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WO1992012992A2 - Basic structural immunogenic polypeptides having epitopes for hcv, antibodies, polynucleotide sequences, vaccines and methods - Google Patents

Basic structural immunogenic polypeptides having epitopes for hcv, antibodies, polynucleotide sequences, vaccines and methods Download PDF

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Publication number
WO1992012992A2
WO1992012992A2 PCT/US1992/000356 US9200356W WO9212992A2 WO 1992012992 A2 WO1992012992 A2 WO 1992012992A2 US 9200356 W US9200356 W US 9200356W WO 9212992 A2 WO9212992 A2 WO 9212992A2
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recited
hcv
polypeptide
antibody
basic
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PCT/US1992/000356
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French (fr)
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WO1992012992A3 (en
Inventor
Girish J. Kotwal
Bahige M. Baroudy
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James N. Gamble Institute Of Medical Research
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Publication of WO1992012992A3 publication Critical patent/WO1992012992A3/en

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    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1131—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against viruses
    • 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 to biological materials for detecting hepatitis C virus (HCV) and preventing and/or treating HCV disease. More particularly, the instant invention relates to truncated, basic structural immunogenic polypeptides having epitopes for HCV, to polynucleotide sequences encoding the HCV polypeptides and antisense polynucleotide sequences derived therefrom, to antibodies raised against the HCV polypeptides, to anti-idiotype antibodies raised against antibodies to the HCV polypeptides, and HCV vaccines. These biological materials are believed to be effective as screening agents for HCV as well as for acute and chronic HCV infection, and as protective agents against HCV disease. Background
  • Hepatitis is the medical term for inflammation or disease of the liver. Viral hepatitis signifies liver inflammation or disease induced by an infecting hepatotropic virus, and is a major worldwide public health problem. At present, five specific hepatotropic viruses involved in this disease have been isolated and characterized. They are identified as hepatitis A virus (HAV), hepatitis B virus (HBV), hepatitis C virus (HCV), hepatitis D virus (HDV) and hepatitis E virus (HEV). These hepatotropic viruses are believed to cause hepatitis, resulting in illnesses characterized by fever, nausea, vomiting, anorexia (loss of appetite) and, in some instances, jaundice and even death.
  • HAV hepatitis A virus
  • HBV hepatitis B virus
  • HCV hepatitis C virus
  • HDV hepatitis D virus
  • HEV hepatitis E virus
  • HCV is believed to cause the majority (at least about 90%) of the parenterally or blood transmitted NANBH whereas HEV is believed to be responsible for the transmission of enteric
  • HAV (fecal/oral) NANBH.
  • HEV fecal/oral NANBH.
  • HBV fecal/oral NANBH.
  • HCV fecal/oral NANBH.
  • HDV are transmitted principally by the parenteral routes and blood or blood derived products. Even though all five of these hepatotropic viruses
  • HDV is unable to grow and replicate on its own.
  • HDV requires the co-presence of HBV for its replication in humans and therefore is found only in patients infected with HBV.
  • HAV and HEV cases and in over 85% of HBV cases progression to chronic hepatitis from acute HCV is observed in about 25-55% of HCV cases.
  • HCV viruses are known to cause serious liver disease such as cirrhosis of the liver, non-established malignant hepatoma (liver cancer) and the like.
  • HCV parenteral NANBH
  • Clinical diagnosis of HCV has been accomplished heretofore primarily by the exclusion of other hepatitis human viruses such as HAV and HBV.
  • methods used hitherto to detect putative HCV antigens and antibodies are agar-gel diffusion, counter immunoelectrophoresis, immunofluorescence microscopy , immune electron microscopy, radioimmuno assay, and enzyme-linked immunoabsorbent assay (ELISA).
  • ELISA enzyme-linked immunoabsorbent assay
  • one present method utilized to detect HCV involves the detection of anti-C100 antibody to the non-structural region of HCV, i.e., C100 protein (antigen). This is accomplished by a commercially available kit distributed by Ortho Diagnostics under the brand name Ortho HCV Ab ELISA test.
  • this assay for detecting HCV cannot identify all blood samples containing HCV, especially those contaminated with acute HCV infections. For instance, it has been reported that this assay detects false positives approximately 40-60% of the time. See Mimms, L. et al.: The Lancet, (336): 1590-1591 (Dec. 22/29, 1990); Zuck, T.F.
  • RIBA I recombinant immunoblot assay I
  • HCV In yet another present method utilized to detect HCV, it involves the indirect detection of anti-HCV antibody. This is accomplished by a methodology developed by Abbott and is referred to as a neutralization assay. Unfortunately, the neutralization assay for detecting HCV is believed to be unreliable because the test utilizes only a portion of HCV, i.e., the non-structural C100 protein (antigen), while a true HCV neutralization assay requires the utilization of the infectious particle, i.e., the virion, of HCV.
  • the neutralization assay for detecting HCV is believed to be unreliable because the test utilizes only a portion of HCV, i.e., the non-structural C100 protein (antigen), while a true HCV neutralization assay requires the utilization of the infectious particle, i.e., the virion, of HCV.
  • HCV parenteral NANBH
  • Whether this form of NANBH (HCV) is acute or chronic may be determined by examining the liver tissue by biopsy according to the criterion described in Sheila Sherlock, diseases of the Liver and Eiliary System, eighth edition, published by Blackwell Scientific Publications, London, pp. 326-333 (1989).
  • liver biopsies are expensive, painful and may lead to patient complications.
  • this technique for diagnosing HCV obviously is inapplicable for screening blood and blood derived products. Because present tests for the detection of HCV (parenteral NANBH) are ineffective, time
  • HCV parenteral NANBH
  • HCV human immunodeficiency virus
  • HCV parenteral NANBH
  • the present invention alleviates and overcomes certain of the above-referenced problems and shortcomings of the present state of the HCV art througn the discovery of novel immunogenic HCV polypeptides, each having epitopes unique to HCV.
  • the uniqueness of the epitopes have been determined by their immunological reactivity with anti-HCV antibodies and lack of immunological reactivity with antibodies to HBV.
  • novel immunogenic HCV polypeptides of the present invention are truncated structural polypeptides which have been discovered from the structural region of a human HCV genome upon analysis of a putative amino acid sequence translated therefrom.
  • the amino acid sequences of the novel immunogenic HCV polypeptides are believed to contain at least about 15 amino acid residues and more preferably at least about 17 amino acid residues, and lie within the first four percent (4%) of the putative amino acid sequence for the human HCV polyprotein.
  • novel immunogenic HCV polypeptides of the instant invention are basic polypeptides having an estimated pI on the order of about 12, and are believed to include at least about 7 amino acid residues (45%) in their Chou-Fasman (CF) turns, and more particularly between about 7 and 17 amino acid residues (45-100%) in their CF turns.
  • the calculated molecular weights of the novel immunogenic HCV polypeptides of the present invention are at least about 1500 and more particularly in the range of between about 1500 and about 2500.
  • novel immunogenic HCV polypeptides of the instant invention are further characterized as having in their amino acid sequences at least about 4 arginine amino acid residues (25%) and more particularly between about 4 and 7 (25-47%) arginine amino acid residues, at least about 11 hydrophillic amino acid residues (70%) and more particularly between about 11 and 14 hydrophillic amino acid residues (70-87%), and at least about 5 polar amino acid residues (30%) and more particularly between about 5 and 8 polar amino acid residues
  • novel immunogenic HCV polypeptides of the instant invention include within their amino acid sequences at least about 5 basic amino acid residues (30%) and more preferably between about 5 and 7 basic amino acid residues (30-47%).
  • polypeptides of the present invention are designated as FGB1 and FGB2.
  • the FGB1 polypeptide has a calculated molecular weight of about 1908, includes 15 amino acids and has the following sequence:
  • the FGB2 polypeptide has a calculated molecular weight of about 1811, also includes 15 amino acids and has the following sequence:
  • the above-recited amino acid sequences for the novel FGB1 and FGB2 polypeptides are believed to be truncated structural segments derived from a putative amino acid sequence of a human HCV capsid protein which is believed to be encoded within the first 4% of codons following the predicted ATG initiation codon of a human HCV polyprotein. It is further believed that the amino acid sequence for the novel FGB1 polypeptide lies within the putative amino acid sequence for the human HCV capsid protein near its -NH 2 terminal end whereas the amino acid sequence for the novel FGB2 polypeptide lies within the putative amino acid sequence for the human HCV capsid protein near its -COOH terminal end.
  • the FGB1 and FGB2 polypeptides may also include, for example, an additional amino acid
  • the FGB1 and FGB2 polypeptides may include an amino acid residue, such as tyrosine or histidine, at the -COOH terminal end in order to facilitate the labeling of these novel polypeptides with a labeling moiety, such as radioactive iodine or phosphorous.
  • the FGB1 and FGB2 polypeptides have calculated molecular weights of about 2,174 and 2,077, respectively. It is believed that the addition of the cysteine and tyrosine amino acid residues uniquely and advantageously enhances the reactivity of the immunogenic HCV polypeptides of the instant invention with antibodies in, for example, immuno assays.
  • polypeptides in accordance with the present invention that are believed to be immunogenic and have epitopes for HCV are recited hereinafter. These polypeptides of the instant invention have been designated herein throughout as FGB polypeptides, and include:
  • R or Arg represents argenine
  • N or Asn represents asparagine
  • C or Cys represents cysteine
  • Q or Gln represents glutamine
  • K or Lys represents lysine
  • P or Pro represents proline
  • S or Ser represents serine
  • T or Thr represents threonine
  • Y or Tyr represents tyrosine.
  • novel immunogenic HCV polypeptides and especially the FGB1 and FGB2 polypeptides, may be used in diagnostic tests and kits, such as enzyme-linked immunosorbent assays (ELISA, EIA), radioimmunoassays (RIA), immunoblot assays, and the like, to detect the presence of specific HCV antibodies present in blood, sera, semen, or other biological materials of patients with HCV.
  • diagnostic tests and kits such as enzyme-linked immunosorbent assays (ELISA, EIA), radioimmunoassays (RIA), immunoblot assays, and the like, to detect the presence of specific HCV antibodies present in blood, sera, semen, or other biological materials of patients with HCV.
  • ELISA enzyme-linked immunosorbent assays
  • RIA radioimmunoassays
  • immunoblot assays and the like
  • the FGB1 and FGB2 polypeptides utilized in this ELISA included the cysteine and tyrosine amino acid residues at their respective -NH 2 and -COOH terminal ends.
  • the 44 sera were also tested for anti-C100 HCV antibody (EIA, Ortho/Chiron), recombinant immunoblot assay I (RIBA I, Ortho/Chiron), and neutralization (Neut., Abbott). Eight of eight samples that were reactive for C100 EIA, RIBA I and Neut., contained antibodies to FGB1 or FGB2
  • This panel was used to compare the ELISA utilizing FGB1 with an experimental C-200/C-22 ELISA (second generation of Chiron Corp.). Like with the first coded panel, the FGB1 polypeptide utilized in this
  • ELISA included the cysteine and tyrosine amino acid residues at its respective -NH 2 and -COOH terminal ends.
  • the 115 sera were also tested with C-100 ELISA
  • HCV-FGB1 ELISA C-200/C-22 ELISA and HCV-FGB1 ELISA, one was reactive with RIBA-II and one was indeterminate.
  • One serum sample (HCV PANEL II, 1r+) that was reactive with HCV-FGB1 ELISA was non-reactive with both the
  • HCV-FGB1 ELISA HCV PANEL III
  • the FGB1 polypeptide utilized in this ELISA included the cysteine and tyrosine amino acid residues at its respective -NH 2 and -COOH
  • o Recipients were patients who received one or more blood transfusions and were diagnosed as having viral hepatitis.
  • the instant invention identifies new epitopes to the structural region of the human HCV genome to which HCV infected patients elicit antibodies. Based upon the results from the coded HCV panels, it is believed that these antibodies correlate with the presence of true positive HCV antibody as defined by C-100 EIA, C-200/C22 EIA, RIBA I and RIBA II. Moreover, it is believed that assays using the truncated immunogenic HCV polypeptides and in particular the FGB1 and FGB2 polypeptides of the instant invention, which are derived from the structual region relatively near the predicted ATG initiation codon of the human HCV genome, to detect the presence of HCV are more
  • novel immunogenic HCV polypeptides of the instant invention are therefore believed to allow for significantly increased specificity in the detection of antibodies in blood or blood derived products contaminated with HCV or in patients with acute or chronic HCV at a relatively lower cost as compared to existing HCV tests.
  • the present invention also contemplates the detection of passive HCV-specific antibodies which have been transferred to patients via transfused blood containing detectable levels of the specific HCV antibodies. Quite remarkably, it is believed to now be possible to detect passive HCV-specific antibodies almost immediately following the initial transfusion initiated HCV infection when following the teachings of the present invention. Even more remarkable, it is believed to now be possible to detect HCV-specific antibodies, i.e., passive HCV-specific antibodies followed by actively produced HCV-specific antibodies, in transfusion initiated HCV infected patients at relatively high levels for several months, as verified by the fifth coded panel and illustrated in FIGS.
  • HCV-specific antibodies it is meant herein to refer to those antibodies present in donated blood which are specific for HCV antigens and which have been produced by the donor.
  • actively produced antibodies it is meant herein to refer to those antibodies which are specific for HCV antigens but which have been produced by a person following HCV infection, such as a
  • HCV-specific antibody was detected by HCV-FGB1 ELISA in all six recipients and its
  • recipients with the exception of recipient #10805 (See FIG. 11D), on average had detectable HCV-specific active antibody within a month of transfusion
  • FIGS. 11A-11G report the detection of passive HCV-specific antibody at seven days following transfusion, it is believed that passive HCV-specific antibody can be detected in a recipient as early as about 20 minutes or earlier, e.g. whatever the time is required for blood to circulate, following transfusion with donated blood which contains detectable levels of passive
  • polynucleotide sequences which encode the two novel HCV polypeptides, FGB1 and FGB2 , respectively, or degenerate polynucleotides which are complementary or correspond to all or certain nucleotides of the polynucleotide sequences.
  • the present invention therefore contemplates the following novel polynucleotide sequences I and II which are believed to encode the novel FGB1 and FGB2 polyeptides, respectively:
  • novel polynucleotides which are complementary or correspond to all or certain nucleotides thereof; and novel polynucleotide sequences obtained by
  • novel polynucleotide sequences of the instant invention may be inserted into recombinant expression vectors or the like and used to transform cells in order to recombinantly produce the FGB1 and FGB2 polypeptides.
  • novel polynucieotide sequences are their use in kits as probes or primers for the detection of RNA or nucleic acids from HCV in samples or other biological materials, such as sera, ascites, lymph, infected tissue, and cell cultures.
  • the probes or primers which are complementary or correspond to the above-recited polynucieotide sequences should be of a sufficient length which allows for the detection of HCV RNA or DNA, respectively.
  • 6-8 nucleotides complementary to or derived from the above-recited polynucleotide sequences may be a workable length
  • sequences of 10-12 nucleotides complementary thereto or derived therefrom are preferred and about 20-45 nucleotides and in particular about 20 nucleotides complementary or derived therefrom thereto appear to be most optimal.
  • the instant invention is also concerned with antisense single stranded DNA and RNA sequences derived from the polynucleotide sequences encoding the FGB1 or FGB2 polypeptides, which have the ability to bind to HCV RNA and block protein translation and/or prevent HCV RNA replication. Still further, the present invention is concerned with ds DNA fragments which, when inserted into appropriate vectors such as retroviral vectors and introduced into infected liver cells results in the transcription of the antisense RNA sequences.
  • antisense polynucleotide sequences can be designed to include in their construct, for example, ribozymes that may play a role in the therapeutic treatment of HCV by catalytic degradation of the HCV RNA in the infected liver cells.
  • the present invention further contemplates the production of antibodies, polyclonal or monoclonal, raised against the novel immunogenic HCV polypeptides of the instant invention.
  • it is concerned with the production of anti-idiotype antibodies raised against antibodies to the novel immunogenic HCV polypeptides.
  • the novel antibodies of the present invention can be used in diagnostic tests and kits to detect HCV proteins (antigens) or anti-HCV antibodies, respectively.
  • HCV proteins antigens
  • anti-HCV antibodies anti-HCV antibodies
  • Still another aspect of the instant invention is concerned with vaccines as prophylaxis against HCV infection by administering the novel immunogenic HCV polypeptides of the instant invention, either individually or in combination, to individuals.
  • the vaccines of the instant invention comprise an
  • immunogenic HCV polypeptide such as FGB1 and/or FGB2 polypeptides, in pharmacologically effective doses mixed in pharmaceutically acceptable excipients.
  • novel immunogenic HCV polypeptides of the instant invention are conjugated to, for example, carrier proteins, antibodies, or particle-forming proteins, such as those associated with hepatitis B surface antigen, and mixed with an adjuvant in order to elicit protective antibodies when administered to individuals.
  • novel antibodies to the novel immunogenic HCV polypeptides of the present invention may be used for passive immunity in short term therapy against HCV.
  • Fig. 1 is a graphic illustration of the hydrophilicity and hydrophobicity (surface
  • Fig. 2 is a grapnic illustration of the hydrophilicity and hydrophobicity (surface
  • Fig. 5 is a graphic illustration depicting that there are seventeen (17) amino acid residues of the Cys-FGB2-Tyr polypeptide present in its
  • Fig. 7 is a HPLC profile that shows the purity of the Cys-FGB1-Tyr polypeptide which has a retention time of about 12.45 minutes;
  • Fig. 3 is a HPLC profile that shows the purity of the Cys-FGB2-Tyr polypeptide which has a retention time of about 17.73 minutes;
  • Fig. S is a mass spectrum profile that shows the purity of the Cys-FGB1-Tyr polypeptide and the molecular weight thereof
  • Fig. 10 is a mass spectrum profile that shows the purity of the Cys-FGB2-Tyr polypeptide and the molecular weight thereof
  • Figs. 11A-11G are correlations between antibodies to HCV-FGB1 and ALT levels
  • antibodies raised against the immunogenic HCV polypeptides antibodies raised against the immunogenic HCV polypeptides, anti-idiotype antibodies raised against the HCV antibodies to the immunogenic HCV polypeptides, HCV vaccines, and methods.
  • the present invention provides for the discovery of highly immunogenic HCV polypeptides, each having epitopes for HCV.
  • the immunogenic HCV is highly immunogenic HCV polypeptides, each having epitopes for HCV.
  • polypeptides of the instant invention have been derived from the putative amino acid sequence of the HCV polyprotein encoded by the five prime (5') end of a human isolate of a HCV genome.
  • the amino acid residues of the epitopes of the immunogenic HCV polypeptides are believed to lie within the first 4% of the amino acid residues in the putative amino acid sequence of the HCV polyprotein.
  • the immunogenic HCV polypeptides of the instant invention are generally basic, truncated structural polypeptides of at least about 15 amino acids in length and are preferably between about 15 and about 17 amino acids.
  • the immunogenic HCV polypeptides of the present invention have calculated molecular weights of at least about 1500 and preferably of between about 1500 and about 2500, and include at least about 7 amino acid residues (45%) in their Chou-Fasman (CF) turn, as evidenced by Figs. 3-6. As more particularly shown in Figs. 5 and 6, certain of the immunogenic HCV polypeptides of the instant invention may include at least about 12-17 amino acids residues (80%-100%) in their CF turns.
  • At least about 11 of the amino acid residues (70%) within their amino acid sequences are hydrophillic.
  • at least about 5 (30%) and preferably between about 5 and 8 (30-53%) of the amino acid residues of the immunogenic HCV polypeptides of the present invention are polar amino acid residues.
  • the immunogenic HCV polypeptides of the instant invention have at least about 5 basic amino acid residues (30%) and more preferably between about 5 and 7 basic amino acid residues (30-47%). Accordingly, the estimated pI for the HCV polypeptides of the instant invention are typically on the order of about 12.
  • the immunogenic HCV polypeptides have at least about 4 arginine amino acid residues (25%) in their amino acid sequences and more preferably include between about 4 and 7 arginine residues (25-47%).
  • polyprotein derived from a human HCV isolate were subjected to hydrophilicity/hydropnobicity/antigenicity analysis. See Fig. 1. This was accomplished via a protein analysis program marketed under the name Mac Vector by International Bio Technologies Inc., P.O. Box 9558, New Haven, CT 06535. According to the hydrophilicity, surface probacility and antigenic index plots as shown in Fig. 1, the first 144 or so putative amino acids following the predicted ATG initiation codon within the HCV structural gene encoding the putative structural polyprotein derived from the human HCV isolate presented what appears to be a strong antigenic region. As a result, the hydrophilicity, surface probability and antigenic index plots of these first 144 putative amino acids were expanded, as shown in Fig.
  • HCV polypeptides of the instant invention are designated herein throughout as FGB1 and FGB2 and, as indicated hereinbefore, have been derived from the putative amino acid sequence encoded by the five prime (5') end of a human isolate of a HCV genome. Moreover, their epitopes are
  • amino acid sequences for FGB1 and FGB2 correspond to the putative amino acid sequences designated as 5-19 and 102-116, respectively, following the amino acid encoded by a predicted ATG initiation codon of a human HCV genome.
  • amino acid sequences for the novel FGB1 and FGB2 polypeptides, respectively are as follows:
  • Arg (R) represents arginine
  • Asn (N) represents asparagine
  • Cys (C) respresents cysteine
  • Gln (Q) represents glutamine
  • Gly (G) represents glycine
  • Lys (K) represents lysine
  • Pro (P) represents proline
  • Ser (S) represents serine
  • Thr (T) represents threonine
  • Trp (W) represents tryptophan
  • Tyr (Y) represents tyrosine.
  • parentheticals above represent optional but preferred amino acids added either individually or together to the immunogenic HCV polypeptide sequences for
  • immunogenic HCV polypeptides of the instant invention by enhancing their attachment to, for example, the microwell plates possibly by interaction in such a manner that they leave the rest or greater portion of the novel HCV immunogenic polypeptides exposed to the reactive antibodies during, for example, an ELISA type assay.
  • Cys and Tyr represent preferred optional amino acids, other optional amino acid residues or other moieties suitable for the purposes stated herein are envisioned by the instant invention.
  • cystine may be substituted for the cysteine amino acid residue for conjugation purposes whereas histidine (His) may be substituted for the tyrosine amino acid residue for labeling purposes.
  • FGB1 and FGB2 refer broadly to the FGB1 and FGB2 polypeptides as either including or excluding one or both of the optional amino acids or moieties, respectively, when it is specifically intended for such polypeptides to include both of the optional amino acids, i.e., Cys and Tyr, they will be referred to herein as Cys-FGB1-Tyr and Cys-FGB2-Tyr, respectively.
  • the FGB1 polypeptide When the FGB1 polypeptide includes the optional Cys and Tyr amino acid residues at its respective -NH 2 and -COOH terminal ends, it has 17 amino acids in its sequence, a molecular weight of about 2174, a pl of about 12.484, 3 amino acids (53%) in its CF turn (Fig. 3), 14 hydrophillic amino acids (82%), 8 polar amino acids (53%), 7 basic amino acids (41%), 4 actual Arg amino acid residues (24%), and a combination of 4 Arg and 3 Lys amino acid residues (41%).
  • the FGB1 polypeptide when free of such optional, but preferred amino acids, it has 15 amino acids in its sequence, a molecular weight of about 1379, a pI of about 12.958, 7 amino acids (47%) in its CF turn (Fig. 4), 13 hydrophillic amino acid residues (37%), 7 polar amino acid residues (47%), 7 basic amino acid residues (47%), 4 actual Arg amino acid residues (27%), and a combination of 4 Arg and 3 Lys amino acid residues (47%). See Cys-FGB1-Tyr and FGB1 TABLES.
  • the FGB2 polypeptide when it includes the optional, but preferred amino acid residues, i.e., the Cys and Tyr amino acid residues at its respective -NH 2 and -COOH terminal ends, it has 17 amino acids, a molecular weight of about 2077, a pI of about 12.306, 17 amino acids (100%) in its CF turn (Fig. 5), 12 hydrophillic amino acid residues (71%), 7 polar amino acid residues (41%), 5 basic amino acid residues (29%), 5 actual Arg amino acid residues
  • the FGB2 polypeptide when free of the optional Cys and Tyr amino acid residues at its respective -NH 2 and -COOH termini, it has 15 amino acids, a molecular weight of about 1311, a pI of about 12.656, 12 amino acids (80%) in its CF turn (Fig. 6), 11 hydrophillic amino acid residues (73%), 5 polar amino acid residues (33%), 5 basic amino acid residues (33%), 5 actual Arg amino acid residues (33%), and no Lys amino acid residues. See Cys-FGB2-Tyr and FGB2 TABLES.
  • the present invention contemplates other equivalent polypeptides whose fingerprint
  • EpI Estimated pI
  • any such FGB polypeptide or immunogenic HCV polypeptide of the present invention may be altered to delete or include only the cysteine or tyrosine amino acid residue or both such residues at their respective terminal ends in accordance with the teachings of the instant invention.
  • the immunogenic HCV polypeptides and in particular the FGB1 and FGB2 polypeptides of the instant invention can be produced by chemical snythesis, recombinant technology or any other methods available in the art so long as the methodology selected does not interfere with their utilities stated herein.
  • alternative amino acid residues may be substituted for those recited in the above sequences for the FGB1 and FGB2 polypeptides so long as the substituted amino acids do not defeat the utility of such novel immunogenic HCV polypeptides.
  • Lys and Arg amino acid residues may be freely substituted for each other within the amino acid sequences.
  • Ser and Thr amino acid residues may be freely substituted for one another.
  • the reference herein to at least about 4 Arg amino acid residues (25%) means that the reference herein to at least about 4 Arg amino acid residues (25%) means that the
  • sequences of the immunogenic HCV polypeptides of the instant invention include at least about 4 Arg amino acid residues or at least about 4 Lys amino acid residues or a combination of at least about 4 Lys and Arg amino acid residues.
  • the above-recited sequence for the FGB1 polypeptide has about 7 Arg amino acid residues (41%), i.e., 4 Arg and 2 Lys, when the optional amino acids are included and about 7 Arg amino acid residues (47%), i.e., 4 Arg and 3 Lys, when the optional amino acids are excluded.
  • the sequences for the immunogenic HCV polypeptides of the instant invention include at least about 4 actual Arg amino acid
  • amino acids may be added to or deleted from the amino acid sequences of the HCV immunogenic polypeptides of the instant invention to expand or shorten same so long as the objectives of the instant invention are not defeated.
  • Cys and Tyr amino acid residues can be added to the respective amino and carboxyl termini of the FGB1 and FGB2 polypeptides without interfering with their function as HCV epitopes.
  • modified or varied amino acid sequences are considered to be equivalents to the immunogenic HCV polypeptides and in particular the FGB1 and FGB2 polypeptides and are contemplated within the scope of the present invention.
  • immunogenic HCV polypeptides and in particular the FGB1 and FGB2 polypeptides of the instant invention can be any immunogenic HCV polypeptides and in particular the FGB1 and FGB2 polypeptides of the instant invention.
  • Cys-FGB1-Tyr and Cys-FGB2-Tyr polypeptides can be synthesized on an Applied BioSystem Peptide Synthesizer 430 using solid phase strategy, as described in Merrifield, F.B.: J. Am. Chem. Soc., 85:2149-2154 (1963) and Mitchell, A. R. et al.: J. Orq. Chem., 43:2845-2854 (1978). Boc amino acids (t-Boc) are activated as benzotriazole (HOBt) esters and coupling is performed in N-methyl- pyrrolidinone.
  • Boc amino acids t-Boc
  • HOBt benzotriazole
  • the side chain protecting groups are Asp (OBzl), Ser (OBzl), Thr (OBzl), Lys (Cl-z), Arg (Tos), Tyr (Br-z), Trp (CHO), Cys (p-MeBzl). Syntheses can be performed starting with 0.5 mmole of BocTyr (BrZ) Pam resin. Deprotection and cleavage of the Cys-FGB1-Tyr and Cys-FGB2-Tyr polypeptides from the resin can be performed using hydrogen fluoride. See Tables 2, 3, 4 and 5. The Cys-FGB1-Tyr and
  • Cys-FGB2-Tyr polypeptides can be purified to greater than 95% using reversed-phase HPLC.
  • Analytical HPLC using the applied Biosystems 130A separation system with a microbore column show the Cys-FGB1-Tyr and Cys-FGB2-Tyr polypeptides to be greater than 98% pure. See Figs. 7 and 8. The authenticity of the
  • Cys-FGB1-Tyr and Cys-FGB2-Tyr polypeptides can be confirmed by amino acid analysis using Applied Biosystems 420A automated hydrolyzer, see Tables 6 and 7, and by molecular weight determination using the
  • Trp (W) 0, 00 0.00 0.00
  • the HCV polypeptides including the FGB1 and FGB2 polypeptides of the present invention also can be produced by recombinant techniques well known to those of skill in the art. That is, they can be produced by gene expression of a ds DNA comprising at least one of the polynucleotide sequences which encode the polypeptides and especially the FGB1 and/or the FGB2 polypeptides of the instant invention, or a degenerate polynucleotide sequence obtained therefrom by, for example, substituting at least one nucleotide in one of the polynucleotide sequences encoding the FGB1 or FGB2 polypeptides in accordance with degeneracy of genetic code.
  • a polynucieotide sequence encoding the FGB1 polypeptide is:
  • A represents a deoxyriboadenylic acid residue (dAMP)
  • G represents a deoxyriboguanylic acid residue (dGMP)
  • C represents a deoxyribocytidylic acid residue (dCMP)
  • T represents a thymidylic acid residue (TMP)
  • A represents a riboadenylic acid residue (AMP)
  • G represents a riboguanylic acid residue (GMP)
  • C represents a ribocytidylic acid (CMP)
  • U which is substituted for T, represents a ribouridylic acid residue (UMP).
  • the appropriate codons must be included in the expressing polynucleotide sequences, and such polynucleotide sequences are within the contemplation of the instant invention.
  • Such codons which encode for Cys and Tyr amino acid residues are well known to those of skill in the art and include, for example, TGT or TGC (DNA) or UGU or UGC (RNA) for Cys and TAT or TAC (DNA) or UAU or UAC (RNA) for Tyr.
  • substitution or addition of a nucleotide of either of the above-mentioned oligonucleotide sequences can be performed by the methods of, for example, Saiki, et al.: Science, 230:1350-1354 (1985), and Saiki, et al.: Science, 239:487-491 (1988).
  • a DNA library can be prepared by customary methods. That is, the polynucleotide sequences can be individually ligated to replicable cloning vectors to thereby obtain DNA libraries.
  • replicable cloning vector any known or commercially available vectors, such as phage genes, cosmids, plasmids and animal virus genes nay te used.
  • the in-vitro packaging of each of the polynucieotide sequences inserted vectors is conducted by a customary method.
  • the DNA-inserted vectors are obtained in the form of recombinant phage particles.
  • the obtained phage particles are used as a DNA library for DNA cloning.
  • plasmid when used as a replicable vector, the above-mentioned polynucleotide sequences, either individually or in combination, can be inserted in the plasmid vectors and the resultant DNA-inserted vectors are then individually introduced into host cells, such as cells of Escherichia coli, Bacillus subtili, yeast or the like, according to a customary method.
  • host cells such as cells of Escherichia coli, Bacillus subtili, yeast or the like.
  • the thus obtained transformants are used as a DNA library for DNA cloning.
  • any of the above-mentioned polynucleotide sequences may be either individually or in combination inserted in the virus gene vectors and the resultant recombinant viruses are then individually transfected into animal cells according to a standard method and multiplied in the cells.
  • the obtained recombinant viruses as such can be used as a DNA library.
  • the transformants When the DNA library is comprised of transformants, the transformants can be cultured on a standard agar medium to form colonies.
  • the DNA libraries are comprised of recombinant phage particles or recombinant viruses, these phage particles or recombinant viruses can be used to infect known host cells , such as Escherichia coli ,
  • the polynculeotides of the instant invention may be further used to recombinantly express the novel immunogenic HCV polypeptides including the FGB1 and FGB2 polypeptides or variations thereof, respectively.
  • the polynucleotide sequences encoding the FGB1 and FGB2 polypeptides especially on a commercial scale, the polynucleotide sequences encoding the FGB1 and FGB2 polypeptides, whether in fused or free form, and whether or not containing a signal sequence to permit secretion, can be ligated into a replicable expression vector by a customary method which is suitable to any convenient host.
  • Both eukaryotic and prokaryotic host systems can be used in forming the recombinant FGB1 and FGB2 polypeptides or variations thereof.
  • the polynucieotide sequences can be synthetically produced or derived from clones.
  • an individual polypeptide can be produced by gene expression.
  • a polypeptide in combination with a DNA fragment is utilized to form a recombinant, a polypeptide can be expressed in the form of a fused or free polypeptide comprising peptides encoded by the inserted polynucieotide sequence and DNA fragment.
  • a recombinant which is capable of expressing a polypeptide of the instant invention includes a coding sequence comprised of a polynucleotide sequence of the instant invention which is operably linked to a control sequence that is compatible with a desired host.
  • a recombinant may be simply referred to herein as a recombinant expression system.
  • control sequence it is meant herein to refer a to polynucleotide sequence which effects expression of a coding sequence to which it is ligated.
  • the nature of such a control sequence may differ depending upon the host organism.
  • control sequences generally include promoters, ribosomal binding sites, and terminators.
  • control sequences generally include promoters, terminators and, in some instances, enhancers.
  • control sequence is therefore intended to include, at a minimum, all components whose presence is necessary for expression, and may also include additional components whose presence is advantageous, such as, leader sequences.
  • operably linked refers herein to a juxtaposition wherein the components so described are in a relationship permitting them to function in their intended manner.
  • a control sequence "operably linked" to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under conditions compatible with the control sequence.
  • coding sequence refers to a polynucleotide sequence which may be transcribed into RNA and/or translated into a polypeptide when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by an initiation codon at the 5'-terminus and a termination codon at the 3'-terminus.
  • a coding sequence may include, but is not limited to RNA, DNA, and recombinant
  • polynucleotide sequences By the term “open reading frame”, it is meant herein to refer to a region of a polynucleotide sequence which encodes a peptide or polypeptide. This region may represent a portion of a coding sequence or a total coding sequence.
  • any conventionally known or commercially available expression vector can be used.
  • expression vectors include plasmid vector pSN508 for enterobacteria, U.S. Patent No. 4,703,005, plasmid vector pBH103 for yeast, and its series, Japanese Patent Application Laid-Open Specification No.
  • the expression vectors containing inserted immunogenic HCV polynucleotides of the instant invention can be individually introduced or transfected into host cells sensitive to the vector according to conventional methods, to obtain transformants.
  • the FGB1 and FGB2 polypeptides can be produced in the culture of the transformants or the recombinant viruses and purified therefrom in accordance with standard techniques on a commercial scale.
  • the FGB1 and FGB2 polypeptides recombinantly produced may be isolated from lysed cells or from the culture medium of the transformants or recombinant viruses and purified to the extent needed for their intended use. Purification may be by any appropriate combination of customary techniques known in the art and selected from, for example, salt fractionation; adsorption and desorption using biogel; differential extraction; precipitation by an inorganic solvent;
  • the FGB1 and FGB2 polypeptides are purified from the culture of an E. coli transformant or a yeast transformant, from a viewpoint of effective removal of allergens derived from E. coli and yeast which cause the quality of the final product of the FGB1 and FGB2 polypeptides to be markedly lowered, it is preferred that the purification be conducted by, for example, the steps of adsorption and elution using a biogel, such as a sepharose or agarose biogel.
  • a recombinant virus e.g., the culture of recombinant virus-infected cells, high purity FGB1 or FGB2
  • polypeptide can be obtained by subjecting a crude solution containing the polypeptide to purification by ultracentrifugation and density gradient
  • a solution containing purified FGB1 or FGB2 polypeptide of the present invention is cotained. If desired, the solution may be lyophilized to obtain purified FGB1 or FGB2
  • polypeptide in a dry form is a polypeptide in a dry form.
  • Such recombinantly expressed FGB1 and FGB2 polypeptides can be used as diagnostics or formulated into vaccines. Antibodies raised against these recombinantly expressed polypeptides can also be used as diagnostics, or for passive immunotherapy. In addition, antibodies to these recombinantly expressed polypeptides or anti-idictype antibodies, which are raised against such antibodies, are useful for isolating and identifying HCV.
  • the polynucleotide seguences of the present invention encoding the FGB1 and FGB2 polypeptides may also be used for diagnosing HCV hepatitis by nucleic acid and in situ hybridization technique. More particularly, the polynucieotide sequences described herein permits the construction of probes which are useful for detecting HCV RNA in biological samples, including blood, lympn, ascites, hepatocytes, etc.
  • probe it is used herein to refer to a polynucleotide which forms a hybrid structure with a sequence in a target region, due to complementarity of at least one sequence in the probe with a sequence in the target region.
  • target region refers to a region of nucleic acids which is to be amplified and/or detected.
  • RNA or DNA oligomers of approximately 6 nucleotides or more which are complementary to the disclosed RNA polynucleotide sequences, either by excision or synthetically, which hybridize with the HCV genome and are useful in identifcation of HCV RNA in, for
  • the crooes for HCV should be of a length which allows the detection of HCV RNA or DNA sequences by hybridization. While 6-8 nucleotides complementary or corresponding to the disclosed polynucleotide sequences may be of a workable length, sequences of 10-12 nucleotides complementary or corresponding thereto are prefered and about 20-45 nucleotides and in particular 20 nucleotides
  • probes of the instant invention can be prepared using routine methods, including automated oligonucieotide synthetic methods, such as described in Warner: DNA, 3:401 (1984), which is incorporated herein by reference in its entirety.
  • useful probes for example, are those derived from the polynucleotide sequences, the novel clones disclosed herein, as well as the various oligomers useful in the DNA libraries, set forth herein. It should be
  • the biological sample to be analyzed such as blood, serum ascites, lymph and hepatocytes, may be processed, if desired, to extract HCV nucleic acids contained therein.
  • the resulting nucleic acids from the sample may be subjected to gel electrophoresis or other size separation techniques; alternatively, the nucleic acid sample may be dot blotted without size separation.
  • the probes of the instant invention can then be labeled with a labeling moiety.
  • the probes may be labeled with 32 P by treatment with polynucleotide kinase in the presence of gamma
  • Suitable other labels include, for example, other radioactive labels such as radioactive iodine, biotin, alkaline phosphatase, fluorescent probes and
  • chemiluminescent probes The labeling of the probes may be performed by standard techniques known in the art such as by using commercially available nick translation kits or multiprime kits, such as those distributed by Amersham International, England and Nippon Gene Co., Ltd., Japan.
  • the nucleic acids extracted from the sample can then be treated with the labeled probe under hybridization conditions of suitable stringencies, and polynucleotide duplexes containing the probe are detected.
  • RNA and DNA probes of the instant invention are believed to be highly complementary to the HCV RNA genome and cDNA, respectively. Therefore, usually high stringency conditions are not required during hybridization. Nevertheless, to ensure against false positives, conditions of high stringency should be used.
  • the stringency of hybridization is
  • the labeled probe is introduced, for example, in a plastic bag.
  • the amount of the labeled probe placed in the plastic bag is generally between about 1 and 100 nanograms per bag.
