WO1994013699A1 - Hepatitis c virus (hcv) non-structural-3 peptides, antibodies thereto and methods for the detection of hcv - Google Patents
Hepatitis c virus (hcv) non-structural-3 peptides, antibodies thereto and methods for the detection of hcv Download PDFInfo
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- WO1994013699A1 WO1994013699A1 PCT/EP1993/003422 EP9303422W WO9413699A1 WO 1994013699 A1 WO1994013699 A1 WO 1994013699A1 EP 9303422 W EP9303422 W EP 9303422W WO 9413699 A1 WO9413699 A1 WO 9413699A1
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- hcv
- antibodies
- peptide
- hepatitis
- peptides
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- 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
- C07K14/01—DNA viruses
- C07K14/02—Hepadnaviridae, e.g. hepatitis B virus
-
- 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
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/576—Immunoassay; Biospecific binding assay; Materials therefor for hepatitis
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/576—Immunoassay; Biospecific binding assay; Materials therefor for hepatitis
- G01N33/5767—Immunoassay; Biospecific binding assay; Materials therefor for hepatitis non-A, non-B hepatitis
-
- 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
- HCV Hepatitis C Virus
- the invention relates to peptides which react immunochemically with antibodies directed against the Hepatitis C virus and to nucleic acid sequences encoding these peptides.
- Antibodies directed against HCV are also part of the present invention.
- the invention also relates to methods for the detection of HCV or anti-HCV in a test fluid and to immunochemical reagents and a test kits for carrying out said detection methods.
- Hepatitis C virus is a 9.4-kb, single stranded polyadenylated RNA virus which has been recognized as one of the causative agents of NANB hepatitis (Non-A, Non-B) . It causes acute and chronic liver disease and is implicated in hepatocellular carcinoma.
- HCV hepatitis A virus
- HBV hepatitis B virus
- HDV hepatitis delta virus
- CMV cytomegalovirus
- EBV Epstein-Barr virus
- Non-A, Non-B Hepatitis was first identified in transfused individuals. Transmission from man to chimpanzee and serial passage in chimpanzees provided evidence that Non-A, Non-B Hepatitis is due to a transmissible infectious agent or agents.
- Non-A, Non-B Hepatitis exist: the water-borne epidemic type; the blood or needle associated type; and the sporadically occurring (community acquired) type.
- the viral genome of HCV encodes a polyprotein of approximately 3010 amino acids that undergoes extensive posttranslational processing.
- the viral structural region is located upstream from the nonstructural region and putatively includes a highly conserved 19-kDa nucleocapsid protein, and two extensively glycosylated envelope polypeptides, gp 33 (El) and gp72 (E2/NS1).
- HCV envelope may be under strong immune selection.
- a variety of presumed nonstructural proteins are processed from the remainder of the HCV polyprotein, including a membrane-bound 23-kDa protein, NS2, and a soluble protein of approximately 60 kDa, NS3, which corresponds to the viral helicase and may contain a N-terminal serine protease domain, currently thought to be involved in the processing of the NS proteins.
- the function of the NS4 protein is presently unknown, but it comprises the 5-1-1 fragment that contains immunodominant antibody binding sites (Kuo G.
- NS5 contains the viral replicase.
- Clinical studies have shown that, following exposure to HCV, antibodies to conserved regions of the viral nucleoprotein and NS3 may appear several weeks before seroconversion to anti-clOO-3, a recombinant protein encompassing the C-terminus of NS3 and part of the NS4 protein.
- serological assays incorporating the highly-conserved HCV nucleocapsid protein as well as NS3 are likely to become useful diagnostic markers of acute HCV infection.
- An objective of ,the current invention is to provide small peptides useful for the diagnosis and monitoring of HCV infection.
- the objective of present invention is to provide peptides with a length small enough to have the advantages of small synthetic peptides but at the same time large enough to be immunoreactive with antibodies against HCV.
- HCV NS3-geno e Smaller peptides (12-mers) from the region encoded by the putative HCV NS3-gene were found to be not particularly useful to detect antibodies against HCV. Larger polypeptides are unpractical because they cannot be easily expressed as fusion proteins, they are prone to endogenous proteolysi ⁇ and there are increased chances on false positive reactivities. Larger polypeptides are also difficult to synthesize, difficult to purify and might be infectious. In this invention, regions of the HCV NS3- geno e are identified which form an optimal synthesis between length and immunoreactivity.
- the present invention includes peptides with amino acid sequences selected from the group of sequences depicted in SEQ ID No. : 6, 7, 8 , 9 and 10, and combinations thereof or fragments of said group of sequences or analogues of said group of sequences which are immunochemically reactive with HCV-antibodies.
- a library can be constructed consisting of DNA fragments from a recombinant clone encoding an antigen which covers most if not all of the putative HCV NS3 gene. These fragments could range in size from approximately 50 to 300 nucleotides and when expressed in the appropriate reading frame encoded HCV polypeptides ranging from approximately 17 to 100 amino acids. In this way a library can be constructed containing enough different recombinants to ensure that any possible fragment in the range of 17 to 100 amino acids is contained at least once. Recombinants which express exceptionally reactive antigens can be selected using an appropriate antibody as a probe and DNA sequence encoding the exceptionally reactive peptides.
- the peptides according to the invention are located in the putative NS3 region of the HCV genome.
- the peptides according to the invention have been found to be exceptionally immunochemically reactive with HCV-antibodies.
- An advantage of this reactivity is that the use of one or more of the peptides according to the invention will increase the specificity of the immunological assay when compared to the use of large recombinant fragments.
- Another advantage is that the use of one or more of the peptides will increase the sensivity of the immunological assay.
- the invention also comprises fragments of said peptides which are still immunochemically reactive with HCV-antibodies.
- fragment as used herein means an amino acid sequence comprising a subsequence of the peptide of the invention. Said fragment is a peptide having one or more immunogenic determinants of the HCV NS3-antigen. Fragments can inter alia be produced by enzymatic cleavage of precursor molecules, using restriction endonucleases for the DNA and proteases for the polypeptides. Other methods include chemical synthesis of the fragments or the expression of polypeptide fragments by DNA fragments.
- Analogues or derivatives of the peptides according to SEQ ID No. 6-10 are also included in the invention.
- analogues refers for instance to post-expression modifications of a peptide, for example, glycosylations, acetylations, phosphorylations etc. Without specifically being incorporated in the claims, it is self-evident that several amino acids in the peptides according to the invention can be deleted or inserted or substituted by other amino acids or amino acid analogues or derivatives due to strain-to-strain variations among different isolates of HCV without affecting the immunochemical activity of the peptides in question.
- analogues of these peptides are also meant acid addition salts of the peptides, amides of the peptides and specifically the C-terminal amides, esters and specifically C-terminal esters and N-acyl derivatives specifically N-terminal acyl derivatives and in particular N-acetyl derivatives.
- the preparation of the peptides according to the invention can be effected adapting one of the known organic chemical methods for peptide synthesis or with the aid of recombinant DNA techniques.
- This latter method involves the preparation of the desired peptide by means of expressing a recombinant polynucleotide with the aid of a suitable vector containing a polynucleotide sequence which is coding for one or more of the peptides in question and introducing the vector in a suitable host.
- the organic chemical methods for peptide synthesis are considered to include the coupling of the required amino acids by means of a condensation reaction, either in homogeneous phase or with the aid of a so-called solid phase.
- the condensation reaction can be carried out as follows: a) condensation of a compound (amino acid, peptide) with a free carboxyl group and protected other reactive groups with a compound (amino acid, peptide) with a free amino group and protected other reactive groups, in the presence of a condensation agent; b) condensation of a compound (amino acid, peptide). with an activated carboxyl group and free or protected other reaction groups with a compound (amino acid, peptide) with a free amino group and free or protected other reactive groups.
