EP0542970A1 - Nucleotide and peptide sequences of an isolate of the hepatitis c virus, diagnostic and therapeutic applications thereof - Google Patents

Nucleotide and peptide sequences of an isolate of the hepatitis c virus, diagnostic and therapeutic applications thereof

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Publication number
EP0542970A1
EP0542970A1 EP92911801A EP92911801A EP0542970A1 EP 0542970 A1 EP0542970 A1 EP 0542970A1 EP 92911801 A EP92911801 A EP 92911801A EP 92911801 A EP92911801 A EP 92911801A EP 0542970 A1 EP0542970 A1 EP 0542970A1
Authority
EP
European Patent Office
Prior art keywords
sequence
peptide sequence
hcv
seq
leu
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Application number
EP92911801A
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German (de)
French (fr)
Inventor
Christian Brechot
Dina Kremsdorf
Colette Porchon
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Institut Pasteur de Lille
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Institut Pasteur de Lille
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Publication of EP0542970A1 publication Critical patent/EP0542970A1/en
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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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • 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
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S435/00—Chemistry: molecular biology and microbiology
    • Y10S435/81—Packaged device or kit
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S530/00—Chemistry: natural resins or derivatives; peptides or proteins; lignins or reaction products thereof
    • Y10S530/808—Materials and products related to genetic engineering or hybrid or fused cell technology, e.g. hybridoma, monoclonal products

Definitions

  • the present invention relates to nucleotide and peptide sequences of a European, more particularly French, strain of the hepatitis C virus, as well as to the diagnostic and therapeutic applications of these sequences.
  • the hepatitis C virus is a major etiological agent of virus infections formerly known as the "Non-A Non-B" virus. Infections with virus C now represent the most frequent forms of acute hepatitis and chronic hepatitis Non-A Non-B (Alter et al., D), Choo et al., (3); Hopf et al., (5); Kuo et al., (8); Miyamura et al., (11). In addition there is asso ⁇ ciation (whose meaning is still not com- socket) between the presence of HCV antibodies and the development of primary liver cancer. The hepatitis C virus has also been shown to be involved in both chronic and acute Non-A Non-B hepatitis linked to transfusions of blood products or sporadic origin.
  • the hepatitis C virus genome has been cloned and the nucleotide sequence of an American isolate has been described in EP-A-0 318 216, EP-A-0 363 025, EP-A-0 388 232 and WO -A-90/14436.
  • data are currently available on nucleotide sequences from several Japanese isolates relating to both the structural and non-structural regions of the virus (O amoto et al., ( 12), Enomoto et al., (4) , Kato et al., (6); Takeuchi et al., (15 and 16).
  • the virus has certain homologies with the group of Flavi and Pestiviruses; however it seems to form a class apart, different from the viruses known until 'now (Miller and Purcell, (10)). Despite the considerable progress represented by the cloning of HCV, several problems persist:
  • HCV E1 a French HCV isolate
  • the present invention is based on new nucleotide and polypeptide sequences hepatitis C virus which have not been described in the aforementioned prior art.
  • the present invention thus relates to a DNA sequence of HCV E1 comprising a DNA sequence chosen from the nucleotide sequences of at least 10 nucleotides comprised between the following nucleotides (n): n 118 to n 138 ; n 177 to n 202 ; n 233 to n 247 ; n 254 and n 272 272 year year 288 re ésentés P r on the sequence SEQ ID No. 2, and No. 156 to No.
  • n 170; n 170 to n 217 ; n 267 to n 283 and n 310 year 334 represented on the sequence SEQ ID N c 3; as well as analogous nucleotide sequences resulting from the degeneration of the genetic code.
  • the subject of the invention is in particular the following nucleotide sequences: SEQ ID No. 2, SEQ ID No. 3 and SEQ ID No. 4.
  • oligonucleotide sequences can advantageously be synthesized by the Applied Bio System technique.
  • the invention also relates to a peptide sequence of HCV E1 comprising a peptide sequence chosen from the sequences of at least 7 amino acids comprised between the following amino acids (aa): aa 5 g to aagg; aa 7 g to represented on the peptide sequence SEQ ID No. 2, aa 4 g to aa 7 g; aag to aa lll ' aa 123 a aa133' aa 140 to aa 149 represented on the peptide sequence SEQ ID N c 3; as well as homologous peptide sequences which do not induce any modification of the biological and immunological properties.