  • the labeled probe may be contained in the plastic bag in the form of a solution.
  • the diagnosis of HCV hepatitis by the use of the labeled probe is conducted by a standard hybridization method. That is, total RNA extracted from plasma, serum leukocytes, ascites fluid, lymph, or hepatocytes obtained from a patient is first electroohoresed and blotted or simply blotted on nitrocellulose. The blots of HCV RNA are then hybridized with the labeled probes in the bag, and the hybridized labeled probes are detected by, for example, autoradiogram in the positive HCV samples.
  • the probes can be packaged into diagnostic kits. Diagnostic kits include the DNA or RNA probes, which may be labeled; alternately, the probes of the instant invention may be unlabeled and the ingredients for labeling may be included in the kit in separate containers.
  • the kit may also contain other suitably packaged reagents and materials needed for the particular hybridization protocol, for example, standards, as well as instructions for conducting the test.
  • the polynucleotides of the instant invention may be further used to prepare anti-HCV viral agents, such as antisense polynucleotide fragments, which may interfere with HCV RNA replication, transcription or translation.
  • Antisense polynucleotide fragments as used herein constitute single stranded DNA or RNA fragments derived from the polynucleotide sequences encoding the FGB1 or FGB2 polypeptides, e.g., complementary thereto, which permits them to bind specifically to designated regions of HCV RNAs.
  • Antisense polynucleotide fragments of the instant invention include, for example, single stranded DNA or RNA fragments that have the ability to bind to HCV RNA to block protein translation and/or prevent replication of HCV RNA by polymerase.
  • the instant invention contemplates double stranded (ds) DNA fragments which, when ligated into an appropriate expression vectors such as a retroviral vector and introduced into infected liver cells, transcribe complementary antisense RNA fragments that will bind to HCV RNA to block protein translation and/or HCV replication.
  • antisense single stranded polynucleotides which bind to HCV RNA
  • the antisense polynucleotides of the instant invention may be further designed to include, for instance, ribozymes which catalyze with high specificity, the degradation of HCV RNA. Hence, they may be delivered in specialized systems, for example, liposomes or by retroviral vectors used in gene therapy. In addition, they may include bonding between modified bases, analogs, etc.
  • the antisense single stranded polynucleotide fragments of the present invention may also include molecules which carry agents (non-covalently attached or covalently bound) for enhancing their stability and effectiveness to degrade HCV RNA. Still further, the antisense polynucleotides of the instant invention may also bind to cellular RNA which enhance and/or are required for viral infectivity, replicative ability, or chronicity.
  • antisense single stranded RNA or DNA fragments contemplated by the instant invention include: SEQ ID NOS : 25-26
  • ds DNA fragments which when ligated in frame into appropriate vectors such as retroviral vectors and introduced into infected liver cells, transcribe the above antisense polynucleotide RNA sequences, include:
  • in frame refers to the proper positioning of a desired sequence of nucleotides within a DNA fragment or coding
  • the immunogenic HCV polypeptides in accordance with the present invention can be prepared as discrete polypeptides or incorporated into larger polypeptides.
  • the FGB1 or FGB2 polypeptides can be prepared as unfused polypeptides or fuse ⁇ polypeptides in combination with one another, cr the FGB1 and/or FGB2 polypeptides can be prepared in combination with other polypeptides to form heterologous fusion polypeptides.
  • Useful heterologous polypeptide sequences include polypeptide sequences that provide for secretion from a recombinant host, enhance the immunological reacitivity of the HCV epitope(s), facilitate the coupling of the polypeptides to immuneassay supports or vaccine carriers, or enhance the solubility of the polypeptides. See, e.g., EPO Pub. No. 116,201; U.S. Pat. No. 4,722,840; EPO Pub. No. 259,149; U.S. Pat. No. 4,629,733.
  • immunogenic HCV polypeptides and especially the FGB1 and FGB2 polypeptides of the instant invention therefore can be prepared as discrete or combination polypeptides or incorporated into larger polypeptides, and may find use as described herein.
  • the HCV polypeptides and in particular the FGB1 and FGB2 polypeptides of the present invention may advantageously be used as active ingredients in vaccines for HCV.
  • Vaccine preparations which contain immunogenic polypeptides as active ingredients, are well known to those of skill in the art. Typically, such vaccines are injectibles, either as liquid solutions or suspensions; solid forms suitable for solution or suspension in liquid prior to injection may also be prepared.
  • the preparation may also be emulsified, or the FGB1 or FGB2 polypeptides encapsulated in liposomes.
  • the active immunogenic FGB1 and/or FGB2 polypeptides can be mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol, or the like and combinations thereof.
  • the vaccines may also contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, and/or adjuvants which enhance the effectiveness of the vaccine.
  • aluminum hydroxide gel is added as an adjuvant so that the concentration of the added gel becomes about 0.1 to about 1.0 mg per ml.
  • an adjuvant there may also be employed precipitating depositary adjuvants such as calcuim phosphate gel, aluminum phosphate gel , aluminum sulfate, alumina hydroxide, alumina and bentonite.
  • Examples of still further adjuvants include but are not limited to: N-acetylmuramyl-L-threonine-D-isoglutamine (thr-MDP),
  • nor-MDP N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1'-2'-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine
  • CGP 19835A referred to as MTP-PE
  • RIBI which contains three components extracted from bacteria, i.e., monophosphoryl lipid A, trehalose dimycolate and cell wall skeleton (MPL + TDM + CWS) in a 2% squalene/Tween 80 emulsion.
  • the effectiveness of an adjuvant may be determined by measuring the amount of antibodies directed against the FGB1 or FGB2 polypeptide resulting from the administration of any of these polypeptides and vaccines in combination with various adjuvants.
  • the immunogenic HCV polypeptides and in particular the FGB1 and FGB2 polypeptides of the instant invention are too small to encompass the total immunogenicity of the structural region of the putative HCV structural protein, they may be linked to a suitable carrier.
  • a number of techniques for obtaining such linkage are known in the art, including the formation of disulfide linkages using N-succinimidyl-3-(2-pyridylthio)propionate(SPDP) and succinimidyl 4-(N-maleimidomethyl)cyclohexane-1- carboxylate(SMCC) available through Pierce Company, Rockford, IL.
  • the FGB1 and FGB2 polypeptides lack a sulfhydryl group, this can be accomplished by providing, for example, a Cys amino acid residue or a cystine moiety on one protein (e.g., the FGB1 or FGB2 polypeptide) and an amide linkage through an epsilonamino on a lysine or other free amino group in the other (e.g., carrier).
  • a variety of such disulfide/ amide-forming agents are known. See, for example, Harlow and Lane: Antibodies. A Laboratory Manual, pp. 130-131, Cold Spring Harbor Laboratory, Cold
  • bifunctional coupling agents form a thioether rather than a disulfide linkage.
  • Many of the thio-ether-forming agents are commercially available and include reactive esters of 6-maieimidocapric acid, 2-bromoacetic acid, 2-iodacetic acid, 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid, and the like.
  • the carboxyl groups can be activated by combining them with succinimide or 1-hydroxyl-2-nitro-4-sulfonic acid, sodium salt.
  • any carrier may be used which preferably does not itself induce the production of antibodies harmful to the host.
  • Suitable carriers are typically large, slowly metabolized macromolecules such as polypeptides, polysaccharides, such as latex functionalized sepharose, agarose, cellulose, cellulose beads and the like;
  • polymeric amino acids such as polyglutamic acid, polylysine, and the like; amino acid copolymers; and inactive virus particles.
  • Especially useful polypeptides substrates are serum albumins, keyhole limpet hemocyanin protein (KLH), immunoglobulin molecules, thyroglobulin, ovalbumin, tetanus toxoid, and other proteins well known to those versed in the art.
  • KLH keyhole limpet hemocyanin protein
  • immunoglobulin molecules thyroglobulin
  • ovalbumin ovalbumin
  • tetanus toxoid and other proteins well known to those versed in the art.
  • the vaccines can be conventionally administered parenterally, by injection, for example, either subcutaneously or intramuscularly.
  • Additional formulations which are suitable for other modes of administration include suppositories and, in some cases, oral formulations.
  • suppositories traditional binders and carriers may include, for example, polyalkylene glycols or triglycerides; said suppositories may be formed from mixtures containing the active ingredient in the range of 0.5% to 10%, preferably 1%-2%.
  • Oral formulations include such normally employed excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, glucose, fructose, galactose, sucrose, lactose, albumin, gelatin, and amino acids such as glycine, alanine, lysine,
  • compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained released formulations or powders and may contain, for example, 10%-95% of active ingredient, and preferably 25%-70%.
  • the FGB1 and FGB2 polypeptides may be formulated into a vaccine as neutral or salt forms.
  • Pharmaceutically acceptable salts include the acid addition salts (formed with free amino groups of the peptide) and which are formed with inorganic acid, such as, for example, hydrochloric or phosphoric acids, or such organic acids such as acetic, oxalic, tartaric, maleic, and the like. Salts formed with the free carboxyl groups may also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, and ferric hydroxides, and such organic bases as isopropylamine, trimethylamine,
  • a vaccine of the instant invention may also be prepared in the form of a mixed vaccine which contains adsorbed HCV polypeptides, preferably
  • the adsorbed FGB1 and/or FGB2 polypeptides mentioned above and at least one antigen other than the present FGB1 and/or FGB2 polypeptide.
  • the antigen other than the present FGB1 and/or FGB2 polypeptides there may be employed any antigens that are conventionally used as active ingredients of the corresponding vaccines in so far as the side effects and adverse reactions caused by such other antigens and the FGB1 and/or FGB2 polypeptides are not additively or synergistically increased by the use of the FGB1 and/or FGB2 polypeptides and such other antigens in combination, and the antigenicities and immunogenicities of the FGB1 and/or FGB2 polypeptides and such other antigens are not reduced by the interference between the FGB1 and/or FGB2 polypeptides and other antigens.
  • the number and the types of the antigens which may be mixed with the FGB1 and/or FGB2 polypeptides are not limited in so far as the side effects and adverse reactions are not increased additively or synergistically and the antigenicity and immunogenicity of each of the FGB1 and FGB2 polypeptides and such antigens are not reduced as mentioned above.
  • two to six or more types of antigens may be mixed with the FGB1 and/or FGB2 polypeptides.
  • antigens which may possibly be mixed with the present FGB1 and/or FGB2 polypeptides, include detoxified antigens, inactivated antigens or toxoids which are derived from encephalitis virus, hfrs virus (hemorrhagic fever with renal syndrome), influenza virus, parainfluenza virus, HAV, HBV, dengue fever virus, AIDS virus, Bordetella pertussis, Diptheria bacillus, Tetanus bacillus, meningococcus, pneumococcus, and the like.
  • detoxified antigens inactivated antigens or toxoids which are derived from encephalitis virus, hfrs virus (hemorrhagic fever with renal syndrome), influenza virus, parainfluenza virus, HAV, HBV, dengue fever virus, AIDS virus, Bordetella pertussis, Diptheria bacillus, Tetanus bacillus, meningococcus, pneumococcus, and the like
  • the immunogencity of the HCV polypeptides including the FGB1 and FGB2 polypeptides may also be enhanced by preparing them in mammalian or yeast systems fused with or assembled with particle-forming proteins such as, for example, that associated with hepatitis B surface antigen. Constructs wherein, for example, the FGB1 and/or FGB2 polypeptides are linked directly to the particle-forming protein coding sequences produce hybrids which are HCV immunogenic.
  • the vectors may be prepared so that they include epitopes specific to HBV, having various degrees of immunogencity, such as, for example, the pre-S peptide.
  • particles constructed from particle-forming proteins which include for instance the FGB1 and/or FGB2 polypeptides are immunogenic with respect to HCV and HBV.
  • Hepatitis surface antigen has been shown to be formed and assembled into particles in S. cerevisiae, Valenzuela, P. et al.: Nature, 298:344 (1982), as well as in, for example, mammalian cells Valenzuela, P. et al.: IN: Hepatitis B, Millman et al., ed, Plenum Press, pp. 225-236 (1984). The formation of such particles has been shown to enhance the immunogencity of the monomer subunit.
  • the constructs may also include the immunodominant epitope of HBSAg, comprising the 55 amino acids of the presurface (pre-S) region. Neurath et al.: Science, 224:392 (1984). Constructs of the pre-S-HBSAg particle expressible in yeast are disclosed in EPO Pub. No.
  • hybrids including heterologous viral sequences for yeast expression are disclosed in EPO Pub. No. 175,261.
  • These constructs may also be expressed in mammalian cells such as Chinese hamster ovary (CHO) cells using an SV40-dihydrofolate reductase vector disclosed in Michelle, M.T. et al.: Viral Hepatitis and Liver Disease, Ed. Vyas, G.N., Dienstag, J.L. and Hoofnagle J.H., Abstract No. 8A.16, p. 659, Grune & Stratten, Inc., (1984).
  • a vaccine comprising, for example, the FGB1 and/or FGB2 polypeptides of the present invention may be contained and sealed in a vial, an ampule or the like.
  • the vaccines of the present invention may be administered in the form of a liquid or suspension.
  • the vaccine is disolved or suspended in sterilized-distilled water or normal saline before administration, the amount of distilled water or normal saline being such that the volume becomes the original volume before being subjected to lyophilization.
  • the vaccines should be administered in a manner compatible with the dosage formulation, and in such an amount as will be prophylactically and/or therapeutically effective.
  • the quantity to be administered which is believed to be generally in the amount of about 5 micrograms to about 250 micrograms or more of FGB1 or FGB2 per dose, depends on the subject to be treated, capacity of the subject's immune system to synthesize antibodies, and the degree of protection desired. Precise amounts of active ingredient
  • the dose of the vaccine per child may be half as much as that of the vaccine per adult.
  • the vaccine may be given in a single dose schedule, or preferably in a multiple dose schedule.
  • a multiple dose schedule is one in which a primary dose of vaccination may be given in 1-10 separate doses, followed with other doses given at subsequent time intervals required to maintain and/or boost the immune response, for example, at 1-4 months for a second dose, and if needed, a subsequent dose(s) after several months.
  • the dosage regimen will also, at least in part, be determined by the need of the individual, and be dependent upon the judgement of the practitioner.
  • the vaccine containing the novel immunogenic HCV polypeptides such as the FGB1 and FGB2 polypeptides may be administered in conjunction with other immunoregulatory agents, such as, immune globulins, interleukins, etc.
  • novel immunogenic HCV polypeptides and especially the FGB1 and FGB2 polypeptides may also be used for preparing an antibody, such as a polyclonal antibody and a monoclonal antibody, specific for the immunogenic HCV polypeptides.
  • an antibody such as a polyclonal antibody and a monoclonal antibody, specific for the immunogenic HCV polypeptides.
  • a polyclonal antibody specific for a KLH coupled-Cys-FGB1-Tyr or Cys-FGB2-Tyr polypeptide may be prepared by conventional methods as follows.
  • the KLH coupled- Cys-FGB1-Tyr or Cys-FGB2-Tyr polypeptide of the present invention is innoculated subcutaneously, intramuscularly, intraperitoneally or intravenously into an animal such as mouse, guinea pig, rabbit, goat, horse, etc.
  • the innoculation of the FGB1 or FGB2 polypeptide is preferably conducted several times at intervals of 1 to 4 weeks, to thereby completely immunize the animal.
  • a conventionally commercially available adjuvant may be used as described hereinbefore.
  • Serum from the immunized animal is collected and the polyclonal antibody is isolated and purified from the blood serum according to standard techniques well known in the art, such as described in Mayer and Walker, eds. Immunochemical Methods in Cell and
  • Monoclonal antibodies specific for the FGB1 or FGB2 polypeptides may also be prepared by conventional techniques as described in, for example,
  • the splenic cells obtained from a mouse immunized with FGB1 and/or FGB2 polypeptide are fused with commercially available mouse myeloma cells by cell fusion technique, to obtain hybridomas.
  • the hybridomas are screened to obtain a hybridoma capable of producing an antibody reactive with either the FGB1 or the FGB2 polypeptide.
  • the obtained hybridoma is cultured in accordance with standard methods. From the supernatant of the culture, a monoclonal antibody against either the FGB1 or the FGB2 polypeptide is isolated and purified by standard techniques.
  • polyclonal and monoclonal antibodies may also be used as diagnostic agents for diagnosing HCV, and those which are neutralizing are useful in passive immunotherapy.
  • diagnosis of HCV using the antibody may be conducted by immunoassay in substantially the same manner as mentioned above with respect to the diagnosis of HCV using the FGB1 or FGB2 polypeptides.
  • novel antibodies and in particular the novel polyclonal antibodies raised against the immunogenic HCV polypeptides, especially the FGB1 polypeptide, of the instant invention nay be useful for detecting autoimmune liver disease induced by, for example, HCV.
  • the immunogenic HCV polypeptides and in particular the FGB1 polypeptide of the present invention share epitopes with host protein (s) possibly expressed or released by the liver in response to inflammation, trauma or disease.
  • the immunogenic HCV polypeptides and in particular the FGB1 polypeptide of the instant invention are therefore believed to be effective for raising antibodies and in particular polyclonal antibodies capable of cross-reacting with HCV epitopes as well as other epitopes shared between HCV antigen(s) and host protein(s) for detecting autoimmune liver disease in immuno-type assays such as an immunofluorescence assay known to those of skill in the art.
  • Gly-Arg-Arg-Gly-Gln-Lys-Ala-Lys-Ser-Asn-Pro-Asn-Arg-Pro-Leu GOR epitope
  • sequence for the novel FGB1 polypeptide is Lys-Pro-Gln-Arg-Lys-Thr-Lys-Arg-Asn-Thr-Asn-Ara-Arg-Pro-Gln.
  • the underscored amino acids represent distant, but similar amino acids between the two epitopes.
  • Monclonal antibodies in particular, may be used to raise anti-idiotype antibodies.
  • Anti-idiotype antibodies are immunoglobulins which carry an "internal image" of the antigen of the infectious agent against which protection is desired. See, for example
  • anti-idiotype antibodies generated may also be useful for treatment and/or diagnosis of HCV by, for example, reacting with HCV antigen or detecting the presence of anti-HCV antibody in biological samples.
  • antibody refers to a polypeptide or group of polypeptides which are comprised of at least one antibody combining site.
  • An "antibody combining site” or “binding domain” is formed from the folding of variable domains of an antibody molecule(s) to form three-dimensional binding spaces with an internal surface shape and charge distribution complementary to the features of an epitope of an antigen, which allows an immunological reaction with the antigen.
  • An antibody combining site may be formed with a heavy and/or a light chain domain (VH and VL, respectively), which form hypervariable loops which contribute to antigen binding.
  • VH and VL light chain domain
  • the term "antibody” therefore is used broadly herein and includes, for example, vertebrate antibodies, hybrid antibodies, chimeric antibodies, ImmunoZAP TM antibodies, altered antibodies, univalent antibodies, the Fab proteins, and single domain antibodies, as known in the art.
  • FGB1 and FGB2 polypeptides and the antibodies raised against such polypeptides are useful in immunoassays to detect presence of HCV antibodies, or the presence of HCV and/or HCV antigens, in biological samples, such as blood, sera and semen.
  • the immunoassay may utilize one HCV epitope; alternatively, the immunoassay may use a combination of HCV epitopes derived from for instance the FGB1 and FGB2 polypeptides. It may use, for example, a monclonal antibody directed towards either the FGB1 or FGB2 polypeptide, a combination of monoclonal antibodies directed towards the FGB1 and FGB2 polypeptides, polyclonal antibodies directed towards either the FGB1 or FGB2 polypeptides, or a combination of polyclonal antibodies directed toward the FGB1 and FGB2 polypeptides. Protocols may be based, for example, upon competition, direct reaction, or sandwich type assays. Protocols may also, for example, use solid supports, or may be by
  • the assays of the present invention involve the use of antibodies or polypeptides and in particular the FGB1 and/or FGB2 polypeptides of the instant invention labeled with a labeling moiety. It is to be understood that the term
  • labeling moiety is used herein throughout in a broad sense and is meant to include, but not limited to, radioisotopes, enzymes, antibodies, dyes, chemiluminescent or fluorescent molecules, immune complexes, and the like, which may be directly or indirectly attached to the antibody, polypeptide or polynucleotide sequence of the instant invention.
  • labeling moiety In order to radioactively label the FGB1 or FGB2 polypeptides, it is preferable to conjugate a Tyr or His amino acid residue or the like to the carboxyl end thereof as discussed hereinbefore. Techniques for radioactively labeling antibodies and polypeptides are well known to those of skill in the art and may be utilized to label the antibodies and immunogenic HCV polypeptides of the instant invention.
  • immuno assays which may be utilized include enzyme-labeled and radioactive immunoassays, such as ELISA and RIA assays,
  • Kits suitable for immunodiagnosis and containing the appropriate labeled reagents are constructed by packaging the appropriate materials, including the novel immunogenic HCV polypeptides such as the FGB1 and/or FGB2 polypeptides of the invention or antibodies raised against such immunogenic HCV polypeptides or anti-idiotype antibodies raised against antibodies to such immunogenic HCV polypeptides in suitable containers, along with the remaining reagents and materials required to conduct the assay, as well as a suitable set of assay instructions.
  • novel immunogenic HCV polypeptides such as the FGB1 and/or FGB2 polypeptides of the invention or antibodies raised against such immunogenic HCV polypeptides or anti-idiotype antibodies raised against antibodies to such immunogenic HCV polypeptides in suitable containers, along with the remaining reagents and materials required to conduct the assay, as well as a suitable set of assay instructions.
  • Polypeptides FGB1 and FGB2 are used as antigens in an ELISA diagnostic assay as described below to detect antibodies to HCV in biological samples.
  • PBS Phosphate Buffered Saline
  • Cys-FGB1-Tyr and/or Cys-FGB2-Tyr polypeptide in PBS at a concentration of about 1mg/ml Dilute the polypeptide solution to 1:100 in PBS (10 ng of polypeptide). Add 100uls of diluted polypeptide solution to each well to be used. Cover plate with parafilm and incubate for about 8-12 hours on a shaker platform at room temperature. Remove polypeptide solution and wash wells by adding 200 uls PBS:Tween to each, cover plate and incubate on shaker for 4-6 hours at room temperature. Remove wash solution and add 80uls PBS:Tween. Add 20 uls of patient or normal serum to each well. Cover plate and incubate
  • PBS:Tween Dilute rabbit anti-human IgG conjugated to horse radish peroxidae 1:200 in PBS: Tween. Add 100 uls diluted rabbit anti-human IgG to each well. Cover plate and incubate for about 2-6 hours on a shaker platform at room temperature. Remove rabbit
  • the protocol described above may be utilized on a commercial basis by assembling in a kit the materials, polypeptides of the instant invention and reagents required to detect antibodies raised against HCV in biological samples, along with appropriate instruction.
  • Cys-FGB1-Tyr and Cys-FGB2-Tyr polypeptides were chemically synthesized as described hereinbefore and included a cysteine amino acid residue at the amino termini and a tyrosine amino acid residue at the carboxyl termini. The results, expressed as
  • NT non-reactive
  • I means
  • the advantages of using the FGB1 and FGB2 polypeptides of the instant invention in an ELISA test are numerous.
  • the FGB1 and FGB2 polypeptides and espeically the FGB1 polypeptide have an increase in specificity and sensitivity, as compared to Ortho's assay which is concerned with the recombinant C100 peptide (antigen).
  • assays involving the FGB1 and FGB2 are less expensive, and are believed to be capable of diagnosing HCV in the acute phase.
  • the polypeptide FGB1 is water soluble, very stable and binds readily to microwells of ELISA plates in an unconjugated form. Still further, since the FGB1 and FGB2 polypeptides can be prepared by chemical
  • Cys-FGB1-Tyr and Cys-FGB2-Tyr polypeptides were coupled to KLH, a carrier protein, with m-Maleimidobenzoyl-N- hydroxysuccinimide ester (MBS) and administered to separate rabbits as
  • This procedure is an established one to produce polyclonal antibodies to peptides in rabbits, and can be modified for use in other laboratory animals.
  • MFS m-Maleimidobenzoyl-N-hydroxysuccinimide ester
  • Dialyze against PBS to remove uncoupled polypeptides Stir at room temperature for about 3 hours. Divide the 1.5 ml dialized solution into 4 aliquots of about 0.5 ml and 1 each of about 0.33 ml.
  • New Zealand rabbits obtained for the purpose of immunization are quarantined for a period of two weeks and are then injected and bled as follows.
  • Cys-FGB2-Tyr polypeptide mixed with about 9.3 mg of Freunds Adjuvant and test bled.
  • Cys-FGB2-Tyr polypeptide mixed with about 0.3 mg of Freunds Adjuvant and test bled. D. Testing of the Anti-Cys-FGB1-Tyr or
  • Tables 11 and 12 show rabbit serum dilutions and corresponding absorbance readings in an ELISA using the respective Cys-FGB1-Tyr
  • spleen from rabits injected with immunogenic HCV polypeptides and in particular either the FGB1 or FGB2 polypeptides of the instant invention for raising monoclonal antibodies (MAbs) can be accomplished by, for example, the following
  • MAbs Monoclonal antibodies
  • the MAbs can be generated in accordance with standard technology, as recited for instance in Harlow and Lowe: Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1988). More particularly, the MAbs can be prepared by the fusion of myeloma cells with B-lymphocytes obtained from spleens of, for example, mice or other animals injected with for instance either the FGB1 or the FGB2 polypeptide conjugated with a carrier. In the presence of
  • hypoxanthine, aminopterin and thymidine (HAT) medium a few immortal hybridoma cells secreting specific immunoglobins can be selected in accordance with the standard techniques. These hybridomas can be further subdivided into clones by serial dilution of the cultured fusion products in 96 well microtiter plates in the presence of HAT media. The detail procedure can be carried out as described hereinbefore.
  • Monoclonal antibodies can also be generated by a four step procedure.
  • the first and second steps involve the construction and separation of heavy and light chain cDNA libraries from poly A mRNA isolated from spleen tissue, peripheral blood, or lymphnode derived from animals inoculated with, for example, the FGB1 or FGB2 polypeptides.
  • the third and fourth steps involve randomly combining the heavy and light chain libraries and screening for antigen binding. This procedure is outlined in detail in the following publications: Antibody Expression Library Technology, distributed by Stratacyte, 11099 N. Torrey Pines Road,
  • Plasmids are constructed using existing vectors such as pSC11, Chakrabarti et al.: Molecular and Cell Biology, 5:3403-3409 (1985), pUV1, Falkner et al.: Nucleic Acids Res., 15:7192 (1987), pTM3, Moss et al.: Nature, (348):91-92 (1990), depending on the level of expression desired and on whether a stable recombinant virus is required. All three vectors are pUC based with the vaccinia virus thymidine kinase gene flanking inserted sequences.
  • pSCll may have, for example, the FGB1 and/or FGB2 polypeptides or other polypeptides of the instant invention expressed from an early/late promoter.
  • pUVl may have for instance the FGB1 and/or FGB2 polypeptides under the control of a strong late promoter.
  • pTM3 has a bacteriophage T7 promoter and an EMC leader and is believed to be the best available vector for transient expression either in a cell line expressing T7 RNA polymerase or by coinfection of the cells with two viruses, on carrying the T7 RNA polymerase and the other carrying, for example, the FGB1 and/or FGB2 polypeptide of interest under the control of the T7 promoter.
  • vectors are constructed from synthetic promoters whose sequence is derived from mutagenesis studies of the promoter.
  • the DNA encoding, for example, the FGB1 and/or FGB2 polypeptides are mutagenized at the nucleotides corresponding to the NH 2 -terminus and the COOH-terminus of the open readin frame (ORF) to have proper restriction sites for insertion into any particular vector.
  • Vaccinia virus recombinants are generated as described previously in Kotwal, G. J. et al:
  • the recombinants are picked off either TK-cells in the presence of 6-bromouridine, Chakrabarti et al: Mol. Cell. Biol., 5:3403-3409 (1985), or in the presence of mycophenolic acid if the gpt gene is inserted in tandem with the foreign gene. Blue color selection is used with lac Z containing vectors, Falkner and Moss: J. Virol., 62:1849-1854 (1988).
  • Recombinant vaccinia viruses are purified according to Joklik: Virology, 18:9-18, (1962).
  • HeLa cells are grown in suspension in MEM spinner medium containing about 5% horse serum in large Bellco 36L spinner flasks with an overhead drive and a teflon paddle impeller assembly at a density of about 5 ⁇ 10 5 cells/ml.
  • Up to about 100 1 of HeLa cells are then infected with trypsinized crude virus made under selective conditions at a multiplicity of infection of about 10 and at a cell density of about 2 ⁇ 10 7 cells/ml for about h hour. After about h hour, the cell density is brought back to about 5x10 5 cells/ml and is incubated at about 37°C for about 48 hours. At the end of about 48 hours, the cells are harvested, washed in PBS and resuspended in about 11 ⁇ 2 volumes of about
  • the virus band is then aspirated and repelleted in about 25% sucrose by spinning in an SW
  • the virus pellet is then resuspended in about 1mM Tris
  • the virus is titered on BSC-1 cells under selective and nonselective conditions.
  • the titer is read at about 48 hours after different dilutions of the virus are placed on confluent BSC-1 cells on a 6 well plate.
  • DNA is estimated from the absorbance of the virus preparation and is isolated by phenol: chloroform extraction of the proteinase K treated and detergent lysed suspension. It is then digested by Hind III and SA1 I and transferred to nitrocellulose and probed with the nick translated FGB1 and/or FGB2
  • CV-1 cells are infected at an m.o.i. of 30 (for WBA) and an m.o.i. of 10 (for IF) and the lysed cells are separated by 12.5% PAGE, transferred to nitrocellulose, Sambrook, J. et al.: Molecular
  • Purified IgG antibody is obtained from the hyperimmune serum (polyclonal antibody) of Example III. More particularly, the hyperimmune sera, i.e., either anti-Cys-FGB1-Tyr or anti-FGB2-Tyr, obtained from the hyperimmunized rabbits are obtained and subjected to ammonium sulfate precipitation, and the precipitate is obtained as an IgG fraction. The obtained IgG fraction is subjected to gel
  • anti-FGB IgG anti-FGB IgG
  • the purified anti-FGB IgG is labeled with, for example, 125 I by the chloramine T method, as described in Greenwood et al.: Biochemical
  • T is dropwise added to the mixture and stirred for about 5 minutes to advance a reaction.
  • About 1 ml of about 200 ug/ml aqueous sodium methabisulfite is added to the resultant mixture to terminate the reaction.
  • the resultant mixture is dialyzed to remove the unreacted 125 I, to thereby obtain 125 I-labeled
  • Cys-FGB2-Tyr polypeptide an antibody against HCV in serum samples obtained from patients having HCV hepatitis can be detected by radioimmuno assay (RIA).
  • RIA radioimmuno assay
  • Example I and further introducing about 0.1 ml of a serum sample to all but three control wells of the reaction plate.
  • about 0.1 ml of a control serum positive for HCV is added.
  • about 0.1 ml of a control serum negative for HCV is added.
  • PBS:Tween was added to all wells of the reaction plate.
  • about 0.1 ml of the appropriate 125 I-labeled anti-FGB IgG is added and allowed to stand overnight at room temperature to advance a reaction. The resultant wells are sufficiently washed with PBS:
  • the radioactivity (CPM) of each well is measured using, for example, an auto-gamma counter.
  • IgG and the Cys-FGB1-Tyr or Cys-FGB2-Tyr polypeptide bound in the wells is calculated using standard techniques.
  • radioactivity of the wells ascribed to the labeled antibody bonded to the appropriate polypeptide bound in the wells will be relatively low in those serum samples derived from patients having antibodies against HCV. This can be confirmed by comparing the radioactivity between the serum wells and the three control wells.
  • Cys-FGB1-Tyr or Cys-FGB2-Tyr polypeptide is labeled with, for example, 125 I by the chloramine T method outlined in Greenwood, et al.: Biochemical J., 80:114 (1963).
  • the anti-FGB-IgG are bound to the wells by simply adding the appropriate anti-FGB-IgG to the wells of the reaction plate.
  • Cys-FGB1-Tyr or Cys-FGB2-Tyr polypeptide and the appropriate anti-FGB-IgG antibody, HCV antigen present in serum samples obtained from patients infected with
  • HCV hepatitis can be detected in substantially the same manner as mentioned above.
  • RIA Thereafter replacing the above solution with about 200 ul PBS:Tween (Example I), and further introducing about 0.1 ml of a serum to all but three control wells of the reaction plate. In one of the three serum-free control wells of the reaction plate, about 0.1 ml of a control serum positive for HCV is added. In a second serum-free control well, about 0.1 ml of a control serum negative for HCV is added.
  • Cys-FGB1-Tyr or Cys-FGB2-Tyr polypeptide and the appropriate anti-FGB-IgG bound to the wells is calculated using standard techniques.
  • Cys-FGB2-Tyr polypeptide bonded to the anti-FGB-IgG bound to the wells will be relatively low in those samples derived from patients having HCV antigen.
  • Polynucieotide GB2 is the complementary sequence of GBl.
  • the GB1 and GB2 polynucleotides are annealed according to the methods of Sambrook , J . , et al . :
  • GB1 and GB2 are denatured in about 5 mis of annealing buffer (0.1 M NaCl, 10 mM Tris-Cl (pH 7.8), 1.0 mM EDTA) at about 95° C. for about 5 minutes and allowed to cool at room temperature.
  • annealing buffer 0.1 M NaCl, 10 mM Tris-Cl (pH 7.8), 1.0 mM EDTA
  • Two volumes (about 10 mis) of 100% ethanol are added and the annealed polynucleotides (GB1-GB2) are allowed to precipitate overnight at about -20° C.
  • the annealed polynucleotides (GB1-GB2) are centrifuged at about 14,000 ⁇ g, dried and
  • Nuclease is a single stranded specific endonuclease which hydrolyzes single stranded RNA or DNA into
  • the result of the electrophoresis indicates that approximately 90-95% of the GBl, GB2 is annealed yielding the desired double stranded (ds) GB1-GB2 fragment.
  • the sequence of the annealed ds GB1-GB2 fragment contains two Sma I restriction enzyme sites, CCCGGG, which are 98 nucleotides apart as well as an initiation codon ATG and a stop codon TAG, both of which are positioned between the two Sma I
  • the annealed ds GB1-GB2 fragment contains the polynucleotide sequence which encodes the FGB1 polypeptide, and flanking nucleotide sequences which lie upstream and downstream from the respective 5' end and 3' end Sma I restriction sites.
  • the two underscored polynucleotide sequences designated as CCCGGG in the above annealed ds GB1-GB2 represent restriction sites for the enzyme Sma I.
  • the two restriction sites CCCGGG are digested by the Sma I enzyme (BRL, Gaithersburg, MD, catalog no. 52285A) between the CCC and GGG nucleotides as indicated in the above-recited annealed ds GB1-GB2 fragment.
  • CTCACG represents the flanking complementary nucleotide sequence which lies upstream from the 5' end of the Sma I restriction site whereas the 7 complementary nucleotide sequence in
  • ACCGCCA represents the flanking complementary nucleotide sequence which lies downstream from the 3' end Sma I restriction site.
  • polynucleotide sequences ATG and TAG represent initiation and stop codons, respectively.
  • polypeptide which includes the amino acid sequence for the FGB1 polypeptide.
  • polynucleotide sequence in the above annealed ds GB1-GB2 fragment which lies between the 5' end restriction site CCCGGG and the initiation codon ATG is optional, and therefore can be deleted, modified, shortened or lengthened so long as such modification(s) does not cause the annealed ds GB1-GB2 fragment encoding FGB1 to be out of phase.
  • restriction sites and other flanking sequences may be employed so long as they permit the annealed ds GB1-GB2 fragment to be ligated into a replicating expression vector and do not cause the annealed ds GB1-GB2 fragment encoding the polypeptide which contains the amino acid sequence of FGB1 to be out of phase.
  • the above annealed ds GB1-GB2 is digested with Sma I restriction enzyme and purified as a 98 nucleotide ds fragment from a low melt 1.5% agarose gel containing about 1 ug of Ethidium Bromide
  • aqueous layers from both tubes are pooled together (13 ml total, some loss occurs in each step of the way) and about 1 ml of about 0.3 M sodium acetate (Mallinckrodt, Paris, KY) is added and the total volume of about 14 ml is transferred to four tubes (about 3.5 ml/tube).
  • about 7 mis of 100% ethanol is added, shaken and allowed to sit at about -20°C overnight.
  • the four tubes are then centrifuged at about 4°C for about 30 minutes in a beckman Model J2-21M Induction Drive Centrifuge (Beckman Instruments, Inc. Spinco Division, Palto Alto, CA catalog no. 341737).
  • the liquid portion in each tube is discarded and the pellet is allowed to dry at room temperature.
  • the pellets in the four tubes are combined in a total of about 40 ul of water, about 1.0 ul of which is subjected to electrophoresis on 1.5% agarose gel with standard markers in order to estimate the purity and yield of this particular batch of the 98 bp SmaI-Sma I fragment.
  • the Sma I-Sma I 98 base pair fragment is ligated into the Sma I site of the linearilized, dephosphorylated pGEM TM-3Zf(-) vector, about 0.1 ug
  • JM 109 Promega, Madison, WI, under catalog No. L2001
  • the competent E. coli cells are thawed on ice for about 30 min. Following thawing, the ligated ds material is mixed with the thawed competent cells for about 30 min. on ice prior to heat shock treatment.
  • the tube containing the mixture is placed in a water bath having a temperature at about 42°C for about 45 seconds.
  • about 900 ul of LB Lauria -Bertani Medium, i.e., Peptone 140 10 g/1, and Select Yeast Extract 5 g/1, Gibco-BRL, Gaithersburg, MD, catalog nos. M00392B and M00393B, respectively, and Sodium Chloride 10 g/1, Sigma, P.O. Box 14508, St. Louis, MO 63178, catalog no. S3014
  • LB Lauria -Bertani Medium, i.e., Peptone 140 10 g/1, and Select Yeast Extract 5 g/1, Gibco-BRL, Gaithersburg, MD, catalog nos. M00392B and M00393B, respectively
  • Sodium Chloride 10 g/1, Sigma, P.O. Box 14508, St. Louis, MO 63178, catalog no. S3014 is added to the heat shocked mixture
  • the agar ampicillin LB plates are made as follows and in accordance with Sambrook, J. et al.: Molecular Cloning. A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
  • agar ampicillin LB plates are prepared by pouring the liquid media from the flask into the plates. Approximately 30-35 ml of liquid media is poured into each 85-mm petri dish. If bubbles occur, the surface of the liquid media can be flamed with a bunsen burner before solidification of the agar.