- Activation of the carboxyl group can take place, inter alia, by converting the carboxyl group to an acid halide, azide, anhydride, imidazolide or an activated ester, such as the N- hydroxy-succinimide, N-hydroxy-benzotriazole or p-nitrophenyl ester.
- the peptides according to the invention are prepared with the aid of recombinant DNA techniques.
- Peptides can, for example, be incorporated in a repeating sequence ("in tandem") or can be prepared as a constituent of a (much larger) protein or polypeptide.
- a polynucleotide with a specific nucleic acid sequence can be used which codes for the peptide according to the invention.
- a polynucleotide of this type which is coding for the peptide according to the invention, and a recombinant DNA in which this polynucleotide is incorporated likewise fall within the scope of the inve tion.
- the invention also relates to an immunochemical reagent, which reagent comprises at least one of the peptides.
- an "immunochemical reagent" according to the invention may comprise one or more peptides according to the invention and a suitable support or a labelling substance.
- Supports which can be used are, for example, the inner wall of a microtest well or a cuvette, a tube or capillary, a membrane, filter, test strip or the surface of a particle such as, for example, a latex particle, an erythrocyte, a dye sol, a metal sol or metal compound as sol particle, a carrier protein such as BSA or KLH.
- Labelling substances which can be used are, inter alia, a radioactive isotope, a fluorescent compound, an enzyme, a dye sol, metal sol or metal compound as sol particle.
- the invention further encompasses nucleic acid sequences encoding the peptides according to the invention preferably a nucleic acid sequence containing at least part of the DNA sequence shown in SEQ ID No. 1, 2, 3, 4, 5.
- Nucleic acid sequence refers to a polymeric form of nucleotides of any length, both to ribonucleic acid sequences and to deoxyribonucleic acid sequences. In principle, this term refers to the primary structure of the molecule. Thus, this term includes double and single stranded DNA, as well as double and single stranded RNA, and modifications thereof.
- a nucleic acid sequence according to the present invention can be ligated to various replication effecting DNA sequences with which it is not associated or linked in nature resulting in a so called recombinant vector molecule which can be used for the transformation of a suitable host.
- Useful recombinant vector molecules are preferably derived from, for example plasmids, bacteriophages, cosmids or viruses.
- vectors or cloning vehicles which can be used to clone nucleic acid sequences according to the invention are known in the art and include inter alia plasmid vectors such as pBR322, the various pUC, pGEM and Bluescript plasmids, bacteriophages, e.g. kgt-Wes, Charon 28 and the Ml3 derived phages or viral vectors such as SV40, adenovirus or polyoma virus (see also Rodriquez, R.L. and D.T. Denhardt, ed. , Vectors: A survey of molecular cloning vectors and their uses, Butterworths, 1988; Lenstra, J.A. et al. , Arch. Virol.
- the insertion of the nucleic acid sequence according to the invention into a cloning vector can easily be achieved when both the genes and the desired cloning vehicle have been cut with the same restriction enzyme(s) as complementary DNA termini are thereby produced.
- the recombinant vector molecules according to the invention may additionally contain one or more marker activities that may be used to select for desired transformants, such as ampicillin and tetracycline resistance in pBR322, as for example ampicillin resistance and ⁇ -peptide of ⁇ - galactosidase in pUC8.
- the invention also comprises (a) host cell(s) transformed with a nucleic acid sequence or recombinant expression vector molecule described above, capable of producing the peptides according to the invention by expression of the corresponding nucleic acid sequence.
- a suitable host cell is a microorganism or cell which can be transformed by a nucleic acid sequence encoding a polypeptide or by a recombinant vector molecule comprising such a nucleic acid sequence and which can if desired be used to express said polypeptide encoded by said nucleic acid sequence.
- the host cell can be of procaryotic origin, e.g. bacteria such as Escherichia coli, Bacillus subtilis and Pseudomonas species; or of eucaryotic origin such as yeasts, e.g. Saccharomyces cerevisiae or higher eucaryotic cells such as insect, plant or mammalian cells, including HeLa cells and Chinese hamster ovary (CHO) cells.
- Insect cells include the Sf9 cell line of Spodoptera frugiperda (Luckow et al., Bio-technology 6., 47-55, 1988).
- Information with respect to the cloning and expression of the nucleic acid sequence of the present invention in eucaryotic cloning systems can be found in Esser, K. et al. (Plasmids of Eukaryotes, Springer-Verlag, 1986).
- prokaryotes are preferred for the construction of the recombinant vector molecules useful in the invention.
- E.coli K12 strains are particularly useful such as DH5 ⁇ or MC1061k.
- nucleic acid sequences of the present invention are introduced into an expression vector, i.e. said sequences are operably linked to expression control sequences.
- control sequences may comprise promoters, enhancers, operators, inducers, ribosome binding sites etc. Therefore, the present invention provides a recombinant vector molecule comprising a nucleic acid sequence encoding the peptides identified above operably linked to expression control sequences, capable of expressing the DNA sequences contained therein in (a) transformed host cell(s) .
- nucleotide sequences inserted at the selected site of the cloning vector may include only a fragment of the complete nucleic acid sequence encoding for the peptides according to the invention as long as the transformed host will produce a polypeptide having at least one or more immunogenic determinants.
- illustrative useful expression control sequences include the Trp promoter and operator (Goeddel, et al., Nucl. Acids Res. 8., 4057, 1980); the lac promoter and operator (Chang, et al., Nature 275, 615, 1978); the outer membrane protein promoter (Nakamura, K. and Inouge, M. , EMBO J. 1 , 771-775, 1982); the bacteriophage kpromoters and operators (Remaut, E. et al., Nucl. Acids Res. .11, 4677- 4688, 1983); the ⁇ -amylase (B.
- subtilis subtilis promoter and operator, termination sequence and other expression enhancement and control sequences compatible with the selected host cell.
- illustrative useful expression control sequences include, e.g., ⁇ - mating factor.
- the polyhedrin or plO promoters of baculoviruses can be used (Smith, G.E. et al., Mol. Cell. Biol. 3 , 2156-65, 1983).
- illustrative useful expression control sequences include, e.g., the SV-40 promoter (Berman, P.W. et al., Science 222, 524-527, 1983) or, e.g.
- the preparation of cell lines producing monoclonal antibodies may occur by, for example, transformation with Epstein-Barr Virus, the Kohler and Milstein technique (Kohler and Milstein devised the techniques that resulted in the formation monoclonal antibody-producing hybridomas (G. Kohler and C. Milstein, 1975, Nature 256:495-497; 1976, Eur. J. Immunol. 6:511- 519)), or a direct transformation technique of B- lymphocytes with oncogenic DNA, or a direct fusion of human B-lymphocytes with a fusion partner being either a human or a mouse-human hybrid myeloma cell line, or a direct fusion of an EBV-transformed B cell line with said myeloma cell lines.
- Epstein Barr virus is capable of transforming and immortalizing human B- lymphocytes. With the aid of the Epstein Barr virus immortalized human B-lymphocytes can be obtained without the need of a myeloma partner cell.
- Epstein Barr virus can be obtained from a variety of sources. The most common used source of EBV is the B95-8 marmoset cell line. The B95-8 cell line spontaneously releases Epstein Barr virus into the medium.
- PBMC peripheral blood mononuclear cells
- fibroblasts a variety of cell types has been suggested to provide good feeder layers for the cloning of EBV transformed cells. The most commonly used are peripheral blood mononuclear cells (PBMC) and fibroblasts.
- PBMC peripheral blood mononuclear cells
- fibroblasts a variety of cell types has been suggested to provide good feeder layers for the cloning of EBV transformed cells. The most commonly used are peripheral blood mononuclear cells (PBMC) and fibroblasts.
- PBMCs consist of monocytes, T lymphocytes and B-lymphocytes (5-10%). Because not all B- lymphocytes will provide antibodies of the right specificity, it is advantageous to enrich PBMCs for appropriate cells.
