incomplete Freund's adjuvant (IF A), GIBCO.
- Day 28 2nd boost, with 80 ⁇ g/mouse of EN-80-2 protein, in PBS.
- IF A incomplete Freund's adjuvant
- EIA enzyme immunoassay
- a rat anti-mouse:HRPO conjugate was added to the wells of a microtiter plate that had been coated with the following antigens or combinations of antigens: a.) NS5 (EN-80-1 antigen); b.) core antigen-envelope protein (EN-80-2 antigen); c.) partial core protein (EN-80-5 antigen); d.) NS5 and core antigen- envelope protein; e.) core antigen-envelope protein and partial core; and, f.) NS5, core antigen-envelope protein, and partial core.
- the samples used in the second EIA were as follows: 0-2 (50X diluted, from day 28); 0-2 (500X diluted, from day 28); 2-2 (2500X diluted, from day 28); 3-1 (12500X diluted, from day 39); 3-4 (2500X diluted, from day 39); 3-5 (2500X diluted, from day 39); 3-6 (2500X diluted, from day 39); and, 3-6
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AU59243/96A AU5924396A (en) | 1995-05-22 | 1996-05-22 | Compositions and methods for the diagnosis of, and vaccinati on against, hepatitis c virus (hcv) |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US44727695A | 1995-05-22 | 1995-05-22 | |
US08/447,276 | 1995-05-22 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO1996037606A1 true WO1996037606A1 (fr) | 1996-11-28 |
Family
ID=23775707
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/US1996/007378 WO1996037606A1 (fr) | 1995-05-22 | 1996-05-22 | Procedes et compositions de diagnostic d'infections par le virus de l'hepatite c et de vaccination contre le virus de l'hepatite c |
Country Status (3)
Country | Link |
---|---|
AU (1) | AU5924396A (fr) |
IL (1) | IL118364A0 (fr) |
WO (1) | WO1996037606A1 (fr) |
Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP1056762A1 (fr) * | 1998-01-30 | 2000-12-06 | The General Hospital Corporation | Immunisation genetique avec des proteines non structurales du virus de l'hepatite c |
WO2004046176A1 (fr) * | 2002-11-15 | 2004-06-03 | Glaxo Group Limited | Vaccin contre hcv |
US7078500B1 (en) | 1998-01-30 | 2006-07-18 | The General Hospital Corporation | Genetic immunization with nonstructural proteins of hepatitis C virus |
WO2020082145A1 (fr) * | 2018-10-22 | 2020-04-30 | Fundação Oswaldo Cruz | Polypeptide, cassette d'expression, vecteur d'expression, cellule hôte, trousse pour le triage immunologique du vhc et/ou le diagnostic de l'hépatite c, composition, utilisation d'au moins un polypeptide, et méthodes pour produire un polypeptide, pour le triage immunologique du vhc et le diagnostic de l'hépatite c |
Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0442394A2 (fr) * | 1990-02-16 | 1991-08-21 | United Biomedical, Inc. | Peptides synthétiques spécifiques pour la détection d'anticorps contre HVC, diagnostic des infections par HVC et prévention de celles-ci comme vaccins |
EP0450931A1 (fr) * | 1990-04-04 | 1991-10-09 | Chiron Corporation | Combinaisons d'antigènes de l'hépatitis C virus (HCV) pour usage dans des échantillons immunologiques pour anticorps anti-HCV |
EP0463848A2 (fr) * | 1990-06-25 | 1992-01-02 | The Research Foundation for Microbial Diseases of Osaka University | Particules du virus de l'hépatite non-A non-B |
WO1993017110A2 (fr) * | 1992-02-21 | 1993-09-02 | The Wellcome Foundation Limited | Polypeptide de recombinaison du virus de l'hepatite c |
WO1994025486A1 (fr) * | 1993-04-30 | 1994-11-10 | Lucky Limited | Agents ameliores pour diagnostiquer la presence de vhc |
-
1996
- 1996-05-22 WO PCT/US1996/007378 patent/WO1996037606A1/fr active Application Filing
- 1996-05-22 IL IL11836496A patent/IL118364A0/xx unknown
- 1996-05-22 AU AU59243/96A patent/AU5924396A/en not_active Abandoned
Patent Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0442394A2 (fr) * | 1990-02-16 | 1991-08-21 | United Biomedical, Inc. | Peptides synthétiques spécifiques pour la détection d'anticorps contre HVC, diagnostic des infections par HVC et prévention de celles-ci comme vaccins |
EP0450931A1 (fr) * | 1990-04-04 | 1991-10-09 | Chiron Corporation | Combinaisons d'antigènes de l'hépatitis C virus (HCV) pour usage dans des échantillons immunologiques pour anticorps anti-HCV |
EP0463848A2 (fr) * | 1990-06-25 | 1992-01-02 | The Research Foundation for Microbial Diseases of Osaka University | Particules du virus de l'hépatite non-A non-B |
WO1993017110A2 (fr) * | 1992-02-21 | 1993-09-02 | The Wellcome Foundation Limited | Polypeptide de recombinaison du virus de l'hepatite c |
WO1994025486A1 (fr) * | 1993-04-30 | 1994-11-10 | Lucky Limited | Agents ameliores pour diagnostiquer la presence de vhc |
Cited By (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP1056762A1 (fr) * | 1998-01-30 | 2000-12-06 | The General Hospital Corporation | Immunisation genetique avec des proteines non structurales du virus de l'hepatite c |
EP1056762A4 (fr) * | 1998-01-30 | 2003-07-23 | Gen Hospital Corp | Immunisation genetique avec des proteines non structurales du virus de l'hepatite c |
US7078500B1 (en) | 1998-01-30 | 2006-07-18 | The General Hospital Corporation | Genetic immunization with nonstructural proteins of hepatitis C virus |
CN100335637C (zh) * | 1998-01-30 | 2007-09-05 | 总医院有限公司 | 采用丙型肝炎病毒非结构蛋白进行的基因免疫 |
WO2004046176A1 (fr) * | 2002-11-15 | 2004-06-03 | Glaxo Group Limited | Vaccin contre hcv |
WO2004046175A1 (fr) * | 2002-11-15 | 2004-06-03 | Glaxo Group Limited | Vaccin contre l'hepatite c |
WO2020082145A1 (fr) * | 2018-10-22 | 2020-04-30 | Fundação Oswaldo Cruz | Polypeptide, cassette d'expression, vecteur d'expression, cellule hôte, trousse pour le triage immunologique du vhc et/ou le diagnostic de l'hépatite c, composition, utilisation d'au moins un polypeptide, et méthodes pour produire un polypeptide, pour le triage immunologique du vhc et le diagnostic de l'hépatite c |
Also Published As
Publication number | Publication date |
---|---|
IL118364A0 (en) | 1996-09-12 |
AU5924396A (en) | 1996-12-11 |
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