  • the agar ampicillin LB plates are stored at room temperature for
  • the ampicillin is prepared as follows. Ampicillin

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Abstract

Novel basic immunogenic polypeptides having epitopes for HCV are disclosed. The novel basic immunogenic polypeptides are truncated polypeptides derived from the structural region of a human HCV isolate. Two preferred novel basic immunogenic polypeptides are designated as FGB1 and FGB2. The FGB1 and FGB2 polypeptides are believed to be derived from near the N- and C-terminals, respectively, of a putative (C) protein of HCV. In an ELISA, eight serum samples reactive for anti-C100 HCV antibody (EIA, Ortho/Chiron), recombinant immunoblot assay I (RIBA I, Ortho/Chiron) and neutralization (Neut., Abbot Labs.) contained antibodies to the FGB1 and FGB2 polypeptides. In addition, the FGB1 and FGB2 polypeptides and RIBA I were non-reactive with twelve serum samples which were reactive with EIA. Still further, two serum samples from a patient clinically diagnosed as NANBH, which were non-reactive with EIA and RIBA I, were reactive with FGB1 and/or FGB2. Novel polynucleotide sequences encoding the basic immunogenic polypeptides are also disclosed. The present invention further includes the application of these new sequences, polypeptides, and antibodies raised against the novel polypeptides in HCV detection, such as immunoassays, diagnostics, PCR technology and gene therapy. Included within the invention also are novel immunogenic HCV polypeptides encoded within recombinant expression systems and clones, novel transformants, novel probes, novel methods for producing the immunogenic HCV polypeptides, novel anti-idiotype antibodies, novel antisense polynucleotides, and novel kits.

Description

BASIC STRUCTURAL IMMUNOGENIC
POLYPEPTIDES HAVING EPITOPES FOR HCV, ANTIBODIES, POLYNUCLEOTIDE
SEQUENCES, VACCINES AND METHODS
Related Applications
This is a continuation-in-part application of U.S. patent application, Serial No. 37,369,809, filed on January 14, 1991.
Field of the Invention
The present invention relates to biological materials for detecting hepatitis C virus (HCV) and preventing and/or treating HCV disease. More particularly, the instant invention relates to truncated, basic structural immunogenic polypeptides having epitopes for HCV, to polynucleotide sequences encoding the HCV polypeptides and antisense polynucleotide sequences derived therefrom, to antibodies raised against the HCV polypeptides, to anti-idiotype antibodies raised against antibodies to the HCV polypeptides, and HCV vaccines. These biological materials are believed to be effective as screening agents for HCV as well as for acute and chronic HCV infection, and as protective agents against HCV disease. Background
Hepatitis is the medical term for inflammation or disease of the liver. Viral hepatitis signifies liver inflammation or disease induced by an infecting hepatotropic virus, and is a major worldwide public health problem. At present, five specific hepatotropic viruses involved in this disease have been isolated and characterized. They are identified as hepatitis A virus (HAV), hepatitis B virus (HBV), hepatitis C virus (HCV), hepatitis D virus (HDV) and hepatitis E virus (HEV). These hepatotropic viruses are believed to cause hepatitis, resulting in illnesses characterized by fever, nausea, vomiting, anorexia (loss of appetite) and, in some instances, jaundice and even death.
In the less severe and more ordinary cases, patients infected with a hepatitis virus normally experience nausea, vomiting, loss of appetite and fever. Jaundice with the appearance of bilirubin in the urine and/or death are uncommon. The symptoms associated with the mild form of hepatitis typically continue for approximately 2-6 weeks and the damage to the liver is normally restored within 8-12 weeks following the prodromal period. While complete recovery occurs in most cases, weariness and fatigue may continue for several months even after the symptoms have disappeared. In the most severe cases of viral hepatitis, the prognosis is much more serious. Generally, the attacks are sudden and the longevity of the illnesses is very acute. The patients infected with this form of the disease usually experience vomiting, severe jaundice, bilirubin in the urine, delirium, coma and death.
Since at least as early as 1973, HAV and HBV have been detected and partially characterized, and reliable diagnostic assays for their detection and diagnosis have been available. Notwithstanding this advancement in the area of viral hepatitis , the continual appearance of hepatitis in patients negative for HAV and HBV led researchers and the medical community to believe that other hepatotropic agents existed. It was learned through research dating back to as early as about 1974 that these unknown hepatotropic agents principally spread through either fecal/oral routes or blood, and they came to be known as non-A, non-B hepatitis (NANBH) agents.
Over the past two years, intense research relative to the NANBH agents has lead to the isolation and identification of the fourth and fifth hepatotropic viruses, namely, HCV and HEV, respectively. See Choo, Q.-L. et al.: Science, 244:359-362 (1989) and Reyes, G.R. et al.: Science, 247:1335-1339
(1990). HCV is believed to cause the majority (at least about 90%) of the parenterally or blood transmitted NANBH whereas HEV is believed to be responsible for the transmission of enteric
(fecal/oral) NANBH. Like HEV, HAV is principally spread by the fecal/oral routes, whereas HBV, HCV and
HDV are transmitted principally by the parenteral routes and blood or blood derived products. Even though all five of these hepatotropic viruses
apparently are somewhat related, only HDV is unable to grow and replicate on its own. HDV requires the co-presence of HBV for its replication in humans and therefore is found only in patients infected with HBV.
Moreover, HDV infection in the presence of HBV
(co-infection or super-infection) is believed to cause the most severe form of viral hepatitis. A high percentage of super-infection cases end in death within a few months from the onset of the disease.
While complete recovery normally occurs in
HAV and HEV cases and in over 85% of HBV cases, progression to chronic hepatitis from acute HCV is observed in about 25-55% of HCV cases. Moreover, HCV viruses are known to cause serious liver disease such as cirrhosis of the liver, non-established malignant hepatoma (liver cancer) and the like.
Clinical diagnosis of HCV (parenteral NANBH) has been accomplished heretofore primarily by the exclusion of other hepatitis human viruses such as HAV and HBV. Among the methods used hitherto to detect putative HCV antigens and antibodies are agar-gel diffusion, counter immunoelectrophoresis, immunofluorescence microscopy , immune electron microscopy, radioimmuno assay, and enzyme-linked immunoabsorbent assay (ELISA). However, none of these assays heretofore available has proved to be sufficiently sensitive, specific, and reproducible to be used as reliable diagnostic tests for HCV, especially acute HCV infections, or for screening blood and blood derived products for HCV contamination.
For example, one present method utilized to detect HCV involves the detection of anti-C100 antibody to the non-structural region of HCV, i.e., C100 protein (antigen). This is accomplished by a commercially available kit distributed by Ortho Diagnostics under the brand name Ortho HCV Ab ELISA test. Unfortunately, it has been found that this assay for detecting HCV cannot identify all blood samples containing HCV, especially those contaminated with acute HCV infections. For instance, it has been reported that this assay detects false positives approximately 40-60% of the time. See Mimms, L. et al.: The Lancet, (336): 1590-1591 (Dec. 22/29, 1990); Zuck, T.F. et al.: Transfusion, 30(8): 759-761 (1990); Getzug, T. et al.: AASLD Abstracts of Papers. Hepatology, 12(4) Pt.2, Abstract No. 32, p. 845 (Oct., 1990); and Attard, L. et al.: JOHEEC, (11) Supplement 2, Abstract No. 10, S3 (1990). But see Baldi, M., et al.: JOHEEC, (11) Supplement 2, Abstract No. 16, S4 (1990). It also has been reported that the Ortho HCV Ab ELISA assay fails to detect HCV infection approximately 10% of the time. See Getzug, T. et al.: AASLD Abstracts of Papers. Hepatology, 12(4) Pt.2, Abstract No. 32, p. 845 (Oct., 1990).
In another present method utilized to detect HCV, it involves the detection of anti-C100 antibody to the non-structural HCV C100 protein (antigen).
This is accomplished by a methodology developed by Ortho/Chiron and is referred to as recombinant immunoblot assay I (RIBA I). While the RIBA I assay for detecting HCV has been found to be more reliable than the Ortho HCV Ab ELISA test, it nevertheless is a qualitative test which is believed to be indeterminant for a significant number of HCV antibody containing samples. See, for example, Alberti, A. et al.:
JOHEEC, (11) Supplement 2, Abstract No. 3, S1 (1990).
In yet another present method utilized to detect HCV, it involves the indirect detection of anti-HCV antibody. This is accomplished by a methodology developed by Abbott and is referred to as a neutralization assay. Unfortunately, the neutralization assay for detecting HCV is believed to be unreliable because the test utilizes only a portion of HCV, i.e., the non-structural C100 protein (antigen), while a true HCV neutralization assay requires the utilization of the infectious particle, i.e., the virion, of HCV.
In still another present method utilized to detect HCV, it involves the detection of HCV-RNA in sera in chronic NANBH by the polymerase chain reaction (PCR) using specific HCV synthetic oligonucleotides. Bartolome, J. et al.: JOHEEC, (11) Supplement 2, Abstract No. 13, S5 (1990). Unfortunately, Bartolome, J. et al. report therein that this assay detects HCV RNA in only 44% of the anti-HCV chronic nepatitis sera, and that there remains the need for a more sensitive test to detect HCV infection.
Presently, the more reliable means to diagnose HCV (parenteral NANBH) infection is by biopsy and differential diagnosis. Whether this form of NANBH (HCV) is acute or chronic may be determined by examining the liver tissue by biopsy according to the criterion described in Sheila Sherlock, diseases of the Liver and Eiliary System, eighth edition, published by Blackwell Scientific Publications, London, pp. 326-333 (1989). Unfortunately, liver biopsies are expensive, painful and may lead to patient complications. Moreover, this technique for diagnosing HCV obviously is inapplicable for screening blood and blood derived products. Because present tests for the detection of HCV (parenteral NANBH) are ineffective, time
consuming, expensive and/or complex, blood and blood products remain potential sources for transmission of HCV (parenteral NANBH) to recipients. In the 1970s, the frequency of post-transfusion NANBH ranged from approximately 7% to 12%. However, in the 1980s, improvements in transfusion medicine, due to the necessity to screen blood supplies for antibody to human immunodeficiency virus (HIV), has significantly reduced the frequency of post-transfusion hepatitis to approximately 5%. See Dienstag, J.L.:
Gastroenterology, 99:1177-1180 (1990). Notwithstanding this reduction in the frequency of post-transfusion hepatitis, it is presently believed that HCV (parenteral NANBH) accounts for up to 90% of these cases, as indicated hereinbefore. It is estimated that in the USA alone, HCV (parenteral NANBH) affects about 200,000-300,000 people annually.
For these reasons, there is a significant demand for sensitive, specific methods for inexpensively and effectively screening and identifying carriers of both acute and chronic HCV infections and HCV contaminated blood or blood derived products.
Patient care as well as the prevention of transmission of HCV by blood and blood products or by close personal contact require reliable screening, diagnostic and prognostic tools to reliably detect nucleic acids, antigens and antibodies related to HCV. Moreover, there is an urgent need for effective vaccines and immunotherapeutic agents for the prevention and/or treatment of HCV disease.
Summary of the Invention
In brief, the present invention alleviates and overcomes certain of the above-referenced problems and shortcomings of the present state of the HCV art througn the discovery of novel immunogenic HCV polypeptides, each having epitopes unique to HCV. The uniqueness of the epitopes have been determined by their immunological reactivity with anti-HCV antibodies and lack of immunological reactivity with antibodies to HBV.
The novel immunogenic HCV polypeptides of the present invention are truncated structural polypeptides which have been discovered from the structural region of a human HCV genome upon analysis of a putative amino acid sequence translated therefrom. The amino acid sequences of the novel immunogenic HCV polypeptides are believed to contain at least about 15 amino acid residues and more preferably at least about 17 amino acid residues, and lie within the first four percent (4%) of the putative amino acid sequence for the human HCV polyprotein. The novel immunogenic HCV polypeptides of the instant invention are basic polypeptides having an estimated pI on the order of about 12, and are believed to include at least about 7 amino acid residues (45%) in their Chou-Fasman (CF) turns, and more particularly between about 7 and 17 amino acid residues (45-100%) in their CF turns. The calculated molecular weights of the novel immunogenic HCV polypeptides of the present invention are at least about 1500 and more particularly in the range of between about 1500 and about 2500. The novel immunogenic HCV polypeptides of the instant invention are further characterized as having in their amino acid sequences at least about 4 arginine amino acid residues (25%) and more particularly between about 4 and 7 (25-47%) arginine amino acid residues, at least about 11 hydrophillic amino acid residues (70%) and more particularly between about 11 and 14 hydrophillic amino acid residues (70-87%), and at least about 5 polar amino acid residues (30%) and more particularly between about 5 and 8 polar amino acid residues
(30-53%). Consistent with the estimated pi, the novel immunogenic HCV polypeptides of the instant invention include within their amino acid sequences at least about 5 basic amino acid residues (30%) and more preferably between about 5 and 7 basic amino acid residues (30-47%). Two novel preferred immunogenic HCV
polypeptides of the present invention are designated as FGB1 and FGB2. The FGB1 polypeptide has a calculated molecular weight of about 1908, includes 15 amino acids and has the following sequence:
Lys - Pro - Gln - Arg - Lys - Thr - Lys - Arg - Asn - Thr - Asn - Arg - Arg - Pro - Gln.
The FGB2 polypeptide has a calculated molecular weight of about 1811, also includes 15 amino acids and has the following sequence:
Ser - Arg - Pro - Ser - Trp - Gly - Pro - Thr - Asp - Pro - Arg - Arg - Arg - Ser - Arg.
The above-recited amino acid sequences for the novel FGB1 and FGB2 polypeptides are believed to be truncated structural segments derived from a putative amino acid sequence of a human HCV capsid protein which is believed to be encoded within the first 4% of codons following the predicted ATG initiation codon of a human HCV polyprotein. It is further believed that the amino acid sequence for the novel FGB1 polypeptide lies within the putative amino acid sequence for the human HCV capsid protein near its -NH2 terminal end whereas the amino acid sequence for the novel FGB2 polypeptide lies within the putative amino acid sequence for the human HCV capsid protein near its -COOH terminal end. The FGB1 and FGB2 polypeptides may also include, for example, an additional amino acid
residue, such as cysteine, or a cystine moiety at the -NH2 terminal end in order to facilitate for instance the conjugation of carrier polypeptides or molecules, such as keyhole limpet hemocyanin (KLH) or ovalbumin, for antibody production in animals. Further, the FGB1 and FGB2 polypeptides may include an amino acid residue, such as tyrosine or histidine, at the -COOH terminal end in order to facilitate the labeling of these novel polypeptides with a labeling moiety, such as radioactive iodine or phosphorous. When both the cysteine and tyrosine amino acid residues are added, the FGB1 and FGB2 polypeptides have calculated molecular weights of about 2,174 and 2,077, respectively. It is believed that the addition of the cysteine and tyrosine amino acid residues uniquely and advantageously enhances the reactivity of the immunogenic HCV polypeptides of the instant invention with antibodies in, for example, immuno assays. It is believed that the added cysteine and tyrosine amino acid residues may enhance the ability of the immunogenic HCV polypeptides to attach to the microwell plates possibly by interaction, especially between the tyrosine amino acid residues and such plates, in a manner that leaves the remainder or a greater portion of the immunogenic HCV polypeptides exposed to the reacting antibodies. Examples of other novel basic HCV
polypeptides in accordance with the present invention that are believed to be immunogenic and have epitopes for HCV are recited hereinafter. These polypeptides of the instant invention have been designated herein throughout as FGB polypeptides, and include:
FGB3-CRPQRKTKRNTNRRPQY; FGB4-CKPQRRTKRNTNRRPQY;
FGB5-CKPQRKTRRNTNRRPQY; FGB6-CKPQKKTKRNTNRRPQY;
FGB7-CKPQRKTKKNTNRPPQY; FGB8-CKPQRKTKRNTNKRPQY;
FGB9-CKPQRKTKRNTNRKPQY; FGB10-CKPQRKSKRNTNRRPQY;
FGB11-CKPQRKTKRNSNRRPQY; FGB12-CRKTKRNTNRRPQY;
FGB13-CKKTKRNTNRRPQY; FGB14-CRRTKRNTNRRPQY;
FGB15-CRKTRRNTNRRPQY; FGB16-CRKSKRNTNRRPQY;
FGB17-CRKTKKNTNRRPQY; FGB18-CRKTKRNTNKRPQY;
FGB19-CRKTKRNTNRKPQY; FGB20-CKQKKTKRSTNRRPQY;
FGB21-KQKKTKRSTNRRPQY; FGB22-KPQKKTKRSTNRRPQY;
FGB23-KPQKKNKRSTNRRPQY; FGB24-CKQRKTKRSTNRRPQ;
FGB25-KQRKTKRSTNRRPQY; FGB26-CKQRKTKRSTNRRPQY;
FGB27-KPQRKTKRSTNRRPQY; FGB28-CKQRKTKRNTNRRQY;
FGB29-KQRKTKRNTNRRQ; FGB30-KQRKTKRNTNRR;
FGB31-KPQRKNKRNTNRRPQ; FGB32-CKQRKTKRNTNRRPQY;
FGB33-KQRKTKRNTNRRPQ; FGB34-CKPQKKNKRNTNRRPQY;
FGB35-CKPQRKNKRNTNRRPQY; FGB36-KPQRKTKRNTNRRPQ;
FGB37-CKPQRKTKRNTNRRPQY; FGB38-KPQKKNKRNTNRRPQ.
In the above-recited amino acid sequences for the FGB3-FGB38 polypeptides, R or Arg represents argenine, N or Asn represents asparagine, C or Cys represents cysteine, Q or Gln represents glutamine, K or Lys represents lysine, P or Pro represents proline, S or Ser represents serine, T or Thr represents threonine and Y or Tyr represents tyrosine.
The novel immunogenic HCV polypeptides, and especially the FGB1 and FGB2 polypeptides, may be used in diagnostic tests and kits, such as enzyme-linked immunosorbent assays (ELISA, EIA), radioimmunoassays (RIA), immunoblot assays, and the like, to detect the presence of specific HCV antibodies present in blood, sera, semen, or other biological materials of patients with HCV. For example, a coded HCV panel of 44 sera was tested in an ELISA using the FGB1 and FGB2
polypeptides. The FGB1 and FGB2 polypeptides utilized in this ELISA included the cysteine and tyrosine amino acid residues at their respective -NH2 and -COOH terminal ends. The 44 sera were also tested for anti-C100 HCV antibody (EIA, Ortho/Chiron), recombinant immunoblot assay I (RIBA I, Ortho/Chiron), and neutralization (Neut., Abbott). Eight of eight samples that were reactive for C100 EIA, RIBA I and Neut., contained antibodies to FGB1 or FGB2
polypeptides. None of the six sera that were EIA reactive, but RIBA I indeterminate had antibodies to the FGB1 and FGB2 polypeptides. More significantly, however, two sera from a patient who had been
clinically diagnosed as having non-A, non-B hepatitis (NANBH) and were EIA and RIBA I non-reactive were reactive to the novel FGB1 and FGB2 polypeptide. One of the two such sera was reactive to the novel FGB2 polypeptide. These results are summarized in the HCV PANEL I and in TABLES 9 and 10 in greater detail.
HCV PANEL I
No. of C100
Sera Samples EIA RIBA I Neut. FGB1 FGB2
8 3r 8r 3y 8r 8r
6 6r 6i 3y 6nr 6nr
12 12r 12nr 6y 12nr 12nr
13 13nr nt nt 13nr 7nr
2* 2nr 2nr nt 2r lr
2 2r nt nt 2r 2nr
1 nt nt nt lnr lr r-reactive; i-indeterminate; nr-non-reactive; nt-not tested; y-positive.
*clinically diagnosed NANBH patient, non-reactive with C100 EIA and RIBA I.
In a second coded HCV panel, 115 sera from patients with clinical evidence of NANB (type C) viral hepatitis or volunteer blood donors with or without evidence of NANB (type C) viral hepatitis were tested in an ELISA using the FGB1 polypeptide (HCV PANEL II).
This panel was used to compare the ELISA utilizing FGB1 with an experimental C-200/C-22 ELISA (second generation of Chiron Corp.). Like with the first coded panel, the FGB1 polypeptide utilized in this
ELISA included the cysteine and tyrosine amino acid residues at its respective -NH2 and -COOH terminal ends. The 115 sera were also tested with C-100 ELISA
(Ortho/Chiron Corp.) and recombinant immunoblot assay II (RIBA II, Chiron Corp.) for HCV antibodies. All 115 sera were initially tested with the C-100 ELISA and all were reactive. Fifty-two sera (HCV PANEL II) that were reactive with C-200/C-22 ELISA and HCV-FGB1 ELISA were verified to be reactive with RIBA-II, with the exception of three sera that were indeterminate (one single band was reactive on RIBA-II). Forty sera non-reactive with C-200/C-22 ELISA and HCV-FGB1 ELISA were also found to be non-reactive with RIBA-II. Of two sera (HCV PANEL II, 2nrs) non-reactive with
C-200/C-22 ELISA and HCV-FGB1 ELISA, one was reactive with RIBA-II and one was indeterminate. One serum sample (HCV PANEL II, 1r+) that was reactive with HCV-FGB1 ELISA was non-reactive with both the
C-200/C-22 ELISA and RIBA-II. On the other hand, two serum sample (HCV PANEL II, 1nr°) that were reactive with both C-200/C-22 ELISA and RIBA-II tested negative with HCV-FGB1 ELISA. Both of these samples became reactive upon retesting with HCV-FGB1 ELISA. Finally, 18 sera which were reactive with C-200/C-22 ELISA tested negative with HCV-FGB1; 11 were negative with RIBA-II while 7 were indeterminate. While the
majority (11/18 or 61%) appear to be false positive by the C-200/C-22 ELISA, the 7 (39%) that were
indeterminate were reactive with one non-structural antigen, (C-100), of RIBA II, and therefore are most probably false positive by C-200/C-22 ELISA. HCV PANEL II
Results of Comparative Testing of Coded Panel 2 (115 sera Reactive by the C-100 ELISA)
HCV
C-200
No. of HCV-SP C-22
Samples ELISA RIBA-II ELISA
52 52r 49r + 3i 52r
40 40nr 40nr 40nr
18 18nr 11nr + 7i 18r
2 2nr# 1r + 1i 2nr
1 1r+ 1nr 1nr
2 2nr° 2r 2r
115
r-reactive; nr-non-reactive; i-indeterminate (only one band out of four was reactive)
RIBA-II
Sample 5-1-1 C-100 C33c C22
#HCV055 - 1+ - -
HCV203 1+ 1+ - -
+HCV225 HCV-SP ELISA ( S/CO= 1.425) o HCV214, HCV234 became reactive upon retesting with
HCV-FGB1 ELISA.
In a third coded HCV panel, 1076 sera from volunteer, healthy, blood donors were also tested with the HCV-FGB1 ELISA (HCV PANEL III). As with the first two coded panels, the FGB1 polypeptide utilized in this ELISA included the cysteine and tyrosine amino acid residues at its respective -NH2 and -COOH
terminal ends. Eleven of the 1,076 samples reactive with HCV-FGB1 ELISA were further tested with RIBA-II and C-200/C-22 ELISA. One of the eleven sera was reactive by all three tests; 10 sera were repeat positives only by HCV-FGB1 ELISA. At this stage, it is unknown whether this reflects the rate of false positive tests (less than 1%) using HCV-FGB1 ELISA or whether the HCV-FGB1 ELISA can detect antibody to HCV undetected by the other two tests, i.e., C-200/C-22 ELISA and RIBA II.
HCV PANEL III
Results of Testing Health Blood Donors for Anti-HCV Antibodies
HCV HCV-FGB1 C-200
NO. of REPEAT C-22
DONORS REACTIVE RIBA-II ELISA
1076 11° 1* 1*
º Only the 11 samples identified reactive by HCV-FGB1 ELISA were tested by RIBA-II and the C-200/C22 ELISA. * This sample was among those that were repeat
reactive with HCV-FGB1 ELISA.
In a fourth coded HCV panel obtained from the Transfusion Transmitted Virus Study (TTVS) at the National, Heart, Lung and Blood Institute (NHLBI) , Bethesda (MD, Hollinger , F. B. et al . , 1982 in Viral Hepatitis: 1981 International Symposium, ed. Szmunes, W., Alter, H.J. and Maynard, J.E., Franklin Institute Press, Philadelphia, pp. 361-376), 94 sera obtained from serial bleeds were tested with the HCV-FGB1
ELISA, C-100 ELISA and RIBA II (HCV PANEL IV). These samples were selected from among those collected from blood transfusion recipients at 2 week intervals for 6 months, 3 week intervals for the next three months, and then at 10 months. A serum sample was also obtained, in most cases, on day 7 after the first transfusion (recipients) or after surgery (controls). After reporting the results on this fourth coded panel, it was revealed that this panel consisted of 18 transfusion recipients and 10 control subjects
(hospitalized, but not transfused). The transfusion recipients had been previously diagnosed as cases of acute or chronic hepatitis B and/or NANB hepatitis. Again, the FGB1 polypeptide utilized in this ELISA included the cysteine and tyrosine amino acid residues at its respective -NH2 and -COOH terminal ends. The second generation C-200/C-22 ELISA was unavailable for this fourth coded panel. The results on each of these samples were matched to the different transfused patients or control subjects in this panel (HCV PANEL IV). Six control (non-transfused) subjects without evidence of hepatitis tested negative by all three assays. Four subjects (non-transfused) with a
clinical diagnosis of NANB hepatitis tested negative by HCV-FGB1 ELISA; three of these four subjects also tested negative by the two other tests. Of the six transfusion recipients with clinical and laboratory diagnosed HBV hepatitis, the two also diagnosed with NANBH tested positive for HCV by all three tests. The remaining four recipients with HBV alone tested negative with HCV-FGB1 ELISA. Nine of nine recipients with diagnosed NANB hepatitis who tested positive with HCV-FGB1 ELISA were reactive on RIBA-II. In contrast, three other recipients with NANB hepatitis tested negative by HCV-FGB1 ELISA, HCV C-100 ELISA; two of the three recipients were negative by RIBA-II, and one was indeterminate.
HCV PANEL IV
HCV-FGB1 ELISA, C-100 ELISA and RIBA-II Results in Recipients and Control Subjects from TTVS-NHLBI
Total No. FGB1 C-100
Subjects ELISA ELISA RIBA-II
Control+ - no NANB 6 NR NR NR Control+ - NANB 3 NR NR NR Control+ - NANB 1 NR R R
Recipientº - HBV 2 NR NR NR
1 NR R NR 1 NR R I
Recipientº - HBV 2 R R R
and NANB
Recipientº - NANB 8 R R R
(type HCV)
Recipientº - NANB 1 R NR R
(type HCV)
Recipientº - NANB 2 NR NR NR
(non HCV)
NR NR I
+ Controls were hospitalized patients who did not
receive a blood transfusion and were diagnosed as having or not having viral hepatitis.
º Recipients were patients who received one or more blood transfusions and were diagnosed as having viral hepatitis.
It should therefore now be apparent that the instant invention identifies new epitopes to the structural region of the human HCV genome to which HCV infected patients elicit antibodies. Based upon the results from the coded HCV panels, it is believed that these antibodies correlate with the presence of true positive HCV antibody as defined by C-100 EIA, C-200/C22 EIA, RIBA I and RIBA II. Moreover, it is believed that assays using the truncated immunogenic HCV polypeptides and in particular the FGB1 and FGB2 polypeptides of the instant invention, which are derived from the structual region relatively near the predicted ATG initiation codon of the human HCV genome, to detect the presence of HCV are more
specific and sensitive than assays using the
recombinant C100 polypeptide, which is derived from the non-structual region of the HCV gene as well as the RIBA I or Neut. assays. Remarkably, the novel immunogenic HCV polypeptides of the instant invention are therefore believed to allow for significantly increased specificity in the detection of antibodies in blood or blood derived products contaminated with HCV or in patients with acute or chronic HCV at a relatively lower cost as compared to existing HCV tests.
The present invention also contemplates the detection of passive HCV-specific antibodies which have been transferred to patients via transfused blood containing detectable levels of the specific HCV antibodies. Quite remarkably, it is believed to now be possible to detect passive HCV-specific antibodies almost immediately following the initial transfusion initiated HCV infection when following the teachings of the present invention. Even more remarkable, it is believed to now be possible to detect HCV-specific antibodies, i.e., passive HCV-specific antibodies followed by actively produced HCV-specific antibodies, in transfusion initiated HCV infected patients at relatively high levels for several months, as verified by the fifth coded panel and illustrated in FIGS.
11A-11G. Moreover, it is believed that the appearance of actively produced HCV-specific antibodies coincides with the first elevation of ALT levels. This is believed to have been confirmed using serial specimens from patients infected with transfusion associated NANB hepatitis. (Coded Panel 5). By "passive
HCV-specific antibodies, it is meant herein to refer to those antibodies present in donated blood which are specific for HCV antigens and which have been produced by the donor. By "actively produced antibodies", it is meant herein to refer to those antibodies which are specific for HCV antigens but which have been produced by a person following HCV infection, such as a
recipient of donated HCV-infected blood.
In order to document the time of appearance of HCV specific antibody responses in certain
recipients from the above TTVS-NHLBI study who
developed NANB hepatitis, we tested, using HCV-FGB1 ELISA, a coded panel of serial serum samples from six recipients and one control subject (Coded Panel 5) and correlated these results with the ALT levels. As with the other coded panels, the FGB1 polypeptide utilized in this ELISA included the cysteine and tyrosine amino acid residues at its respective -NH2 and -COOH
terminal ends. HCV-specific antibody was detected by HCV-FGB1 ELISA in all six recipients and its
appearance was found to coincide with the earliest elevation of ALT levels. Five out of the six
recipients, with the exception of recipient #10805 (See FIG. 11D), on average had detectable HCV-specific active antibody within a month of transfusion
transmitted HCV infection. (FIGS. 11B-11G).
Significant levels of passive HCV-specific antibody, probably passively transferred from a donor, were detected during the first week post-transfusion for 3 recipients, i.e., recipient #s 30587, 21354 and 20621 (FIGS. 11F, 11C and G, respectively), with marginal presence in two other recipients, i.e., recipient ss 20959 and 10762 (FIGS. 11B and 11E, respectively). This same coded panel was tested subsequently under code with the C-200/C-22 ELISA. With the exception of a few test results, the data relative to the
C-200/C-22 ELISA coincided with the HCV-SP specific antibody response, as reported in the description of FIGS. 11A-11G hereinafter. While FIGS. 11A-11G report the detection of passive HCV-specific antibody at seven days following transfusion, it is believed that passive HCV-specific antibody can be detected in a recipient as early as about 20 minutes or earlier, e.g. whatever the time is required for blood to circulate, following transfusion with donated blood which contains detectable levels of passive
HCV-specific antibody. It should be understood by those versed in this art that all five coded panels were tested with an HCV-FGB1 ELISA as described in Example I.
In another aspect of the present invention, it is concerned with the polynucleotide sequences which encode the two novel HCV polypeptides, FGB1 and FGB2 , respectively, or degenerate polynucleotides which are complementary or correspond to all or certain nucleotides of the polynucleotide sequences. The present invention therefore contemplates the following novel polynucleotide sequences I and II which are believed to encode the novel FGB1 and FGB2 polyeptides, respectively:
(I)
5'-AAACCTCAAAGAAAAACCAAACGTAACACCAACCGTCGCCCACAG-3';
(U) (U) (U)
and
(II)
5'-TCTCGGCCTAGTTGGGGCCCCACGGACCCCCGGCGTAGGTCGCGC-3'.
(U U) (UU) (U) (U)
novel polynucleotides which are complementary or correspond to all or certain nucleotides thereof; and novel polynucleotide sequences obtained by
substituting at least one nucleotide in each codon for a corresponding amino acid of said polynucleotide sequences I and II, in accordance with degeneracy of the genetic code. The novel polynucleotide sequences of the instant invention may be inserted into recombinant expression vectors or the like and used to transform cells in order to recombinantly produce the FGB1 and FGB2 polypeptides. In another application of the novel polynucieotide sequences is their use in kits as probes or primers for the detection of RNA or nucleic acids from HCV in samples or other biological materials, such as sera, ascites, lymph, infected tissue, and cell cultures. The probes or primers which are complementary or correspond to the above-recited polynucieotide sequences should be of a sufficient length which allows for the detection of HCV RNA or DNA, respectively. For example, while 6-8 nucleotides complementary to or derived from the above-recited polynucleotide sequences may be a workable length, sequences of 10-12 nucleotides complementary thereto or derived therefrom are preferred and about 20-45 nucleotides and in particular about 20 nucleotides complementary or derived therefrom thereto appear to be most optimal.
The instant invention is also concerned with antisense single stranded DNA and RNA sequences derived from the polynucleotide sequences encoding the FGB1 or FGB2 polypeptides, which have the ability to bind to HCV RNA and block protein translation and/or prevent HCV RNA replication. Still further, the present invention is concerned with ds DNA fragments which, when inserted into appropriate vectors such as retroviral vectors and introduced into infected liver cells results in the transcription of the antisense RNA sequences. As a further advantage, it is believed that such antisense polynucleotide sequences can be designed to include in their construct, for example, ribozymes that may play a role in the therapeutic treatment of HCV by catalytic degradation of the HCV RNA in the infected liver cells.
The present invention further contemplates the production of antibodies, polyclonal or monoclonal, raised against the novel immunogenic HCV polypeptides of the instant invention. In another aspect of the instant invention, it is concerned with the production of anti-idiotype antibodies raised against antibodies to the novel immunogenic HCV polypeptides. The novel antibodies of the present invention can be used in diagnostic tests and kits to detect HCV proteins (antigens) or anti-HCV antibodies, respectively. Moreover, it is believed that such antibodies of the present invention may have the unique ability to cross-react with host protein(s) to detect autoimmune liver disease induced by, for example, HCV. Still another aspect of the instant invention is concerned with vaccines as prophylaxis against HCV infection by administering the novel immunogenic HCV polypeptides of the instant invention, either individually or in combination, to individuals. The vaccines of the instant invention comprise an
immunogenic HCV polypeptide, such as FGB1 and/or FGB2 polypeptides, in pharmacologically effective doses mixed in pharmaceutically acceptable excipients.
Preferably, the novel immunogenic HCV polypeptides of the instant invention are conjugated to, for example, carrier proteins, antibodies, or particle-forming proteins, such as those associated with hepatitis B surface antigen, and mixed with an adjuvant in order to elicit protective antibodies when administered to individuals. In addition, novel antibodies to the novel immunogenic HCV polypeptides of the present invention may be used for passive immunity in short term therapy against HCV.
Accordingly, it can now be appreciated that the present invention is believed to provide a
solution to the HCV art that has long sought effective and inexpensive means to treat and reliably detect HCV, including HCV infection in acute and chronic phases.
The above features and advantages will be better understood with reference to the Figs., Examples and Detailed Description set out hereinbelow. It will also be understood that the biological
materials of this invention are exemplary only and are not to be regarded as limitations of this invention.
Brief Description of the Figs.
Reference is now made to the accompanying Figs, in which are shown characteristics corresponding to the novel immunogenic HCV polypeptides of the instant invention from which certain of their novel features and advantages will be apparent:
Fig. 1 is a graphic illustration of the hydrophilicity and hydrophobicity (surface
probability) profiles and the antigenic index of about the first 500 putative amino acids encoded by the structual gene of a human HCV isolate following the predicted ATG initiation codon;
Fig. 2 is a grapnic illustration of the hydrophilicity and hydrophobicity (surface
probability) profiles and the antigenic index of about the first 144 putative amino acids encoded by the structual gene of the human HCV isolate of Fig. 1 following the predicted ATG initiation codon;
Fig. 3 is a graphic illustration depicting that there are eight (8) amino acid residues of the Cys-FGB1-Tyr polypeptide present in its Chou-Fasman turn (C = cysteine, K = lysine, P = proline, Q = glutamine, R = arginine, T = threonine, N =
asparagine, Y = tyrosine);
Fig. 4 is a graphic illustration depicting that there are seven (7) amino acid residues of the FGB1 polypeptide present in its Chou-Fasman turn (K = lysine, P = proline, Q = glutamine, R = arginine, T = threonine, N = asparagine);
Fig. 5 is a graphic illustration depicting that there are seventeen (17) amino acid residues of the Cys-FGB2-Tyr polypeptide present in its
Chou-Fasman turn (C = cysteine, S = serine, R = arginine, P = proline, W = tryptophan, G = glycine, T = threonine, D = aspartic acid, Y = tyrosine);
Fig. 6 is a graphic illustration depicting that there are twelve (12) amino acid residues of the FGB2 polypeptide present in its Chou-Fasman turn (S = serine, R = arginine, P = proline, W = tryptophan, G = glycine, T = threonine, D = aspartic acid);
Fig. 7 is a HPLC profile that shows the purity of the Cys-FGB1-Tyr polypeptide which has a retention time of about 12.45 minutes;
Fig. 3 is a HPLC profile that shows the purity of the Cys-FGB2-Tyr polypeptide which has a retention time of about 17.73 minutes;
Fig. S is a mass spectrum profile that shows the purity of the Cys-FGB1-Tyr polypeptide and the molecular weight thereof; Fig. 10 is a mass spectrum profile that shows the purity of the Cys-FGB2-Tyr polypeptide and the molecular weight thereof; and
Figs. 11A-11G are correlations between antibodies to HCV-FGB1 and ALT levels
Figure imgf000032_0001
on serial samples from a control
Figure imgf000032_0002
patient without hepatitis, i.e., recipient =10521 (Fig. 11A), and six transfusion recipients with NANB hepatitis. The number of units received by the recipients is indicated in parenthesis. The sample to cutoff (5/CO) ratio of 1.0 for the HCV-FGB1 ELISA is indicated by the horizontal line where values above the line indicate reactivity while those below the line are non-reactive. Results on the C-200/C-22 ELISA which differed from those of the HCV-FGB1 ELISA were as follows: All the serum samples of recipient #10805 (Fig. 11D) including the one withdrawn prior to receiving blood transfusion were found to be positive with C-200/C22 ELISA. The other differences were that, in recipient #s 21354 and 30587 (Figs. 11C and 11F, respectively), the passive HCV-specific
antibodies, i.e., passively transferred HCV-specific antibodies from the donors, persisted for a longer time than the HCV-FGB1 specific antibodies, especially on days 21 and 17 respectively, and the serum sample withdrawn on day 29 for the recipient =21354 (Fig. 11C) was negative with C-200/C-22 ELISA, but positive with the HCV-FGB1 ELISA.
Detailed Description
By way of illustrating and providing a more complete appreciation of the present invention and many of the attendant advantages thereof, the following detailed description is provided concerning the novel immunogenic HCV polypeptides, polynucleotide sequences encoding the immunogenic HCV polypeptides and antisense polynucieotide sequences derived
therefrom, antibodies raised against the immunogenic HCV polypeptides, anti-idiotype antibodies raised against the HCV antibodies to the immunogenic HCV polypeptides, HCV vaccines, and methods.
The present invention provides for the discovery of highly immunogenic HCV polypeptides, each having epitopes for HCV. The immunogenic HCV
polypeptides of the instant invention have been derived from the putative amino acid sequence of the HCV polyprotein encoded by the five prime (5') end of a human isolate of a HCV genome. The amino acid residues of the epitopes of the immunogenic HCV polypeptides are believed to lie within the first 4% of the amino acid residues in the putative amino acid sequence of the HCV polyprotein. The immunogenic HCV polypeptides of the instant invention are generally basic, truncated structural polypeptides of at least about 15 amino acids in length and are preferably between about 15 and about 17 amino acids. In addition, the immunogenic HCV polypeptides of the present invention have calculated molecular weights of at least about 1500 and preferably of between about 1500 and about 2500, and include at least about 7 amino acid residues (45%) in their Chou-Fasman (CF) turn, as evidenced by Figs. 3-6. As more particularly shown in Figs. 5 and 6, certain of the immunogenic HCV polypeptides of the instant invention may include at least about 12-17 amino acids residues (80%-100%) in their CF turns.