- T-lymphocytes can be removed, prior to EBV infection (with, for example, the supernatant from the EBV-productive B95-8 cell line), by treating the cells with washed sheep red blood cells (In: "Antibodies Vol.I, a practicle approach". Editor: Catty D, IRL Press, Oxford, England. 1988, Ch. 41.
- the antibodies produced by immortalized B cell lines were considered monoclonal if they satisfied the following requirements:
- the present invention is further directed to a method for the detection of antibodies directed against HCV in a test fluid, wherein a peptide according to the invention is brought into contact with the test fluid and the presence of immune complexes formed between the peptide and antibodies in the test fluid is detected.
- the presence of immune complexes formed between the peptide and antibodies in the test fluid is detected and by this detection the presence of antibodies to HCV in the test fluid is known and can be determined.
- the immunochemical reaction that takes place can be a so called sandwich reaction, an agglutination reaction, a competition reaction or an inhibition reaction.
- a particularly suitable method for the detection of HCV in a test fluid is based on a competition reaction between a peptide according to the invention provided with a labelling substance and a HCV antigen (present in the test fluid) whereby the peptide and the antigen are competing with the antibody directed against HCV attached to a solid support.
- the antibody coated on the support can, for example be a monoclonal antibody according to the invention.
- the invention is further directed to a method for the detection of Hepatitis C virus in a sample comprising contacting the sample with a monoclonal antibody according to the invention, and detecting immune complexes formed between the monoclonal antibody and a Hepatitis C antigen.
- a sandwich reaction for the detection of HCV in a test sample the test kit to be used comprises a monoclonal antibody according to the invention coated on a solid support, for example the inner wall of a microtest well, and either a labelled monoclonal antibody or fragment thereof as conjugate.
- a further example of an immuno assay that can be used for the detection of HCV is an inhibition assay using human monoclonal antibodies as labelled reagent.
- the binding of this reagent to antigen on a solid phase can be competed by antibodies in the test sample.
- monoclonal antibodies according to the invention are very suitable in diagnosis, while those antibodies which are neutralizing are very useful in passive immunotherapy.
- the invention also relates to a test kit for carrying out an immuno-assay, said test kit containing at least an immunochemical reagent according to the invention.
- a test kit according to the invention comprises as an essential constituent an immunochemical reagent as described above.
- This immunochemical reagent may comprise an antibody or a peptide according to the invention.
- Test kits comprising a combination of different immunochemical reagents according to the invention, for example a peptide coated on a solid support and an antibody provided with a label, are of course within the scope of this invention.
- the test kit may comprise, for example, a peptide according to the invention coated to a solid support, for example the inner wall of a microtest well, and either a labelled peptide according to the invention or a labelled anti-antibody.
- a sandwich reaction test format is the detection of HCV antigen whereby monoclonal antibodies according to the invention are coated to a solid support and monoclonal antibodies are used as conjugate.
- sandwich reactions are described in our American patents with regard to the enzyme immuno assay viz. RE 31.006 and RE 32.696 (Schuurs et al.).
- the test kit may comprise a peptide according to the invention coated to a solid support, and a labelled antibody directed against HCV preferably a monoclonal antibody directed against said peptide.
- the test kit comprises an immunochemical reagent which may comprise a peptide according to the invention coated to particles or sols.
- test kit is, for example, the use of a labelled peptide according to the invention as immunochemical reagent in a competition reaction with a HCV antigen to be detected for a binding site on the antibody directed against HCV, which is coated to a solid support.
- a part of the present invention is a peptide or fragment thereof according to the invention which can be used in suitable pharmaceutical dosage forms in the prevention and/or treatment of NANB Hepatitis-disease.
- the preparation of vaccines thus obtained using such a peptide or fragment thereof as active ingredients, can be accomplished by one skilled in the art.
- Example 1 Construction and screening of lambda ⁇ t-11 library
- the sequence coding for a part of the NS-3 gene of HCV has been multiplied by PCR using specific primers.
- Starting material was obtained from a clone constructed by rt-PCR from chimpanzeeserum infected with the prototype HCV-strain.
- the PCR products were isolated from TBE-polyacrylamide gel (8% PAGE) by electroelution.
- Portions (20 ⁇ l out of 80 ⁇ l) of this PCR-material have been digested under controlled conditions (25 °C, 10- 60 minutes, in an endvolume of 25 ⁇ l containing 1 mM MnCl 2 , 20 mM Tris-HCl (pH 7.5) and DNAse-1 (Worthington 2635 units per mg, end concentration: 0.6 units)).
- the digestions were stopped in phenol/chloroform-isoamylalcohol and extracted.
- the DNAse digestions were controlled by nicktranslation. Fragments with the length from approximately fifty till twohundred basepairs were isolated after 8% PAGE by diffusion (J. Sambrook, E.F. Fritsch, T.
- the polynucleotide which codes for the peptide according to SEQ ID No. 6, was tailed with oligo- dG following recommendations of the supplier (GIBCO/BRL) .
- PCR was performed on the tailed product using as a primer poly-C with a terminal EcoRl-site attached. After EcoRl digestion and phenol extraction the products were cloned in lambda gt-11 arms and transfected into E.coli as detailed by the supplier (Promega) .
- PCR on the libraries using lambda gtll-primers revealed smears with lengths concordant with the lengths of the inserted fragments.
- the libraries were screened on duplo filters using standard procedures with human monoclonal antibody (1:50) of which the reaction was detected with alkaline phosphatase-conjugated goat anti-human IgG.
- the positive phages were rescreened to positively identify their contents and thereafter their inserts were transferred to the vector pGEM7Zf(+), (Promega).
- the inserts in this vector were sequenced using a commercial kit (Pharmacia T7-sequencing kit) according to the recommendations of the supplier.
- ⁇ -Galactosidase fusion proteins encoded by the recombinants, obtained as described above, were purified according to standard procedures using anti- ⁇ -galactosidase affinity columns.
- ID No. 6 is dissolved to 7.5 ⁇ g/ml in 100 mM phosphate buffer pH 9.6 and 135 ⁇ l of the above peptide solution is placed into each well of a NUNC microtiter plate. Binding of the peptide to the microtiter plate is allowed to proceed overnight at 4° C.
- the plates are blocked with a solution of 0.05% Tween 20 (R) in 0.2 M Tris pH 7.4/0.2 M NaCl for 5 min. at room temperature. Plates are then washed once with 0.2 M Tris pH 7.4/0.2 M NaCl, twice with 0.04 M Tris pH 7.4, at 250 ⁇ l per well and dried.
- the serum samples are diluted in sample diluent (phosphate buffered saline (PBS)/20% normal goat serum/1% Triton X100) pipetted into the well (100 ⁇ l per well) and incubated for 1 h at 37 °C.
- sample diluent phosphate buffered saline (PBS)/20% normal goat serum/1% Triton X100
- the peptides according to the sequence of SEQ ID No. 7, 8, 9 and 10 are prepared using above-mentioned recombinant-DNA technique. The same set-up as described above is used for testing said peptides in an immunoassay. With sera from patients with Non-A Non-B hepatitis the specific recognition is positive whereas the results of 20 normal human sera is negative (Table 1).