  • the peptide sequence is chosen from the following amino acid sequences: aa5g to aagg; aa g to aa- j _ Q i represented on the peptide sequence SEQ ID No. 2, aa 4 g to aa 7 g; aagg to aa-m; aa 123 a aa 133 and aa 140 aa 149 represented on the SEQ ID N ° 3 peptide sequence.
  • the peptide sequence is also advantageously chosen from the peptide sequences SEQ ID No 2, SEQ ID No 3 and SEQ ID No 4.
  • the invention also relates to a nucleotide sequence coding for a peptide sequence as defined above.
  • the invention further relates to a polynucleotide probe comprising a DNA sequence as defined above.
  • the invention also relates to an immunogenic peptide comprising a peptide sequence as defined above.
  • the peptide sequences according to the invention can be obtained by conventional synthetic methods or by application of genetic engineering techniques comprising the insertion of a DNA sequence coding for a peptide sequence according to the invention in a vector. expression such as a plasmid and the transformation of cells with this expression vector and culture of these cells.
  • the invention also relates to plasmids or expression vectors comprising a DNA sequence coding for a peptide sequence as defined above as well as hosts transformed with this vector.
  • the preferred plasmids are those deposited at the CNCM on June 5, 1991 under the numbers 1-1105, 1-1106 and 1-1107.
  • the invention also relates to monoclonal antibodies directed against a peptide sequence according to the invention or an immunogenic sequence of such a polypeptide.
  • the monoclonal antibodies according to the invention can be prepared according to a conventional technique.
  • the polypeptides can be coupled if necessary to an immunogenic agent, such as tetanus toxoid, by a coupling agent such as glutaraldehyde, a carbodiimide or a bis diazotized benzidine.
  • the present invention also encompasses the fragments and derivatives of monoclonal antibodies according to the invention.
  • These fragments are in particular the fragments F (ab ') 2 which can be obtained by enzymatic cleavage of the antibody molecules with pepsin, the Fab' fragments which can be obtained by reduction of the disulfide bridges of the fragments F (ab ') 2 and the Fab fragments which can be obtained by enzymatic cleavage of the antibody molecules with papain in the presence of a reducing agent.
  • These fragments, as well as Fc fragments can also be obtained by genetic engineering.
  • the derivatives of monoclonal antibodies are for example antibodies or fragments of these antibodies, to which markers such as a radioisotope are linked.
  • Monoclonal antibody derivatives are also antibodies or fragments of these antibodies to which therapeutically active molecules are linked.
  • the subject of the invention is also an analysis kit for the detection of nucleotide sequences specific for the strain HVC El comprising one or more probes as defined above.
  • the subject of the invention is also an in vitro diagnostic procedure by highlighting specific antigens of HCVEl in a sample.
  • a biological which may contain said antigens, in which the biological sample is brought into contact with an antibody or an antibody frage, as defined above; as well as a diagnostic kit for implementing the process.
  • the subject of the invention is also a method of in vitro diagnosis by detecting antibodies specific for HCVEl in a biological sample which may contain said antibodies, in which a biological sample is brought into contact with an antigen comprising an epitope corresponding to a sequence peptide as well as a diagnostic kit for the detection of specific antibodies, comprising an antigen comprising an epitope corresponding to a peptide sequence as defined above.
  • the invention also relates to a therapeutic composition
  • a therapeutic composition comprising monoclonal antibodies or fragments of monoclonal antibodies or derivatives of monoclonal antibodies as defined above.
  • the derivatives of monoclonal antibodies are monoclonal antibodies or fragments of these antibodies linked to a therapeutically active molecule.
  • the subject of the invention is also an immunogenic composition comprising an immunogenic sequence as defined above possibly linked to a carrier protein, said immunogenic sequence being capable of inducing protective antibodies or cytotoxic T cells.
  • carrier proteins toxoids such as tetanus toxoid can be used.
  • immunogens constituted according to the MAP (Multiple Antigenic Peptide) technique can be used.
  • the immunogenic composition can contain an adjuvant endowed with immunostimulatory properties.