See Chou, P.Y. and Fasman, G.D.: Biochemistry,
13:211-222 (1974); Chou, P.Y. and Fasman, G.D.:
Biochemistry, 13:222-245 (1974); Chou, P.Y. and
Fasman, G.D.: Ann. Rev. Biochem., 47:251-275 (1978); and Chou, P.Y. and Fasman, G.D.: Adv. Enzymol. Relat. Areas Mol. Biol., 47:45-148 (1978). Still further, at least about 11 of the amino acid residues (70%) within their amino acid sequences are hydrophillic. Preferably, however, between about 11 and 14 (70-87%) of the amino acid residues are hydrophillic. Still further, at least about 5 (30%) and preferably between about 5 and 8 (30-53%) of the amino acid residues of the immunogenic HCV polypeptides of the present invention are polar amino acid residues. Moreover, the immunogenic HCV polypeptides of the instant invention have at least about 5 basic amino acid residues (30%) and more preferably between about 5 and 7 basic amino acid residues (30-47%). Accordingly, the estimated pI for the HCV polypeptides of the instant invention are typically on the order of about 12. In addition, the immunogenic HCV polypeptides have at least about 4 arginine amino acid residues (25%) in their amino acid sequences and more preferably include between about 4 and 7 arginine residues (25-47%).
In discovering the polypeptides of the instant invention, the first 500 or so putative amino acids following a predicted ATG initiation codon of a structural gene encoding a putative structural
polyprotein derived from a human HCV isolate were subjected to hydrophilicity/hydropnobicity/antigenicity analysis. See Fig. 1. This was accomplished via a protein analysis program marketed under the name Mac Vector by International Bio Technologies Inc., P.O. Box 9558, New Haven, CT 06535. According to the hydrophilicity, surface probacility and antigenic index plots as shown in Fig. 1, the first 144 or so putative amino acids following the predicted ATG initiation codon within the HCV structural gene encoding the putative structural polyprotein derived from the human HCV isolate presented what appears to be a strong antigenic region. As a result, the hydrophilicity, surface probability and antigenic index plots of these first 144 putative amino acids were expanded, as shown in Fig. 2. The expanded index plots of the first 144 putative amino acids, as depicted in Fig. 2, revealed that these first 144 putative amino acids further presented several smaller regions which appear to be strongly antigenic. The results of this computer analysis provided the basis for the discovery of the novel highly immunogenic, basic truncated structural HCV polypeptides of the instant invention. It is understood by those of skill in the art, however, that such computer analysis of hydrophilicity, surface probability and antigenicity does not always identify epitopes that actually exist, and can also incorrectly identify regions of a
putative amino acid sequence as containing epitopes.
In particular, the present invention
provides for the discovery of two highly immunogenic, basic truncated structural HCV polypeptides, each having epitopes for HCV. These HCV polypeptides of the instant invention are designated herein throughout as FGB1 and FGB2 and, as indicated hereinbefore, have been derived from the putative amino acid sequence encoded by the five prime (5') end of a human isolate of a HCV genome. Moreover, their epitopes are
believed to lie within the first 4% of amino acids found in the putative amino acid sequence of the human HCV polyprotein. Still further, the amino acid sequences for FGB1 and FGB2 correspond to the putative amino acid sequences designated as 5-19 and 102-116, respectively, following the amino acid encoded by a predicted ATG initiation codon of a human HCV genome. The amino acid sequences for the novel FGB1 and FGB2 polypeptides, respectively are as follows:
SEQ ID NOS: 1-4
(Cys) - Lys - Pro - Gln - Arg - Lys - Thr - Lys -
Arg - Asn - Thr - Asn - Arg - Arg - Pro - Gln - 'Tyr; ; ana
SEQ ID NOS: 5-8
(Cys) - Ser - Arg - Pro - Ser - Trp - Gly - Pro - Thr - Asp - Pro - Arg - Arg - Arg - Ser - Arg - (Tyr).
In the above-recited amino acid sequences, Arg (R) represents arginine, Asn (N) represents asparagine, Cys (C) respresents cysteine, Gln (Q) represents glutamine, Gly (G) represents glycine, Lys (K) represents lysine, Pro (P) represents proline, Ser (S) represents serine, Thr (T) represents threonine, Trp (W) represents tryptophan and Tyr (Y) represents tyrosine. The amino acids designated in
parentheticals above represent optional but preferred amino acids added either individually or together to the immunogenic HCV polypeptide sequences for
conjugation and labelling. Moreover, such optional amino acids and in particular the tyrosine amino acid are belived to enhance the reactivity of the
immunogenic HCV polypeptides of the instant invention by enhancing their attachment to, for example, the microwell plates possibly by interaction in such a manner that they leave the rest or greater portion of the novel HCV immunogenic polypeptides exposed to the reactive antibodies during, for example, an ELISA type assay. While Cys and Tyr represent preferred optional amino acids, other optional amino acid residues or other moieties suitable for the purposes stated herein are envisioned by the instant invention. For example, cystine may be substituted for the cysteine amino acid residue for conjugation purposes whereas histidine (His) may be substituted for the tyrosine amino acid residue for labeling purposes. Moreover, while the terms FGB1 and FGB2 as used herein refer broadly to the FGB1 and FGB2 polypeptides as either including or excluding one or both of the optional amino acids or moieties, respectively, when it is specifically intended for such polypeptides to include both of the optional amino acids, i.e., Cys and Tyr, they will be referred to herein as Cys-FGB1-Tyr and Cys-FGB2-Tyr, respectively.
When the FGB1 polypeptide includes the optional Cys and Tyr amino acid residues at its respective -NH2 and -COOH terminal ends, it has 17 amino acids in its sequence, a molecular weight of about 2174, a pl of about 12.484, 3 amino acids (53%) in its CF turn (Fig. 3), 14 hydrophillic amino acids (82%), 8 polar amino acids (53%), 7 basic amino acids (41%), 4 actual Arg amino acid residues (24%), and a combination of 4 Arg and 3 Lys amino acid residues (41%). On the other hand, when the FGB1 polypeptide is free of such optional, but preferred amino acids, it has 15 amino acids in its sequence, a molecular weight of about 1379, a pI of about 12.958, 7 amino acids (47%) in its CF turn (Fig. 4), 13 hydrophillic amino acid residues (37%), 7 polar amino acid residues (47%), 7 basic amino acid residues (47%), 4 actual Arg amino acid residues (27%), and a combination of 4 Arg and 3 Lys amino acid residues (47%). See Cys-FGB1-Tyr and FGB1 TABLES.
CYS-FGB1-TYR TABLE
Amino Acid Composition:
Non-polar: Number Percent
Ala 0 0.00 Val 0 0.00 Leu 0 0. 00 Ile 0 0.00 Pro 2 11.76 Met 0 0.00 Phe 0 0.00 Trp 0 0.00
Polar: Number Percent
Gly 0 0.00 Ser 0 0.00 Thr 2 11.76 Cys 1 5.88 Tyr 1 5.88 Asn 2 11.76 Gln 2 11.76
Acidic: Number Percent
Asp 0 0.00 Glu 0 0.00
Basic: Number Percent
Lys 3 17.65 Arg 4 23.53
His 0 0.00
Calculated Molecular Weight = 2174.310
Estimated pI = 12.484
FGB1 TABLE
Amino Acid Comoosition:
Non-polar: Number Percent
Ala 0 0.00
Val 0 0.00
Leu 0 0.00 Ile 0 0.00
Pro 2 13.33
Met 0 0.00
Phe 0 0.00
Trp 0 0.00
Polar: Number Percent
Gly 0 0.00
Ser 0 0.00
Thr 2 13.33
Cys 0 0.00
Tyr 0 0.00
Asn 2 13.33 Gln 2 13.33
Acidic: Number Percent
Asp 0 0.00
Glu 0 0.00
Basic: Number Percent
Lys 3 20.00
Arg 4 26.67
His 0 0.00
Calculated Molecular Weight = 1908. 010
Estimated pI = 12 . 958
With respect to the FGB2 polypeptide, when it includes the optional, but preferred amino acid residues, i.e., the Cys and Tyr amino acid residues at its respective -NH2 and -COOH terminal ends, it has 17 amino acids, a molecular weight of about 2077, a pI of about 12.306, 17 amino acids (100%) in its CF turn (Fig. 5), 12 hydrophillic amino acid residues (71%), 7 polar amino acid residues (41%), 5 basic amino acid residues (29%), 5 actual Arg amino acid residues
(29%), and no Lys amino acid residues. However, when the FGB2 polypeptide is free of the optional Cys and Tyr amino acid residues at its respective -NH2 and -COOH termini, it has 15 amino acids, a molecular weight of about 1311, a pI of about 12.656, 12 amino acids (80%) in its CF turn (Fig. 6), 11 hydrophillic amino acid residues (73%), 5 polar amino acid residues (33%), 5 basic amino acid residues (33%), 5 actual Arg amino acid residues (33%), and no Lys amino acid residues. See Cys-FGB2-Tyr and FGB2 TABLES.
CYS-FGB2-TYR TABLE
Amino Acid Composition
Non-polar: Number Percent
Ala 0 0.00 Val 0 0.00 Leu 0 0.00 Ile 0 0.00 Pro 3 17.65 Met 0 0.00 Phe 0 0, 00 Trp 1 5, 88
Polar: Number Percent
Gly 1 5.88 Ser 3 17.65
Thr 1 5.88 Cys 1 5.88 Tyr 1 5.88 Asn 0 0.00 Gln 0 0.00
Acidic: Number Percent
Asp 1 5.88 Glu 0 0.00
Basic: Number Percent
Lys 0 0.00 Arg 5 29.41 His 0 0.00
Calculated Molecular Weight = 2077. 210
Estimated pI = 12.306
FGB2 TABLE
Amino Acid Composition:
Non-polar: Number Percent
Ala 0 0. 00 Val o 0.00 Leu 0 0.00 Ile 0 0.00 Pro 3 20.00 Met 0 0.00 Phe 0 0.00 Trp 1 6.67
Polar: Number Percent
Gly 1 6.67 Ser 3 20.00
Thr 1 6. 67 Cys 0 0.00 Tyr 0 0.00 Asn 0 0.00 Gln 0 0.00
Acidic: Number Percent
Asp 1 6.67 Glu 0 0.00
Basic: Number Percent
Lys 0 0. 00 Arg 5 33.33 His 0 0.00
Calculated Molecular weight = 1810.910
Estimated pI = 12.656
Other examples of novel FGB HCV polypeptides in accordance with the present invention that are believed to be immunogenic and have epitopes for HCV are recited hereinafter. It should be understood that while the FGB1 and FGB2 polypeptides as well as those FGB polypeptides having, inter alia, at least about 15 amino acid residues, at least about 7 amino acid residues in their Chou-Fasman Turn and at least about 4 arginine amino acid residues are presently
preferred, the present invention contemplates other equivalent polypeptides whose fingerprint
characteristics may fall somewhat outside of the parameters set forth herein to define the basic, immunogenic HCV polypeptides.
FGB TABLE
CMW EpI AA CFT R HP P B K
FGB3 2202.36 12.658 17 8 5 15 3 7 2
FGB4 2202.36 12.658 17 8 5 15 3 7 2
FGB5 2202.36 12.658 17 10 5 15 3 7 2
FGB6 2146.26 12.195 17 8 3 15 8 7 4
FGB7 2146.26 12.195 17 8 1 15 8 7 4
FGB8 2146.26 12.195 17 8 3 15 3 7 4
FGB9 2146.26 12.195 17 5 3 15 8 7 4
FGB10 2160.28 12.484 17 11 4 15 3 1 3
FGB11 2160.28 12.484 17 10 4 15 3 1 3
FGB12 1820.94 12.484 14 3 4 13 7 6 2
FGB13 1792.89 12.192 14 8 3 13 7 6 3
FGB14 1348.99 12.658 14 13 5 13 7 6 1
FGB15 1848.99 12.658 14 10 5 13 7 6 1 FGB16 1806.91 12.484 14 14 4 13 7 6 2
FGB17 1792.89 12.192 14 8 3 13 7 6 3
FGB18 1792.39 12.192 14 3 3 13 7 6 3
FGB19 1792.39 12.192 14 5 3 13 7 6 3
FGB20 2022.12 12.195 16 11 3 15 8 7 4
FGB21 1918.99 12.195 15 11 3 14 7 7 4
FGB22 2016.1 12.195 16 11 3 14 7 7 4
FGB23 2029.1 12.195 16 13 3 14 7 7 4
FGB24 1887 12.958 15 10 4 14 7 7 3
FGB25 1947.04 12.484 15 11 4 14 7 7 3
FGB26 2050.17 12.484 16 11 4 15 8 7 3
FGB27 2044.15 12.484 16 11 4 14 7 7 3
FGB28 1980.09 12.484 15 7 4 15 8 7 3
FGB29 1713.79 12.958 13 6 4 13 6 7 3
FGB30 1585.66 12.958 12 5 4 12 5 7 3
FGB31 1921.01 12.958 15 12 4 13 6 7 3
FGB32 2077.2 12.484 16 8 4 15 8 7 3
FGB33 1810.9 12.958 14 7 4 13 6 7 3
FGB34 2159.26 12.195 17 13 3 15 8 7 4
FGB35 2187.21 12.484 17 13 4 15 3 7 3
FGB36 1908.01 12.958 15 7 4 13 6 7 3
FGB37 2174.31 12.484 17 8 4 15 8 7 3
FGB38 1892.96 12.784 15 12 3 13 6 7 4
CMW = Calculated Molecular Weight
EpI = Estimated pI
AA = Amino Acids
CFT = Chou-Fasman Turn
R = Arginine
HP = Hydrophilic Amino Acids
P = Polar Amino Acids
B = Basic Amino Acids
K = Lysine Examples of other FGB polypeptides include,
FGB3-CRPQRKTKRNTNRRPQY; FGB4-CKPQRRTKRNTNRRPQY;
FGB5-CKPQRKTRRNTNRRPQY; FGB6-CKPQKKTKRNTNRRPQY;
FGB7-CKPQRKTKKNTNRPPQY; FGB8-CKPQRKTKRNTNKRPQY;
FGB9-CKPQRKTKRNTNRKPQY ; FGB10-CKPQRKSKRNTNRRPQY;
FGB11-CKPQRKTKRNSNRRPQY ; FGB12-CRKTKRNTNRRPQY;
FGB13-CKKTKRNTNRRPQY; FGB14-CRRTKRNTNRRPQY;
FGB15-CRKTRRNTNRRPQY; FGB16-CRKSKRNTNRRPQY;
FGB17-CRKTKKNTNRRPQY ; FGB18-CRKTKRNTNKRPQY ;
FGB19-CRKTKRNTNRKPQY; FGB20-CKQKKTKRSTNRRPQY;
FGB21-KQKKTKRSTNRRPQY; FGB22-KPQKKTKRSTNRRPQY;
FGB23-KPQKKNKRSTNRRPQY; FGB24-CKQRKTKRSTNRRPQ;
FGB25-KQRKTKRSTNRRPQY; FGB26-CKQRKTKRSTNRRPQY ;
FGB27-KPQRKTKRSTNRRPQY; FGB28-CKQRKTKRNTNRRQY ;
FGB29-KQRKTKRNTNRRQ; FGB30-KQRKTKRNTNRR;
FGB31-KPQRKNKRNTNRRPQ; FGB32-CKQRKTKRNTNRRPQY;
FGB33-KQRKTKRNTNRRPQ; FGB34-CKPQKKNKRNTNRRPQY;
FGB35-CKPQRKNKRNTNRRPQY; FGB36-KPQRKTKRNTNRRPQ;
FGB37-CKPQRKTKRNTNRRPQY; FGB38-KPQKKNKRNTNRRPQ.
It should be further understood that while the
sequences to certain of the above FGB polypeptides include the cysteine and/or tyrosine amino acid residue at their respective -NH2 and -COOH terminal ends, any such FGB polypeptide or immunogenic HCV polypeptide of the present invention may be altered to delete or include only the cysteine or tyrosine amino acid residue or both such residues at their respective terminal ends in accordance with the teachings of the instant invention.
The immunogenic HCV polypeptides and in particular the FGB1 and FGB2 polypeptides of the instant invention can be produced by chemical snythesis, recombinant technology or any other methods available in the art so long as the methodology selected does not interfere with their utilities stated herein. Moreover, alternative amino acid residues may be substituted for those recited in the above sequences for the FGB1 and FGB2 polypeptides so long as the substituted amino acids do not defeat the utility of such novel immunogenic HCV polypeptides. For example, it is believed that Lys and Arg amino acid residues may be freely substituted for each other within the amino acid sequences. It is likewise believed that Ser and Thr amino acid residues may be freely substituted for one another. Thus, it is to be understood that the reference herein to at least about 4 Arg amino acid residues (25%) means that the
sequences of the immunogenic HCV polypeptides of the instant invention include at least about 4 Arg amino acid residues or at least about 4 Lys amino acid residues or a combination of at least about 4 Lys and Arg amino acid residues. For example, the above-recited sequence for the FGB1 polypeptide has about 7 Arg amino acid residues (41%), i.e., 4 Arg and 2 Lys, when the optional amino acids are included and about 7 Arg amino acid residues (47%), i.e., 4 Arg and 3 Lys, when the optional amino acids are excluded. Preferably, however, it means that the sequences for the immunogenic HCV polypeptides of the instant invention include at least about 4 actual Arg amino acid
residues (25%) and more preferably about 4 actual Arg amino acid residues and up to about 3 actual Lys amino acid residues (up to about 47%) . Likewise, selected amino acids may be added to or deleted from the amino acid sequences of the HCV immunogenic polypeptides of the instant invention to expand or shorten same so long as the objectives of the instant invention are not defeated. For instance, as already indicated hereinbefore, Cys and Tyr amino acid residues can be added to the respective amino and carboxyl termini of the FGB1 and FGB2 polypeptides without interfering with their function as HCV epitopes. Thus, such modified or varied amino acid sequences are considered to be equivalents to the immunogenic HCV polypeptides and in particular the FGB1 and FGB2 polypeptides and are contemplated within the scope of the present invention.
As previously stated, the immunogenic HCV polypeptides and in particular the FGB1 and FGB2 polypeptides of the instant invention can be
synthetically produced. For example, the Cys-FGB1-Tyr and Cys-FGB2-Tyr polypeptides can be synthesized on an Applied BioSystem Peptide Synthesizer 430 using solid phase strategy, as described in Merrifield, F.B.: J. Am. Chem. Soc., 85:2149-2154 (1963) and Mitchell, A. R. et al.: J. Orq. Chem., 43:2845-2854 (1978). Boc amino acids (t-Boc) are activated as benzotriazole (HOBt) esters and coupling is performed in N-methyl- pyrrolidinone. The side chain protecting groups are Asp (OBzl), Ser (OBzl), Thr (OBzl), Lys (Cl-z), Arg (Tos), Tyr (Br-z), Trp (CHO), Cys (p-MeBzl). Syntheses can be performed starting with 0.5 mmole of BocTyr (BrZ) Pam resin. Deprotection and cleavage of the Cys-FGB1-Tyr and Cys-FGB2-Tyr polypeptides from the resin can be performed using hydrogen fluoride. See Tables 2, 3, 4 and 5. The Cys-FGB1-Tyr and
Cys-FGB2-Tyr polypeptides can be purified to greater than 95% using reversed-phase HPLC. Analytical HPLC using the applied Biosystems 130A separation system with a microbore column show the Cys-FGB1-Tyr and Cys-FGB2-Tyr polypeptides to be greater than 98% pure. See Figs. 7 and 8. The authenticity of the
Cys-FGB1-Tyr and Cys-FGB2-Tyr polypeptides can be confirmed by amino acid analysis using Applied Biosystems 420A automated hydrolyzer, see Tables 6 and 7, and by molecular weight determination using the
Bio-Ion mass analyzer. See Figs. 9 and 10. It should be understood that the Cys-FGB1-Tyr and Cys-FGB2-Tyr polypeptides referenced in TABLES 1-7, Figs. 3, 5, 7-10 and Examples I-III specifically include the optional Cys amino acid residues at the amino termini and Tyr amino acid residues at the carboxyl termini.
TABLE 2
Cys-FGB1-Tyr Polypeptide
CYCLE AMINO REACTION CONCENTRATOR ACTIVATOR
NUMBER ACID VESSEL VESSEL TEMP VESSEL BEG RBEG11 R CBEG 11 10c ABEG11
1 Gln RBOC 11R CBOC 11 10C ABOC 12
2 Pro RBOC 11R CBOC 11 10c ABOC 11
3 Arg RBOC 11R CBOC 11 10c ABOC 14
4 Arg RBOC11DR CBOC 11D 10c ABOC 14D
5 Asn RBOC11DR CBOC 11D 10c ABOC 13D
6 Thr RBOC 11R CBOC 11 10c ABOC 11
7 Asn RBOC11DR CBOC 11D 10c ABOC 13D
8 Arg RBOC 11R CBOC 11 10c ABOC 14
9 Lys RBOC11DR CBOC 11D 10c ABOC 13D
10 Thr RBOC11DR CBOC 11D 10c ABOC 11D
11 Lys RBOC 11R CBOC 11 10c ABOC 13
12 Arg RBOC11DR CBOC 11D 10c ABOC 14D
13 Gln RBOC 11R CBOC 11 10c ABOC 12
14 Pro RBOC11DR CBOC 11D 10c ABOC 11D
15 Lys RBOC 11R CBOC 11 10c ABOC 13
16 Cys RBOC 11R CBOC 11 10c ABOC 11
END RNH211 R CEND 11 10c AEND11
TABLE 3
Cys-FGB1-Tyr POLYPEPTIDE
RES. AMINO BOC MW. TOTAL WEIGHT OF SUBSTITUTION
NUMBER ACID OF ACID PEPTIDE RESIN (mmol/g) COMMENT
0.630 INITIAL SUB
17 Tyr 000 00.796 0.630
16 Gln 246 00.860 0.581
15 Pro 215 00.908 0.550
14 Arg 429 01.064 0.470 (TOS)
13 Arg 429 01.219 0.410 (TOS)
12 Asn 232 01.276 0.392
11 Thr 309 01.372 0.364 (Bzl)
10 Asn 232 01.429 0.350
09 Arg 429 01.584 0.316 (TOS)
08 Lys 415 01.733 0.289 (Cl-Z)
07 Thr 309 01.828 0.274 (Bzl)
06 Lys 415 01.977 0.253 (Cl-Z)
05 Arg 429 02.132 0.235 (TOS)
04 Gln 246 02.196 0.228
03 Pro 215 02.245 0.223
02 Lys 415 02.393 0.209 (Cl-Z)
01 Cys 325 02.497 0.200 (4-MeBzl)
TABLE
Cys-FGB2-Tyr POLYPEPTIDE
CYCLE AMINO REACTION CONCENTRATOR ACTIVATOR
NUMBER ACID VESSEL VESSEL TEMP VESSEL BEG RBEG11 R CBEG 11 10c ABEG11
1 Arg RBOC 11R CBOC 11 10c ABOC 14
2 Ser RBOC 11R CBOC 11 IOC ABOC 11
3 Arg RBOC 11R CBOC 11 10c ABOC 14
4 Arg RBOC11DR CBOC 11D 10c ABOC 14D
5 Arg RBOC11DR CBOC 11D 10c ABOC 14D
6 Pro RBOC11DR CBOC 11D 10c ABOC 11D
7 Asp RBOC 11R CBOC 11 10c ABOC 11
8 Thr RBOC11DR CBOC 11D 10c ABOC 11D
9 Pro RBOC11DR CBOC 11D 10c ABOC 11D
10 Gly RBOC 11R CBOC 11 10c ABOC 11
11 Trp RBOC 11R CBOC 11 10c ABOC 13
12 Ser RBOC 11R CBOC 11 10c ABOC 11
13 Pro RBOC11DR CBOC 11D 10c ABOC 11D
14 Arg RBOC11DR CBOC 11D 10c ABOC 14D
15 Ser RBOC 11R CBOC 11 10c ABOC 11
16 Cys RBOC 11R CBOC 11 10c ABOC 11
END RNH211 R CEND 11 10c AEND11
TABLE 5
Cys-FGB2-Tyr POLYPEPTIDE
RES. AMINO BOC MW. TOTAL WEIGHT OF SUBSTITUTION
NUMBER ACID OF ACID PEPTIDE RESIN immol/g) COMMENT
0.630 INITIAL SUB
17 Tyr 000 00.796 0.630
16 Arg 429 00.951 0.525 (TOS)
15 Ser 295 01.040 0.481 (Bzl)
14 Arg 429 01.195 0.418 (TOS)
13 Arg 429 01.351 0.370 (TOS)
12 Arg 429 01.506 0.332 (TOS)
11. Pro 215 01.555 0.322
10 Asp 323 01.657 0.302 (OBzl)
09 Thr 309 01.753 0.285 (Bzl)
08 Pro 215 01.801 0.278
07 Gly 175 01.830 0.273
06 Trp 332 01.937 0.258 CHO
05 Ser 295 02.026 0.247 (Bzl)
04 Pro 215 02.074 0.241
03 Arg 429 02.230 0.224 (TOS)
02 Ser 295 02.318 0.216 (Bzl)
01 Cys 325 02.422 0.206 (4-MeBzl)
TABLE 6
AMINO ACID ANALYSIS OF Cys-FGBl-Tyr NANOMOLES THEORETICAL ACTUAL
Asp ( D) 0.47 2.00 2.05
Glu (E) 0.45 2.00 1.90
Ser ( S) 0.00 0.00 0.00
Gly ( G) 0.00 0.00 0.00
His ( H) 0.00 0. 00 0.00
Arg ( R) 0. 89 4.00 3.90
Thr ( T) 0.45 2.00 1. 90
Ala ( A) 0.00 0.00 0.00
Pro ( P) 0.49 2.00 2.10
Tyr (Y) 0.24 1.00 1.00
Val (V) 0.00 0.00 0.00
Met (M) 0.00 0.00 0.00
Cys (C)* 0.00 0.00 0.00 Ile (I) 0.00 0.00 0.00
Leu (L) 0.00 0.00 0.00
Phe (F) 0. 00 0.00 0.00
Trp (W) 0, 00 0.00 0.00
Lys (K) 0.67 3.00 2.90
Ave. 0.23
Molecular weight: 2,174.5
*Cys amino acid residues are undetectable by an Applied Biosystems 420 A automated hydrolyzer.
TABLE 7
AMINO ACID ANALYSIS of CVS -FGB2-Tyr NANOMOLES I THEORETICAL ACTUAL
Asp (D) 0.23 1.00 0.90
Glu (E) 0.00 0.00 0.00
Ser (S) 0.67 3.00 2.80
Gly (G) 0.24 1.00 1.00
His (H) 0.00 0.00 0.00
Arg (R) 1.23 5.00 5.20
Thr (T) 0.23 1.00 0.90
Ala (A) 0.00 0.00 0.00
Pro (P) 0.73 3.00 3.10
Tyr (Y) 0 . 23 1 . 00 0 . 90
Val (V) 0.00 0.00 0.00
Met (M) 0.00 0.00 0.00
Cys (C)* 0.00 0.00 0.00 Ile (I) 0.00 0.00 0.00
Leu (L) 0.00 0.00 0.00
Phe (F) 0.00 0.00 0.00
Trp (W) 0.00 0.00 0.00
Lys (K) 0.00 0.00 0.00
Ave. 0.24
Molecular weight: 2,077.32
*Cys amino acid residues are undetectable by an Applied Biosystems 420A automated hydrolizer.
The HCV polypeptides including the FGB1 and FGB2 polypeptides of the present invention also can be produced by recombinant techniques well known to those of skill in the art. That is, they can be produced by gene expression of a ds DNA comprising at least one of the polynucleotide sequences which encode the polypeptides and especially the FGB1 and/or the FGB2 polypeptides of the instant invention, or a degenerate polynucleotide sequence obtained therefrom by, for example, substituting at least one nucleotide in one of the polynucleotide sequences encoding the FGB1 or FGB2 polypeptides in accordance with degeneracy of genetic code. As recited hereinbefore, a polynucieotide sequence encoding the FGB1 polypeptide is:
SEQ ID NOS: 9-16
5'-(TGT) AAACCTCAAAGAAAAACCAAACGTAACACCAACCGTCGCCC (TGC) (U) (U) (U)
(UGU)
(UGC)
ACAG (TAT)-3'
(TAC)
(UAU)
(UAC)
whereas a polynucleotide sequence encoding the FGB2 polypeptide is:
SEQ ID NOS: 17-24
5'-(TGT) TCTCGGCCTAGTTGGGGCCCCAGGGACCCCCGGCGTAGGTC (TGC) (U U) (U) (UU) (U) (U)
(UGU)
(UGC)
GCGC (TAT)-3'.
(TAC)
(UAU)
(UAC) In the above-mentioned polynucleotide sequences and herein throughout, when in the form of DNA, A represents a deoxyriboadenylic acid residue (dAMP), G represents a deoxyriboguanylic acid residue (dGMP), C represents a deoxyribocytidylic acid residue (dCMP), and T represents a thymidylic acid residue (TMP), and when in the form of RNA, A represents a riboadenylic acid residue (AMP), G represents a riboguanylic acid residue (GMP), C represents a ribocytidylic acid (CMP), and U, which is substituted for T, represents a ribouridylic acid residue (UMP). The left and right ends of each sequence recited in the above-mentioned polynucleotide sequences represent the 5'-hydroxyl group side and 3'-hydroxyl group side, respectively. It of course should be understood that in the event the immunogenic HCV polypeptides
including the FGB1 and FGB2 polypeptides are to be recombinantly expressed with the Cys and Tyr amino acid residues, the appropriate codons must be included in the expressing polynucleotide sequences, and such polynucleotide sequences are within the contemplation of the instant invention. Such codons which encode for Cys and Tyr amino acid residues are well known to those of skill in the art and include, for example, TGT or TGC (DNA) or UGU or UGC (RNA) for Cys and TAT or TAC (DNA) or UAU or UAC (RNA) for Tyr. The substitution or addition of a nucleotide of either of the above-mentioned oligonucleotide sequences can be performed by the methods of, for example, Saiki, et al.: Science, 230:1350-1354 (1985), and Saiki, et al.: Science, 239:487-491 (1988).
Using the polynucleotide sequences disclosed herein, a DNA library can be prepared by customary methods. That is, the polynucleotide sequences can be individually ligated to replicable cloning vectors to thereby obtain DNA libraries. As a replicable cloning vector, any known or commercially available vectors, such as phage genes, cosmids, plasmids and animal virus genes nay te used. When a phage gene or a cosmid is used as a replicable vector, in order to attain high stability and high transforming ability of the vector after the DNA sequences have been individually inserted therein, the in-vitro packaging of each of the polynucieotide sequences inserted vectors is conducted by a customary method. Thus, the DNA-inserted vectors are obtained in the form of recombinant phage particles. The obtained phage particles are used as a DNA library for DNA cloning. On the other hand, when a plasmid is used as a replicable vector, the above-mentioned polynucleotide sequences, either individually or in combination, can be inserted in the plasmid vectors and the resultant DNA-inserted vectors are then individually introduced into host cells, such as cells of Escherichia coli, Bacillus subtili, yeast or the like, according to a customary method. The thus obtained transformants are used as a DNA library for DNA cloning. Further, when an animal virus gene is used as a replicable vector, any of the above-mentioned polynucleotide sequences may be either individually or in combination inserted in the virus gene vectors and the resultant recombinant viruses are then individually transfected into animal cells according to a standard method and multiplied in the cells. In the case of a recombinant virus, the obtained recombinant viruses as such can be used as a DNA library.
When the DNA library is comprised of transformants, the transformants can be cultured on a standard agar medium to form colonies. On the other hand, when the DNA libraries are comprised of recombinant phage particles or recombinant viruses, these phage particles or recombinant viruses can be used to infect known host cells , such as Escherichia coli ,
Bacillus subtilli, yeast, animal cell culture and the like, and culture to form a plaque, or to multiply hhe infected cells.
The polynculeotides of the instant invention may be further used to recombinantly express the novel immunogenic HCV polypeptides including the FGB1 and FGB2 polypeptides or variations thereof, respectively. In order to recombinantly express the polynucleotide sequences encoding the FGB1 and FGB2 polypeptides especially on a commercial scale, the polynucleotide sequences encoding the FGB1 and FGB2 polypeptides, whether in fused or free form, and whether or not containing a signal sequence to permit secretion, can be ligated into a replicable expression vector by a customary method which is suitable to any convenient host. Both eukaryotic and prokaryotic host systems can be used in forming the recombinant FGB1 and FGB2 polypeptides or variations thereof. The polynucieotide sequences can be synthetically produced or derived from clones. When a polynucleotide sequence of the instant invention is inserted into an expression vector to form a recombinant which is introduced in a compatible host, an individual polypeptide can be produced by gene expression. When such a polynucieotide sequence in combination with a DNA fragment is utilized to form a recombinant, a polypeptide can be expressed in the form of a fused or free polypeptide comprising peptides encoded by the inserted polynucieotide sequence and DNA fragment. A recombinant which is capable of expressing a polypeptide of the instant invention includes a coding sequence comprised of a polynucleotide sequence of the instant invention which is operably linked to a control sequence that is compatible with a desired host. In this form, such a recombinant may be simply referred to herein as a recombinant expression system.
By the term "control sequence," it is meant herein to refer a to polynucleotide sequence which effects expression of a coding sequence to which it is ligated. The nature of such a control sequence may differ depending upon the host organism. For example, in prokaryotes, such control sequences generally include promoters, ribosomal binding sites, and terminators. In eukaryotes, however, such control sequences generally include promoters, terminators and, in some instances, enhancers. The term "control sequence" is therefore intended to include, at a minimum, all components whose presence is necessary for expression, and may also include additional components whose presence is advantageous, such as, leader sequences. By the term "operably linked", it refers herein to a juxtaposition wherein the components so described are in a relationship permitting them to function in their intended manner. A control sequence "operably linked" to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under conditions compatible with the control sequence. The term "coding sequence" as used herein refers to a polynucleotide sequence which may be transcribed into RNA and/or translated into a polypeptide when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by an initiation codon at the 5'-terminus and a termination codon at the 3'-terminus. A coding sequence may include, but is not limited to RNA, DNA, and recombinant
polynucleotide sequences. By the term "open reading frame", it is meant herein to refer to a region of a polynucleotide sequence which encodes a peptide or polypeptide. This region may represent a portion of a coding sequence or a total coding sequence.
As a replicable expression vector which may be used, any conventionally known or commercially available expression vector can be used. Examples of expression vectors include plasmid vector pSN508 for enterobacteria, U.S. Patent No. 4,703,005, plasmid vector pBH103 for yeast, and its series, Japanese Patent Application Laid-Open Specification No.
63-22098, piasmid pJM105, Japanese Patent Application Laid-Open Specification No. 62-286930, an attenuated chickenpox virus gene, Japanese Patent Application Laid-Open Specification No. 53-41202, an attenuated Marek's disease virus, J. Jap. Soc. Vet., 27:20-24 (1974) and Gan Monograph on Cancer Res., 10:91-107 (1971), plasmid pSC11, Chakrabarri et al.: Mol. Cell. Biol., 5:3403-3409 (1985), plasmid pUV1, Falkner et al.: Nucleic Acid. Res., 15:7192 (1987), plasmid pTM3 , Moss et al.: Nature, 348:91-92 (1990), plasmid pTTQ series which is sold by Amersham International,
England, plasmid pSIV series which is sold by
Pharmacia LKB, Sweden, plasmid pGEM TM-3ZF(-) sold by
Promego, Madison, WI, and the like, depending on the level of expression desired. The expression vectors containing inserted immunogenic HCV polynucleotides of the instant invention can be individually introduced or transfected into host cells sensitive to the vector according to conventional methods, to obtain transformants. By culturing the transformants or recombinant viruses obtained above according to customary methods, the FGB1 and FGB2 polypeptides can be produced in the culture of the transformants or the recombinant viruses and purified therefrom in accordance with standard techniques on a commercial scale.
The FGB1 and FGB2 polypeptides recombinantly produced may be isolated from lysed cells or from the culture medium of the transformants or recombinant viruses and purified to the extent needed for their intended use. Purification may be by any appropriate combination of customary techniques known in the art and selected from, for example, salt fractionation; adsorption and desorption using biogel; differential extraction; precipitation by an inorganic solvent;
fractionation by ultracentrifugation; chromatography on ion exchange resins; affinity chromatography;
high-performance liquid chromatography; electrophoresis, and the like. When the FGB1 and FGB2 polypeptides are purified from the culture of an E. coli transformant or a yeast transformant, from a viewpoint of effective removal of allergens derived from E. coli and yeast which cause the quality of the final product of the FGB1 and FGB2 polypeptides to be markedly lowered, it is preferred that the purification be conducted by, for example, the steps of adsorption and elution using a biogel, such as a sepharose or agarose biogel. When the FGB1 and FGB2 polypeptides are purified from the culture of a recombinant virus, e.g., the culture of recombinant virus-infected cells, high purity FGB1 or FGB2
polypeptide can be obtained by subjecting a crude solution containing the polypeptide to purification by ultracentrifugation and density gradient
centrifugation repeatly. Thus, a solution containing purified FGB1 or FGB2 polypeptide of the present invention is cotained. If desired, the solution may be lyophilized to obtain purified FGB1 or FGB2
polypeptide in a dry form.
Such recombinantly expressed FGB1 and FGB2 polypeptides can be used as diagnostics or formulated into vaccines. Antibodies raised against these recombinantly expressed polypeptides can also be used as diagnostics, or for passive immunotherapy. In addition, antibodies to these recombinantly expressed polypeptides or anti-idictype antibodies, which are raised against such antibodies, are useful for isolating and identifying HCV.
The polynucleotide seguences of the present invention encoding the FGB1 and FGB2 polypeptides may also be used for diagnosing HCV hepatitis by nucleic acid and in situ hybridization technique. More particularly, the polynucieotide sequences described herein permits the construction of probes which are useful for detecting HCV RNA in biological samples, including blood, lympn, ascites, hepatocytes, etc. By the term "probe," it is used herein to refer to a polynucleotide which forms a hybrid structure with a sequence in a target region, due to complementarity of at least one sequence in the probe with a sequence in the target region. As used herein, the term "target region" refers to a region of nucleic acids which is to be amplified and/or detected.
For example, it is possible to prepare RNA or DNA oligomers of approximately 6 nucleotides or more which are complementary to the disclosed RNA polynucleotide sequences, either by excision or synthetically, which hybridize with the HCV genome and are useful in identifcation of HCV RNA in, for
example, donated blood, sera of suojects suspected of harboring the virus, or cell culture systems in which the viruses replicate. The crooes for HCV should be of a length which allows the detection of HCV RNA or DNA sequences by hybridization. While 6-8 nucleotides complementary or corresponding to the disclosed polynucleotide sequences may be of a workable length, sequences of 10-12 nucleotides complementary or corresponding thereto are prefered and about 20-45 nucleotides and in particular 20 nucleotides
complementary or corresponding thereto appear optimal.