- Chiron Riba Chiron Riba HCV test system
- HCV Hepatitis C Virus
- ORGANISM Escherichia coli
- STRAIN JM101
- MOLECULE TYPE DNA (genomic)
- CTATCCCAGA CAAAGCAGAG TGGGGAGAAC CTTCCTTACC 40 TGGTAGCGTA CCAAGCCACC GTGTGCGCTA GGGCTCAAGC 80 CCCTCCCCCA TCGTGGGACC AGATGTGGAA GTGTTTGATT 120 CGCCTCAAGC CCACCCTCCA TGGGCCAACA CCCCTGCT 158
- MOLECULE TYPE DNA (genomic)
- ORGANISM Escherichia coli
- MOLECULE TYPE DNA (genomic)
- ORGANISM Escherichia Coli
- MOLECULE TYPE DNA (genomic)
Abstract
The invention concerns peptides which react immunochemically with antibodies directed against HCV. A preferred peptide according to the invention comprises an HCV specific epitope of the NS-3 protein. The invention further relates to antibodies that specifically react with the NS-3 protein of HCV. Methods for the detection of HCV or HCV antibodies, and a method for the detection of antibodies specifically reactive with the NS-3 antigen are also part of the present invention.
Description
Title: Hepatitis C Virus (HCV) Non-εtructural-3 peptides, antibodies thereto and methods for the detection of HCV.
The invention relates to peptides which react immunochemically with antibodies directed against the Hepatitis C virus and to nucleic acid sequences encoding these peptides.
Antibodies directed against HCV are also part of the present invention.
The invention also relates to methods for the detection of HCV or anti-HCV in a test fluid and to immunochemical reagents and a test kits for carrying out said detection methods.
Hepatitis C virus (HCV) is a 9.4-kb, single stranded polyadenylated RNA virus which has been recognized as one of the causative agents of NANB hepatitis (Non-A, Non-B) . It causes acute and chronic liver disease and is implicated in hepatocellular carcinoma.
It can be distinguished from other forms of viral-associated liver diseases, including those caused by known hepatitis viruses, i.e., hepatitis A virus (HAV) , hepatitis B virus (HBV) , and hepatitis delta virus (HDV) , as well as the hepatitis induced by cytomegalovirus (CMV) or Epstein-Barr virus (EBV). Evidence based on hydrophobicity plots and sequence homologies suggests that HCV may be distantly related to the family Flaviviridae (Houghton M. et al.f Hepatoloσv. 14:381. 19911.
Non-A, Non-B Hepatitis was first identified in transfused individuals. Transmission from man to chimpanzee and serial passage in chimpanzees provided evidence that Non-A, Non-B Hepatitis is
due to a transmissible infectious agent or agents.
Epidemiologic evidence is suggestive that three types of Non-A, Non-B Hepatitis exist: the water-borne epidemic type; the blood or needle associated type; and the sporadically occurring (community acquired) type. The viral genome of HCV encodes a polyprotein of approximately 3010 amino acids that undergoes extensive posttranslational processing. The viral structural region is located upstream from the nonstructural region and putatively includes a highly conserved 19-kDa nucleocapsid protein, and two extensively glycosylated envelope polypeptides, gp 33 (El) and gp72 (E2/NS1). Recent studies indicate that substantial sequence heterogeneity exists among virtually all HCV isolates in the N-terminal region of E2/NS1, suggesting that this region of the HCV envelope may be under strong immune selection. A variety of presumed nonstructural proteins are processed from the remainder of the HCV polyprotein, including a membrane-bound 23-kDa protein, NS2, and a soluble protein of approximately 60 kDa, NS3, which corresponds to the viral helicase and may contain a N-terminal serine protease domain, currently thought to be involved in the processing of the NS proteins. The function of the NS4 protein is presently unknown, but it comprises the 5-1-1 fragment that contains immunodominant antibody binding sites (Kuo G. et al., Science 244:362, 1991; Cerino A. et al.J-Immunol♦ . 147:2692) ; NS5 contains the viral replicase. Clinical studies have shown that, following exposure to HCV, antibodies to conserved regions of the viral nucleoprotein and NS3 may appear several weeks before
seroconversion to anti-clOO-3, a recombinant protein encompassing the C-terminus of NS3 and part of the NS4 protein.
Thus, serological assays incorporating the highly-conserved HCV nucleocapsid protein as well as NS3 are likely to become useful diagnostic markers of acute HCV infection.
For the development of a specific and sensitive method to enable a reliable diagnosis to be made in various phases of the infection with HCV it is of great importance to identify immunodominant viral epitopes of this type.
An objective of ,the current invention is to provide small peptides useful for the diagnosis and monitoring of HCV infection.
Long recombinant antigens encoding at least part of the putative HCV NS3 antigen are reactive with antibodies against HCV but have substantial disadvantages as outlined above. Small synthetic peptides circumvent these disadvantages but are not satisfactory im unoreactive. The objective of present invention is to provide peptides with a length small enough to have the advantages of small synthetic peptides but at the same time large enough to be immunoreactive with antibodies against HCV.
Smaller peptides (12-mers) from the region encoded by the putative HCV NS3-gene were found to be not particularly useful to detect antibodies against HCV. Larger polypeptides are unpractical because they cannot be easily expressed as fusion proteins, they are prone to endogenous proteolysiε and there are increased chances on false positive reactivities. Larger polypeptides are also difficult to synthesize, difficult to purify and might be infectious.
In this invention, regions of the HCV NS3- geno e are identified which form an optimal synthesis between length and immunoreactivity.
It is a further object of the present invention to provide a peptide comprising a HCV specific NS-3 antigenic sequence.
The present invention includes peptides with amino acid sequences selected from the group of sequences depicted in SEQ ID No. : 6, 7, 8 , 9 and 10, and combinations thereof or fragments of said group of sequences or analogues of said group of sequences which are immunochemically reactive with HCV-antibodies.
A library can be constructed consisting of DNA fragments from a recombinant clone encoding an antigen which covers most if not all of the putative HCV NS3 gene. These fragments could range in size from approximately 50 to 300 nucleotides and when expressed in the appropriate reading frame encoded HCV polypeptides ranging from approximately 17 to 100 amino acids. In this way a library can be constructed containing enough different recombinants to ensure that any possible fragment in the range of 17 to 100 amino acids is contained at least once. Recombinants which express exceptionally reactive antigens can be selected using an appropriate antibody as a probe and DNA sequence encoding the exceptionally reactive peptides.
The peptides according to the invention are located in the putative NS3 region of the HCV genome. The peptides according to the invention have been found to be exceptionally immunochemically reactive with HCV-antibodies. An advantage of
this reactivity is that the use of one or more of the peptides according to the invention will increase the specificity of the immunological assay when compared to the use of large recombinant fragments. Another advantage is that the use of one or more of the peptides will increase the sensivity of the immunological assay.
The invention also comprises fragments of said peptides which are still immunochemically reactive with HCV-antibodies.
The term "fragment" as used herein means an amino acid sequence comprising a subsequence of the peptide of the invention. Said fragment is a peptide having one or more immunogenic determinants of the HCV NS3-antigen. Fragments can inter alia be produced by enzymatic cleavage of precursor molecules, using restriction endonucleases for the DNA and proteases for the polypeptides. Other methods include chemical synthesis of the fragments or the expression of polypeptide fragments by DNA fragments.
Analogues or derivatives of the peptides according to SEQ ID No. 6-10 are also included in the invention.
The term "analogues" refers for instance to post-expression modifications of a peptide, for example, glycosylations, acetylations, phosphorylations etc. Without specifically being incorporated in the claims, it is self-evident that several amino acids in the peptides according to the invention can be deleted or inserted or substituted by other amino acids or amino acid analogues or derivatives due to strain-to-strain variations among different isolates of HCV without affecting
the immunochemical activity of the peptides in question.
In addition, with analogues of these peptides are also meant acid addition salts of the peptides, amides of the peptides and specifically the C-terminal amides, esters and specifically C-terminal esters and N-acyl derivatives specifically N-terminal acyl derivatives and in particular N-acetyl derivatives.
The preparation of the peptides according to the invention can be effected adapting one of the known organic chemical methods for peptide synthesis or with the aid of recombinant DNA techniques. This latter method involves the preparation of the desired peptide by means of expressing a recombinant polynucleotide with the aid of a suitable vector containing a polynucleotide sequence which is coding for one or more of the peptides in question and introducing the vector in a suitable host.