  • adjuvants that can be used are mineral salts such as aluminum hydroxide, hydrophobic compounds of surfactants such as incomplete Freund's adjuvant, squalane or liposomes, polynucleotides syn-- * thetics, microorganisms or components of microorganisms such as marabutide, synthetic artificial molecules such as imuthiol or levamisole, or even cytokines such as interferons A, p,> ' or interleukins.
  • the invention also relates to a process for assaying a peptide sequence as defined above comprising the use of monoclonal antibodies directed against this peptide sequence.
  • the subject of the invention is also a method for preparing a peptide sequence as defined above, comprising the insertion of a DNA sequence coding for the peptide sequence into an expression vector, the transformation of cells with this expression vector and cell culture.
  • FIG. 2 represents the comparison of the nucleotide sequence of HCVEl (1), in the non-coding region, with the sequences of an American isolate (2) and two Japanese isolates: HCJ1 (3) and HCJ4 (4 ) described respectively in WO-A-90/14436 and by Okamoto et al. (12);
  • FIG. 3 represents the comparison of the nucleotide sequence of HCVEl (1), in the El region, with the sequences of an American isolate (HCVpt) (2) described in WO 90/14436 and three Japanese isolates: HCVJ- 1 (3), HCJ1 (4) and HCJ4 (5) described in Takeuchi et al.
  • FIG. 6 represents the comparison of the amino acid sequence, in the E2 / NS1 region, of HCVEl (1) with the American isolate HCVpt (2) and the Japanese isolates HCJ1 (3), HCJ4 (4) and HCVJ1 (5); the variable regions are framed;
  • FIG. 7 shows the hydrophilicity profile of HCVEl in the E2 / NS1 region; the hydrophobic regions are located below the midline;
  • FIG. 8 shows the comparison of the sequence nucleotide, in the NS3 / NS4 region, of HCVEl (1) with the American isolate HCVpt (2) described in WO-A-
  • FIG. 9 shows the comparison of the amino acid sequence, in the NS3 / NS4 region, of HCVEl (1) with the American isolate HCVpt (2) and the Japanese isolate
  • the HCVE1 RNA was prepared as previously described in EP-A-0 318 216 from the serum of a French blood donor suffering from chronic hepatitis, anti-HCV positive (anti-ClOO) ( Kubo et al., (7).
  • 100 ⁇ l of serum was diluted in a final volume of 1 ml, in the following extraction buffer: 50 mM tris-HCl, pH.8, 1 mM EDTA, 100 mM NaCl, 1 mg / ml of proteinase K, and 0.5% SDS.
  • the proteins were extracted with a volume of phenol saturated with TE (10 mM Tris-HCl, pH.8, 1 mM EDTA).
  • the aqueous phase was then extracted 2 times with 1 volume of phenol / chloroform (1: 1) and 1 time with 1 volume of chloroform.
  • the aqueous phase was then brought to a final concentration of 0.2 M sodium acetate and the nucleic acids were precipitated by the addition of two volumes of ethanol. After centrifugation the nucleic acids were suspended in 30 ⁇ l of sterile distilled water treated with DEPC.
  • a complementary DNA was synthesized using as oligonucleotide primer HCV-specific cleotides shown in Table I below, ie a mixture of non-specific hexanucleotides of HCV and the murine reverse transcriptase.
  • a PCR Polymerase Chain Reaction
  • HCVpt The different HCV primers were made from the sequence of the prototype HCV (HCVpt), isolated from a chronically infected chimpanzee (Bradley et al., (2); Alter et al., (1), EP -A-0 318 216).
  • the nucleotide sequence of the 5 ′ region of the E2 / NS1 gene was obtained using a strategy derived from the amplification technique from a single primer independent of the sequence "Sequence Independent Single Primer Amplifi ⁇ cation "(SISPA) described by Reyes et al., (13).
  • NCA (+) 5 'CCATGGCGTTAGTATGAGT (-259- -239)
  • NCB (-) 5 'GCAGGTCTACGAGACCTC (-4- -23)
  • NSIB / C (+) 5'AGGTCGGGCGCGCCCACCTACAGCTGGGGTGAAAATGATA (210-248)
  • the amplification products were cloned into M13 mpl9 or into bacteriophage lambda gt 10 as described by Thiers et al. (17).
  • the probes used for screening the DNA sequences are shown in Table I above.