The probes of the instant invention can be prepared using routine methods, including automated oligonucieotide synthetic methods, such as described in Warner: DNA, 3:401 (1984), which is incorporated herein by reference in its entirety. Among useful probes, for example, are those derived from the polynucleotide sequences, the novel clones disclosed herein, as well as the various oligomers useful in the DNA libraries, set forth herein. It should be
appreciated that when the nucleotide sequences of the instant invention are used as probes, complete
complementarity is desirable, although it may be unnecessary as the length of the probe is increased as indicated hereinbefore.
For use of such probes as diagnostics, the biological sample to be analyzed, such as blood, serum ascites, lymph and hepatocytes, may be processed, if desired, to extract HCV nucleic acids contained therein. The resulting nucleic acids from the sample may be subjected to gel electrophoresis or other size separation techniques; alternatively, the nucleic acid sample may be dot blotted without size separation. The probes of the instant invention can then be labeled with a labeling moiety. For example, the probes may be labeled with 32P by treatment with polynucleotide kinase in the presence of gamma
32 P-ATP, using standard conditions for the reaction, or by nick translation. Suitable other labels are known in the art, and include, for example, other radioactive labels such as radioactive iodine, biotin, alkaline phosphatase, fluorescent probes and
chemiluminescent probes. The labeling of the probes may be performed by standard techniques known in the art such as by using commercially available nick translation kits or multiprime kits, such as those distributed by Amersham International, England and Nippon Gene Co., Ltd., Japan. The nucleic acids extracted from the sample can then be treated with the labeled probe under hybridization conditions of suitable stringencies, and polynucleotide duplexes containing the probe are detected.
The RNA and DNA probes of the instant invention are believed to be highly complementary to the HCV RNA genome and cDNA, respectively. Therefore, usually high stringency conditions are not required during hybridization. Nevertheless, to ensure against false positives, conditions of high stringency should be used. The stringency of hybridization is
determined by a number of factors during hybridization and during the washing procedure, including
temperature, ionic strength, length of time, and concentration of formamide. These factors are
outlined in, for example, Sambrook, J. et al.:
Molecular Cloning. A Laboratory Manual, 2nd., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989).
In carrying out such an assay, the labeled probe is introduced, for example, in a plastic bag. The amount of the labeled probe placed in the plastic bag is generally between about 1 and 100 nanograms per bag. The labeled probe may be contained in the plastic bag in the form of a solution. The diagnosis of HCV hepatitis by the use of the labeled probe is conducted by a standard hybridization method. That is, total RNA extracted from plasma, serum leukocytes, ascites fluid, lymph, or hepatocytes obtained from a patient is first electroohoresed and blotted or simply blotted on nitrocellulose. The blots of HCV RNA are then hybridized with the labeled probes in the bag, and the hybridized labeled probes are detected by, for example, autoradiogram in the positive HCV samples.
The probes can be packaged into diagnostic kits. Diagnostic kits include the DNA or RNA probes, which may be labeled; alternately, the probes of the instant invention may be unlabeled and the ingredients for labeling may be included in the kit in separate containers. The kit may also contain other suitably packaged reagents and materials needed for the particular hybridization protocol, for example, standards, as well as instructions for conducting the test.
The polynucleotides of the instant invention may be further used to prepare anti-HCV viral agents, such as antisense polynucleotide fragments, which may interfere with HCV RNA replication, transcription or translation. Antisense polynucleotide fragments as used herein constitute single stranded DNA or RNA fragments derived from the polynucleotide sequences encoding the FGB1 or FGB2 polypeptides, e.g., complementary thereto, which permits them to bind specifically to designated regions of HCV RNAs. Antisense polynucleotide fragments of the instant invention include, for example, single stranded DNA or RNA fragments that have the ability to bind to HCV RNA to block protein translation and/or prevent replication of HCV RNA by polymerase. In addition, the instant invention contemplates double stranded (ds) DNA fragments which, when ligated into an appropriate expression vectors such as a retroviral vector and introduced into infected liver cells, transcribe complementary antisense RNA fragments that will bind to HCV RNA to block protein translation and/or HCV replication.
As indicated hereinbefore, antisense single stranded polynucleotides which bind to HCV RNA
according to the present invention are designed based upon the polynucleotide sequences encoding the FGB1 or FGB2 polypeptides provided herein. The antisense polynucleotides of the instant invention may be further designed to include, for instance, ribozymes which catalyze with high specificity, the degradation of HCV RNA. Hence, they may be delivered in specialized systems, for example, liposomes or by retroviral vectors used in gene therapy. In addition, they may include bonding between modified bases, analogs, etc. The antisense single stranded polynucleotide fragments of the present invention may also include molecules which carry agents (non-covalently attached or covalently bound) for enhancing their stability and effectiveness to degrade HCV RNA. Still further, the antisense polynucleotides of the instant invention may also bind to cellular RNA which enhance and/or are required for viral infectivity, replicative ability, or chronicity.
Examples of antisense single stranded RNA or DNA fragments contemplated by the instant invention include: SEQ ID NOS : 25-26
UUUGGAGUUUCUUUUUGGUUUGCAUUGUGGUUGGCAGCGGGUGUC; and (TTT) (TTT TTTTT) (TTT) (TT T) (TT) (T T)
SEQ ID NOS: 27-28
AGAGCCGGAUCAACCCCGGGGUCCCUGGGGGCCGCAUCCAGCGCG.
(T) (T) (T) (T)
Examples of ds DNA fragments, which when ligated in frame into appropriate vectors such as retroviral vectors and introduced into infected liver cells, transcribe the above antisense polynucleotide RNA sequences, include:
SEQ ID NO: 29
(I) 5'-GACACCCGCTGCCAACCACAATGCAAACCAAAAAGAAAC
3'-CTGTGGGCGACGGTTGGTGTTACGTTTGGTTTTTCTTTG
TCCAAA-3'
AGGTTT-5'; and
SEQ ID NO: 30
(II) 5'-CGCGCTGGATGCGGCCCCCAGGGACCCCGGGGTTGA .
3'-GCGCGACCTACGCCGGGGGTCCCTGGGGCCCCAACT
TCCGGCTCT-3'
AGGCCGAGA-5' .
By the term "in frame" as used herein throughout, it refers to the proper positioning of a desired sequence of nucleotides within a DNA fragment or coding
sequence operably linked to a control sequence that results in optimal transcription or translation.
It of course should be understood that the above antisense single stranded DNA and RNA fragments and ds DNA fragments transcribing the antisense single stranded RNA fragments can be shortened, expanded cr modified as indicated hereinbefore so long as such modifications do not interfere with the ability of the antisense fragments to block HCV protein translation and/or HCV replication.
The immunogenic HCV polypeptides in accordance with the present invention can be prepared as discrete polypeptides or incorporated into larger polypeptides. For example, the FGB1 or FGB2 polypeptides can be prepared as unfused polypeptides or fuseα polypeptides in combination with one another, cr the FGB1 and/or FGB2 polypeptides can be prepared in combination with other polypeptides to form heterologous fusion polypeptides. Useful heterologous polypeptide sequences include polypeptide sequences that provide for secretion from a recombinant host, enhance the immunological reacitivity of the HCV epitope(s), facilitate the coupling of the polypeptides to immuneassay supports or vaccine carriers, or enhance the solubility of the polypeptides. See, e.g., EPO Pub. No. 116,201; U.S. Pat. No. 4,722,840; EPO Pub. No. 259,149; U.S. Pat. No. 4,629,733. The abovereferenced immunogenic HCV polypeptides and especially the FGB1 and FGB2 polypeptides of the instant invention therefore can be prepared as discrete or combination polypeptides or incorporated into larger polypeptides, and may find use as described herein. Further, the HCV polypeptides and in particular the FGB1 and FGB2 polypeptides of the present invention may advantageously be used as active ingredients in vaccines for HCV. Vaccine preparations, which contain immunogenic polypeptides as active ingredients, are well known to those of skill in the art. Typically, such vaccines are injectibles, either as liquid solutions or suspensions; solid forms suitable for solution or suspension in liquid prior to injection may also be prepared. The preparation may also be emulsified, or the FGB1 or FGB2 polypeptides encapsulated in liposomes. The active immunogenic FGB1 and/or FGB2 polypeptides can be mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol, or the like and combinations thereof. If desired, the vaccines may also contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, and/or adjuvants which enhance the effectiveness of the vaccine. For example, to a solution containing a FGB1 and/or FGB2 polypeptide concentration of about 1 to about 500 micrograms per ml, aluminum hydroxide gel is added as an adjuvant so that the concentration of the added gel becomes about 0.1 to about 1.0 mg per ml. As an adjuvant, there may also be employed precipitating depositary adjuvants such as calcuim phosphate gel, aluminum phosphate gel , aluminum sulfate, alumina hydroxide, alumina and bentonite.
Examples of still further adjuvants include but are not limited to: N-acetylmuramyl-L-threonine-D-isoglutamine (thr-MDP),
N-acetyl-normuramyl-L-alanyl-D-isoglutamine (CGP
11637, referred to as nor-MDP), N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1'-2'-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine (CGP 19835A, referred to as MTP-PE), and RIBI, which contains three components extracted from bacteria, i.e., monophosphoryl lipid A, trehalose dimycolate and cell wall skeleton (MPL + TDM + CWS) in a 2% squalene/Tween 80 emulsion. The effectiveness of an adjuvant may be determined by measuring the amount of antibodies directed against the FGB1 or FGB2 polypeptide resulting from the administration of any of these polypeptides and vaccines in combination with various adjuvants.
In the event that the immunogenic HCV polypeptides and in particular the FGB1 and FGB2 polypeptides of the instant invention are too small to encompass the total immunogenicity of the structural region of the putative HCV structural protein, they may be linked to a suitable carrier. A number of techniques for obtaining such linkage are known in the art, including the formation of disulfide linkages using N-succinimidyl-3-(2-pyridylthio)propionate(SPDP) and succinimidyl 4-(N-maleimidomethyl)cyclohexane-1- carboxylate(SMCC) available through Pierce Company, Rockford, IL. Since the FGB1 and FGB2 polypeptides lack a sulfhydryl group, this can be accomplished by providing, for example, a Cys amino acid residue or a cystine moiety on one protein (e.g., the FGB1 or FGB2 polypeptide) and an amide linkage through an epsilonamino on a lysine or other free amino group in the other (e.g., carrier). A variety of such disulfide/ amide-forming agents are known. See, for example, Harlow and Lane: Antibodies. A Laboratory Manual, pp. 130-131, Cold Spring Harbor Laboratory, Cold
Spring Harbor, N.Y. (1988). Other bifunctional coupling agents form a thioether rather than a disulfide linkage. Many of the thio-ether-forming agents are commercially available and include reactive esters of 6-maieimidocapric acid, 2-bromoacetic acid, 2-iodacetic acid, 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid, and the like. The carboxyl groups can be activated by combining them with succinimide or 1-hydroxyl-2-nitro-4-sulfonic acid, sodium salt.
Additional methods of coupling antigens employs the rotavirus/"binding peptide" system described in, for example, EPO Pub. No. 259,149. The foregoing list is not meant to be exhaustive, and modifications of the recited compounds can clearly be used. In addition to cysteine, it should be understood that any other amino acid residue or moiety, such as cystine, may be used that is suitable for this purpose so long as the objectives of the instant invention are not defeated.
It should also be understood that any carrier may be used which preferably does not itself induce the production of antibodies harmful to the host. Suitable carriers are typically large, slowly metabolized macromolecules such as polypeptides, polysaccharides, such as latex functionalized sepharose, agarose, cellulose, cellulose beads and the like;
polymeric amino acids, such as polyglutamic acid, polylysine, and the like; amino acid copolymers; and inactive virus particles. Especially useful polypeptides substrates are serum albumins, keyhole limpet hemocyanin protein (KLH), immunoglobulin molecules, thyroglobulin, ovalbumin, tetanus toxoid, and other proteins well known to those versed in the art.
The vaccines can be conventionally administered parenterally, by injection, for example, either subcutaneously or intramuscularly. Additional formulations which are suitable for other modes of administration include suppositories and, in some cases, oral formulations. For suppositories, traditional binders and carriers may include, for example, polyalkylene glycols or triglycerides; said suppositories may be formed from mixtures containing the active ingredient in the range of 0.5% to 10%, preferably 1%-2%. Oral formulations include such normally employed excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, glucose, fructose, galactose, sucrose, lactose, albumin, gelatin, and amino acids such as glycine, alanine, lysine,
arginine, glutamine, and the like. These compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained released formulations or powders and may contain, for example, 10%-95% of active ingredient, and preferably 25%-70%.
The FGB1 and FGB2 polypeptides may be formulated into a vaccine as neutral or salt forms. Pharmaceutically acceptable salts include the acid addition salts (formed with free amino groups of the peptide) and which are formed with inorganic acid, such as, for example, hydrochloric or phosphoric acids, or such organic acids such as acetic, oxalic, tartaric, maleic, and the like. Salts formed with the free carboxyl groups may also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, and ferric hydroxides, and such organic bases as isopropylamine, trimethylamine,
2-ethylamino ethanol, histidine, procaine, and the like. A vaccine of the instant invention may also be prepared in the form of a mixed vaccine which contains adsorbed HCV polypeptides, preferably
adsorbed FGB1 and/or FGB2 polypeptides, mentioned above and at least one antigen other than the present FGB1 and/or FGB2 polypeptide. As the antigen other than the present FGB1 and/or FGB2 polypeptides, there may be employed any antigens that are conventionally used as active ingredients of the corresponding vaccines in so far as the side effects and adverse reactions caused by such other antigens and the FGB1 and/or FGB2 polypeptides are not additively or synergistically increased by the use of the FGB1 and/or FGB2 polypeptides and such other antigens in combination, and the antigenicities and immunogenicities of the FGB1 and/or FGB2 polypeptides and such other antigens are not reduced by the interference between the FGB1 and/or FGB2 polypeptides and other antigens. The number and the types of the antigens which may be mixed with the FGB1 and/or FGB2 polypeptides are not limited in so far as the side effects and adverse reactions are not increased additively or synergistically and the antigenicity and immunogenicity of each of the FGB1 and FGB2 polypeptides and such antigens are not reduced as mentioned above. Generally, two to six or more types of antigens may be mixed with the FGB1 and/or FGB2 polypeptides. Examples of antigens which may possibly be mixed with the present FGB1 and/or FGB2 polypeptides, include detoxified antigens, inactivated antigens or toxoids which are derived from encephalitis virus, hfrs virus (hemorrhagic fever with renal syndrome), influenza virus, parainfluenza virus, HAV, HBV, dengue fever virus, AIDS virus, Bordetella pertussis, Diptheria bacillus, Tetanus bacillus, meningococcus, pneumococcus, and the like.
The immunogencity of the HCV polypeptides including the FGB1 and FGB2 polypeptides may also be enhanced by preparing them in mammalian or yeast systems fused with or assembled with particle-forming proteins such as, for example, that associated with hepatitis B surface antigen. Constructs wherein, for example, the FGB1 and/or FGB2 polypeptides are linked directly to the particle-forming protein coding sequences produce hybrids which are HCV immunogenic. In addition, the vectors may be prepared so that they include epitopes specific to HBV, having various degrees of immunogencity, such as, for example, the pre-S peptide. Thus, particles constructed from particle-forming proteins which include for instance the FGB1 and/or FGB2 polypeptides are immunogenic with respect to HCV and HBV.
Hepatitis surface antigen (HBSAg) has been shown to be formed and assembled into particles in S. cerevisiae, Valenzuela, P. et al.: Nature, 298:344 (1982), as well as in, for example, mammalian cells Valenzuela, P. et al.: IN: Hepatitis B, Millman et al., ed, Plenum Press, pp. 225-236 (1984). The formation of such particles has been shown to enhance the immunogencity of the monomer subunit. The constructs may also include the immunodominant epitope of HBSAg, comprising the 55 amino acids of the presurface (pre-S) region. Neurath et al.: Science, 224:392 (1984). Constructs of the pre-S-HBSAg particle expressible in yeast are disclosed in EPO Pub. No.
174,444 whereas hybrids including heterologous viral sequences for yeast expression are disclosed in EPO Pub. No. 175,261. These constructs may also be expressed in mammalian cells such as Chinese hamster ovary (CHO) cells using an SV40-dihydrofolate reductase vector disclosed in Michelle, M.T. et al.: Viral Hepatitis and Liver Disease, Ed. Vyas, G.N., Dienstag, J.L. and Hoofnagle J.H., Abstract No. 8A.16, p. 659, Grune & Stratten, Inc., (1984).
Generally, a vaccine comprising, for example, the FGB1 and/or FGB2 polypeptides of the present invention may be contained and sealed in a vial, an ampule or the like. The vaccines of the present invention may be administered in the form of a liquid or suspension. In the case where the vaccine is in a dried form, the vaccine is disolved or suspended in sterilized-distilled water or normal saline before administration, the amount of distilled water or normal saline being such that the volume becomes the original volume before being subjected to lyophilization. The vaccines should be administered in a manner compatible with the dosage formulation, and in such an amount as will be prophylactically and/or therapeutically effective. The quantity to be administered, which is believed to be generally in the amount of about 5 micrograms to about 250 micrograms or more of FGB1 or FGB2 per dose, depends on the subject to be treated, capacity of the subject's immune system to synthesize antibodies, and the degree of protection desired. Precise amounts of active ingredient
required to be administered may depend on the judgement of the practitioner and may be peculiar to each subject. The dose of the vaccine per child may be half as much as that of the vaccine per adult. The vaccine may be given in a single dose schedule, or preferably in a multiple dose schedule. A multiple dose schedule is one in which a primary dose of vaccination may be given in 1-10 separate doses, followed with other doses given at subsequent time intervals required to maintain and/or boost the immune response, for example, at 1-4 months for a second dose, and if needed, a subsequent dose(s) after several months. The dosage regimen will also, at least in part, be determined by the need of the individual, and be dependent upon the judgement of the practitioner.
In addition, the vaccine containing the novel immunogenic HCV polypeptides such as the FGB1 and FGB2 polypeptides may be administered in conjunction with other immunoregulatory agents, such as, immune globulins, interleukins, etc.
The novel immunogenic HCV polypeptides and especially the FGB1 and FGB2 polypeptides may also be used for preparing an antibody, such as a polyclonal antibody and a monoclonal antibody, specific for the immunogenic HCV polypeptides. For example, a polyclonal antibody specific for a KLH coupled-Cys-FGB1-Tyr or Cys-FGB2-Tyr polypeptide may be prepared by conventional methods as follows. The KLH coupled- Cys-FGB1-Tyr or Cys-FGB2-Tyr polypeptide of the present invention is innoculated subcutaneously, intramuscularly, intraperitoneally or intravenously into an animal such as mouse, guinea pig, rabbit, goat, horse, etc. The innoculation of the FGB1 or FGB2 polypeptide is preferably conducted several times at intervals of 1 to 4 weeks, to thereby completely immunize the animal. In order to enhance the
immunizing effect, a conventionally commercially available adjuvant may be used as described hereinbefore. Serum from the immunized animal is collected and the polyclonal antibody is isolated and purified from the blood serum according to standard techniques well known in the art, such as described in Mayer and Walker, eds. Immunochemical Methods in Cell and
Molecular Biology, Academic Press, London (1987).
Monoclonal antibodies specific for the FGB1 or FGB2 polypeptides may also be prepared by conventional techniques as described in, for example,
Harlow, E. and Lane, D.: Antibodies. A Laboratory
Manual, pp. 139-244, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1988), and by ImmunoZAP TM technology as reported in Antibody Expression Library Technology, distributed by Stratacyte, 11099 N. Torey Pines Rd., Suite 400, La Jolla, CA 92037. See also Marx, J.: Science, 246:1250-1251 (Dec. 8, 1989); and Huse, W.D. et al.: Science, 246:1275-1281 (Dec. 8, 1989). For example, when using conventional techniques, the splenic cells obtained from a mouse immunized with FGB1 and/or FGB2 polypeptide are fused with commercially available mouse myeloma cells by cell fusion technique, to obtain hybridomas. The hybridomas are screened to obtain a hybridoma capable of producing an antibody reactive with either the FGB1 or the FGB2 polypeptide. The obtained hybridoma is cultured in accordance with standard methods. From the supernatant of the culture, a monoclonal antibody against either the FGB1 or the FGB2 polypeptide is isolated and purified by standard techniques.
The above-mentioned polyclonal and monoclonal antibodies may also be used as diagnostic agents for diagnosing HCV, and those which are neutralizing are useful in passive immunotherapy. The diagnosis of HCV using the antibody may be conducted by immunoassay in substantially the same manner as mentioned above with respect to the diagnosis of HCV using the FGB1 or FGB2 polypeptides. By the use of the polyclonal or the monoclonal antibodies, the identification and quantification of HCV antigen peptides present in, for example, a liver tissue, blood, sera, semen, lymph and ascites can be
determined.
In addition, the novel antibodies and in particular the novel polyclonal antibodies raised against the immunogenic HCV polypeptides, especially the FGB1 polypeptide, of the instant invention nay be useful for detecting autoimmune liver disease induced by, for example, HCV. In other words, it is believed that the immunogenic HCV polypeptides and in particular the FGB1 polypeptide of the present invention share epitopes with host protein (s) possibly expressed or released by the liver in response to inflammation, trauma or disease. Thus, the immunogenic HCV polypeptides and in particular the FGB1 polypeptide of the instant invention are therefore believed to be effective for raising antibodies and in particular polyclonal antibodies capable of cross-reacting with HCV epitopes as well as other epitopes shared between HCV antigen(s) and host protein(s) for detecting autoimmune liver disease in immuno-type assays such as an immunofluorescence assay known to those of skill in the art.
Recently, it has been reported that an amino acid sequence
Gly-Arg-Arg-Gly-Gln-Lys-Ala-Lys-Ser-Asn-Pro-Asn-Arg-Pro-Leu (GOR epitope), derived from the sequence of a clone which was isolated from the plasma of a laboratory chimpanzee infected with human parenteral NANBH (HCV) , detected what was believed to be an auto-antibody believed to have been generated by patients in response to acute or chronic parenteral NANBH (HCV) infection. See Mishiro, S. et al.: The Lancet, 336:1400-1403 (Dec. 8, 1990) and The Lancet,
336:1414-1415 (Dec. 8, 1990). With respect to autoimmune disease, it was also reported that if dissimilar amino acid(s) in the epitopes shared between the host protein and the foreign antigen constitute a radical substitution so as to interfere with antibody binding, virtual homology between the two may not lead to a cross-reacting immune response. See Oldstone, M.B.A.: Cell, 50:819-820 (Sept. 11, 1987). For example, Oldstone reports that a high degree of homology between epitopes of the acetylcholine receptor alpha chain (Pro-Glu-Ser-Asp-Gln-Pro-Asp-Leu) and polyoma virus middle T antigen
(Pro-Glu-Ser-Asp-Gln-Asp-Gln-Leu) fails to cause cross-reacting antibodies, whereas a more distant similarity between the acetylcholine receptor sequence and herpes simplex virus glycoprotein D
(Pro-Asn-Ala-Thr-Gln-Pro-Glu-Leu) induces strong immunological cross-reacitivity. The underscored amino acids represent similar amino acids. See
Oldstone, M.B.A.: Cell, 50:819-820 (Sept. 11, 1987) and Dyrberg and Oldstone: Curr. Topics Microbiol.
Immunol., 130:25-37 (1986).
With respect to the novel FGB1 polypeptide of the instant invention, it has a distant similarity to the recently reported GOR epitope by Mishiro, S. et al. from the plasma of a laboratory chimpanzee
infected with human parenteral NANBH (HCV). As already indicated herein, the GOR epitope has a reported sequence
Gly-Arg-Arg-Gly-Gln-Lys-Ala-lys-Ser-Asn-Pro-Asn-Arg-Pro-Leu,
whereas the sequence for the novel FGB1 polypeptide is Lys-Pro-Gln-Arg-Lys-Thr-Lys-Arg-Asn-Thr-Asn-Ara-Arg-Pro-Gln. The underscored amino acids represent distant, but similar amino acids between the two epitopes.
Monclonal antibodies, in particular, may be used to raise anti-idiotype antibodies. Anti-idiotype antibodies are immunoglobulins which carry an "internal image" of the antigen of the infectious agent against which protection is desired. See, for
example, Nisonoff, A. et al.: Clin. Immunol. Immunopathol., 21:397-406 (1981) and Dreesman et al.: J. Infect. Disease, 151:761 (1985). Techniques for raising anti-idiotype antibodies are well known in the art. See, for example, Grych et al.: Nature, 316:74 (1985), MacNamara et al.: Science, 226:1325 (1984), and Vytdehaag et al.: J. Immunol., 134:1225 (1985). The anti-idiotype antibodies generated may also be useful for treatment and/or diagnosis of HCV by, for example, reacting with HCV antigen or detecting the presence of anti-HCV antibody in biological samples.
It should also be recoginized by one of ordinary skill in the art that a variety of types of antibodies directed against the immunogenic HCV polypeptides including the FGB1 or FGB2 polypeptides may be produced. As used herein, the term "antibody" refers to a polypeptide or group of polypeptides which are comprised of at least one antibody combining site. An "antibody combining site" or "binding domain" is formed from the folding of variable domains of an antibody molecule(s) to form three-dimensional binding spaces with an internal surface shape and charge distribution complementary to the features of an epitope of an antigen, which allows an immunological reaction with the antigen. An antibody combining site may be formed with a heavy and/or a light chain domain (VH and VL, respectively), which form hypervariable loops which contribute to antigen binding. The term "antibody" therefore is used broadly herein and includes, for example, vertebrate antibodies, hybrid antibodies, chimeric antibodies, ImmunoZAP TM antibodies, altered antibodies, univalent antibodies, the Fab proteins, and single domain antibodies, as known in the art.
Both the FGB1 and FGB2 polypeptides and the antibodies raised against such polypeptides are useful in immunoassays to detect presence of HCV antibodies, or the presence of HCV and/or HCV antigens, in biological samples, such as blood, sera and semen.
Design of the immunoassays is subject to a great deal of variation, and a variety of these are well known in the art. For example, the immunoassay may utilize one HCV epitope; alternatively, the immunoassay may use a combination of HCV epitopes derived from for instance the FGB1 and FGB2 polypeptides. It may use, for example, a monclonal antibody directed towards either the FGB1 or FGB2 polypeptide, a combination of monoclonal antibodies directed towards the FGB1 and FGB2 polypeptides, polyclonal antibodies directed towards either the FGB1 or FGB2 polypeptides, or a combination of polyclonal antibodies directed toward the FGB1 and FGB2 polypeptides. Protocols may be based, for example, upon competition, direct reaction, or sandwich type assays. Protocols may also, for example, use solid supports, or may be by
immunoprecipitation.
Preferably, the assays of the present invention involve the use of antibodies or polypeptides and in particular the FGB1 and/or FGB2 polypeptides of the instant invention labeled with a labeling moiety. It is to be understood that the term
"labeling moiety" is used herein throughout in a broad sense and is meant to include, but not limited to, radioisotopes, enzymes, antibodies, dyes, chemiluminescent or fluorescent molecules, immune complexes, and the like, which may be directly or indirectly attached to the antibody, polypeptide or polynucleotide sequence of the instant invention. In order to radioactively label the FGB1 or FGB2 polypeptides, it is preferable to conjugate a Tyr or His amino acid residue or the like to the carboxyl end thereof as discussed hereinbefore. Techniques for radioactively labeling antibodies and polypeptides are well known to those of skill in the art and may be utilized to label the antibodies and immunogenic HCV polypeptides of the instant invention. For example, the techniques disclosed in Harlow and Lane: Antibodies. A Laboratory Manual, Chapter 9, pp 324-339, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1988), and Weir, D.M.: Radioimmunoassay,
Chapter 17, (IN):Immunochemistry, Vol. 1, 2nd ed., Blackwell Scientific Publications, London (1973) may be employed to label the antibodies and immunogenic HCV polypeptides of the present invention with radioiodine. Examples of immuno assays which may be utilized include enzyme-labeled and radioactive immunoassays, such as ELISA and RIA assays,
respectively.
Kits suitable for immunodiagnosis and containing the appropriate labeled reagents are constructed by packaging the appropriate materials, including the novel immunogenic HCV polypeptides such as the FGB1 and/or FGB2 polypeptides of the invention or antibodies raised against such immunogenic HCV polypeptides or anti-idiotype antibodies raised against antibodies to such immunogenic HCV polypeptides in suitable containers, along with the remaining reagents and materials required to conduct the assay, as well as a suitable set of assay instructions.
It is to be understood by those versed in this art that the practice of the present invention employs, unless otherwise indicated herein , conventional techniques of molecular biology , biochemistry, microbiology, virology, recombinant DNA, and immunology, which are within the skill of the art. All patents, patent applications, and publications cited herein, both supra and infra, are hereby incorporated herein by reference in their entireties.
Examples of various embodiments of the present invention will now be further illustrated with reference to the following Examples.
Example I
Enzyme-Linked Immunosorbent Assay (ELISA) for
Detection of HCV Antibodies and Diagnostic Kit
Polypeptides FGB1 and FGB2 are used as antigens in an ELISA diagnostic assay as described below to detect antibodies to HCV in biological samples.
Materials and Reagents
1. Phosphate Buffered Saline (PBS) (SIGMA, P.O. Box 14508, St. Louis, Mo. 53178, catalog no. D6650)
2. 96 well microtiter plates (Dynatech Immunolon U shaped wells, Dynatech Laboratories, Inc., 900 Slaters Lane, Alexandria, Va 22314).
3. Parafilm (or other cover for plates)
4. Tween 20 (Biorad Laboratories, 1414 Harbour Way South, Richmond, Ca 94804)
5. Rabbit anti-human IgG conjugated to horse radish peroxidase (Dako Corporation, 6392 Via Real, Carpinteria, Ca 93013)
6. OPD (1,2-phenylenedianine, dihydrochloride)
tablets (2mg/tablet) (Dako Corporation, 6392 Via Real, Carpinteria, Ca 93013)
7. 0.1 M Citric Acid-Phosphate Buffer pH 5.0
-Citric Acid*H2
-Na2HPO4*12H2O
8. 30% Hydrogen Peroxide (H2O2)
9. Plastic Forceps or Tweezers
10. Aluminum Foil
11. 1 M Sulfuric Acid (H2SO4)
PBS:Tween (Wash Solution)
PBS containing 0.1% Tween 20 Citric Acid-Phosphate Buffer
Dissolve 7.3g Citric Acid and 23.88g Na2HPO4 in 1 L H2O. Store at 4°C.
Substrate Solution
Bring OPD tablets and Citric Acid-Phosphate Buffer to room temperature. Use plastic forceps or tweezers to retrieve OPD tablets. For every 96 well microtiter plate, dissolve 4 OPD tablets in 12 mis of 0.1 M Citric Acid-Phosphate Buffer pH 5.0. Add 5 uls 30% H2O2. The solution is maintained at room
temperature and used within one hour of preparation. Since it is light sensitive, the container is kept wrapped in aluminum foil until used.
Procedure
Dissolve Cys-FGB1-Tyr and/or Cys-FGB2-Tyr polypeptide in PBS at a concentration of about 1mg/ml. Dilute the polypeptide solution to 1:100 in PBS (10 ng of polypeptide). Add 100uls of diluted polypeptide solution to each well to be used. Cover plate with parafilm and incubate for about 8-12 hours on a shaker platform at room temperature. Remove polypeptide solution and wash wells by adding 200 uls PBS:Tween to each, cover plate and incubate on shaker for 4-6 hours at room temperature. Remove wash solution and add 80uls PBS:Tween. Add 20 uls of patient or normal serum to each well. Cover plate and incubate
overnight on a shaker platform at room temperature. Remove samples and wash 3 times with 100 uls
PBS:Tween. Dilute rabbit anti-human IgG conjugated to horse radish peroxidae 1:200 in PBS: Tween. Add 100 uls diluted rabbit anti-human IgG to each well. Cover plate and incubate for about 2-6 hours on a shaker platform at room temperature. Remove rabbit
anti-human IgG and wash 3 times with 100 ul PBS: Tween. Add 100 uls Substrate Solution to each well. Cover plate and incubate for about 5 min. at room
temperature. Add 50 uls or 1 M H2SO4 to each well. Read absorbance of yellow colored end-product by a microwell plate reader at 450nm/492nm.
The protocol described above may be utilized on a commercial basis by assembling in a kit the materials, polypeptides of the instant invention and reagents required to detect antibodies raised against HCV in biological samples, along with appropriate instruction.
Example II
Assay of Coded HCV Panel I
A coded panel containing 44 serum samples that have been tested by the kit from Ortho
Diagnostics Co. , Raritan, NJ, and the RIBA I
(Recombinant ImmunoBlot Assay) and neutralization assays were received from The Center for Blood
Research, 1631 Stockton Boulevard, Suite 250,
Sacramento, California 95816-7089. At the time the coded HCV panel was received, the results of these above-mentioned tests on the 44 serum samples were unavailable. The 44 serum samples were tested with an ELISA utilizing Cys-FGB1-Tyr and Cys-FGB2-Tyr
polypeptides, as described in Example I. The
Cys-FGB1-Tyr and Cys-FGB2-Tyr polypeptides were chemically synthesized as described hereinbefore and included a cysteine amino acid residue at the amino termini and a tyrosine amino acid residue at the carboxyl termini. The results, expressed as
absorbance at 450 nm, are recited in Table 8 (the cutoff value was set at 1.0, derived by addition of 0.4 to the mean of 5 negative controls), and are compared with those obtained at the Center for Blood Research, Sacramento in Tables 9 and 10. Of the two polypeptides, Cys-FGB1-Tyr appears to correlate better with those of the Ortho and RIBA I tests. The results are in agreement with those obtained at the Center for Blood Research, Sacramento, except for sample =40. While sample #40 initially tested as reactive, when retested, however, it was non-reactive (NR) . Sample #40 was a cross with samples #12 and #20, meaning that it was the same serum sample placed in three different tubes in the coded panel.
There are advantages for using the ELISA of the instant invention such as described in Example I versus the assays that were used by the Center for Blood Research, Sacramento, as mentioned herein. As observed in Table 9, forty-one percent (41%), i.e., eighteen out of forty-four samples (4, 5, 9, 11, 12, 18, 19, 20, 24, 33, 37, 33, 39, 40, 41, 42, 43 and 44), are false positives due to inconsistences between the Ortho and the RIBA I test. The results of the ELISA of this Example II correspond well with the RIBA I test, as evidenced by Tables 9 and 10.
Of significance are samples =6 and =13.
These two samples were derived from a patient with clinically diagnosed NANBH, but tested negative with the Ortho test and RIBA I assay for HCV. While both samples were positive with the FGB1 ELISA test of the instant invention as shown in Table 9 (Cys-FGB1-Tyr), one sample was positive with the FGB2 ELISA test of the present invention as shown in Table 10
(Cys-FGB2-Tyr). This suggests that the ELISA tests and in particular the FGB1 ELISA test of the present invention appear to be effective in the diagnosis of HCV due to acute or chronic phase infection. In
Tables 9 and 10, R means reactive, NR means
non-reactive, NT means not tested, I means
indeterminant, Y means yes or positive, and N means no or negative.
The advantages of using the FGB1 and FGB2 polypeptides of the instant invention in an ELISA test are numerous. First, it is believed that the FGB1 and FGB2 polypeptides and espeically the FGB1 polypeptide have an increase in specificity and sensitivity, as compared to Ortho's assay which is concerned with the recombinant C100 peptide (antigen). It is also believed that assays involving the FGB1 and FGB2 are less expensive, and are believed to be capable of diagnosing HCV in the acute phase. Further, the polypeptide FGB1 is water soluble, very stable and binds readily to microwells of ELISA plates in an unconjugated form. Still further, since the FGB1 and FGB2 polypeptides can be prepared by chemical
synthesis, they are easier to produce on a large scale without the variability found with recombinant
proteins. Still further, the tests involving the FGB1 and FGB2 polypeptides are quantitative, allowing one to monitor the progress of the disease. TABLE 8
No. Cys-FGB1-TYr Cys-FGB2-Tyr
1 0.531 1.653
2 0.762 0.606
3 2.632 2.530
4 0.614 0.782
5 0.514 0.757
6 1.483 0.892
7 2.030 0.988
8 0.312 2.417
9 0.594 0.72310 2.715 2.619 11 0.685 0.607 12 0.642 0.83613 1.955 1.86814 0.606 2.402
15 2.357 0.906 16 0.655 1.249
17 1.993 1.749
18 0.628 0.634
19 0.564 0.819 20 0.557 0.69221 2.406 2.51122 0.511 0.499
23 0.781 1.092
24 0.430 0.459
25 0.594 1.699
26 0.701 0.876
27 0.563 0.468
28 0.563 0.652
29 0.651 0.588
30 0.408 1.212
31 2.362 2.401
32 0.960 0.515
33 0.601 0.663
34 2.324 1.910
35 2.570 2.541
36 2.587 2.559
37 0.327 0.602
38 0.771 0.840
39 0.659 0.794
40 2.606 0.967
41 0.985 0.929
42 0.652 0.580
43 0.792 0.768
44 0.690 1.090
Cutoff Value = 1. 0 TABLE 9
NO. CROSS1 CROSS2 ORTHO RIBA NEUT.
Cys-FGB1-Tyr
1 8 25 NR NT NT NR
2 28 NR NT NT NR
3 10 31 R R Y R
4 11 43 R I Y NR
5 38 39 R N Y NR
6 13 NR N NT R
7 R NT NT R
8 1 25 NR NT NT NR
9 41 44 R N N NR 10 3 31 R R Y R 11 4 43 R I Y NR 12 20 40 R N N NR 13 6 NR N NT R 14 29 NR NT NT NR 15 R NT NT R 16 NT NT NT NR 17 34 R R Y R 18 42 43 R I N NR 19 24 37 R N Y NR20 12 40 R N N NR21 35 36 R R Y R 22 27 32 NR NT NT NR 23 30 NR NT NT NR 24 19 37 R N Y NR25 1 8 NR NT NT NR 26 16 NR NT NT NR27 22 32 NR NT NT NR 28 2 NR NT NT NR29 14 NR NT NT NR30 23 NR NT NT NR31 S 10 R R Y R32 22 27 NR NT NT NR33 18 42 R I N NR34 17 R R Y R 35 21 36 R R Y R36 21 35 R R Y R37 19 24 R N Y NR38 5 39 R N Y NR39 5 38 R N Y NR 40 12 20 R N N R41 9 44 R N N NR42 18 33 R I N NR43 4 11 R I Y NR44 9 41 R N N NR TABLE 10
NO. CROSS1 CROSS2 ORTHO RIBA NEUT.