The organic chemical methods for peptide synthesis are considered to include the coupling of the required amino acids by means of a condensation reaction, either in homogeneous phase or with the aid of a so-called solid phase. The condensation reaction can be carried out as follows: a) condensation of a compound (amino acid, peptide) with a free carboxyl group and protected other reactive groups with a compound (amino acid, peptide) with a free amino group and protected other reactive groups, in the presence of a condensation agent; b) condensation of a compound (amino acid, peptide). with an activated carboxyl group and
free or protected other reaction groups with a compound (amino acid, peptide) with a free amino group and free or protected other reactive groups. Activation of the carboxyl group can take place, inter alia, by converting the carboxyl group to an acid halide, azide, anhydride, imidazolide or an activated ester, such as the N- hydroxy-succinimide, N-hydroxy-benzotriazole or p-nitrophenyl ester.
The most common methods for the above condensation reactions are: the carbodiimide method, the azide method, the mixed anhydride method and the method using activated esters, such as described in The Peptides, Analysis, Synthesis, Biology Vol. 1-3 (Ed. Gross, E. and Meienhofer, J.) 1979, 1980, 1981 (Academic Press, Inc. ) .
Alternatively, the peptides according to the invention are prepared with the aid of recombinant DNA techniques.
Peptides can, for example, be incorporated in a repeating sequence ("in tandem") or can be prepared as a constituent of a (much larger) protein or polypeptide. For this purpose, as a constituent of a recombinant DNA, a polynucleotide with a specific nucleic acid sequence can be used which codes for the peptide according to the invention.
A polynucleotide of this type, which is coding for the peptide according to the invention, and a recombinant DNA in which this polynucleotide is incorporated likewise fall within the scope of the inve tion.
The invention also relates to an immunochemical reagent, which reagent comprises at least one of the peptides.
An "immunochemical reagent" according to the invention may comprise one or more peptides according to the invention and a suitable support or a labelling substance.
Supports which can be used are, for example, the inner wall of a microtest well or a cuvette, a tube or capillary, a membrane, filter, test strip or the surface of a particle such as, for example, a latex particle, an erythrocyte, a dye sol, a metal sol or metal compound as sol particle, a carrier protein such as BSA or KLH. Labelling substances which can be used are, inter alia, a radioactive isotope, a fluorescent compound, an enzyme, a dye sol, metal sol or metal compound as sol particle.
The invention further encompasses nucleic acid sequences encoding the peptides according to the invention preferably a nucleic acid sequence containing at least part of the DNA sequence shown in SEQ ID No. 1, 2, 3, 4, 5.
"Nucleic acid sequence" as used herein refers to a polymeric form of nucleotides of any length, both to ribonucleic acid sequences and to deoxyribonucleic acid sequences. In principle, this term refers to the primary structure of the molecule. Thus, this term includes double and single stranded DNA, as well as double and single stranded RNA, and modifications thereof.
A nucleic acid sequence according to the present invention can be ligated to various replication effecting DNA sequences with which it is not associated or linked in nature resulting
in a so called recombinant vector molecule which can be used for the transformation of a suitable host. Useful recombinant vector molecules, are preferably derived from, for example plasmids, bacteriophages, cosmids or viruses.
Specific vectors or cloning vehicles which can be used to clone nucleic acid sequences according to the invention are known in the art and include inter alia plasmid vectors such as pBR322, the various pUC, pGEM and Bluescript plasmids, bacteriophages, e.g. kgt-Wes, Charon 28 and the Ml3 derived phages or viral vectors such as SV40, adenovirus or polyoma virus (see also Rodriquez, R.L. and D.T. Denhardt, ed. , Vectors: A survey of molecular cloning vectors and their uses, Butterworths, 1988; Lenstra, J.A. et al. , Arch. Virol. 110, 1-24, 1990). The methods to be used for the construction of a recombinant vector molecule according to the invention are known to those of ordinarily skill in the art and are inter alia set forth in Maniatis, T. et al. (Molecular Cloning A Laboratory Manual, second edition; Cold Spring Harbor Laboratory, 1989).
For example, the insertion of the nucleic acid sequence according to the invention into a cloning vector can easily be achieved when both the genes and the desired cloning vehicle have been cut with the same restriction enzyme(s) as complementary DNA termini are thereby produced.
The recombinant vector molecules according to the invention may additionally contain one or more marker activities that may be used to select for desired transformants, such as ampicillin and tetracycline resistance in pBR322, as for example ampicillin resistance and α-peptide of β- galactosidase in pUC8.
The invention also comprises (a) host cell(s) transformed with a nucleic acid sequence or recombinant expression vector molecule described above, capable of producing the peptides according to the invention by expression of the corresponding nucleic acid sequence.
A suitable host cell is a microorganism or cell which can be transformed by a nucleic acid sequence encoding a polypeptide or by a recombinant vector molecule comprising such a nucleic acid sequence and which can if desired be used to express said polypeptide encoded by said nucleic acid sequence. The host cell can be of procaryotic origin, e.g. bacteria such as Escherichia coli, Bacillus subtilis and Pseudomonas species; or of eucaryotic origin such as yeasts, e.g. Saccharomyces cerevisiae or higher eucaryotic cells such as insect, plant or mammalian cells, including HeLa cells and Chinese hamster ovary (CHO) cells. Insect cells include the Sf9 cell line of Spodoptera frugiperda (Luckow et al., Bio-technology 6., 47-55, 1988). Information with respect to the cloning and expression of the nucleic acid sequence of the present invention in eucaryotic cloning systems can be found in Esser, K. et al. (Plasmids of Eukaryotes, Springer-Verlag, 1986).
In general, prokaryotes are preferred for the construction of the recombinant vector molecules useful in the invention. For example E.coli K12 strains are particularly useful such as DH5α or MC1061k.
For expression nucleic acid sequences of the present invention are introduced into an expression vector, i.e. said sequences are operably linked to expression control sequences.
Such control sequences may comprise promoters, enhancers, operators, inducers, ribosome binding sites etc. Therefore, the present invention provides a recombinant vector molecule comprising a nucleic acid sequence encoding the peptides identified above operably linked to expression control sequences, capable of expressing the DNA sequences contained therein in (a) transformed host cell(s) . It should, of course, be understood that the nucleotide sequences inserted at the selected site of the cloning vector may include only a fragment of the complete nucleic acid sequence encoding for the peptides according to the invention as long as the transformed host will produce a polypeptide having at least one or more immunogenic determinants.
When the host cells are bacteria, illustrative useful expression control sequences include the Trp promoter and operator (Goeddel, et al., Nucl. Acids Res. 8., 4057, 1980); the lac promoter and operator (Chang, et al., Nature 275, 615, 1978); the outer membrane protein promoter (Nakamura, K. and Inouge, M. , EMBO J. 1 , 771-775, 1982); the bacteriophage kpromoters and operators (Remaut, E. et al., Nucl. Acids Res. .11, 4677- 4688, 1983); the α-amylase (B. subtilis) promoter and operator, termination sequence and other expression enhancement and control sequences compatible with the selected host cell. When the host cell is yeast, illustrative useful expression control sequences include, e.g., α- mating factor. For insect cells the polyhedrin or plO promoters of baculoviruses can be used (Smith, G.E. et al., Mol. Cell. Biol. 3 , 2156-65, 1983). When the host cell is of mammalian origin illustrative useful expression control sequences
include, e.g., the SV-40 promoter (Berman, P.W. et al., Science 222, 524-527, 1983) or, e.g. the metallothionein promoter (Brinster, R.L. , Nature 296, 39-42, 1982) or a heat shock promoter (Voellmy et al., Proc. Natl. Acad. Sci. USA 81, 4949-53, 1985). Alternatively, also expression control sequences present in HCV may be applied. For maximizing expression, see also Roberts and Lauer (Methods in Enzymology 68./ 473, 1979).