  • the nucleotide sequence of the inserts was determined by the method using dideoxy-nucleotides described by Sanger et al., (14). II - Study of the nucleotide sequences of the French isolate (HCVEl)
  • FIG. 1 The location of the various amplification products which have made it possible to obtain the nucleotide sequence of the HCV El isolate in non-structural and structural regions as well as in the non-coding region of the virus, is shown diagrammatically in FIG. 1.
  • SEQ ID No. 1 Nucleotide sequence of HCV El in the 5 ′ non-coding region
  • SEQ ID No. 1 The 5 ′ non-coding region of HCV El amplified and sequenced is called SEQ ID No. 1. It corresponds to a fragment of 256 base pairs (bp) localized in position -259 to -4 on the HCV pt as described in WO-A-90/14436. The comparison of the HCV E1 sequence with those previously published shows a very high conservation in nucleic acids (Fig. 2).
  • the nucleotide sequences probably correspond to two regions coding for the envelope proteins of the virus (currently designated, such as the El and E2 / NS1 regions).
  • the sequence obtained for the HCV E1 corresponds to the 3 'half of the gene. She was designated SEQ ID N ° 2. This 501 bp sequence is located at position 470 and 973 on the HCV pt sequence as described in WO-A-90/14436. The comparison of this sequence with those previously described shows significant genetic variability (Fig. 3). Indeed, depending on the isolates studied, a divergence of 10 to 27% in nucleic acids and 7 to 20% in amino acids can be observed, as shown in Table II below. In addition, the comparison of the peptide sequence highlights the existence of two hyper-variable regions framed in FIG. 4.
  • the HCV E1 sequence data were obtained from three overlapping amplification products (Fig.l).
  • the consensus sequence thus obtained (1210 bp) contains the entire E2 / NS1 gene, and has been designated SEQ ID No. 3.
  • the sequence of the E2 / NS1 region of HCVE1 is located at position 999 and 2209 relative to the sequence of HCV Pt described in WO-A-90/14436.
  • the comparison of the sequences of HCVEl with the isolates previously described shows a divergence of 13 to 33% as regards the nucleic acids and of 11 to 30% as regards the amino acids (FIGS. 5 and 6, Table II). The greatest variability is observed in 5 'of the E2 / NS1 gene (Fig. 5).
  • the amino acid comparison shows the existence of four hyper variable regions framed in Fig. 6.
  • the hydrophilicity profile of the E2 / NS1 region (Kyte and Dolittle, (9)) is given in Figure 7.
  • a hydrophilic region surrounded by two hydrophobic regions are observed.
  • the two hydrophobic regions probably correspond to the signal sequence as well as to the transmembrane segment.
  • the central region has ten potential sites glycolization (NXT / S), preserved on the different isolates (Fig. 6).
  • HCV E1 in the non-structural region corresponds to the 3 ′ and 5 ′ terminal parts of the NS3 and NS4 genes, respectively (FIG. 1).
  • the sequence obtained for the HCV El (943 bp) is located in position 4361 to 5303 on the sequence of the HCV pt and has been designated SEQ ID No. 4.
  • the sequence homology is 95% with the HCV pt isolate and 78.6% with a Japanese isolate (Fig. 8, Table II above).
  • amino acid comparison 98% and 93% homology was observed with the HCV pt and Japanese isolates, respectively (Fig. 8, Table II above).
  • hyper variable regions and conserved regions can lead to:
  • peptides for determining type-specific epitopes are advantageously defined in a region comprised between amino acids 75 to 100 (FIG. 4).
  • peptides for the E2 / NS1 protein allow the characterization of specific epitopes are synthesized in regions preferably between amino acids 50 to 149, (FIG. 6).
  • Kubo, Y. Takeuchi, K., Boonmar, S., Katayama, T., Choo, Q. -L., Kuo, G., Weiner, AJ, Bradley, DW, Houghton, M., Saito, I. & Miyamura, T. (1989).
  • Hepatitis C virus shares amino acid sequence similarity with pestiviruses and flaviviruses as well as members of two plant virus super groups. Proceedings of the National Academy of Sciences, U.S.A. 87, 2057-2061.