Cys-FGB2-Tyr
1 8 25 NR NT NT R
2 28 NR NT NT NR
3 10 31 R R Y R
4 11 43 R I Y NR
5 38 39 R N Y NR
6 13 NR N NT NR
7 R NT NT NR
8 1 25 NR NT NT R
9 41 44 R N N NR
10 3 31 R R Y R
11 4 43 R I Y NR
12 20 40 R N N NR
13 6 NR N NT R
14 29 NR NT NT R
15 R NT NT NR
16 NT NT NT R
17 34 R R Y R
18 42 43 R I N NR
19 24 37 R N Y NR
20 12 40 R N N NR
21 35 36 R R Y R
22 27 32 NR NT NT NR
23 30 NR NT NT R
24 19 37 R N Y NR
25 1 8 NR NT NT R
26 16 NR NT NT NR
27 22 32 NR NT NT NR
28 2 NR NT NT NR
29 14 NR NT NT NR
30 23 NR NT NT R
31 3 10 R R Y R
32 22 27 NR NT NT NR
33 18 42 R I N NR
34 17 R R Y R
35 21 36 R R Y R
36 21 35 R R Y R
37 19 24 R N Y NR
38 5 39 R N Y NR
39 5 38 R N Y NR
40 12 20 R N N NR
41 9 44 R N N NR
42 18 33 R I N NR
43 4 11 R I Y NR
44 9 41 R N N R Example III
Generation of Anti-Cys-FGB1-Tyr and Anti-Cys-FGB2-Tyr Polypeptide Antibodies (Polyclonal) in Rabbits
The Cys-FGB1-Tyr and Cys-FGB2-Tyr polypeptides were coupled to KLH, a carrier protein, with m-Maleimidobenzoyl-N- hydroxysuccinimide ester (MBS) and administered to separate rabbits as
described below. Before coupling, however, a cysteine amino acid residue was added to the amino end and a tyrosine amino acid residue was added to the carboxyl end of each polypeptide according to standard
techniques as for example described hereinbefore.
This procedure is an established one to produce polyclonal antibodies to peptides in rabbits, and can be modified for use in other laboratory animals.
Coupling of the Cys-FGB1-Tyr and Cys-FGB2-Tyr
polypeptides to carrier proteins with
m-Maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) was done in accordane with Kitagawa and Aikawa: J. Biochem., 79:233-236 (1976), Lia et al.: Biochemistry, 18:690-697 (1979), and Green et al.: Cell, 28:477-487 (1982).
A.) Materials and Reagents
1. 10mM phosphate buffer pH 7.0 (1.6 gm KH PO to 1 L, adjust pH with NaOH)
2. 50 mM phosphate buffer pH 6.0 (6.8 gm KH PO to 1 L, adjust pH with NaOH) 3. 15 mg/ml solution of MBS in dimethyl fomamide (DMF)
4. PBS 0.15 M NaCl in 10 mM phosphate buffer pH 7.3
5. MBS from Pierce, P.O. Box 117, Rockford, IL 61105.
6. DMF from Fisher, 711 Forbes Ave., Pittsburgh, PA 15219.
7. Keyhole limpet Protein (KLH) from Pierce,
P.O. Box 117, Rockford, IL 61105.
8. Ovalbumin from Pierce, P.O. Box 117, Rockford, IL 61105.
9. Sephadex PD-10 from Pharmacia, 800 Centennial
Ave., Piccataway, NJ 08854.
10. PBS from Sigma, P.O. Box 14508, St. Louis, MO
63178.
B.) Coupling to KLH (may be substituted with any
other effective carrier)
Dissolve about 20 mg KLH in about 1.25 ml of about 10 mM phosphate buffer about pH 7.0 and stir overnight at room temperature. Spin down, remove supernatent. Read absorbance at 280 nm. Estimate the amount of KLH per ml using the ratio of 1 A280 which corresponds to about 2.26 mg KLH per ml. Dilute about
5 mg soluble KLH to about 500 ul in the 10 mM phophate buffer pH 7.0. Dissolve about 1-2 mg MBS at about 15 mg/ml in DMF. Add about 63 ul MBS solution dropwise with stirring to KLH solution. Stir the solution for about 30 minutes at room temperature. Load the solution on G-25 column equilibrated with about 50 mM phosphate buffer about pH 6.0 using the sephadex PD-10 column. Dissolve about 5.0 mg pure polypeptide in about 1.0 ml PBS. Add the MBS-KLH pool off the G-25 column and adjust pH to about 7.0-7.5 with NaOH.
Dialyze against PBS to remove uncoupled polypeptides. Stir at room temperature for about 3 hours. Divide the 1.5 ml dialized solution into 4 aliquots of about 0.5 ml and 1 each of about 0.33 ml.
C.) Injection of Rabbits
New Zealand rabbits obtained for the purpose of immunization are quarantined for a period of two weeks and are then injected and bled as follows.
DAY 01 Pre-bled and inject with a homogeneous
emulsion of about 0.5 ml of coupled
Cys-FGB1-Tyr or Cys-FGB2-Tyr polypeptide mixed with about 0.5 mg of Freunds Adjuvant and formed.
DAY 14 Inject with about 0.33 ml of the coupled
Cys-FGB1-Tyr or Cys-FGB2-Tyr polypeptide mixed with about 0.3 mg of Freunds Adjuvant.
DAY 28 Boost again with about 0.33 ml of the
coupled Cys-FGB1-Tyr or
Cys-FGB2-Tyr polypeptide mixed with about 9.3 mg of Freunds Adjuvant and test bled.
DAY 60 Boost again with about 0.33 ml of the
coupled Cys-FGB1-Tyr or
Cys-FGB2-Tyr polypeptide mixed with about 0.3 mg of Freunds Adjuvant and test bled. D. Testing of the Anti-Cys-FGB1-Tyr or
Anti-Cys-FGB2-Tyr Polypeptide Antibodies
in Rabbits
The procedure that may be used is essentially the same as that for testing of patient serum described in Example II, and the results are reported in Tables 11 and 12. Tables 11 and 12 show rabbit serum dilutions and corresponding absorbance readings in an ELISA using the respective Cys-FGB1-Tyr
(Table 11) and Cys-FGB2-Tyr (Table 12) polypeptides.
TABLE 11
SERUM PRE-BLEED 3 MONTH BLEED AVERAGE OF
DILUTION Cys-FGB1-Tyr Cys-FGB1-Tyr 3MO.DUP.-PREBLEED
A450 A450
1/10,000 0.226 2.703 2.667 2.459
1/25,000 0.142 2.659 2.667 2.521
1/50,000 0.169 2.659 2.639 2.480
1/75,000 0.154 2.620 2.639 2.475
1/100,000 0.134 2.620 2.588 2.470
1/250,000 0.149 2.218 2.153 2.036
1/375,000 0.167 1.775 2.051 1.746
1/500,000 0.193 1.384 1.685 1.341
1/750,000 0.185 1.087 1.277 0.997
1/1,000,000 0.252 0.806 0.753 0.527
TABLE 12
SERUM PRE-BLEED 3 MONTH BLEED AVERAGE OF
DILUTION Cys-FGB1-Tyr Cys-FGB1-Tyr 3MO.PUP.-PREBLEED
A450 A450
1/10,000 0.112 2.320 2.254 2.175
1/25,000 0.088 2.330 2.342 2.248
1/50,000 0.102 2.362 2.131 2.145
1/75,000 0.083 2.158 2.166 2.079
1/100,000 0.074 2.077 2.332 2.130
1/250,000 0.067 1.782 1.771 1.710
1/375,000 0.068 1.477 1.513 1.427
1/500,000 0.085 1.321 1.548 1.350
1/750,000 0.071 1.173 1.135 1.083
1/1,000,000 0.084 0.806 0.805 0.721
Example IV
Monoclonal Antibodies to HCV
The use of spleen from rabits injected with immunogenic HCV polypeptides and in particular either the FGB1 or FGB2 polypeptides of the instant invention for raising monoclonal antibodies (MAbs) can be accomplished by, for example, the following
procedures.
1. Hybridoma Technology
Monoclonal antibodies (MAbs) can be generated in accordance with standard technology, as recited for instance in Harlow and Lowe: Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1988). More particularly, the MAbs can be prepared by the fusion of myeloma cells with B-lymphocytes obtained from spleens of, for example, mice or other animals injected with for instance either the FGB1 or the FGB2 polypeptide conjugated with a carrier. In the presence of
hypoxanthine, aminopterin and thymidine (HAT) medium, a few immortal hybridoma cells secreting specific immunoglobins can be selected in accordance with the standard techniques. These hybridomas can be further subdivided into clones by serial dilution of the cultured fusion products in 96 well microtiter plates in the presence of HAT media. The detail procedure can be carried out as described hereinbefore.
Monoclonal antibodies on conjugate groups to toxins (such as pseudomonas endotoxin), drugs, isotopes, dyes or the like could be used as diagnostics and/or in therapy. 2. ImmunoZAP TM Technology
Monoclonal antibodies can also be generated by a four step procedure. The first and second steps involve the construction and separation of heavy and light chain cDNA libraries from poly A mRNA isolated from spleen tissue, peripheral blood, or lymphnode derived from animals inoculated with, for example, the FGB1 or FGB2 polypeptides. The third and fourth steps involve randomly combining the heavy and light chain libraries and screening for antigen binding. This procedure is outlined in detail in the following publications: Antibody Expression Library Technology, distributed by Stratacyte, 11099 N. Torrey Pines Road,
Suite 400, La Jolla, CA 92037; Marx, J.: Science,
246:1250-1251 (Dec. 8, 1989); and Huse, W.D. et al.:
Science, 246:1275-1281 (Dec. 8, 1989).
Example V
1. Construction of Vaccinia Recombinant Viruses
Containing Nucleotide Sequences which Encode the Immunogenic HCV Polypeptides and in Particular the FGB1 and/or FGB2 Polypeptides
(a) Construciton of Recombinant Plasmids.
Plasmids are constructed using existing vectors such as pSC11, Chakrabarti et al.: Molecular and Cell Biology, 5:3403-3409 (1985), pUV1, Falkner et al.: Nucleic Acids Res., 15:7192 (1987), pTM3, Moss et al.: Nature, (348):91-92 (1990), depending on the level of expression desired and on whether a stable recombinant virus is required. All three vectors are pUC based with the vaccinia virus thymidine kinase gene flanking inserted sequences. pSCll may have, for example, the FGB1 and/or FGB2 polypeptides or other polypeptides of the instant invention expressed from an early/late promoter. pUVl may have for instance the FGB1 and/or FGB2 polypeptides under the control of a strong late promoter. pTM3 has a bacteriophage T7 promoter and an EMC leader and is believed to be the best available vector for transient expression either in a cell line expressing T7 RNA polymerase or by coinfection of the cells with two viruses, on carrying the T7 RNA polymerase and the other carrying, for example, the FGB1 and/or FGB2 polypeptide of interest under the control of the T7 promoter. In addition, vectors are constructed from synthetic promoters whose sequence is derived from mutagenesis studies of the promoter. The DNA encoding, for example, the FGB1 and/or FGB2 polypeptides are mutagenized at the nucleotides corresponding to the NH2-terminus and the COOH-terminus of the open readin frame (ORF) to have proper restriction sites for insertion into any particular vector.
(b) Generation of Vaccinia Virus Recombinants.
Vaccinia virus recombinants are generated as described previously in Kotwal, G. J. et al:
Virology, 171:579-587 (1989). CV-1 cells in
monolayers (25 cm2 flask) are infected with about 1 ml of vaccinia virus at about 0.05 pfu. The virus inoculum is left on cells for about 2 h at about 37°C with rocking. Calcium phosphate precipitated DNA is prepared by gently mixing about 5-15 ug of plasmid DNA in about 1 ml of hepes buffered saline, pH about 7.05,
(NaCl 0.14 M, KCl 5 mM, Na2HPO4*2H2O 1 mM, Dextrose
0.1%, Hepes 20 mM). About 50 ul of about 2.5 mM CaCl is added and after gentle mixing is left at room temperature for about 30 min. The virus inoculum is aspirated off and about 1 ml of the DNA precipitate is placed over the cells at room temperature for about 30 min. About 9 ml of prewarmed Eagles MEM containing about 8% FBS is then added to the DNA precipitate and the incubation is carried out for about 3.5 h at about 37°C. The medium is aspirated off and replaced with about 10 mis of fresh Eagles MEM containing about 8% FBS and left at about 37°C for 2 days. After about 48 h the cells are harvested by scraping from bottles, and resuspended in about 0.5 ml Eagles MEM and frozen at about -20°C.
(c) Selection of Vaccinia Virus Recominants.
Depending on the vector used, the recombinants are picked off either TK-cells in the presence of 6-bromouridine, Chakrabarti et al: Mol. Cell. Biol., 5:3403-3409 (1985), or in the presence of mycophenolic acid if the gpt gene is inserted in tandem with the foreign gene. Blue color selection is used with lac Z containing vectors, Falkner and Moss: J. Virol., 62:1849-1854 (1988).
2. Large Scale Production of Recombinant Vaccinia
Viruses.
(a) Growth and Purification of Vaccinia Virus Recombinants.
Recombinant vaccinia viruses are purified according to Joklik: Virology, 18:9-18, (1962). HeLa cells are grown in suspension in MEM spinner medium containing about 5% horse serum in large Bellco 36L spinner flasks with an overhead drive and a teflon paddle impeller assembly at a density of about 5×10 5 cells/ml. Up to about 100 1 of HeLa cells are then infected with trypsinized crude virus made under selective conditions at a multiplicity of infection of about 10 and at a cell density of about 2×107 cells/ml for about h hour. After about h hour, the cell density is brought back to about 5x10 5 cells/ml and is incubated at about 37°C for about 48 hours. At the end of about 48 hours, the cells are harvested, washed in PBS and resuspended in about 1½ volumes of about
10mM Tris HCl (pH 9.0) and homogenized in a douce homogenizer placed in ice using about 25 strokes, five at a time. The homogenate is then centrifuged at about 2500 rpm for about five minutes. The
supernatant is then vortexed and sonicated for about
60 seconds and placed on an equal volume of about 36% sucrose and centrifued at about 13,500 rpm in a SW27 rotor for about 80 minutes. The pellet is resuspended in about 10mM Tris HCl (pH 9.0), sonicated and placed over a sterile sucrose step gradient (ranging from 25%
- 40%) left over-night at about 4ºC. The gradient is then spun in SW 27 rotor at about 12,000 rpm for about
45 minutes. The virus band is then aspirated and repelleted in about 25% sucrose by spinning in an SW
27 rotor at about 13,500 rpm for about 60 minutes.
The virus pellet is then resuspended in about 1mM Tris
HCl (pH 9.0) and distributed into screwtop microfuge tubes in about 150ul aliquots. A titer of about 1 × 10 11/ml is the yield expected for each titer of cells. (b) Quantitation of Virus.
The following conversion table is used for estimating the amount of virus particles:
1 OD260 = 64 ug virus particles
1 OD260 = 2.5 ug DNA
(c) Titration of Virus.
The virus is titered on BSC-1 cells under selective and nonselective conditions. The titer is read at about 48 hours after different dilutions of the virus are placed on confluent BSC-1 cells on a 6 well plate.
(d) Southern Blot Analysis.
DNA is estimated from the absorbance of the virus preparation and is isolated by phenol: chloroform extraction of the proteinase K treated and detergent lysed suspension. It is then digested by Hind III and SA1 I and transferred to nitrocellulose and probed with the nick translated FGB1 and/or FGB2
polynucleotide sequence that is expected to be
inserted in the genome. Both its presence and
location are determined. Essentially the protocol of Sambrook, J. et al.: Molecular Cloning. A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989) is followed. (e) Western Blot Analysis (WBA) and/or
Immunofluorescence (IF).
CV-1 cells are infected at an m.o.i. of 30 (for WBA) and an m.o.i. of 10 (for IF) and the lysed cells are separated by 12.5% PAGE, transferred to nitrocellulose, Sambrook, J. et al.: Molecular
Cloning. A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
(1989), and probed with patient's serum or specific antibody if available. In case of immunofluorescence, the cells are grown on 8 well slides and 24 hour post infection treated with the antibody and then with FITC conjugated antibody, Kotwal, G.J. et al.: Modern Approaches to New Vaccines Including Prevention of AIDS, pp. 389-392, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1989). The fluorescence indicates the intracellular localization of, for example, the FGB1 and/or FGB2 polypeptides.
Example VI
Radioimmuno Assay (RIA)
A.) Detection of Antibodies against HCV in Sera
Purified IgG antibody is obtained from the hyperimmune serum (polyclonal antibody) of Example III. More particularly, the hyperimmune sera, i.e., either anti-Cys-FGB1-Tyr or anti-FGB2-Tyr, obtained from the hyperimmunized rabbits are obtained and subjected to ammonium sulfate precipitation, and the precipitate is obtained as an IgG fraction. The obtained IgG fraction is subjected to gel
chromatography using Sephacryl S200, distributed by Pharmacia Fine Chemicals AB, Sweden, to obtain a purified anti-Cys-FGB1-Tyr IgG or anti-Cys-FGB2-Tyr
IgG (anti-FGB IgG). The purified anti-FGB IgG is labeled with, for example, 125I by the chloramine T method, as described in Greenwood et al.: Biochemical
J., 80:114 (1963). Illustratively stated, about 4 ml of the purified anti-FGB IgG (5 mg/ml) is cooled in ice water, and about 1 mCi/10 ul of 125I is added to the purified anti-FGB IgG while gently stirring.
Then, about 1 ml of about 200 ug/ml aqueous chloramine
T is dropwise added to the mixture and stirred for about 5 minutes to advance a reaction. About 1 ml of about 200 ug/ml aqueous sodium methabisulfite is added to the resultant mixture to terminate the reaction.
The resultant mixture is dialyzed to remove the unreacted 125I, to thereby obtain 125I-labeled
anti-FGB IgG.
Using the above obtained 125I-labeled anti-FGB IgG and the appropriate Cys-FGB1-Tyr or
Cys-FGB2-Tyr polypeptide, an antibody against HCV in serum samples obtained from patients having HCV hepatitis can be detected by radioimmuno assay (RIA).
This can be accomplished by, for example, binding about 0.1 ml of appropriate Cys-FGB1-Tyr or
Cys-FGB2-Tyr polypeptide (1 ug/ml) to each well of the reaction plate for the RIA. Thereafter, the above solution is replaced with about 200 ul of PBS:Tween
(Example I), and further introducing about 0.1 ml of a serum sample to all but three control wells of the reaction plate. In one of the three serum-free control wells of the reaction plate, about 0.1 ml of a control serum positive for HCV is added. In a second serum-free control well, about 0.1 ml of a control serum negative for HCV is added. After about 4 hours of incubation at room temperature, the serum samples are removed and the reaction plate is washed several times with PBS:Tween. To all wells of the reaction plate, about 0.1 ml of the appropriate 125I-labeled anti-FGB IgG is added and allowed to stand overnight at room temperature to advance a reaction. The resultant wells are sufficiently washed with PBS:
Tween. The radioactivity (CPM) of each well is measured using, for example, an auto-gamma counter.
From the radioactivity, an inhibition (%) of the reaction between the appropriate 125I-labeled anti-FGB
IgG and the Cys-FGB1-Tyr or Cys-FGB2-Tyr polypeptide bound in the wells is calculated using standard techniques.
When the serum samples contain antibody against HCV, the reaction between the appropriate polypeptide bound to the wells and the 125I-labeled anti-FGB IgG is inhibited by the reaction between the polypeptide bound in the wells and the anti-HCV antibody present in the serum. Therefore, the
radioactivity of the wells ascribed to the labeled antibody bonded to the appropriate polypeptide bound in the wells will be relatively low in those serum samples derived from patients having antibodies against HCV. This can be confirmed by comparing the radioactivity between the serum wells and the three control wells.
B.) Detection of HCV Antigen in Sera
125I-labeled Cys-FGB1-Tyr or Cys-FGB2-Tyr polypeptide and anti-FGB-IgG bound in the wells of the reaction plate are prepared in substantially the same manner as mentioned above. The appropriate
Cys-FGB1-Tyr or Cys-FGB2-Tyr polypeptide is labeled with, for example, 125I by the chloramine T method outlined in Greenwood, et al.: Biochemical J., 80:114 (1963). The anti-FGB-IgG are bound to the wells by simply adding the appropriate anti-FGB-IgG to the wells of the reaction plate.
Using the above-obtained 125I-labeled
Cys-FGB1-Tyr or Cys-FGB2-Tyr polypeptide, and the appropriate anti-FGB-IgG antibody, HCV antigen present in serum samples obtained from patients infected with
HCV hepatitis can be detected in substantially the same manner as mentioned above.
This is accomplished by, for example, introducing about 0.1 ml of the appropriate
anti-FGB-IgG in each well of the reaction plate for
RIA. Thereafter replacing the above solution with about 200 ul PBS:Tween (Example I), and further introducing about 0.1 ml of a serum to all but three control wells of the reaction plate. In one of the three serum-free control wells of the reaction plate, about 0.1 ml of a control serum positive for HCV is added. In a second serum-free control well, about 0.1 ml of a control serum negative for HCV is added.
After about 4 hrs. of incubating at room temperature, the serum samples are removed from the wells and the plate is washed several times in PBS:Tween. To all wells of the reaction plate, about 0.1 ml of the appropriate 125I-labeled-Cys-FGB1-Tyr or Cys-FGB2-Tyr polypeptide (1 ug/ml) is added and allowed to stand overnight at room temperature to advance a reaction. The resultant wells are sufficiently washed with PBS:Tween. The radioactivity (CPM) of each well is measured using, for example, an auto-gamma counter.
From the radioactivity, an inhibition (%) of the reaction between the appropriate 125I-labeled
Cys-FGB1-Tyr or Cys-FGB2-Tyr polypeptide and the appropriate anti-FGB-IgG bound to the wells is calculated using standard techniques.
When the serum samples contain HCV antigen, the reaction between the 125I-labeled Cys-FGB1-Tyr or
Cys-FGB2-Tyr polypeptide and the appropriate
anti-FGB-IgG bound to the wells is inhibited by the reaction between the HCV antigen and the anti-FGB-IgG bound in the wells. Therefore, the radioactivity of the wells ascribed to the 125I-labeled Cys-FGB1-Tyr or
Cys-FGB2-Tyr polypeptide bonded to the anti-FGB-IgG bound to the wells will be relatively low in those samples derived from patients having HCV antigen.
This can be confirmed by comparing the radioactivity between the serum wells and the three control wells.
Example VII
FGBl Clone
Two polynucleotide DNA sequences designated as GB1 and GB2 , respectively, each 117 nucleotides in length are synthesized on a Gene Assembler Plus
(Pharmacia LKB, Sweden) in accordance with the
Pharmacia Manual and reagents supplied therewith. The polynucleotide sequences for GB1 and GB2 are as follows:
SEQ ID NOS: 31-31
GB1 5'-GAGTGCCCCGGGAGGTCTCGTAGACCGTGCACCATGAGCACGATT
CCCAAACCTCAAAGAAAAACCAAACGTAACACCAACCGTCGCCCA
CAGGACGTCTAGTTCCCGGGTGGCGGT-3'; and
GB2 5'-CTCACGGGGCCCTCCAGAGCATCTGGCACGTGGTACTCGTGCTAA
GGGTTTGGAGTTTCTTTTTGGTTTGCATTGTGGTTGGCAGCGGGT
GTCCTGCAGATCAAGGGCCCACCGCCA-3'.
See also Table 13 (GBl) and Table 14(GB2).
Polynucieotide GB2 is the complementary sequence of GBl. The GB1 and GB2 polynucleotides are annealed according to the methods of Sambrook , J . , et al . :
Molecular Cloning . A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., (1989), with slight modifications. Approximately 5-10 ng/ml of final concentration of GB1 and GB2 are denatured in about 5 mis of annealing buffer (0.1 M NaCl, 10 mM Tris-Cl (pH 7.8), 1.0 mM EDTA) at about 95° C. for about 5 minutes and allowed to cool at room temperature. Two volumes (about 10 mis) of 100% ethanol are added and the annealed polynucleotides (GB1-GB2) are allowed to precipitate overnight at about -20° C. The annealed polynucleotides (GB1-GB2) are centrifuged at about 14,000 × g, dried and
resuspended in about 100 ul of sterile water. About 1.0 ul of the annealed polynucleotides is treated with SI Nuclease (BRL, Gaithersburg, MD under catalog nos. 80015A or 80015B) and electrophoresed on a 1.5% agarose gel next to a 1.0 ul control sample that is not subjected to treatment with S1 Nuclease. S1
Nuclease is a single stranded specific endonuclease which hydrolyzes single stranded RNA or DNA into
5'-mononucleotides. The result of the electrophoresis indicates that approximately 90-95% of the GBl, GB2 is annealed yielding the desired double stranded (ds) GB1-GB2 fragment. The sequence of the annealed ds GB1-GB2 fragment contains two Sma I restriction enzyme sites, CCCGGG, which are 98 nucleotides apart as well as an initiation codon ATG and a stop codon TAG, both of which are positioned between the two Sma I
restriction enzyme sites. In addition, the annealed ds GB1-GB2 fragment contains the polynucleotide sequence which encodes the FGB1 polypeptide, and flanking nucleotide sequences which lie upstream and downstream from the respective 5' end and 3' end Sma I restriction sites.
Annealed ds GB1-GB2 Fragment
SEQ ID NOS: 33-34
Sma I
↓
GB1 5'-(GAGTGC) CCCGGGAGGTCTCGTAGACCGTGCACCATGAGCACG GB2 3'-(CTCACG) GGGCCCTCCAGAGCATCTGGCACGTGGTACTCGTGC GB1 ATTCCCAAACCTCAAAGAAAAACCAAACGTAACACCAACCGTCGCCC GB2 TAAGGGTTTGGAGTTTCTTTTTGGTTTGCATTGTGGTTGGCAGCGGG
Sma I
↓
GB1 ACAGGACGTCTAGTTCCCGGG(TGGCGGT)-3'
GB2 TGTCCTGCAGATCAAGGGCCC(ACCGCCA)-5'
The two underscored polynucleotide sequences designated as CCCGGG in the above annealed ds GB1-GB2 represent restriction sites for the enzyme Sma I. The two restriction sites CCCGGG are digested by the Sma I enzyme (BRL, Gaithersburg, MD, catalog no. 52285A) between the CCC and GGG nucleotides as indicated in the above-recited annealed ds GB1-GB2 fragment. The 6 complementary nucelotide sequence in
parentheticals (GAGTGC)
(CTCACG) represents the flanking complementary nucleotide sequence which lies upstream from the 5' end of the Sma I restriction site whereas the 7 complementary nucleotide sequence in
parentheticals (TGGCGGT)
(ACCGCCA) represents the flanking complementary nucleotide sequence which lies downstream from the 3' end Sma I restriction site.
Moreover, the polynucleotide sequences ATG and TAG represent initiation and stop codons, respectively. The annealed ds GB1-GB2 fragment between the initiation codon ATG and termination codon TAG
encodes a polypeptide which includes the amino acid sequence for the FGB1 polypeptide. It should be understood that the polynucleotide sequence in the above annealed ds GB1-GB2 fragment which lies between the 5' end restriction site CCCGGG and the initiation codon ATG is optional, and therefore can be deleted, modified, shortened or lengthened so long as such modification(s) does not cause the annealed ds GB1-GB2 fragment encoding FGB1 to be out of phase. In addition, other restriction sites and other flanking sequences may be employed so long as they permit the annealed ds GB1-GB2 fragment to be ligated into a replicating expression vector and do not cause the annealed ds GB1-GB2 fragment encoding the polypeptide which contains the amino acid sequence of FGB1 to be out of phase.
The above annealed ds GB1-GB2 is digested with Sma I restriction enzyme and purified as a 98 nucleotide ds fragment from a low melt 1.5% agarose gel containing about 1 ug of Ethidium Bromide
(International Biotechnologies, Inc., New Haven, CT, catalog no. 40060) per ml of agarose (FMC Bioproducts, Rockland, ME, catalog no. 50004) in TAE buffer (0.04 M Tris-acetate 0.001 M EDTA). After the electrophoresis is stopped, long wavelength ultraviolet light is shined over the gel and the band containing the 98 bp Sma I-Sma I fragment is cut with a razor blade into small pieces and introduced into a 17 × 100 mm polypropylene round bottom tube (Becton Dickinson Labware, Lincoln Park, NJ, catalog no. 2059). The tube with the small fragments is weighed to determine the net weight of the fragments (came up to 2.4 g). About 12 ml of the TE buffer (lOmM Tris-HCl, pH 7.5, 0.1 mM EDTA) is added to this tube and placed in a water bath at about 65°C for about 15 minutes with occasional shaking. After 15 minutes, the tube is transferred to the bench and allowed to reach room temperature in about 0.75 hr. At this time, about 0.4 ml of sodium acetate (Mallinckrodt, Paris, KY) is added. Half of the contents of this tube is
transferred into another identical tube and about 7 ml of phenol (BRL Life Technologies, Inc., Gaithersburg, MD catalog no. 5509UA) is added to each tube and the contents shaken vigorously for about 3 minutes at which time they are centrifuged in a Beckman GPR
Centrifuge (Beckman Instruments, Inc., Spinco
Division, Palto Alto, CA catalog no. 349702) at about 3800 rpm for about 5 minutes. The aqueous layer is removed and transferred into a new tube and 7 ml of a 50/50 mixture of phenol and chloroform is added to each tube and again shaken vigorously and centrifuged as above. Again the aqueous layer is removed and transferred into a new tube and about 7 ml of chloroform (Mallinckrodt, Paris, KY) is added to each tube and again shaken vigorously and centrifuged as above. The aqueous layers from both tubes are pooled together (13 ml total, some loss occurs in each step of the way) and about 1 ml of about 0.3 M sodium acetate (Mallinckrodt, Paris, KY) is added and the total volume of about 14 ml is transferred to four tubes (about 3.5 ml/tube). To each of the four tubes, about 7 mis of 100% ethanol is added, shaken and allowed to sit at about -20°C overnight. The four tubes are then centrifuged at about 4°C for about 30 minutes in a beckman Model J2-21M Induction Drive Centrifuge (Beckman Instruments, Inc. Spinco Division, Palto Alto, CA catalog no. 341737). At the end of centrifugation, the liquid portion in each tube is discarded and the pellet is allowed to dry at room temperature. The pellets in the four tubes are combined in a total of about 40 ul of water, about 1.0 ul of which is subjected to electrophoresis on 1.5% agarose gel with standard markers in order to estimate the purity and yield of this particular batch of the 98 bp SmaI-Sma I fragment.
Sma I-Sma I
98 Base Pair Fragment
SEQ ID NOS: 35-36
GB1 5'-GGGAGGTCTCGTAGACCGTGCACCATGAGCACGATTCCCAAACCTC GB2 3'-CCCTCCAGAGCATCTGGCACGTGGTACTCGTGCTAAGGGTTTGGAG GB1 AAAGAAAAACCAAACGTAACACCAACCGTCGCCCACAGGACGTCTA GB2 TTTCTTTTTGGTTTGCATTGTGGTTGGCAGCGGGTGTCCTGCAGAT GB1 GTTCCC-5'
GB2 CAAGGG-3'
The Sma I-Sma I 98 base pair fragment, about 0.5 ug, is ligated into the Sma I site of the linearilized, dephosphorylated pGEM TM-3Zf(-) vector, about 0.1 ug
(Promega, Madison, WI, under catalog no. P2241), in ligation buffer (about 66 mM TRIS-Cl, about pH 7.6, about 6.6 mM MgCl2, about 10 mM DTP, and about 66 uM ATP) in the presence of about 1 unit of T4 DNA Ligase
(BRL, Gaithersburg, MD, catalog no. 5224SB) for about 4 hours at room temperature. After incubation, about 1 ul of the ligation mixture is then introduced into a competent, high efficiency strain of E. coli bacterial cells designated as JM 109 (Promega, Madison, WI, under catalog No. L2001), which have been stored at -70°C. Before introducing the ligated Sma I-Sma I 98 base pair fragment into the competent E. coli JM 109 cells, the competent E. coli cells are thawed on ice for about 30 min. Following thawing, the ligated ds material is mixed with the thawed competent cells for about 30 min. on ice prior to heat shock treatment. To heat shock, the tube containing the mixture is placed in a water bath having a temperature at about 42°C for about 45 seconds. After heat shocking, about 900 ul of LB (Lauria -Bertani Medium, i.e., Peptone 140 10 g/1, and Select Yeast Extract 5 g/1, Gibco-BRL, Gaithersburg, MD, catalog nos. M00392B and M00393B, respectively, and Sodium Chloride 10 g/1, Sigma, P.O. Box 14508, St. Louis, MO 63178, catalog no. S3014) is added to the heat shocked mixture and allowed to incubate at about 37°C for about 60 min. Thereafter, about 100 ul of the bacteria is plated out on agar ampicillin LB plates and incubated at about 37 "C overnight to generate transformants containing the FGB1 recombinant plasmid, i.e., the FGB1 clones.
The agar ampicillin LB plates are made as follows and in accordance with Sambrook, J. et al.: Molecular Cloning. A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
(1989) with slight modification. Prior to
autoclaving, about 15 g of Select Agar (Gibco-BRL, Gaithersburg, MD, catalog no. M00391B) is added to about 1.0 liter of LB. The liquid media in the flask is autoclaved for about 45 min. and then allowed to equilibrate in a water bath to about 58°C before adding the ampicillin thereto. Once equilibrated, ampicillin is added to the liquid media to a final concentration of about 100 ug/ml. The agar ampicillin LB plates are prepared by pouring the liquid media from the flask into the plates. Approximately 30-35 ml of liquid media is poured into each 85-mm petri dish. If bubbles occur, the surface of the liquid media can be flamed with a bunsen burner before solidification of the agar. The agar ampicillin LB plates are stored at room temperature for
approximately 1-2 days before use to minimize
condensation and sweating. If excessive moisture is present, the bacterial colonies may streak. The ampicillin is prepared as follows. Ampicillin
(obtained from International Biotechnologies, Inc., New Haven, CT, catalog no. 02040) is dissolved in water to make a stock solution having a concentration of about 50 mg/ml of the sodium salt of ampicillin. The ampicillin stock solution is sterilized by filtration and stored in selected 1.0 ml aliquots at about -20°C.
Following incubation, single colonies are randomly selected and allowed to grow in about 10 ml LB containing about 100 ug/ml ampicillin overnight at about 37°C with vigorous shaking (about 225 rpm).
Mini-preparations are made of each cultivated colony in accordance with Sambrook, J. et al. Molecular
Cloning, A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
(1989) with slight modification. About 1.5 ml of the overnight culture is then poured into an Eppendorf tube. The culture is centrifuged at about 14,000 rpm for about 1 min. in an Eppendorf centrifuge. The remainder of the overnight culture is stored at about 4°C. The centrifuged medium is removed by aspiration, leaving the bacterial pellet as dry as possible. The dried pellet is then resuspended in the same tube by vortexing in about 100 ul of an ice-cold solution of about 500 mM glucose, 10 mM EDTA and 25 mM Tris-Cl (about pH 8.0). The resuspended pellet is allowed to stand for about 5 min. at room temperature. To the Eppendorf tube, approximately 200 ul of a freshly prepared solution containing about 0.2 N NaOH and about 1% SDS is added. The Eppendorf tube is then closed and the contents thereof are mixed by inverting the Eppendorf tube quickly for about 2-3 min. Do not vortex the Eppendorf tube during this step. The
Eppendorf tube is then stored on ice for approximately 5 min. At this point, about 150 ul of an ice-cold potassium acetate solution, about pH 4.8 (about 11.5 ml of glacial acetic acid and about 28.5 ml of water are added to about 60 ml of 5 M potassium acetate), is added to the Eppendorf tube. The potassium acetate solution is about 3 M potassium and 5 M acetate. The Eppendorf tube is then closed and mixed gently in an inverted position for about 10 seconds and then allowed to stand on ice for approximately 5 min. The Eppendorf tube is then centrifuged at about 14,000 rpm for about 10 min. in an Eppendorf centrifuge at about 4°C. The supernatant is then transferred to a new Eppendorf tube. An equal volume of phenol is added to the Eppendorf tube and mixed by vortexing. The
Eppendorf tube is then centrifuged at about 14,000 rpm for about 1 min. in an Eppendorf centrifuge, and the aqueous layer is transferred to another Eppendorf tube where an equal volume of chloroform is added and mixed by vortexing. The Eppendorf tube is centrifuged for about 1 min. in an Eppendorf centrifuge at about
14,000 rpm, and the aqueous layer is removed and transferred to a new Eppendorf tube. Two volumes of ethanol, about 1 ml , are added to the Eppendorf tube at room temperature and mixed by vortexing. The resultant solution is stored at -20°C for about 1 hr. Thereafter, the Eppendorf tube is centrifuged in an Eppendorf centrifuge at about 4°C for approximately 10 min. at about 14,000 rpm. The supernatant is then removed and the remaining contents in the Eppendorf tube are dried in a Speed Vac Concentrator (Model SVC 100H distribued by Savant Instruments, Inc.,
Farmandale, N.Y.). Approximately 20 ul of TE buffer (10 mM Tris-HCl, pH 7.5, 0. l mM EDTA), containing about 20-40 ug/ml DNase-free pancreatic RNase
(Boehringer Mannheim Biochemicals, Indianapolis, IN) is added to the dried pellet in the Eppendorf tube and is mixed briefly and allowed to incubate at about 37°C for about 20 min. About 2 ul of the solution is then transferred to a new Eppendorf tube wherein about 2 ul of React 6 buffer (50 mM Tris-HCl, pH 7.4, 6 mM MgCl2, 50 mM KC1, 50 mM NaCl), 1 unit of Pvu II restrictive enzyme (BRL, Gaithersburg, MD, under catalog no.
54125A) and about 15 ul of water is added. The resultant solution is incubated for about 45 min. at about 37°C. to yield ds DNA fragments. The remainder of the untransferred preparation can be stored at about -20°C.
It should of course be appreciated by those of skill in this art that digestion of plasmids obtained by minipreparations yields ds DNA fragments that can be cleaved to completion with appropriate restriction enzymes. Nonetheless, since certain batches of some enzymes may be less effective with these minipreparations of the plasmids than with highly purified DNA, this may be overcome by
increasing the amount of enzyme by two-three fold and/or by increasing the time of incubation by
approximately 6-12 hrs.
The resultant ds DNA fragments after digestion with Pvu II can be analyzed by gel
electrophoresis in 1% agarose gel (FMC Bioproducts, Rockland, ME, catalog no. 50004). The clones that contain the inserted HCV polynucleotide sequence will have one of its ds DNA fragments migrate with an increase in mobility by about 98 nucleotides as compared with a Pvu II digest of pGEM TM-3ZF(-) plasmids and standard DNA markers electrophoresed in the same 1% agarose gel. This confirms the insertion of the Sma I-Sma I 98 base pair fragment into the pGEM TM-3ZF(-) plasmid and the existence of an FGB1 clone.
To further confirm the insertion of the
Sma I-Sma I 98 base pair fragment into the
pGEM TM-3ZF(-) plasmid and the existence of the FGB1 clone, direct DNA sequencing of a single stranded clone designated as UR-23 using Reagent Kit for DNA
Sequencing with Sequenase, United States Biochemical
Corporation, Cleveland, OH, catalog no. 70721
(Sequenase T7 DNA Polymerase, U.S. Patent No.
4,795,699) is performed. To prepare the single stranded UR-23 clone, an overnight culture of a single colony containing pGEM TM-3ZF(-) plasmid (Promega,
Madison, WI, catalog no. P2261) with the Sma I-Sma I
98 nucleotides fragment insert is grown in about
50-100 ug/ml ampicillin (International
Biotechnologies, Inc., New Haven, CT, catalog no.