It is further object of the present invention to provide novel monoclonal antibodies that specifically react with the NS3 protein of the HCV virus and are particularly useful in immunodiagnostic tests for the detection of the presence or absence of HCV in clinical specimen.
The preparation of cell lines producing monoclonal antibodies may occur by, for example, transformation with Epstein-Barr Virus, the Kohler and Milstein technique (Kohler and Milstein devised the techniques that resulted in the formation monoclonal antibody-producing hybridomas (G. Kohler and C. Milstein, 1975, Nature 256:495-497; 1976, Eur. J. Immunol. 6:511- 519)), or a direct transformation technique of B- lymphocytes with oncogenic DNA, or a direct fusion of human B-lymphocytes with a fusion partner being either a human or a mouse-human hybrid myeloma cell line, or a direct fusion of an EBV-transformed B cell line with said myeloma cell lines.
The Epstein Barr virus (EBV) is capable of transforming and immortalizing human B- lymphocytes. With the aid of the Epstein Barr virus immortalized human B-lymphocytes can be
obtained without the need of a myeloma partner cell. The Epstein Barr virus can be obtained from a variety of sources. The most common used source of EBV is the B95-8 marmoset cell line. The B95-8 cell line spontaneously releases Epstein Barr virus into the medium.
A variety of cell types has been suggested to provide good feeder layers for the cloning of EBV transformed cells. The most commonly used are peripheral blood mononuclear cells (PBMC) and fibroblasts.
PBMCs consist of monocytes, T lymphocytes and B-lymphocytes (5-10%). Because not all B- lymphocytes will provide antibodies of the right specificity, it is advantageous to enrich PBMCs for appropriate cells. To prevent the generation of cytotoxic T-cells against EBV-transformed cells, T-lymphocytes can be removed, prior to EBV infection (with, for example, the supernatant from the EBV-productive B95-8 cell line), by treating the cells with washed sheep red blood cells (In: "Antibodies Vol.I, a practicle approach". Editor: Catty D, IRL Press, Oxford, England. 1988, Ch. 41.
The antibodies produced by immortalized B cell lines were considered monoclonal if they satisfied the following requirements:
1) Stability of antibody secretion over time (>6 months) ,
2) Secretion of only one IgG (H and L) functional molecule,
3) Specificity and stability of antibody secretion following at least 2 sequential subcloning procedures (100% of growing colonies secreting IgG with specificity and genetic phenotype identical to the parental line) .
The present invention is further directed to a method for the detection of antibodies directed against HCV in a test fluid, wherein a peptide according to the invention is brought into contact with the test fluid and the presence of immune complexes formed between the peptide and antibodies in the test fluid is detected.
The presence of immune complexes formed between the peptide and antibodies in the test fluid is detected and by this detection the presence of antibodies to HCV in the test fluid is known and can be determined.
Depending on the nature and further characteristics of the immunochemical reagents used the immunochemical reaction that takes place can be a so called sandwich reaction, an agglutination reaction, a competition reaction or an inhibition reaction.
A particularly suitable method for the detection of HCV in a test fluid is based on a competition reaction between a peptide according to the invention provided with a labelling substance and a HCV antigen (present in the test fluid) whereby the peptide and the antigen are competing with the antibody directed against HCV attached to a solid support. The antibody coated on the support can, for example be a monoclonal antibody according to the invention.
The invention is further directed to a method for the detection of Hepatitis C virus in a sample comprising contacting the sample with a monoclonal antibody according to the invention, and detecting immune complexes formed between the monoclonal antibody and a Hepatitis C antigen.
Carrying out, for instance, a sandwich reaction for the detection of HCV in a test sample the test kit to be used comprises a monoclonal antibody according to the invention coated on a solid support, for example the inner wall of a microtest well, and either a labelled monoclonal antibody or fragment thereof as conjugate.
A further example of an immuno assay that can be used for the detection of HCV is an inhibition assay using human monoclonal antibodies as labelled reagent. The binding of this reagent to antigen on a solid phase can be competed by antibodies in the test sample. As already mentioned monoclonal antibodies according to the invention are very suitable in diagnosis, while those antibodies which are neutralizing are very useful in passive immunotherapy.
The invention also relates to a test kit for carrying out an immuno-assay, said test kit containing at least an immunochemical reagent according to the invention. A test kit according to the invention comprises as an essential constituent an immunochemical reagent as described above. This immunochemical reagent may comprise an antibody or a peptide according to the invention. Test kits comprising a combination of different immunochemical reagents according to the invention, for example a peptide coated on a solid support and an antibody provided with a label, are of course within the scope of this invention.
Carrying out a sandwich reaction, for the detection of HCV antibodies the test kit may
comprise, for example, a peptide according to the invention coated to a solid support, for example the inner wall of a microtest well, and either a labelled peptide according to the invention or a labelled anti-antibody. Another sandwich reaction test format is the detection of HCV antigen whereby monoclonal antibodies according to the invention are coated to a solid support and monoclonal antibodies are used as conjugate. For instance sandwich reactions are described in our American patents with regard to the enzyme immuno assay viz. RE 31.006 and RE 32.696 (Schuurs et al.).
For carrying out a competition reaction, the test kit may comprise a peptide according to the invention coated to a solid support, and a labelled antibody directed against HCV preferably a monoclonal antibody directed against said peptide. In an agglutination reaction the test kit comprises an immunochemical reagent which may comprise a peptide according to the invention coated to particles or sols.
Another embodiment of a test kit is, for example, the use of a labelled peptide according to the invention as immunochemical reagent in a competition reaction with a HCV antigen to be detected for a binding site on the antibody directed against HCV, which is coated to a solid support.
Also a part of the present invention is a peptide or fragment thereof according to the invention which can be used in suitable pharmaceutical dosage forms in the prevention and/or treatment of NANB Hepatitis-disease. The preparation of vaccines thus obtained using such
a peptide or fragment thereof as active ingredients, can be accomplished by one skilled in the art.
EXAMPLES
Example 1: Construction and screening of lambda αt-11 library
The sequence coding for a part of the NS-3 gene of HCV (nucleotides 3573-4890) has been multiplied by PCR using specific primers. Starting material was obtained from a clone constructed by rt-PCR from chimpanzeeserum infected with the prototype HCV-strain. The PCR products were isolated from TBE-polyacrylamide gel (8% PAGE) by electroelution. Portions (20 μl out of 80 μl) of this PCR-material have been digested under controlled conditions (25 °C, 10- 60 minutes, in an endvolume of 25 μl containing 1 mM MnCl2, 20 mM Tris-HCl (pH 7.5) and DNAse-1 (Worthington 2635 units per mg, end concentration: 0.6 units)). The digestions were stopped in phenol/chloroform-isoamylalcohol and extracted. The DNAse digestions were controlled by nicktranslation. Fragments with the length from approximately fifty till twohundred basepairs were isolated after 8% PAGE by diffusion (J. Sambrook, E.F. Fritsch, T. Maniatis, Molecular Cloning, second edition, Cold Spring HarborLaboratory Press, 1989). The polynucleotide, which codes for the peptide according to SEQ ID No. 6, was tailed with oligo- dG following recommendations of the supplier (GIBCO/BRL) . PCR was performed on the tailed product using as a primer poly-C with a terminal EcoRl-site attached. After EcoRl digestion and phenol extraction the products were cloned in lambda gt-11 arms and transfected into E.coli as detailed by the supplier (Promega) . PCR on the libraries using lambda gtll-primers revealed
smears with lengths concordant with the lengths of the inserted fragments. The libraries were screened on duplo filters using standard procedures with human monoclonal antibody (1:50) of which the reaction was detected with alkaline phosphatase-conjugated goat anti-human IgG. The positive phages were rescreened to positively identify their contents and thereafter their inserts were transferred to the vector pGEM7Zf(+), (Promega). The inserts in this vector were sequenced using a commercial kit (Pharmacia T7-sequencing kit) according to the recommendations of the supplier. β-Galactosidase fusion proteins encoded by the recombinants, obtained as described above, were purified according to standard procedures using anti-β-galactosidase affinity columns. These purified antigens were coated onto Enzym- Linked Immuno Sorbent Assay (ELISA)-plates and allowed to react with sera from patients with non-A, non-B hepatitis. The use of this ELISA allowed us to discriminate between these patients sera and the normal human serum controles (Table 1). The procedure is further detailed below. The peptide according to the sequence of SEQ
ID No. 6 is dissolved to 7.5 μg/ml in 100 mM phosphate buffer pH 9.6 and 135 μl of the above peptide solution is placed into each well of a NUNC microtiter plate. Binding of the peptide to the microtiter plate is allowed to proceed overnight at 4° C.