  • TYPE Nucleotide sequence
  • ORGANISM hepatitis C virus CHARACTERISTICS
  • TYPE Nucleotide sequence and its corresponding protein
  • CHARACTERISTIC DNA coding for a peptide sequence belonging to the El envelope protein (3 'half of the El gene)
  • Trp Glu Gly Val Phe Thr Gly Leu Thr His Ile Asp Ala 116

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Abstract

L'invention concerne des séquences nucléotidiques et peptidiques spécifiques d'un isolat français du virus de l'hépatite C, ainsi que les applications diagnostiques et thérapeutiques de ces séquences.The invention relates to nucleotide and peptide sequences specific for a French isolate of the hepatitis C virus, as well as to the diagnostic and therapeutic applications of these sequences.

Description

Séquences nucléotidiques et peptidiques d'un isolât de virus de l'hépatite C, applications diagnostiques et thérapeutiques. Nucleotide and peptide sequences of a hepatitis C virus isolate, diagnostic and therapeutic applications.
La présente invention se rapporte à des séquences nucléotidiques et peptidiques d'une souche européenne, plus particulièrement française, du virus de l'hépatite C, ainsi qu'aux applications diagnos- tiques et thérapeutiques de ces séquences.The present invention relates to nucleotide and peptide sequences of a European, more particularly French, strain of the hepatitis C virus, as well as to the diagnostic and therapeutic applications of these sequences.
Le virus de l'hépatite C est un agent étio- logique majeur des infections par les virus auparavant appelé virus "Non-A Non-B". Les infections par le virus C représentent en fait maintenant les formes les plus fréquentes d'hépatites aiguës et d'hépatites chroniques Non-A Non-B (Alter et coll., d), Choo et coll., (3) ; Hopf et coll., (5) ; Kuo et coll., (8) ; Miyamura et coll., (11). De plus il existe une asso¬ ciation (dont la signification est encore mal com- prise) entre la présence d'anticorps anti-HCV et le développement de cancers primitifs du foie. Il a été également montré que le virus de 1'hépatite C était impliqué à la fois dans des hépatites chroniques ou aiguës Non-A Non-B liées à des transfusions de produits sanguins ou d'origine sporadique.The hepatitis C virus is a major etiological agent of virus infections formerly known as the "Non-A Non-B" virus. Infections with virus C now represent the most frequent forms of acute hepatitis and chronic hepatitis Non-A Non-B (Alter et al., D), Choo et al., (3); Hopf et al., (5); Kuo et al., (8); Miyamura et al., (11). In addition there is asso ¬ ciation (whose meaning is still not com- socket) between the presence of HCV antibodies and the development of primary liver cancer. The hepatitis C virus has also been shown to be involved in both chronic and acute Non-A Non-B hepatitis linked to transfusions of blood products or sporadic origin.
Le génome du virus de 1'hépatite C a été clone et la séquence nucléotidiquc d'un isolât américain a été décrite dans EP-A-0 318 216, EP-A-0 363 025, EP-A-0 388 232 et WO-A-90/14436. En outre, on dispose actuellement de données de séquences nucléotidiques de plusieurs isolats japonais concer¬ nant à la fois la région structurale et la région non structurale du virus (O amoto et coll., (12), Enomoto et coll., (4), Kato et coll., (6) ; Takeuchi et coll., (15 et 16). Le virus présente certaines homologies avec le groupe des Flavi et des Pestivirus ; cependant il semble bien former une classe à part, différente des virus connus jusqu'à présent (Miller and Purcell, (10)). Malgré 1'avancée considérable qu'à représen¬ té le clonage de l'HCV, plusieurs problèmes persis¬ tent :The hepatitis C virus genome has been cloned and the nucleotide sequence of an American isolate has been described in EP-A-0 318 216, EP-A-0 363 025, EP-A-0 388 232 and WO -A-90/14436. In addition, data are currently available on nucleotide sequences from several Japanese isolates relating to both the structural and non-structural regions of the virus (O amoto et al., ( 12), Enomoto et al., (4) , Kato et al., (6); Takeuchi et al., (15 and 16). The virus has certain homologies with the group of Flavi and Pestiviruses; however it seems to form a class apart, different from the viruses known until 'now (Miller and Purcell, (10)). Despite the considerable progress represented by the cloning of HCV, several problems persist:
- il existe une variabilité génétique importante dans certaines régions du virus qui a permis de décrire l'existence de deux groupes de virus.- there is significant genetic variability in certain regions of the virus which has made it possible to describe the existence of two groups of viruses.