02040). About 5 ml of LB with 50 ug/ml ampicillin is inoculated with about 100 ul of overnight culture. It is shaken for about 1-2 hours at 37°C at 225 rpm. The culture is infected with about 80 ul of about 5 × 10 10 pfu/ml of helper phage R408 (Promega, Madison, WI, catalog no. P2291), and the shaking is continued overnight at 225 rpm. Supernatant is collected by spining the cells at about 12,000 g for about 15 min. at about 4°C. The supernatant is removed, transferred to a new tube and recentrifuged at about 12,000 g for about 15 min. The phage is precipitated by adding about 0.25 ml volume of 20% PEG (Polyethylene glycol Sigma, St. Louis, MO, catalog no. P-5413) and about 2.5 M sodium chloride at room temperature for about 1 hr. and is then centrifuged at about 12,000 g for about 15 min. at about 4°C. The supernatant is discarded and the pellet is resuspended in about 400 ul of a buffer comprised of about 10 mM Tris-HCl pH 8.0 (International Biotechnologies, Inc., New Haven, CT, catalog no. 70142) and 0.1 mM EDTA (International Biotechnologies, Inc., New Haven, CT, catalog no.
70182). The resuspended pellet is first extracted with an equal volume of phenol (BRL Life Technologies, Inc., Gaithersburg, MD, catalog no. 5509UA) and then with an equal volume of chloroform (Mallinckrodt, Paris, KY). Several phenol/chloroform extractions are performed until the interface between the aqueous and organic phases is clear. DNA is precipitated by addition of about 1/10 volume of about 3 M Sodium Acetate (Mallinckrodt, Paris, KY) and about 2.5 ml of absolute ethanol (Aaper Alcohol and Chemical Company). Purity of the DNA of the single stranded UR-23 clone is examined upon electrophoresis of about 2 ul on about 1.2% agarose gel (FMC Bioproducts, Rockland, ME, catalog no. 50004).
The results of the direct sequencing of the single stranded UR-23 clone reveal that the
Sma I-Sma I 98 base pair fragment is inserted into the pGEM TM-3ZF(-) plasmid, and that such plasmid is introduced into a competent, high efficiency E. coli bacterial cell of the JM 109 strain, but with slight modification. According to the direct sequencing results, two bases in the GB1 Sma I-Sma I 98
nucleotides fragment and their complements in the GB2 fragment are changed by the JM 109 bacterial cells.
More particularly, the base pairs designated at the
20th and 66th positions downstream from the 5' Sma I end of the transcribing GB1 strand are changed from a dGMP(G)-dCMP(C) to a TMP(T)-dAMP(A) and from a
dCMP(C)-dGMP(G) to a dAMP(A)-TMP(T), respectively, as illustrated below:
Modified Sma I-Sma I 98 Base Pair Fragment and Translation Thereof
SEQ ID NOS: 37-39
Sma I (G)
10 20 30
GB1 5'-GGGAGGTCTCGTAGACCGTTCACCATGAGC
GB2 3'-CCCTCCAGAGCATCTGGCAAGTGGTACTCG
(C) METSer
40 50 60
GB1 ACGATTCCCAAACCTCAAAGAAAAACCAAA
GB2 TGCTAAGGGTTTGGAGTTTCTTTTTGGTTT
ThrIleProLysProGlnArgLysThrLys
Sma I
(C) 70 80 90
GB1 CGTAAAACCAACCGTCGCCCACAGGACGTCTAGTTCCC-3' GB2 GCATTTTGGTTGGCAGCGGGTGTCCTGCAGATCAATTT-5'
(G)
ArgLysThrAsnArgArgProGlnAspValEND
(Asn)
More particularly, in the modified Sma I-Sma
I 98 base pair fragment, the base pairs designated in parentheticals (G)-(C) and (C)-(G) at the 20th and
66th positions represent the original base pairs which are changed in the GB1 sequence by the JM 109
bacterial cells. The two sequences ATG and TAG are the initiation and termination codons, respectively.
The lysine amino acid residue in parentheticals (Asn) represents the original amino acid residue encoded by the GB1 strand. The amino acid residues underscored in the modified Sma I-Sma I 98 base pair fragment constitute those amino acid residues which correspond to the amino acid sequence for the FGB1 polypeptide, except for the substituted lysine amino acid residue
(Lys). The change at the 20th base pair from the 5' Sma-I end in the transcribing GB1 strand of the modified Sma I-Sma-I 98 base pair fragment appears to be of little importance since it is positioned upstream from the ATG initiation codon. Nevertheless, the effect of the change at the 66th base pair from the 5' Sma I end in the modified GB1 strand appears to be of greater significance since it changes a codon consisting of bases 64-66, which is positioned
downstream from the ATG initiation codon, but upstream from the TAG termination codon. The modified codon in the FGB1 clone substitutes a lysine amino acid residue (Lys) for an asparagine amino acid residue (Asn).
It should be understood by those of skill in the art that the new dAMP(A) -TMP(T) base pair in the modified Sma I-Sma I 98 base pair fragment at position 66 may be converted back to the original
dCMP(C)-dGMP(G) base pair by using the technique of site directed mutagenesis, provided that the JM-109 strain or other bacterial strains utilized will permit this change to remain stable in the newly developed FGB1 clone.
It, of course, should be understood that the present invention contemplates other FGB1 clones prepared by other recombinant techniques known in the art as well as other clones prepared with recombinant DNA fragments which encode amino acid sequences comprising the polypeptides of the instant invention. Moreover, it should be understood that the instant invention contemplates FGB2 clones which may be prepared in a similar manner as mentioned
hereinbefore.
Figure imgf000140_0001
Figure imgf000141_0001
Figure imgf000142_0001
Figure imgf000143_0001
Figure imgf000144_0001
TABLE 14
Gene Assembler Plus
Sequence: GB2
Synthesis: GB2
Scale: 0.2 micromole
Sequence Length: 117
Column: 1
Final Detritylation: Yes
Coupling Efficiency Threshold: 50%
Ace Area Last eff Ave eff
Pos Base ration Peak ht
mins mins %FS %min % %
117 C 0. 39 0.75 372 41. 62 ┄ ┄
116 T 0. 38 0.82 476 57.61 ┄ ┄
115 C 0. 38 0.85 614 73.98 ┄ ┄ 114 A 0.38 0.83 681 76.89 ┄ ┄
113 C 0. 37 0.87 650 80.01 104.0 100.0
112 G 0. 36 0.82 620 69.75 ┄ 100.0 111 G 0.36 0.82 615 70.81 101.5 100.0
110 G 0. 36 0.86 587 70.61 99. 7 100.0 109 G 0.40 0.87 584 71.03 100.6 100.0 108 C 0.40 0.93 612 82.94 100.7 100.0 107 C 0.44 0.94 615 82.33 99.3 100.0 106 C 0.41 0.96 596 82.53 100.2 100.0 105 T 0.45 1.00 602 88.44 104.0 100.0 104 C 0.43 0.99 594 84.77 101.3 100.0 103 C 0.46 1.00 565 83.42 98.4 100.0 102 A 0.41 0.96 530 74.96 99.8 100.0 101 G 0.43 0.92 489 68.96 99.6 100.0 100 A 0.45 1.00 536 78.05 102.0 100.0 99 G 0.46 0.96 474 68.87 99.9 100.0 98 C 0.49 1.00 537 81.87 99.6 100.0
OS Base Retention Duration Peak ht Ace Area Last eff Ave eff mins mins %FS %min % %
97 A 0.43 0.96 505 73.90 98.2 100.0
96 T 0.50 1.00 551 87.19 99.8 100.0
95 C 0.51 1.00 528 81.58 99.9 100.0
94 T 0.51 1.00 523 89.78 101.5 100.0
93 G 0.44 0.97 457 67.55 99.7 100.0
92 G 0.55 0.97 453 68.30 101.1 100.0
91 C 0.54 1.00 497 79.14 99.2 100.0
90 A 0.56 1.00 480 74.19 100.1 100.0
89 C 0.55 1.00 496 79.76 100.4 100.0
88 G 0.48 0.99 431 65.21 98.8 100.0
87 T 0.57 1.00 516 83.81 99.0 100.0
86 G 0.48 1.00 430 65.28 100.1 100.0
85 G 0.58 0.94 429 65.09 99.7 100.0
84 T 0.60 1.00 498 83.19 99.8 100.0
83 A 0.59 1.00 462 73.76 99.9 100.0
82 C 0.58 1.00 480 75.66 99.2 100.0
81 T 0.59 1.00 503 83.05 99.9 100.0
80 C 0.58 1.00 472 77.28 101.1 100.0
79 G 0.51 1.00 400 62.70 99.4 100.0
78 T 0.60 1.00 485 82.91 99.9 100.0
77 G 0.52 1.00 409 62.78 100.1 100.0
76 C 0.60 1.00 450 74.44 99.1 100.0
75 T 0.60 1.00 474 80.50 99.0 100.0
74 A 0.61 1.00 431 70.02 99.4 100.0
73 A 0.61 1.00 409 64.69 92.4 99.9
72 G 0.56 0.99 417 58.83 98.7 99.9
71 G 0.63 1.00 392 62.01 105.4 100.0
70 G 0.63 0.97 374 59.64 96.2 99.9
69 T 0.63 1.00 451 76.83 99.2 99.9
68 T 0.64 1.00 456 79.06 102.9 100.0
67 T 0.63 1.00 461 81.55 103.1 100.0
66 G 0.54 1.00 378 61.28 100.7 100.0
Pos Base Retention Duration Peak ht Ace Area Last eff Ave eff mins mins %FS %min % %
65 G 0.55 1.00 377 62.27 101.6 100.0
64 A 0.62 1.00 387 64.47 100.0 100.0
63 G 0.56 1.00 362 60.26 98.4 100.0
62 T 0.64 1.00 447 78.76 99.3 100.0
61 T 0.65 1.00 437 79.33 100.7 100.0
60 T 0.63 1.00 454 81.60 102.9 100.0
59 C 0.63 1.00 415 72.64 99.9 100.0
58 T 0.63 1.00 438 78.53 98.1 100.0
57 T 0.60 1.00 442 79.53 101.3 100.0
56 T 0.61 1.00 449 80.46 101.2 100.0
55 T 0.61 1.00 449 80.76 100.4 100.0
54 T 0.61 1.00 465 83.66 103.6 100.0
53 G 0.55 1.00 393 67.17 101.1 100.0
52 G 0.55 1.00 401 68.33 101.7 100.0
51 T 0.63 1.00 428 77.66 97.5 100.0
50 T 0.63 1.00 448 80.73 104.0 100.0
49 T 0.64 1.00 437 79.86 98.9 100.0
48 G 0.59 1.00 375 66.23 99.2 100.0
47 C 0.59 1.00 438 75.14 100.3 100.0
46 A 0.56 1.00 413 71.37 100.6 100.0
45 T 0.58 1.00 430 77.83 99.4 100.0
44 T 0.59 1.00 432 78.71 101.1 100.0
43 G 0.55 1.00 429 73.64 102.1 100.0
42 T 0.59 1.00 442 81.70 101.9 100.0
41 G 0.56 1.00 422 70.65 97.9 100.0
40 G 0.55 1.00 416 71.06 100.6 100.0
39 T 0.59 1.00 434 80.87 99.7 100.0
38 T 0.62 1.00 416 78.10 96.6 100.0
37 G 0.57 0.97 390 69.82 99.4 100.0
36 G 0.56 1.00 390 69.72 99.9 100.0
35 C 0.58 1.00 391 71.25 99.6 100.0
34 A 0.59 0.95 360 64.87 99.2 100.0
Pos Base Retention Duration Peak ht Ace Area Last eff Ave eff mins mins %FS %min % %
33 G 0.56 0.93 348 62.91 96.6 100.0
32 C 0.60 1.00 350 67.06 98.0 100.0
31 G 0.57 1.00 337 63.40 100.4 100.0
30 G 0.57 1.00 327 61.10 96.5 100.0
29 G 0.57 1.00 323 61.26 100.1 100.0
28 T 0.62 1.00 335 66.15 98.4 100.0
27 G 0.57 1.00 310 60.34 99.2 100.0
26 T 0.61 1.00 322 66.58 100.3 100.0
25 C 0.62 1.00 310 62.46 99.0 100.0
24 C 0.60 1.00 305 63.62 101.9 100.0
23 T 0.63 1.00 319 65.88 99.6 100.0
22 G 0.59 0.99 298 59.70 99.8 100.0
21 C 0.62 1.00 299 62.66 99.5 100.0
20 A 0.61 0.98 288 58.54 99.3 100.0
19 G 0.58 0.96 281 57.39 98.7 100.0
18 A 0.58 1.00 276 56.57 98.3 100.0
17 T 0.62 1.00 287 61.25 98.8 100.0
16 C 0.60 1.00 277 59.14 98.9 99.9
15 A 0.60 0.98 277 58.47 101.1 100.0
14 A 0.60 0.98 272 57.73 98.7 99.9
13 G 0.56 0.94 261 54.97 99.3 99.9
12 G 0.56 1.00 257 54.71 99.5 99.9
11 G 0.60 1.00 259 54.78 100.1 99.9
10 C 0.60 1.00 258 56.94 99.4 99.9
9 C 0.62 1.00 258 56.08 98.5 99.9
8 C 0.60 1.00 256 57.29 102.2 99.9
7 A 0.61 0.98 253 55.40 99.4 99.9
6 C 0.64 1.00 250 56.66 99.4 99.9
5 C 0.61 1.00 249 56.53 99.8 99.9
4 G 0.58 1.00 242 54.15 99.8 99.9
3 C 0.63 1.00 243 55.26 98.9 99.9
Pos Base Retention Duration Peak ht Ace Area Last eff Ave eff mins mins %FS %min % %
2 C 0.60 1.00 243 56.87 102.9 99.9 1 A 0.62 1.13 243 56.76 100.4 99.9
Total Synthesis yield from start = 94.3%
Examole VIII
The majority of parenterally transmitted non-A, non-B hepatitis (PT-NANBH) cases are believed to be caused by the hepatitis C virus (HCV). See, for example, Choo, Q-L. et al.: Science, 244: 359-362 (1989); Kuo, G. et al.: Science, 244:362-364 (1989); and Choo, Q-L. et al.: Br. Med. Bul., 46:423-441
(1990). Possible sources of infection for a
substantial number of remaining cases, known as sporadic hepatitis C, have been controversial. It has been suggested that heterosexual activity, see for example Alter, M.J. et al.: JAMA, 262:1201-1205
(1989); Alter, M.J. et al.: JAMA, 264: 2231-2235
(1990); and Melbye, M. et al.: Br. Med. J.,
301:210-212 (1990), and possibly homosexual activity, Tedder, R.S. et al.: Br. Med. J., 302: 1299-1302
(1991), may be important, perhaps accounting for up to an additional 11% of all cases seen. In order to obtain more direct evidence for the potential of sexual transmission of HCV, enzyme-linked
immunosorbent assays (ELISA) based upon polyclonal antibodies raised against either the FGB1 or FGB2 polypeptides in New Zealand rabbits were used to detect HCV in semen samples.
The two polypeptide antigens, FGB1 and FGB2, were synthetically constructed as set forth in detail hereinbefore. The FGB1 and FGB2 polypeptides included the cysteine and tyrosine amino acid residues at their respective -NH2 and -COOH terminal ends.
Each antigen was injected into New Zealand rabbits along with Freund's adjuvant using standard protocols, as described in Example III.
In order to determine the titers of the antibodies made, ELISA plates coated separately with about 100 ng of the two antigens were treated with the serial dilutions of the prebleed and the three-month bleeds from the rabbits for 4-6 hr. at room
temperature. The plates then were washed five times with phosphate-buffered saline containing about 0.1%
Tween and treated with goat anti-rabbit conjugated with horse radish peroxidase (diluted 1:200) for about
2-4 hr. The washed plates were then treated with a substrate solution prepared by dissolving four OPD
(1,2-phenylenediamine dihydrochloride) tablets
(2mg/tablet) in about 12 ml of about 0.1 M citric acid-phosphate, about pH 5.0, and further adding about
5 micro liters of about 30% H2O2. The plates then were incubated in the dark for about 5 min. at about room temperature. To stop the reaction, about 50 micro liters of about 1 M H2SO were added to each well and absorbance of the orange-colored end-product was read by a microwell plate reader at 450/492 nm.
TABLES 11 and 12 show the absorbance values obtained for the serial dilution of the prebleed and the three-month bleed for each of the two antigens, i.e., FGB1 and FGB2. The presence of antibodies in a very high dilution (1:1,000,000) suggests that both FGB1 and FGB2 antigens are highly immunogenic and that the antibodies are likely to be very sensitive reagents for the detection of structural components of HCV.
Blood-free semen samples from nine patients with clinical manifestation of NANBH and from five healthy donors were homogenized and treated with about 0.5% NP-40 in phosphate-buffered saline to obtain a final concentration of about 0.1% NP-40 at about room temperature. The contents were centrifuged at about 12,000 rpm for about 15 min. at about 4°C.
The plates were then washed five times with PBS containing about 0.1% Tween 20 and treated with the rabbit antibody at a dilution of about 1:100 for about
6-8 hr. at approximately room temperature. The plates were washed once again and treated as described above with goat anti-rabbit antibody and further with the substrate solution; the absorbance values obtained at
450/492 nm are reported in TABLE 15.
TABLE 15
SEMEN ANALYSIS
Patient FGB2 FGB1
1 0.813 1.519
2 2.052 2.796
3 1.420 2.363
4 1.110 1.930
5 1.910 2.399
6 1.339 2.045
7 1.224 1.622
8 0.S54 1.420
9 0.709 1.513
Mean of 5 Control
Semen 0.500 0.900
Range of Control
Semen 0.380-0.640 0.700-1.200
In order to verify whether patients whose semen contained HCV-specific antigen had HCV-specific antibodies in their serum, we tested the serum from eight of the nine patients. We coated ELISA plates with about 50 ng of either FGB1 or FGB2 and added about 100 micro liters of the eight serum samples from the healthy donors, which were diluted about 1:5 in PBS-Tween to individual wells. The procedure was identical to the one described above except that a mixture of goat anti-human and rabbit anti-human IgG conjugated with horse radish peroxidase was used as a second antibody in place of goat anti-rabbit. The absorbance values are shown in TABLE 16 under the columns of FGB1 and FGB2. The cutoff values were obtained by multiplying by a factor of 1.5 the mean absorbance value obtained by averaging the absorbance values for the five healthy donors. The serum samples were also tested with the commercially available Ortho test kit, i.e., C-100 nonstructural antigen, and the values are also shown in TABLE 16.
TABLE 16
SERUM ANALYSIS
Patient FGB2 FGB1 Ortho Intero.*
Cutoff 0.768 0.220 0.140 R
1 1.042 2.669 2.215 R
2 1.340 0.320 0.300 R
3 3.298 2.545 2.208 R
4 0.633 0.681 0.079 R
5 1.100 0.829 2.262 R
6 2.662 2.713 2.287 R
7 NA NA NA NA
8 1.306 2.761 0.961 R
9 1.112 2.423 0.672 R
*Interp. = Interpretation; R = Reactive;
NA = Not Available
All nine semen samples obtained from NANBH patients had absorbance values higher than the values obtained from the healthy donors employing antibodies to each of the two antigens, FGB1 and FGB2. Significantly higher absorbance values (greater than twice the mean absorbance value for health donors) were obtained in five of the nine semen samples. See TABLE 15.
Available serum from eight patients was reactive. Patient 4 was nonreactive with both the
Ortho test and an ELISA using HCV-FGB1, but was repeatedly positive (A 492 nm1.669 at a cutoff of
0.447) with the ELISA using FGB2 as antigen. Based upon the results, all patients tested are believed to be infected with HCV, and are further believed to contain varying amounts of HCV viral antigen in their semen.
This study is believed to have detected the presence of HCV-specific viral antigens in the semen of nine patients with well-documented HCV infection. In contrast, semen from five healthy controls did not show HCV antigen presence. These findings support the potential for sexual transmission of hepatitis C virus.
The present invention may, of course, be carried out in other specific ways than those herein set forth without departing from the spirit and essential characteristics of the invention. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive and all changes coming within the meaning and equivalency range of the appended claims are intended to be embraced herein.
APPENDIX I
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: Kotwal, Girish J.
Baroudy, Bahige M.
(ii) TITLE OF INVENTION: Basic Structural Immunogenic
Polypeptides having Epitopes for HCV, Antibodies, Polynucieotide Sequences, Vaccines, and Methods
(iii) NUMBER OF SEQUENCES: 75
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Wood, Herron & Evans
(B) STREET: 2700 Carew Tower
(C) CITY: Cincinnati
(D) STATE: Ohio
(E) COUNTRY: U.S.A.
(F) ZIP: 45202
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: Patentin Release #1.0, Version #1.25
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER: US
(B) FILING DATE: 14-JAN-1992
(C) CLASSIFICATION:
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: US 07/639,809
(B) FILING DATE: 14-JAN-1991
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Manso, Peter J.
(B) REGISTRATION NUMBER: 32,264
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (513) 241-2324
(B) TELEFAX: (513) 421-7269
(C) TELEX: 501826 (2) INFORMATION FOR SEQ ID NO:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(v) FRAGMENT TYPE: N-terminal
(ix) FEATURE:
(A) NAME/KEY: Peptide
(B) LOCATION: 1..15
(D) OTHER INFORMATION: /label= Polypeptide
/note= "Polypeptide having epitope for HCV.
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:
Lys Pro Gln Arg Lys Thr Lys Arg Asn Thr Asn Arg Arg Pro Gln 1 5 10 15
(2) INFORMATION FOR SEQ ID NO:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 16 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(v) FRAGMENT TYPE: N-terminal
(ix) FEATURE:
(A) NAME/KEY: Peptide
(B) LOCATION: 1..16
(D) OTHER INFORMATION: /label= Polypeptide
/note= "Polypeptide having epitope for HCV."
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:
Cys Lys Pro Gln Arg Lys Thr Lys Arg Asn Thr Asn Arg Arg Pro Gln 1 5 10 15
(2) INFORMATION FOR SEQ ID NO: 3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 16 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(v) FRAGMENT TYPE: N-terminal ( ix) FEATURE :
(A) NAME/KEY: Peptide
(B) LOCATION: 1..16
(D) OTHER INFORMATION: /label= Polypeptide
/note= "Polypeptide having epitope for HCV."
(Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:
Lys Pro Gln Arg Lys Thr Lys Arg Asn Thr Asn Arg Arg Pro Gln Tyr 1 5 10 15
(2) INFORMATION FOR SEQ ID NO: 4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(v) FRAGMENT TYPE: N-terminal
(ix) FEATURE:
(A) NAME/KEY: Peptide
(B) LOCATION: 1..17
(D) OTHER INFORMATION: /label= Polypeptide
/note= "Polypeptide having epitope for HCV."
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:
Cys Lys Pro Gln Arg Lys Thr Lys Arg Asn Thr Asn Arg Arg Pro Gln 1 5 10 15
Tyr
(2) INFORMATION FOR SEQ ID NO: 5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(v) FRAGMENT TYPE: C-terminal
(ix) FEATURE:
(A) NAME/KEY: Peptide
(B) LOCATION: 1..15
(D) OTHER INFORMATION: /label= Polypeptide
/note= "Polypeptide having epitope for HCV. (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:
Ser Arg Pro Ser Trp Gly Pro Thr Asp Pro Arg Arg Arg Ser Arg 1 5 10 15
(2) INFORMATION FOR SEQ ID NO: 6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 16 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(v) FRAGMENT TYPE: C-terminal
(ix) FEATURE:
(A) NAME/KEY: Peptide
(B) LOCATION: 1..16
(D) OTHER INFORMATION: /label= Polypeptide
/note= "Polypeptide having epitope for HCV."
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:
Cys Ser Arg Pro Ser Trp Gly Pro Thr Asp Pro Arg Arg Arg Ser Arg 1 5 10 15
(2) INFORMATION FOR SEQ ID NO: 7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 16 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(v) FRAGMENT TYPE: C-terminal
(ix) FEATURE:
(A) NAME/KEY: Peptide
(B) LOCATION: 1..16
(D) OTHER INFORMATION: /label= Polypeptide
/note= "Polypeptide having epitope for HCV."
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:
Ser Arg Pro Ser Trp Gly Pro Thr Asp Pro Arg Arg Arg Ser Arg Tyr 1 5 10 15 (2) INFORMATION FOR SEQ ID NO: 8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(v) FRAGMENT TYPE: C-terminal
(ix) FEATURE:
(A) NAME/KEY: Peptide
(B) LOCATION: 1..17
(D) OTHER INFORMATION: /label= Polypeptide
/note= "Polypeptide having epitope for HCV."
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:
Cys Ser Arg Pro Ser Trp Gly Pro Thr Asp Pro Arg Arg Arg Ser Arg
1 5 10 15
Tyr
(2) INFORMATION FOR SEQ ID NO: 9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 45 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS : single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: RNA (genomic)
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: hepatitis C virus
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9:
AAACCUCAAA GAAAAACCAA ACGUAACACC AACCGUCGCC CACAG 45
(2) INFORMATION FOR SEQ ID NO:10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 48 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: RNA (genomic)
(iv) ANTI-SENSE: NO ( ix) FEATURE :
(A) NAME/KEY: misc_RNA
(B) LOCATION: 1..48
(D) OTHER INFORMATION: /function= "Epitope"
/product= "Polypeptide having epitope for HCV"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:
UGYAAACCUC AAAGAAAAAC CAAACGUAAC ACCAACCGUC GCCCACAG 48 (2) INFORMATION FOR SEQ ID NO: 11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 48 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: RNA (genomic)
(iv) ANTI-SENSE: NO
(ix) FEATURE:
(A) NAME/KEY: misc_RNA
(B) LOCATION: 1..48
(D) OTHER INFORMATION: /function= "Epitope"
/product= "Polypeptide having epitope for HCV"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11:
AAACCUCAAA GAAAAACCAA ACGUAACACC AACCGUCGCC CACAGUAY 48 (2) INFORMATION FOR SEQ ID NO: 12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 51 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: RNA (genomic)
(iv) ANTI-SENSE: NO
(ix) FEATURE:
(A) NAME/KEY: misc_RNA
(B) LOCATION: 1..51
(D) OTHER INFORMATION: /function= "Epitope"
/product= "Polypeptide having epitope for HCV" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12:
UGYAAACCUC AAAGAAAAAC CAAACGUAAC ACCAACCGUC GCCCACAGUA Y 51 (2) INFORMATION FOR SEQ ID NO: 13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 45 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13:
AAACCTCAAA GAAAAACCAA ACGTAACACC AACCGTCGCC CACAG 45
(2) INFORMATION FOR SEQ ID NO: 14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 48 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14:
TGYAAACCTC AAAGAAAAAC CAAACGTAAC ACCAACCGTC GCCCACAG 48 (2) INFORMATION FOR SEQ ID NO: 15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 48 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15:
AAACCTCAAA GAAAAACCAA ACGTAACACC AACCGTCGCC CACAGTAY 48
(2) INFORMATION FOR SEQ ID NO: 16:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 51 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16:
TGYAAACCTC AAAGAAAAAC CAAACGTAAC ACCAACCGTC GCCCACAGTA Y 51 (2) INFORMATION FOR SEQ ID NO: 17:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 45 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: RNA (genomic)
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17:
UCUCGGCCUA GUUGGGGCCC CAGGGACCCC CGGCGUAGGU CGCGC 45
(2) INFORMATION FOR SEQ ID NO: 18:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 48 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: RNA (genomic)
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18:
UGYUCUCGGC CUAGUUGGGG CCCCAGGGAC CCCCGGCGUA GGUCGCGC 48 (2) INFORMATION FOR SEQ ID NO: 19:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 48 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: RNA (genomic)
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19:
UCUCGGCCUA GUUGGGGCCC CAGGGACCCC CGGCGUAGGU CGCGCUAY 48 (2) INFORMATION FOR SEQ ID NO: 20:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 51 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: RNA (genomic)
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:
UGYUCUCGGC CUAGUUGGGG CCCCAGGGAC CCCCGGCGUA GGUCGCGCUA Y 51 (2) INFORMATION FOR SEQ ID NO: 21:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 45 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:
TCTCGGCCTA GTTGGGGCCC CAGGGACCCC CGGCGTAGGT CGCGC 45 (2) INFORMATION FOR SEQ ID NO: 22:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 48 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:
TGYTCTCGGC CTAGTTGGGG CCCCAGGGAC CCCCGGCGTA GGTCGCGC 48
(2) INFORMATION FOR SEQ ID NO: 23:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 48 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 23:
TCTCGGCCTA GTTGGGGCCC CAGGGACCCC CGGCGTAGGT CGCGCTAY 48
(2) INFORMATION FOR SEQ ID NO: 24:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 51 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:
TGYTCTCGGC CTAGTTGGGG CCCCAGGGAC CCCCGGCGTA GGTCGCGCTA Y 51 (2) INFORMATION FOR SEQ ID NO: 25:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 45 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: RNA (genomic)
(iv) ANTI-SENSE: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 25:
UUUGGAGUUU CUUUUUGGUU UGCAUUCUGG UUGGCAGCGG GUGUC 45
(2) INFORMATION FOR SEQ ID NO:26:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 45 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iv) ANTI-SENSE: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:
TTTGGAGTTT CTTTTTGGTT TGCATTCTGG TTGGCAGCGG GTGTC 45
(2) INFORMATION FOR SEQ ID NO: 27:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 45 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: RNA (genomic)
(iv) ANTI-SENSE: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:
AGAGCCGGAU CAACCCCGGG GUCCCUGGGG GCCGCAUCCA GCGCG 45 (2) INFORMATION FOR SEQ ID NO: 28:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 45 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iv) ANTI-SENSE: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 28:
AGAGCCGGAT CAACCCCGGG GTCCCTGGGG GCCGCATCCA GCGCG 45
(2) INFORMATION FOR SEQ ID NO: 29:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 45 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iv) ANTI-SENSE: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 29:
GACACCCGCT GCCAACCACA ATGCAAACCA AAAAGAAACT CCAAA 45
(2) INFORMATION FOR SEQ ID NO: 30:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 45 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iv) ANTI-SENSE: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:
CGCGCTGGAT GCGGCCCCCA GGGACCCCGG GGTTGATCCG GCTCT 45 (2) INFORMATION FOR SEQ ID NO: 31:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 117 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iv) ANTI-SENSE: NO
(ix) FEATURE:
(A) NAME/KEY: insertion_seg
(B) LOCATION: 1..117
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 34..102
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 31:
GAGTGCCCCG GGAGGTCTCG TAGACCGTGC ACC ATG AGC ACG ATT CCC AAA CCT 54
Met Ser Thr Ile Pro Lys Pro
1 5
CAA AGA AAA ACC AAA CGT AAC ACC AAC CGT CGC CCA CAG GAC GTC 99 Gln Arg Lys Thr Lys Arg Asn Thr Asn Arg Arg Pro Gln Asp Val
10 15 20
TAGTTCCCGG GTGGCGGT 117
(2) INFORMATION FOR SEQ ID NO: 32:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 32:
Met Ser Thr Ile Pro Lys Pro Gln Arg Lys Thr Lys Arg Asn Thr Asn
1 5 10 15
Arg Arg Pro Gln Asp Val
20
SUBSTITUTE SHEET (2) INFORMATION FOR SEQ ID NO: 33:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 117 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iv) ANTI-SENSE: NO
(ix) FEATURE:
(A) NAME/KEY: insertion_seg
(B) LOCATION: 1..117
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 34..102
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 33:
GAGTGCCCC GGGAGGTCTC GTAGACCGTG CACC ATG AGC ACG ATT CCC AAA 51
Met Ser Thr Ile Pro Lys
1 5
CCT CAA AGA AAA ACC AAA CGT AAC ACC AAC CGT CGC CCA CAG GAC GTC 99 Pro Gln Arg Lys Thr Lys Arg Asn Thr Asn Arg Arg Pro Gln Asp Val
10 15 20
TAGTTCCCG GGTGGCGGT 117
(2) INFORMATION FOR SEQ ID NO: 34:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 34:
Met Ser Thr Ile Pro Lys Pro Gln Arg Lys Thr Lys Arg Asn Thr Asn 1 5 10 15
Arg Arg Pro Gln Asp Val
20
SUBSTITUTE SHEET (2) INFORMATION FOR SEQ ID NO: 35:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 98 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iv) ANTI-SENSE: NO
(ix) FEATURE:
(A) NAME/KEY: insertion_seq
(B) LOCATION: 1..98
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 25..93
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 35:
GGGAGGTCTC GTAGACCGTG CACC ATG AGC ACG ATT CCC AAA CCT CAA AGA AAA 54
Met Ser Thr lie Pro Lys Pro Gln Arg Lys 1 5 10
ACC AAA CGT AAC ACC AAC CGT CGC CCA CAG GAC GTC TAGTTCCC 98
Thr Lys Arg Asn Thr Asn Arg Arg Pro Gln Asp Val
15 20
(2) INFORMATION FOR SEQ ID NO: 36:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 36:
Met Ser Thr Ile Pro Lys Pro Gln Arg Lys Thr Lys Arg Asn Thr Asn
1 5 10 15
Arg Arg Pro Gln Asp Val
20 (2) INFORMATION FOR SEQ ID NO: 37:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 98 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iv) ANTI-SENSE: NO
(ix) FEATURE:
(A) NAME/KEY: insertion_seq
(B) LOCATION: 1..98
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 25..93
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 37:
GGGAGGTCTC GTAGACCGTK CACCATGAGC ACGATTCCCA AACCTCAAAG AAAAACCAAA60 CGTAAMACCA ACCGTCGCCC ACAGGACGTC TAGTTCCC 98
(2) INFORMATION FOR SEQ ID NO: 38:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 38:
Met Ser Thr Ile Pro Lys Pro Gln Arg Lys Thr Lys Arg Asn Thr Asn
1 5 10 15
Arg Arg Pro Gln Asp Val
20
(2) INFORMATION FOR SEQ ID NO: 39:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide (iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 39:
Met Ser Thr lie Pro Lys Pro Gln Arg Lys Thr Lys Arg Lys Thr Asn 1 5 10 15
Arg Arg Pro Gln Asp Val
20
(2) INFORMATION FOR SEQ ID NO: 40:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(v) FRAGMENT TYPE: N-terminal
(ix) FEATURE:
(A) NAME/KEY: Peptide
(B) LOCATION: 1..17
(D) OTHER INFORMATION: /label= Polypeptide
/note= "Polypeptide having epitope for HCV."
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 40:
Cys Arg Pro Gln Arg Lys Thr Lys Arg Asn Thr Asn Arg Arg Pro Gln
1 5 10 15
Tyr
(2) INFORMATION FOR SEQ ID NO: 41:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(v) FRAGMENT TYPE: N-terminal
(ix) FEATURE:
(A) NAME/KEY: Peptide
(B) LOCATION: 1..17
(D) OTHER INFORMATION: /label= peptide
/note= "Polypeptide having epitope for HCV." (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 41:
Cys Lys Pro Gln Arg Arg Thr Lys Arg Asn Thr Asn Arg Arg Pro Gln 1 5 10 15
Tyr
(2) INFORMATION FOR SEQ ID NO: 42:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(v) FRAGMENT TYPE: N-terminal
(ix) FEATURE:
(A) NAME/KEY: Peptide
(B) LOCATION: 1..17
(D) OTHER INFORMATION: /label= Peptide
/note= "Polypeptide having epitope for HCV."
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 42:
Cys Lys Pro Gln Arg Lys Thr Arg Arg Asn Thr Asn Arg Arg Pro Gln 1 5 10 15
Tyr
(2) INFORMATION FOR SEQ ID NO: 43:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(v) FRAGMENT TYPE: N-terminal
(ix) FEATURE:
(A) NAME/KEY: Peptide
(B) LOCATION: 1..17
(D) OTHER INFORMATION: /label= Peptide
/note= "Polypeptide having epitope for HCV."
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 43:
Cys Lys Pro Gln Lys Lys Thr Lys Arg Asn Thr Asn Arg Arg Pro Gln 1 5 10 15
Tyr (2) INFORMATION FOR SEQ ID NO: 44:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(v) FRAGMENT TYPE: N-terminal
(ix) FEATURE:
(A) NAME/KEY: Peptide
(B) LOCATION: 1..17
(D) OTHER INFORMATION: /label= Peptide
/note= "Polypeptide having epitope for HCV."
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 44:
Cys Lys Pro Gln Arg Lys Thr Lys Lys Asn Thr Asn Arg Arg Pro Gln
1 5 10 15
Tyr
(2) INFORMATION FOR SEQ ID NO: 45:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(v) FRAGMENT TYPE: N-terminal
(ix) FEATURE:
(A) NAME/KEY: Peptide
(B) LOCATION: 1..17
(D) OTHER INFORMATION: /label= Peptide
/note= "Polypeptide having epitope for HCV."
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 45:
Cys Lys Pro Gln Arg Lys Thr Lys Arg Asn Thr Asn Lys Arg Pro Gln
1 5 10 15
Tyr (2) INFORMATION FOR SEQ ID NO: 46:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(v) FRAGMENT TYPE: N-terminal
(ix) FEATURE:
(A) NAME/KEY: Peptide
(B) LOCATION: 1..17
(D) OTHER INFORMATION: /label= Peptide
/note= "Polypeptide having epitope for HCV."
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:46:
Cys Lys Pro Gln Arg Lys Thr Lys Arg Asn Thr Asn Arg Lys Pro Gln 1 5 10 15
Tyr
(2) INFORMATION FOR SEQ ID NO: 47:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(v) FRAGMENT TYPE: N-terminal
(ix) FEATURE:
(A) NAME/KEY: Peptide
(B) LOCATION: 1..17
(D) OTHER INFORMATION: /label= Peptide
/note= "Polypeptide having epitope for HCV."
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:47:
Cys Lys Pro Gln Arg Lys Ser Lys Arg Asn Thr Asn Arg Arg Pro Gln 1 5 10 15
Tyr (2) INFORMATION FOR SEQ ID NO: 48:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(v) FRAGMENT TYPE: N-terminal
(ix) FEATURE:
(A) NAME/KEY: Peptide
(B) LOCATION: 1..17
(D) OTHER INFORMATION: /label= Peptide
/note= "Polypeptide having epitope for HCV."
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 48:
Cys Lys Pro Gln Arg Lys Thr Lys Arg Asn Ser Asn Arg Arg Pro Gln
1 5 10 15
Tyr
(2) INFORMATION FOR SEQ ID NO: 49:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(v) FRAGMENT TYPE: N-terminal
(ix) FEATURE:
(A) NAME/KEY: Peptide
(B) LOCATION: 1..14
(D) OTHER INFORMATION: /label= Peptide
/note= "Polypeptide having epitope for HCV."
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 49:
Cys Arg Lys Thr Lys Arg Asn Thr Asn Arg Arg Pro Gln Tyr 1 5 10
(2) INFORMATION FOR SEQ ID NO: 50:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide
(v) FRAGMENT TYPE: N-terminal
(ix) FEATURE:
(A) NAME/KEY: Peptide
(B) LOCATION: 1..14
(D) OTHER INFORMATION: /label= Peptide
/note= "Polypeptide having epitope for HCV."