Subsequently the plates are blocked with a solution of 0.05% Tween 20(R) in 0.2 M Tris pH 7.4/0.2 M NaCl for 5 min. at room temperature. Plates are then washed once with 0.2 M Tris pH 7.4/0.2 M NaCl, twice with 0.04 M Tris pH 7.4, at 250 μl per well and dried. For the determination
of antibodies specific for Non-A, Non-B Hepatitis virus, the serum samples are diluted in sample diluent (phosphate buffered saline (PBS)/20% normal goat serum/1% Triton X100) pipetted into the well (100 μl per well) and incubated for 1 h at 37 °C. After washing the wells with PBS/0.05% Tween 20(R) the bound human antibodies are detected with goat anti-human immunoglobulin labeled with peroxidase (100 μl per well, 1 h at 37 °C) diluted in sample diluent. The plates are washed 4 times with PBS/0.05% Tween 20(R). TMB is added (100 μl per well) as a substrate for the peroxidase enzyme and the reaction is allowed to proceed for 30 min., at room temperature. The reaction is stopped by adding 100 μl 2M H2S0 to each well. The yellow color is read at 450 nm in an Organon Teknika icroelisa reader.
With sera from patients with Non-A Non-B hepatitis positive results can be obtained whereas the results of 20 normal human sera are negative (Table 1).
As a control, the procedure can be repeated with two unrelated peptides. In both cases no significant differences can be observed in the specific recognition obtained with normal human sera and serum samples from patients with NANBH. From above-mentioned results the conclusion seems justified that said polypeptides according to the invention are extremely immunochemically reactive with HCV-antibodies and can be used alone or in combination in a diagnostic test kit.
The peptides according to the sequence of SEQ ID No. 7, 8, 9 and 10 are prepared using above-mentioned recombinant-DNA technique. The same set-up as described above is used for testing said peptides in an immunoassay.
With sera from patients with Non-A Non-B hepatitis the specific recognition is positive whereas the results of 20 normal human sera is negative (Table 1).
TABLE 1:
Table 1; Reactivity of sera from 4 different patients with non-A. non-P hepatitis as tested in 3 commercial assays, compared with their reactivity with the peptides described in the invention.
* Chiron Riba: Chiron Riba HCV test system
** Peptide numbers refer to peptides as shown in SEQ ID list.
Abbreviations:
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT:
(A) NAME: AKZO N.V.
(B) STREET: Velperweg 76
(C) CITY: Arnhem
(D) COUNTRY: The Netherlands
(E) POSTAL CODE (ZIP) 6824 BM
(ii) TITLE OF INVENTION: Hepatitis C Virus (HCV) Non-structural-3 peptides, antibodies thereto and methods for the detection of HCV
(iii) NUMBER OF SEQUENCES: 10
(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) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: EP 92203802
(B) FILING DATE: 07-DEC-1992
(2) INFORMATION FOR SEQ ID NO:l:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 148 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Escherichia coli (B) STRAIN: JM101
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:
TGAGCTCACG CCCGCCGAGA CTACAGTTAG GCTACGAGCG 40 TACATGAACA CCCCGGGGCT TCCCGTGTGC CAGGACCATC 80 TTGAATTTTG GGAGGGCGTC TTTACAGGCC TCACTCATAT 120 AGATGCCCAC TTTCTATCCC AGACAAAG 148
(2) INFORMATION FOR SEQ ID NO:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 158 base pairs
(B) TYPE: nucleic acid (C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(ϋi) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE: (A) ORGANISM: Escherichia coli
(B) STRAIN: JM101
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
CTATCCCAGA CAAAGCAGAG TGGGGAGAAC CTTCCTTACC 40 TGGTAGCGTA CCAAGCCACC GTGTGCGCTA GGGCTCAAGC 80 CCCTCCCCCA TCGTGGGACC AGATGTGGAA GTGTTTGATT 120 CGCCTCAAGC CCACCCTCCA TGGGCCAACA CCCCTGCT 158
(2) INFORMATION FOR SEQ ID NO:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 164 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Escherichia coli
(B) STRAIN: JM101
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
CAAAGCAGAG TGGGGAGAAC CTTCCTTACC TGGTAGCGTA 40 CCAAGCCACC GTGTGCGCTA GGGCTCAAGC CCCTCCCCCA 80 TCGTGGGACC AGATGTGGAA GTGTTTGATT CGCCTCAAGC 120 CCACCCTCCA TGGGCCAACA CCCCTGCTAT ACAGACTGGG 160 CGCT 164
(2) INFORMATION FOR SEQ ID NO:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 148 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Escherichia Coli
(B) STRAIN:, JM101
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:
CAAAGCAGAG TGGGGAGAAC CTTCCTTACC TGGTAGCGTA 40 CCAAGCCACC GTGTGCGCTA GGGCTCAAGC CCCTCCCCCA 80 TCGTGGGACC AGATGTGGAA GTGTTTGATT CGCCTCAAGC 120 CCACCCTCCA TGGGCCAACA CCCCTGCT 148
(2) INFORMATION FOR SEQ ID NO:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 68 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(vi) ORIGINAL SOURCE: (A) ORGANISM: Escherichia coli
(B) STRAIN: JM101
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:
CTATCCCAGA CAAAGCAGAG TGGGGAGAAC CTTCCTTACC 40 TGGTAGCGTA CCAAGCCACC GTGTGCGC 68
(2) INFORMATION FOR SEQ ID NO:6:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 49 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: internal
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:
Glu Leu Thr Pro Ala Glu Thr Thr Val Arg 1 5 10
Leu Arg Ala Tyr Met Asn Thr Pro Gly Leu 15 20
Pro Val Cys Gin Asp His Leu Glu Phe Trp
25 30
Glu Gly Val Phe Thr Gly Leu Thr His lie
35 40 Asp Ala His Phe Leu Ser Gin Thr Lys
45 49
(2) INFORMATION FOR SEQ ID NO:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 52 amino acids (B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:
Leu Ser Gin Thr Lys Gin Ser Gly Glu Asn
1 5 10
Leu Pro Tyr Leu Val Ala Tyr Gin Ala Thr 15 20
Val Cys Ala Arg Ala Gin Ala Pro Pro Pro
25 30
Ser Trp Asp Gin Met Trp Lys Cys Leu lie
35 40 Arg Leu Lys Pro Thr Leu His Gly Pro Thr
45 50 Pro Leu 52
(2) INFORMATION FOR SEQ ID NO:8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 54 amino acids
(B) TYPE: amino acid (C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:
Lys Gin Ser Gly Glu Asn Leu Pro Tyr Leu 1 5 10
Val Ala Tyr Gin Ala Thr Val Cys Ala Arg
15 20
Ala Gin Ala Pro Pro Pro Ser Trp Asp Gin
25 30 Met Trp Lys Cys Leu lie Arg Leu Lys Pro
35 40
Thr Leu His Gly Pro Thr Pro Leu Leu Tyr
45 50
Arg Leu Gly Ala 54
(2) INFORMATION FOR SEQ ID NO:9:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 48 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:
Lys Gin Ser Gly Glu Asn Leu Pro Tyr Leu
1 5 10
Val Ala Tyr Gin Ala Thr Val Cys Ala Arg
15 20 Ala Gin Ala Pro Pro Pro Ser Trp Asp Gin
25 30
Met Trp Lys Cys Leu lie Arg Leu Lys Pro
35 40 Thr Leu His Gly Pro Thr Pro Leu 45 48
(2) INFORMATION FOR SEQ ID NO:10:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:
Leu Ser Gin Thr Lys Gin Ser Gly Glu Asn 1 5 10
Leu Pro Tyr Leu Val Ala Tyr Gin Ala Thr
15 20
Val Cys 22
Claims
1. A peptide comprising a HCV specific epitope of the NS-3 antigen of the Hepatitis C virus.
2. A peptide according to claim 1, comprising an amino acid sequence selected from the group of sequences depicted in SEQ ID No. : 6, 7, 8, 9 and 10, and combinations thereof or fragments of said group of sequences or analogues of said group of sequences which are immunochemically reactive with HCV-antibodies.
3. Immunochemical reagent comprising peptide according to any of claims 1-2.
4. A nucleic acid sequence encoding a peptide according to any of claims 1-2.
5. A nucleic acid sequence according to claim 4, containing at least part of the DNA sequence shown in SEQ ID No. 1, 2, 3 , 4 and 5.
6. A recombinant vector molecule comprising a nucleic acid sequence according to any of claims 4-5.
7. A host cell transformed with a vector according to claim 6.
8. A monoclonal antibody directed against a peptide according to any of claims 1-2.
9. Immunodiagnostic reagent comprising a monoclonal antibody according to claim 8.
10. Method for the detection of antibodies directed against HCV in a test fluid, wherein a peptide according to any of claims 1-2 is brought into contact with the test fluid and the presence of immune complexes formed between the peptide and antibodies in the test fluid is detected.
11. Method for the detection of hepatitis C virus in a sample comprising contacting the sample with a monoclonal antibody according to claim 8, and detecting immune complexes formed between the monoclonal antibody and a hepatitis C antigen.
12. Method for the detection of antibodies directed against a HCV specific epitope of the NS-3 protein of the Hepatitis C virus in a test- fluid, wherein a peptide according to claim 1 or 2 is brought into contact with the test fluid and the presence of immune complexes formed between the peptide and the antibodies in the test fluid is detected.
Priority Applications (5)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| EP94903750A EP0672065A1 (en) | 1992-12-07 | 1993-12-06 | Hepatitis c virus (hcv) non-structural-3 peptides, antibodies thereto and methods for the detection of hcv |
| AU66533/94A AU6653394A (en) | 1992-12-07 | 1993-12-06 | Hepatitis c virus (hcv) non-structural-3 peptides, antibodies thereto and methods for the detection of hcv |
| JP6513756A JPH08505131A (en) | 1992-12-07 | 1993-12-06 | Hepatitis C virus (HCV) nonstructural-3-peptide, antibody to the peptide and method for detecting HCV |
| KR1019950702373A KR950704353A (en) | 1992-12-07 | 1993-12-06 | Hepatitis C Viral (HCV) Non-Struct-3 Peptides, Antibodies To It, and Methods for Detecting HCV (HCV) NON-STRUCTURAC-3 PEPTIDES DETECTION OF HCV |
| FI952778A FI952778A (en) | 1992-12-07 | 1995-06-06 | Hepatitis C virus (HCV) 3-non-structural peptides, antibodies against them and methods for detecting HCV |
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| EP92203802.1 | 1992-12-07 | ||
| EP92203802 | 1992-12-07 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| WO1994013699A1 true WO1994013699A1 (en) | 1994-06-23 |
Family
ID=8211111
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| PCT/EP1993/003422 WO1994013699A1 (en) | 1992-12-07 | 1993-12-06 | Hepatitis c virus (hcv) non-structural-3 peptides, antibodies thereto and methods for the detection of hcv |
Country Status (8)
| Country | Link |
|---|---|
| EP (1) | EP0672065A1 (en) |
| JP (1) | JPH08505131A (en) |
| KR (1) | KR950704353A (en) |
| AU (1) | AU6653394A (en) |
| CA (1) | CA2151128A1 (en) |
| FI (1) | FI952778A (en) |
| WO (1) | WO1994013699A1 (en) |
| ZA (1) | ZA939169B (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB2294690A (en) * | 1994-11-01 | 1996-05-08 | United Biomedical Inc | Polypeptides for the diagnosis and therapy of hepatitis C virus(HCV) |
| WO2002026785A2 (en) * | 2000-09-28 | 2002-04-04 | Immusystems Gmbh | Epitopes of virus hepatitis c specifically cd4+ t lymphocytes |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0318216A1 (en) * | 1987-11-18 | 1989-05-31 | Chiron Corporation | NANBV diagnostics and vaccines |
-
1993
- 1993-12-06 AU AU66533/94A patent/AU6653394A/en not_active Abandoned
- 1993-12-06 EP EP94903750A patent/EP0672065A1/en not_active Withdrawn
- 1993-12-06 CA CA002151128A patent/CA2151128A1/en not_active Abandoned
- 1993-12-06 WO PCT/EP1993/003422 patent/WO1994013699A1/en not_active Application Discontinuation
- 1993-12-06 KR KR1019950702373A patent/KR950704353A/en not_active Application Discontinuation
- 1993-12-06 JP JP6513756A patent/JPH08505131A/en active Pending
- 1993-12-07 ZA ZA939169A patent/ZA939169B/en unknown
-
1995
- 1995-06-06 FI FI952778A patent/FI952778A/en not_active Application Discontinuation
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0318216A1 (en) * | 1987-11-18 | 1989-05-31 | Chiron Corporation | NANBV diagnostics and vaccines |
Non-Patent Citations (2)
| Title |
|---|
| CHOO Q.L. ET AL., PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF USA., vol. 88, 1991, WASHINGTON US, pages 2451 - 2455 * |
| KREMSDORF ET AL., JOURNAL OF GENERAL VIROLOGY, vol. 72, 1991, COLCHESTER GB, pages 2557 - 2561 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB2294690A (en) * | 1994-11-01 | 1996-05-08 | United Biomedical Inc | Polypeptides for the diagnosis and therapy of hepatitis C virus(HCV) |
| GB2294690B (en) * | 1994-11-01 | 1998-10-28 | United Biomedical Inc | Peptides effective for diagnosis and detection of hepatitis C infection |
| WO2002026785A2 (en) * | 2000-09-28 | 2002-04-04 | Immusystems Gmbh | Epitopes of virus hepatitis c specifically cd4+ t lymphocytes |
| EP1195381A1 (en) * | 2000-09-28 | 2002-04-10 | Immusystems GmbH | Hepatitis c virus epitopes specific for cd4+ t-cell lymphocytes |
| WO2002026785A3 (en) * | 2000-09-28 | 2003-11-06 | Immusystems Gmbh | Epitopes of virus hepatitis c specifically cd4+ t lymphocytes |
| US7108856B2 (en) | 2000-09-28 | 2006-09-19 | Immusystems Gmbh | CD4+ T-lymphocyte-specific Hepatitis C virus-epitopes |
Also Published As
| Publication number | Publication date |
|---|---|
| KR950704353A (en) | 1995-11-20 |
| JPH08505131A (en) | 1996-06-04 |
| FI952778A0 (en) | 1995-06-06 |
| ZA939169B (en) | 1994-08-08 |
| FI952778A (en) | 1995-07-19 |
| AU6653394A (en) | 1994-07-04 |
| EP0672065A1 (en) | 1995-09-20 |
| CA2151128A1 (en) | 1994-06-23 |
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