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 50:
Cys Lys Lys Thr Lys Arg Asn Thr Asn Arg Arg Pro Gln Tyr 1 5 10
(2) INFORMATION FOR SEQ ID NO:51:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(v) FRAGMENT TYPE: N-terminal
(ix) FEATURE:
(A) NAME/KEY: Peptide
(B) LOCATION: 1..14
(D) OTHER INFORMATION: /label= Peptide
/note= "Polypeptide having epitope for HCV."
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 51:
Cys Arg Arg Thr Lys Arg Asn Thr Asn Arg Arg Pro Gln Tyr 1 5 10
(2) INFORMATION FOR SEQ ID NO:52:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(v) FRAGMENT TYPE: N-terminal
(ix) FEATURE:
(A) NAME/KEY: Peptide
(B) LOCATION: 1..14
(D) OTHER INFORMATION: /label= Peptide
/note= "Polypeptide having epitope for HCV. " (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 52:
Cys Arg Lys Thr Arg Arg Asn Thr Asn Arg Arg Pro Gln Tyr 1 5 10
(2) INFORMATION FOR SEQ ID NO: 53:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(v) FRAGMENT TYPE: N-terminal
(ix) FEATURE:
(A) NAME/KEY: Peptide
(B) LOCATION: 1..14
(D) OTHER INFORMATION: /label= Peptide
/note= "Polypeptide having epitope for HCV."
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 53:
Cys Arg Lys Ser Lys Arg Asn Thr Asn Arg Arg Pro Gln Tyr 1 5 10
(2) INFORMATION FOR SEQ ID NO: 54:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(v) FRAGMENT TYPE: N-terminal
(ix) FEATURE:
(A) NAME/KEY: Peptide
(B) LOCATION: 1..14
(D) OTHER INFORMATION: /label= Peptide
/note= "Polypeptide having epitope for HCV."
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 54:
Cys Arg Lys Thr Lys Lys Asn Thr Asn Arg Arg Pro Gln Tyr 1 5 10 (2) INFORMATION FOR SEQ ID NO:55:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(v) FRAGMENT TYPE: N-terminal
(ix) FEATURE:
(A) NAME/KEY: Peptide
(B) LOCATION: 1..14
(D) OTHER INFORMATION: /label= Peptide
/note= "Polypeptide having epitope for HCV."
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 55:
Cys Arg Lys Thr Lys Arg Asn Thr Asn Lys Arg Pro Gln Tyr 1 5 10
(2) INFORMATION FOR SEQ ID NO: 56:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(v) FRAGMENT TYPE: N-terminal
(ix) FEATURE:
(A) NAME/KEY: Peptide
(B) LOCATION: 1..14
(D) OTHER INFORMATION: /label= Peptide
/note= "Polypeptide having epitope for HCV."
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 56:
Cys Arg Lys Thr Lys Arg Asn Thr Asn Arg Lys Pro Gln Tyr 1 5 10
(2) INFORMATION FOR SEQ ID NO: 57:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 16 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(v) FRAGMENT TYPE: N-terminal ( ix) FEATURE :
(A) NAME/KEY: Peptide
(B) LOCATION: 1..16
(D) OTHER INFORMATION: /label= Peptide
/note= "Polypeptide having epitope for HCV."
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 57:
Cys Lys Gln Lys Lys Thr Lys Arg Ser Thr Asn Arg Arg Pro Gln Tyr 1 5 10 15
(2) INFORMATION FOR SEQ ID NO:58:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(v) FRAGMENT TYPE: N-terminal
(ix) FEATURE:
(A) NAME/KEY: Peptide
(B) LOCATION: 1..15
(D) OTHER INFORMATION: /label= Peptide
/note= "Polypeptide having epitope for HCV."
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 58
Lys Gln Lys Lys Thr Lys Arg Ser Thr Asn Arg Arg Pro Gln Tyr
1 5 10 15
(2) INFORMATION FOR SEQ ID NO:59:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 16 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(v) FRAGMENT TYPE: N-terminal
(ix) FEATURE:
(A) NAME/KEY: Peptide
(B) LOCATION: 1..16
(D) OTHER INFORMATION: /label= Peptide
/note= "Polypeptide having epitope for HCV." (xi) SEQUENCE DESCRIPTION: SEQ ID NO:59:
Lys Pro Gln Lys Lys Thr Lys Arg Ser Thr Asn Arg Arg Pro Gln Tyr 1 5 10 15
(2) INFORMATION FOR SEQ ID NO:60:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 16 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(v) FRAGMENT TYPE: N-terminal
(ix) FEATURE:
(A) NAME/KEY: Peptide
(B) LOCATION: 1..16
(D) OTHER INFORMATION: /label= Peptide
/note= "Polypeptide having epitope for HCV."
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 60:
Lys Pro Gln Lys Lys Asn Lys Arg Ser Thr Asn Arg Arg Pro Gln Tyr 1 5 10 15
(2) INFORMATION FOR SEQ ID NO: 61:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(v) FRAGMENT TYPE: N-terminal
(ix) FEATURE:
(A) NAME/KEY: Peptide
(B) LOCATION: 1..15
(D) OTHER INFORMATION: /label= Peptide
/note= "Polypeptide having epitope for HCV."
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 61:
Cys Lys Gln Arg Lys Thr Lys Arg Ser Thr Asn Arg Arg Pro Gln
1 5 10 15 (2) INFORMATION FOR SEQ ID NO:62::
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(v) FRAGMENT TYPE: N-terminal
(ix) FEATURE:
(A) NAME/KEY: Peptide
(B) LOCATION: 1..15
(D) OTHER INFORMATION: /label= Peptide
/note= "Polypeptide having epitope for HCV."
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:62:
Lys Gln Arg Lys Thr Lys Arg Ser Thr Asn Arg Arg Pro Gln Tyr
1 5 10 15
(2) INFORMATION FOR SEQ ID NO: 63:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 16 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(v) FRAGMENT TYPE: N-terminal
(ix) FEATURE:
(A) NAME/KEY: Peptide
(B) LOCATION: 1..16
(D) OTHER INFORMATION: /label= Peptide
/note= "Polypeptide having epitope for HCV."
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 63:
Cys Lys Gln Arg Lys Thr Lys Arg Ser Thr Asn Arg Arg Pro Gln Tyr 1 5 10 15
(2) INFORMATION FOR SEQ ID NO:64:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 16 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: N-terminal
(ix) FEATURE:
(A) NAME/KEY: Peptide
(B) LOCATION: 1..16
(D) OTHER INFORMATION: /label= Peptide
/note= "Polypeptide having epitope for HCV. "
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 64:
Lys Pro Gln Arg Lys Thr Lys Arg Ser Thr Asn Arg Arg Pro Gln Tyr 1 5 10 15
(2) INFORMATION FOR SEQ ID NO: 65:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(v) FRAGMENT TYPE: N-terminal
(ix) FEATURE:
(A) NAME/KEY: Peptide
(B) LOCATION: 1..15
(D) OTHER INFORMATION: /label= Peptide
/note= "Polypeptide having epitope for HCV."
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 65:
Cys Lys Gln Arg Lys Thr Lys Arg Asn Thr Asn Arg Arg Gln Tyr
1 5 10 15
(2) INFORMATION FOR SEQ ID NO: 66:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 13 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(v) FRAGMENT TYPE: N-terminal
(ix) FEATURE:
(A) NAME/KEY: Peptide
(B) LOCATION: 1..13
(D) OTHER INFORMATION: /label= Peptide
/note= "Polypeptide having epitope for HCV." (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 66:
Lys Gln Arg Lys Thr Lys Arg Asn Thr Asn Arg Arg Gln 1 5 10
(2) INFORMATION FOR SEQ ID NO: 67:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 12 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(v) FRAGMENT TYPE: N-terminal
(ix) FEATURE:
(A) NAME/KEY: Peptide
(B) LOCATION: 1..12
(D) OTHER INFORMATION: /label= Peptide
/note= "Polypeptide having epitope for HCV,
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 67:
Lys Gln Arg Lys Thr Lys Arg Asn Thr Asn Arg Arg
1 5 10
(2) INFORMATION FOR SEQ ID NO: 68:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(v) FRAGMENT TYPE: N-terminal
(ix) FEATURE:
(A) NAME/KEY: Peptide
(B) LOCATION: 1..15
(D) OTHER INFORMATION: /label= Peptide
/note= "Polypeptide having epitope for HCV."
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 68:
Lys Pro Gln Arg Lys Asn Lys Arg Asn Thr Asn Arg Arg Pro Gln
1 5 10 15 (2) INFORMATION FOR SEQ ID NO: 69:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 16 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(v) FRAGMENT TYPE: N-terminal
(ix) FEATURE:
(A) NAME/KEY: Peptide
(B) LOCATION: 1..16
(D) OTHER INFORMATION: /label= Peptide
/note= "Polypeptide having epitope for HCV."
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 69:
Cys Lys Gln Arg Lys Thr Lys Arg Asn Thr Asn Arg Arg Pro Gln Tyr 1 5 10 15
(2) INFORMATION FOR SEQ ID NO: 70:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(v) FRAGMENT TYPE: N-terminal
(ix) FEATURE:
(A) NAME/KEY: Peptide
(B) LOCATION: 1..14
(D) OTHER INFORMATION: /label= Peptide
/note= "Polypeptide having epitope for HCV."
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:70:
Lys Gln Arg Lys Thr Lys Arg Asn Thr Asn Arg Arg Pro Gln 1 5 10
(2) INFORMATION FOR SEQ ID NO:71:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: N-terminal
(ix) FEATURE:
(A) NAME/KEY: Peptide
(B) LOCATION: 1..17
(D) OTHER INFORMATION: /label= Peptide
/note= "Polypeptide having epitope for HCV."
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 71:
Cys Lys Pro Gln Lys Lys Asn Lys Arg Asn Thr Asn Arg Arg Pro Gln
1 5 10 15
Tyr
(2) INFORMATION FOR SEQ ID NO: 72:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(v) FRAGMENT TYPE: N-terminal
(ix) FEATURE:
(A) NAME/KEY: Peptide
(B) LOCATION: 1..17
(D) OTHER INFORMATION: /label= Peptide
/note= "Polypeptide having epitope for HCV."
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 72:
Cys Lys Pro Gln Arg Lys Asn Lys Arg Asn Thr Asn Arg Arg Pro Gln 1 5 10 15
Tyr
(2) INFORMATION FOR SEQ ID NO: 73:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(v) FRAGMENT TYPE: N-terminal ( ix) FEATURE :
(A) NAME/KEY: Peptide
(B) LOCATION: 1..15
(D) OTHER INFORMATION: /label= Peptide
/note= "Polypeptide having epitope for HCV."
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 73:
Lys Pro Gln Arg Lys Thr Lys Arg Asn Thr Asn Arg Arg Pro Gln 1 5 10 15
(2) INFORMATION FOR SEQ ID NO:74:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(v) FRAGMENT TYPE: N-terminal
(ix) FEATURE:
(A) NAME/KEY: Peptide
(B) LOCATION: 1..17
(D) OTHER INFORMATION: /label= Peptide
/note= "Polypeptide having epitope for HCV."
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:74:
Cys Lys Pro Gln Arg Lys Thr Lys Arg Asn Thr Asn Arg Arg Pro Gln
1 5 10 15
Tyr
(2) INFORMATION FOR SEQ ID NO: 75:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(v) FRAGMENT TYPE: N-terminal
(ix) FEATURE:
(A) NAME/KEY: Peptide
(B) LOCATION: 1..15
(D) OTHER INFORMATION: /label= Peptide
/note= "Polypeptide having epitope for HCV." (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 75:
Lys Pro Gln Lys Lys Asn Lys Arg Asn Thr Asn Arg Arg Pro Gln
1 5 10 15

Claims

Having described our invention, we claim:
1. A basic, immunogenic polypeptide having an epitope for HCV, said basic, immunogenic
polypeptide further having at least about 15 amino acid residues in its sequence of which at least about 4 said residues are arginine amino acid residues and at least about 7 said residues are in a Chou-Fasman turn.
2. A basic, immunogenic polypeptide as recited in claim 1, said basic, immunogenic
polypeptide further having between about 15 and about 17 amino acid residues in its sequence.
3. A basic, immunogenic polypeptide as recited in claim 1, said basic, immunogenic
polypeptide further having between about 7 and about 17 amino acid residues in a Chou-Fasman turn.
4. A basic, immunogenic polypeptide as recited in claim 1, said basic, immunogenic
polypeptide further having a calculated molecular weight of at least about 1500.
5. A basic, immunogenic polypeptide as recited in claim 4, said basic, immunogenic
polypeptide further having a calculated molecular weight of between about 1500 and about 2500.
6. A basic, immunogenic polypeptide as recited in claim 1, said basic, immunogenic
polypeptide further having a pI on the order of about 12.
7. A basic, immunogenic polypeptide as recited in claim 1, said basic, immunogenic
polypeptide further having between about 4 and about 7 arginine amino acid residues in its sequence.
8. A basic, immunogenic polypeptide as recited in claim 7, said basic, immunogenic
polypeptide further having about 4 arginine amino acid residues and up to about 3 lysine amino acid residues in its sequence.
9. A basic, immunogenic polypeptide as recited in claim 1, said basic, immunogenic
polypeptide further having at least about 11
hydrophillic amino acid residues in its sequence.
10. A basic, immunogenic polypeptide as recited in claim 9, said basic, immunogenic
polypeptide further having between about 11 and about 14 hydrophillic amino acid residues in its sequence.
11. A basic, immunogenic polypeptide as recited in claim 1, said basic, immunogenic
polypeptide further having at least about 5 polar amino acid residues in its sequence.
12. A basic, immunogenic polypeptide as recited in claim 11, said basic, immunogenic
polypeptide further having between about 5 and about 8 polar amino acid residues in its sequence.
13. A basic, immunogenic polypeptide as recited in claim 1, said basic, immunogenic
polypeptide further having at least about 5 basic amino acid residues in its sequence.
14. A basic, immunogenic polypeptide as recited in claim 13, said basic, immunogenic
polypeptide further having between about 5 and about 7 basic amino acid residues in its sequence.
15. A basic, immunogenic polypeptide as recited in claim 1, said basic, immunogenic
polypeptide further having an amino acid sequence which corresponds to an amino acid sequence encoded by a polynucleotide sequence that lies within the first 4% of a human HCV genome following an initiation codon encoding for a human HCV polyprotein.
16. A basic, immunogenic polypeptide as recited in claim 1, said basic, immunogenic
polypeptide further having an amino acid sequence which corresponds to an amino acid sequence encoded by a polynucleotide sequence in the structural region of a human HCV genome.
17. A polypeptide comprised of said basic, immunogenic polypeptide as recited in claim 1.
18. A basic, immunogenic polypeptide as recited in claim 1, said basic, immunogenic
polypeptide being attached to a solid substrate.
19. A truncated, basic immunogenic
polypeptide having an epitope for HCV, said truncated basic, immunogenic polypeptide further having an amino acid sequence which corresponds to an amino acid sequence encoded by a polynucleotide sequence that lies within the first 4% of a HCV genome following an initiation codon encoding for a human HCV polyprotein.
20. A truncated, basic immunogenic
polypeptide as recited in claim 19, said truncated, basic, immunogenic polypeptide amino acid sequence corresponding to an amino acid sequence which is encoded by a polynucleotide sequence in the structural region of a HCV genome.
21. FGB1.
22. An immunogenic polypeptide comprising a carrier and said FGB1 as recited in claim 21.
23. FGB1 as recited in claim 22, said carrier being a polypeptide.
24. FGB1 as recited in claim 21, said FGB1 having a labeling moiety.
25. FGB1 as recited in claim 21, said FGB1 further having the following amino acid sequence
Lys-Pro-Gln-Arg-Lys-Thr-Lys- Arg-Asn-Thr-Asn-Arg-Arg-Pro- Gln-Ser-Arg-Pro-Ser-Trp-Gly- Pro-Thr-Asp-Pro-Arg-Arg-Arg- Ser-Arg.
26. FGB1 as recited in claim 21, said FGB1 further having the following amino acid sequence
Ser-Arg-Pro-Ser-Trp-Gly-Pro- Thr-Asp-Pro-Arg-Arg-Arg-Ser- Arg-Lys-Pro-Gln-Arg-Lys-Thr- Lys-Arg-Asn-Thr-Asn-Arg-Arg- Pro-Gln.
27. FGB1 as recited in claim 21, said FGB1 further having the following structure:
NH2-FGB1-COOH.
28. A polypeptide comprised of said FGB1 as recited in claim 21.
29. A polypeptide as recited in claim 28, said FGB1 being FGB1-Tyr.
30. FGB1 as recited in claim 21, said FGB1 being attached to a substrate.
31. FGB2.
32. An immunogenic polypeptide comprising a carrier and said FGB2 as recited in claim 31.
33. FGB2 as recited in claim 32, said carrier being a polypeptide.
34. FGB2 as recited in claim 31, said FGB2 having a labeling moiety.
35. FGB2 as recited in claim 34, said FGB2 further having the following structure:
NH2-FGB2-COOH.
36. A polypeptide comprised of said FGB2 as recited in claim 31.
37. A polypeptide as recited in claim 36, said FGB2 being FGB2-Tyr.
38. FGB2 as recited in claim 31, said FGB2 being attached to a substrate.
39. A basic, immunogenic polypeptide having an epitope for HCV, said basic polypeptide further having:
a) at least about 15 amino acids in its sequence;
b) at least about 7 amino acids in a Chou-Fasman turn;
c) at least about 11 hydrophillic amino acid residues in its sequence;
d) at least about 5 polar amino acid residues in its sequence;
e) at least about 5 basic amino acid residues in its sequence; and
f) a calculated molecular weight of at least about 1500.
40. A basic, immunogenic polypeptide as recited in claim 39, said basic, immunogenic
polypeptide further having:
a.) between about 15 and 17 amino acid residues;
b.) between about 7 and 17 amino acid residues in a Chou-Fasman turn;
c.) between about 11 and 14 hydrophillic amino acid residues;
d.) between about 5 and 8 polar amino acid residues;
e.) between about 5 and 7 basic amino acid residues;
f.) a calculated molecular weight of between about 1500 and 2500; and
g.) a pI on the order of about 12.
41. A polypeptide comprised of said basic, immunogenic polypeptide as recited in claim 40.
42. A basic, immunogenic polypeptide as recited in claim 40, said basic, immunogenic
polypeptide being attached to a substrate.
43. An isolated antibody against FGB1.
44. An isolated antibody as recited in claim 43, said antibody being selected from the group consisting of monoclonal and polyclonal antibodies.
45. An isolated anti-idiotype antibody against said antibody as recited in claim 43.
46. An isolated antibody as recited in claim 43, said FGB1 further comprising a carrier.
47. An isolated antibody as recited in claim 46, said antibody being selected from the group consisting of polyclonal and monoclonal antibodies.
48. An isolated anti-idiotype antibody against said antibody as recited in claim 46.
49. An isolated antibody as recited in claim 46, said antibody having a labeling moiety.
50. An isolated antibody as recited in claim 43, said FGB1 further comprising a polypeptide.
51. An isolated antibody as recited in claim 46, said antibody being attached to a substrate.
52. An isolated antibody as recited in claim 43, said antibody having a labeling moiety.
53. An isolated antibody against FGB2.
54. An isolated antibody as recited in claim 53, said antibody being selected from the group consisting of monoclonal and polyclonal antibodies.
55. An isolated anti-idiotype antibody against said antibody as recited in claim 53.
56. An isolated antibody as recited in claim 53, said FGB2 further comprising a carrier.
57. An isolated antibody as recited in claim 56, said antibody being selected from the group consisting of polyclonal and monoclonal antibodies.
58. An isolated anti-idiotype antibody against said antibody as recited in claim 56.
59. An isolated antibody as recited in claim 56, said antibody having a labeling moiety.
60. An isolated antibody as recited in claim 53, said FGB2 further comprising a polypeptide.
61. An isolated antibody as recited in claim 56, said antibody being attached to a substrate.
62. An isolated antibody as recited in claim
53, said antibody having a labeling moiety.
63. An isolated antibody against a basic, immunogenic polypeptide as recited in claim 1.
64. An isolated antibody against a truncated, basic immunogenic polypeptide as recited in claim 17.
65. An isolated antibody against a truncated, basic immunogenic polypeptide as recited in claim 19.
66. An isolated antibody against a truncated, basic immunogenic polypeptide as recited in claim 20.
67. An isolated antibody against a basic, immunogenic polypeptide as recited in claim 39.
68. An isolated antibody against a basic, immunogenic polypeptide as recited in claim 40.
69. An isolated antibody against a basic, immunogenic polypeptide as recited in claim 41.
70. A polynucieotide sequence comprising
AAACCTCAAAGAAAAACCAAACGTAACACCAACCGTCGCCCACAG.
(ϋ) (U) (U)
71. A probe having at least about 6
nucleotides derived from said polynucleotide sequence as recited in claim 70 or a complement thereto, said probe further having the ability to bind to HCV
polynucleotides in a target region.
72. A polynucleotide sequence as recited in claim 70, said polynucleotide sequence further having the following sequence
(TGT)AAACCTCAAAGAAAAACCAAACGTAACACCAACCGTCGCCCACAG(TAT) (UGU) (U) (U) (U) (UAU)
(TGC) (TAC)
(UGC) (UAC) .
73. An antisense polynucleotide sequence comprised of nucleotides which are complementary to said polynucleotide sequence as recited in claim 70, said antisense polynucleotide sequence having the ability to bind to HCV polynucleotides.
74. An antisense polynucleotide as recited in claim 73, said antisense polynucleotide having the following sequence
TTTGGAGTTTCTTTTTGGTTTGCATTGTGGTTGGCAGCGGGTGTC.
(UUU) (UUU UUUUU) (UUU) (UU U) (UU) (U U)
75. A polynucleotide sequence comprising
TCTCGGCCTAGTTGGGGCCCCAGGGACCCCCGGCGTAGGTCGCGC.
(U U) (U) (UU) (U) (U)
76. A probe having at least about 6
nucleotides derived from said polynucleotide sequence as recited in claim 75 or a complement thereto, said probe further having the ability to bind to HCV
polynucleotides in a target region.
77. A polynucleotide sequence as recited in claim 75, said polynucleotide sequence further having the following sequence
(TGT) TCTCGGCCTAGTTGGGGCCCCAGGGACCCCCGGCGTAGGTCGCGCTAT (TAT) (UGU) (U U) (U) (UU) (U) (U) (U U) (UAU)
(TGC) (TAC)
(UGC) (UAC).
78) An antisense polynucleotide sequence
comprised of nucleotides which are complementary to said polynucleotide sequence as recited in claim 75, said antisense polynucleotide sequence having the
ability to bind to HCV polynucleotides.
79. An antisense polynucleotide sequence as recited in claim 78, said antisense polynucleotide sequence having the following sequence
AGAGCCGGATCAACCCCGGGGTCCCTGGGGGCCGCATCCAGCGCG.
(U) (U) (U) (U)
80. A double stranded DNA fragment having a polynucleotide sequence selected from the group consisting of
GACACCCGCTGCCAACCACAATGCAAACCAAAAAGAAACTCCAAA CTGTGGGCGACGGTTGGTGTTACGTTTGGTTTTTCTTTGAGGTTT and
CGCGCTGGATGCGGCCCCCAGGGACCCCGGGGTTGATCCGGCTCT GCGCGACCTACGCCGGGGGTCCCTGGGGCCCCAACTAGGCCGAGA.
81. A recombinant expression system
containing an in frame sequence operably linked to a control sequence compatible with a desired host, said in frame sequence including said DNA fragment as recited in claim 80.
82. A host transformed with said recombinant expression system as recited in claim 81.
83. An antisense polynucieotide strand expressed by said transformant as recited in claim 82.
84.) A double stranded DNA fragment having the following sequence
(T)
GGGAGGTCTCGTAGACCGTGCACCATGAGCACGATTCCCAAACCTC CCCTCCAGAGCATCTGGCACGTGGTACTCGTGCTAAGGGTTTGGAG
(A)
(A)
AAAGAAAAACCAAACGTAACACCAACCGTCGCCCACAGGACGTCTA TTTCTTTTTGGTTTGCATTGTGGTTGGCAGCGGGTGTCCTGCAGAT
(T)
GTTCCC CAAGGG.
85. A double stranded DNA fragment as recited in claim 84, said DNA fragment further having the following sequence
(T)
GAGTGCCCCGGGAGGTCTCGTAGACCGTGCACCATGAGCACGATTCCCAAACCT CTCACGGGGCCCTCCAGAGCATCTGGCACGTGGTACTCGTGCTAAGGGTTTGGA
(A)
(A)
CAAAGAAAAACCAAACGTAACACCAACCGTCGCCCACAGGACGTCTA GTTTCTTTTTGGTTTGCATTGTGGTTGGCAGCGGGTGTCCTGCAGAT
(T)
GTTCCCGGGTGGCGGT CAAGGGCCCACCGCCA
86. A recombinant expression system
containing an open reading frame operably linked to a control sequence compatible with a desired host, said open reading frame including said DNA fragment as recited in claim 84.
87. A recombinant expression system comprising a pGEM-3Zf(-) plasmid and said DNA fragment as recited in claim 84.
88. A host transformed with said recombinant expression system as recited in claim 86.
89. A recombinant polypeptide expressed by said transformant as recited in claim 88.
90. FGB1 clone.
91. A kit for analyzing a sample for the presence of HCV polynucleotides, said kit comprising a polynucleotide probe as recited in claim 71, said polynucleotide probe being in a suitable carrier.
92. A kit for analyzing a sample for the presence of HCV polynucleotides, said kit comprising a polynucleotide probe as recited in claim 76, said polynucleotide probe being in a suitable carrier.
93. A kit for analyzing a sample for the presence of an HCV antibody, said kit comprising a polypeptide selected from the group consisting of FGB1 and FGB2 polypeptides, said polypeptide being present in a suitable container.
94. A kit as recited in claim 93, said kit further including a labeling moiety for said
polypeptide.
95. A kit as recited in claim 93, said polypeptide further comprising said FGB1 or FGB2 polypeptide and a second polypeptide.
96. A kit as recited in claim 95, said kit further including a labeling moiety for said
polypeptide.
97. A kit for analyzing a sample for the presence of HCV antigen, said kit comprising an antibody raised against a polypeptide selected from the group consisting of FGB1 and FGB2 polypeptides.
98. A kit as recited in claim 97, said polypeptide further comprising a carrier.
99. A kit as recited in claim 97, said polypeptide further comprising a second polypeptide.
100. A kit as recited in claim 97, said kit further including a labeling moiety for said antibody.
101. A kit as recited in claim 98, said kit further including a labeling moiety for said antibody.
102. A kit as recited in claim 99, said kit further including a labeling moiety for said antibody.
103. A kit for analyzing a sample for the presence of HCV antibody, said kit comprising an anti-idiotype antibody raised against an antibody as recited in claim 63.
104. A kit for analyzing a sample for the presence of HCV antibody, said kit comprising an anti-idiotype antibody raised against the antibody as recited in claim 17.
105. A kit for analyzing a sample for the presence of HCV antibody, said kit comprising an anti-idiotype antibody raised against said antibody as recited in claim 20.
106. A kit for analyzing a sample for the presence of HCV antibody, said kit comprising an anti-idiotype antibody raised against said antibody as recited in claim 41.
107. A method for detecting HCV nucleotides in a sample suspected of containing HCV, said method comprising:
a.) reacting nucleotides of the sample with the probe as recited in claim 71 under conditions which permit the nucleotides and the probe to form a polynucleotide duplex; and
b.) detecting the formed polynucleotide duplex.
108. A method for detecting HCV nucleotides in a sample suspected of containing HCV, said method comprising:
a.) reacting nucleotides of the sample with the probe as recited in claim 76 under conditions which permit the nucleotides and the probe to form a polynucleotide duplex; and
b.) detecting the formed polynucleotide duplex.
109. An immunoassay for detecting HCV antigen, said immunoassay comprising:
a.) incubating a sample suspected of containing HCV antigen with an antibody as recited in claim 43 under conditions which permit the HCV antigen and the antibody to form a complex; and
b.) detecting the formed complex.
110. An immunoassay for detecting HCV antigen, said immunoassay comprising:
a.) incubating a sample suspected of containing HCV antigen with an antibody as recited in claim 46 under conditions which permit the HCV antigen and the antibody to form a complex; and
b.) detecting the formed complex.
111. An immunoassay for detecting HCV antigen, said immunoassay comprising:
a.) incubating a sample suspected of containing HCV antigen with an antibody as recited in claim 50 under conditions which permit the HCV antigen and the antibody to form a complex; and
b.) detecting the formed complex.
112. An immunoassay for detecting HCV antigen, said immunoassay comprising:
a.) incubating a sample suspected of containing HCV antigen with an antibody as recited in claim 53 under conditions which permit the HCV antigen and the antibody to form a complex; and
b.) detecting the formed complex.
113. An immunoassay for detecting HCV antigen, said immunoassay comprising:
a.) incubating a sample suspected of containing HCV antigen with an antibody as recited in claim 56 under conditions which permit the HCV antigen and the antibody to form a complex; and
b.) detecting the formed complex.
114. An immunoassay for detecting HCV antigen, said immunoassay comprising:
a.) incubating a sample suspected of containing HCV antigen with an antibody as recited in claim 60 under conditions which permit the HCV antigen and the antibody to form a complex; and
b.) detecting the formed complex.
115. An immunoassay for detecting an antibody directed against HCV, said method comprising:
a.) incubating a sample suspected of containing an anti-HCV antibody with a polypeptide as recited in claim 1 under conditions which permit the anti-HCV antibody and the polypeptide to form a complex; and
b.) detecting the formed
antibody-antigen complex.
116. An immunoassay as recited in claim 115, the polypeptide being selected from the group
consisting of FGB1 and FGB2 polypeptides.
117. An immunoassay as recited in claim 115, the sample being a blood product.
118. An immunoassay as recited in claim 116, the sample being a blood product.
119. An immunoassay for diagnosing autoimmune liver disease, said method comprising:
a.) incubating a sample derived from a patient suspected of having autoimmune liver disease with an antibody as recited in claim 63 under
conditions which allow the antibody to form a complex with an antigen in the sample; and
b.) detecting the formed complex to diagnose autoimmune liver disease.
120. An immunoassay for diagnosing autoimmune liver disease, said method comprising:
a.) incubating a sample derived from a patient suspected of having autoimmune liver disease with an antibody as recited in claim 17 under
conditions which allow the antibody to form a complex with an antigen in the sample; and
b.) detecting the formed complex to diagnose autoimmune liver disease.
121. An immunoassay for diagnosing autoimmune liver disease, said method comprising:
a.) incubating a sample derived from a patient suspected of having autoimmune liver disease with an antibody as recited in claim 46 under
conditions which allow the antibody to form a complex with an antigen in the sample; and
b.) detecting the formed complex to diagnose autoimmune liver disease.
122. An immunoassay for diagnosing autoimmune liver disease, said method comprising:
a.) incubating a sample derived from a patient suspected of having autoimmune liver disease with an antibody as recited in claim 53 under
conditions which allow the antibody to form a complex with an antigen in the sample; and
b.) detecting the formed complex to diagnose autoimmune liver disease.
123. A vaccine for immunizing against HCV infection comprising
a pharmacologically effective dose of a polypeptide selected from the group consisting of FGB1 and FGB2 polypeptides, and
a pharmaceutically acceptable excipient.
124. A vaccine as recited in claim 123, said polypeptide comprising a second polypeptide.
125. A vaccine as recited in claim 123, said polypeptide comprising a carrier.
126. A vaccine as recited in claim 123, said vaccine further including a pharmacologically
effective dose of an adjuvant.
127. A vaccine as recited in claim 124, said vaccine further including a pharmacologically
effective dose of an adjuvant.
128. A vaccine as recited in claim 125, said vaccine further including a pharmacologically
effective dose of an adjuvant.
129. A vaccine for immunizing against HCV infection comprising
a pharmacologically effective dose of a polypeptide selected from the group consisting of a polypeptide as recited in claim 1, a polypeptide comprised of the polypeptide as recited in claim 1 and a polypeptide comprised of the polypeptide as recited in claim 1 and a carrier, and
a pharmaceutically acceptable excipient.
130. A vaccine as recited in claim 129, said vaccine further including a pharmacologically
effective dose of an adjuvant.
131. A method of immunizing against HCV infection, said method comprising
administering to an individual a vaccine as recited in claim 129.
132. A method of immunizing against HCV infection, said method comprising
administering to an individual a vaccine as recited in claim 130.
133. A method of producing a recombinant, basic immunogenic polypeptide having an epitope for HCV, said method comprising:
a.) providing a host transformed with a recombinant expression system comprising a coding sequence containing a DNA fragment encoding the polypeptide as recited in claim 1, said coding
sequence being operably linked to a control sequence compatible with the host, and
b.) incubating the host under conditions which allow expression of the recombinant, basic immunogenic polypeptide.
134. A method as recited in claim 133 wherein the DNA fragment encodes a polypeptide selected from the group consisting of said polypeptide and a second polypeptide and said polypeptide and a carrier.
135. A method as recited in claim 133 wherein the polypeptide is selected from the group consisting of FGB1 and FGB2 polypeptides.
136. A recombinant expression system
comprising a coding sequence including a DNA fragment which encodes the polypeptide as recited in claim 1, said coding sequence being operably linked to a control sequence campatible with a desired host.
137. A host transformed with said recombinant expression system as recited in claim 135.
138. A recombinant expression system as recited in claim 136, said DNA fragment encoding a polypeptide selected from the group consisting of said polypeptide and a second polypeptide, and said
polypeptide and a carrier.
139. A recombinant expression system as recited in claim 136, said DNA fragment being selected from the group consisting of
AAACCTCAAAGAAAAACCAAACGTAACACCAACCGTCGCCCACAG
(U) (U) (U)
and
TCTCGGCCTAGTTGGGGCCCCAGGGACCCCCGGCGTAGGTCGCGC.
(U U) (U) (UU) (U) (U)
140. A recombinant expression system as recited in claim 136, said control sequence being derived from a vaccinia virus.
141. A recombinant expression system as recited in claim 136, said polypeptide being selected from the group consisting of FGB1 and FGB2
polypeptides.
142. A recombinant expression system as recited in claim 140, said polypeptide being selected from the group consisting of FGB1 and FGB2
polypeptides.
143. A transformed host as recited in claim 137, said host being a vaccinia virus.
144. A transformed host as recited in claim 137, said control sequence being derived from a vaccinia virus and said polypeptide being selected from the group consisting of FGB1 and FGB2
polypeptides.
145. An immunoassay for detecting an HCV antigen in semen comprising:
a.) incubating semen suspected of containing an HCV antigen with an antibody directed against an HCV epitope wherein said incubation is under
conditions which permit the HCV antigen and the antibody to form an antigen-antibody complex; and
b.) detecting the formed complex.
146. An immunoassay as recited in claim 145, said antibody being selected from a group consisting of polyclonal and monoclonal antibodies.
147. An immunoassay as recited in claim 145 including the further step of
raising the antibody against an HCV antigen in an animal.
148. An immunoassay as recited in claim 147, said animal being a rabbit.
149. An immunoassay as recited in claim 145 including the further step of
labeling the antibody with a labeling moiety.
150. An immunoassay as recited in claim 145 including the further step of
attaching a substrate to the antibody.
151. An immunoassay as recited in claim 147, said HCV antigen being a basic immunogenic polypeptide having an epitope for HCV.
152. An immunoassay as recited in claim 151, said basic, immunogenic HCV polypeptide being FGB1.
153. An immunoassay as recited in claim 151, said basic, immunogenic HCV polypeptide being FGB2.
154. An immunoassay as recited in claim 151, said basic, immunogenic HCV polypeptide being selected from the group consisting of FGB3, FGB4, FGB5, FGB6, FGB7, FGB8, FGB9, FGB10, FGB11, FGB12, FGB13, FGB14, FGB15, FGB16, FGB17, FGB18, FGB19, FGB20, FGB21, FGB22, FGB23, FGB24, FGB25, FGB26, FGB27, FGB28, FGB29, FGB30, FGB31, FGB32, FGB33, FGB34, FGB35, FGB36, FGB37 and FGB38.
155. An immunoassay as recited in claim 145 including the further step of
attaching the antibody to a solid
substrate.
156. An immunoassay for detecting a passive antibody directed against an HCV antigen comprising:
a.) incubating a sample suspected of containing a passive anti-HCV antibody with an HCV antigen under conditions which permit the passive anti-HCV antibody and the HCV antigen to form a complex; and
b.) detecting the formed passive anti-HCV antibody-HCV antigen complex.
157. An immunoassay as recited in claim 156, said HCV antigen being a basic, immunogenic
polypeptide having an epitope for HCV.
158. An immunoassay as recited in claim 157, said basic, immunogenic HCV polypeptide being FGB1.
159. An immunoassay as recited in claim 157, said basic, immunogenic HCV polypeptide being selected from the group consists of FGB2, FGB3 , FGB4, FGB5, FGB6, FGB7, FGB8, FGB9, FGB10, FGB11, FGB12, FGB13, FGB14, FGB15, FGB16, FGB17, FGB18, FGB19, FGB20, FGB21, FGB22, FGB23, FGB24, FGB25, FGB26, FGB27,
FGB28, FGB29, FGB30, FGB31, FGB32, FGB33, FGB34,
FGB35, FGB36, FGB37 and FGB38.
160. An immunoassay as recited in claim 157 including the further step of labeling the
polypeptide.
161. An immunoassay as recited in claim 157 including the further step of fusing the polypeptide to a second polypeptide.
162. An immunogenic assay as recited in claim 157, said polypeptide including a labeling moiety.
163. An immunogenic assay as recited in claim 157, said polypeptide including a carrier.
164. An immunogenic assay as recited in claim 156 including the further step of attaching the HCV antigen to a solid substrate.
165. An immunogenic assay as recited in claim 156, said sample being a blood product.
166. A basic, immunogenic polypeptide having an epitope for HCV, said polypeptide being selected from a group consisting of FGB3, FGB4, FGB5, FGB6, FGB7, FGB8, FGB9, FGB10, FGB11, FGB12, FGB13, FGB14, FGB15, FGB16, FGB17, FGB18, FGB19, FGB20, FGB21, FGB22, FGB23, FGB24, FGB25, FGB26, FGB27, FGB28, FGB29, FGB30, FGB31, FGB32, FGB33, FGB34, FGB35, FGB36, FGB37 and FGB38.
167. A basic, immunogenic HCV polypeptide as recited in claim 166, said HCV polypeptide further including a carrier.
168. A basic, immunogenic HCV polypeptide as recited in claim 166, said HCV polypeptide further including a labeling moiety.
169. A basic, immunogenic HCV polypeptide as recited in claim 166, said HCV polypeptide being fused to a second polypeptide.
170. A basic immunogenic HCV polypeptide as recited in claim 166, said HCV polypeptide being attached to a solid substrate.
171. A basic immunogenic HCV polypeptide as recited in claim 168, said HCV polypeptide being attached to a solid substrate.
172. A basic immunogenic HCV polypeptide as recited in claim 169, said HCV polypeptide being attached to a solid substrate.
PCT/US1992/000356 1991-01-14 1992-01-14 Basic structural immunogenic polypeptides having epitopes for hcv, antibodies, polynucleotide sequences, vaccines and methods WO1992012992A2 (en)

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