- le diagnostic de l'infection virale reste difficile malgré la possibilité de détecter des anticorps anti-HCV dans le sérum des malades. Cela est dû à l'existence de résultats faussement positifs et à une séroconversion retardée après infection aiguë. Enfin il existe manifestement des cas où seul la détection de l'ARN du virus permet de mettre en évidence l'in¬ fection HCV alors que la sérologie reste négative. Ces problèmes ont des implications impor¬ tantes à la fois en ce qui concerne le diagnostic et la protection contre le virus.- the diagnosis of viral infection remains difficult despite the possibility of detecting anti-HCV antibodies in the serum of the patients. This is due to the existence of false positive results and delayed seroconversion after acute infection. Finally, there are obviously cases where only the detection of the RNA of the virus makes it possible to highlight the HCV infection while the serology remains negative. These problems have important implications for both diagnosis and protection against the virus.
Les auteurs de la présente invention ont réalisé le clonage et ont obtenus la séquence nuclé- otidique partielle d'un isolât français d'HCV (dénommé ci-après HCV El) à partir d'un donneur de sang ayant transmis une hépatite chronique active à un receveur. La comparaison des séquences nucléotidiques et des séquences peptidiques obtenues avec les séquences respectives des isolats américain et japonais a montré qu'il existaitThe authors of the present invention carried out the cloning and obtained the partial nucleotide sequence of a French HCV isolate (hereinafter called HCV E1) from a blood donor having transmitted chronic active hepatitis to a receiver. Comparison of the nucleotide sequences and the peptide sequences obtained with the respective sequences of the American and Japanese isolates showed that there were
- une forte conservation en acides nucléiques dans la région non codante du HCV El, une importante variabilité génétique dans les régions structurales dénommées El et E2/NS1,- a high conservation in nucleic acids in the non-coding region of HCV El, a significant genetic variability in the structural regions called El and E2 / NS1,
- une variabilité génétique moindre dans la région non structurale.- less genetic variability in the non-structural region.
La présente invention repose sur des nouvelles séquences nucléotidiques et polypeptidiques du virus de l'hépatite C qui n'ont pas été décrites dans l'état de la technique précité.The present invention is based on new nucleotide and polypeptide sequences hepatitis C virus which have not been described in the aforementioned prior art.
La présente invention a ainsi pour objet une séquence d'ADN de HCV El comprenant une séquence d'ADN choisie parmi les séquences nucléotidiques d'au moins 10 nucléotides comprises entre les nucléotides (n) suivants : n118 à n138 ; n177 à n202 ; n233 à n247 ; n254 a n272 et n272 a n288 rePrésentés sur la séquence SEQ ID N°2, et, n156 à n170 ; n170 à n217 ; n267 à n283 et n310 a n334 représen és sur la séquence SEQ ID Nc3 ; ainsi que les séquences nucléotidiques analogues résultant de la dégénérescence du code génétique.The present invention thus relates to a DNA sequence of HCV E1 comprising a DNA sequence chosen from the nucleotide sequences of at least 10 nucleotides comprised between the following nucleotides (n): n 118 to n 138 ; n 177 to n 202 ; n 233 to n 247 ; n 254 and n 272 272 year year 288 re ésentés P r on the sequence SEQ ID No. 2, and No. 156 to No. 170; n 170 to n 217 ; n 267 to n 283 and n 310 year 334 represented on the sequence SEQ ID N c 3; as well as analogous nucleotide sequences resulting from the degeneration of the genetic code.
L'invention a en particulier pour objet les séquences nucléotidiques suivantes : SEQ ID N°2, SEQ ID N°3 et SEQ ID N°4.The subject of the invention is in particular the following nucleotide sequences: SEQ ID No. 2, SEQ ID No. 3 and SEQ ID No. 4.
Les séquences oligonucléotidiques peuvent avantageusement être synthétisées par la technique Applied Bio System.The oligonucleotide sequences can advantageously be synthesized by the Applied Bio System technique.
L'invention a également pour objet une séquence peptidique de HCV El comprenant une séquence peptidique choisie parmi les séquences d'au moins 7 aminoacides compris entre les aminoacides (aa) sui¬ vants : aa5g à aagg ; aa7g à représentés sur la séquence peptidique SEQ ID N°2, aa4g à aa7g ; aag à aalll ' aa123 a aa133 ' aa140 a aa149 représentés sur la séquence peptidique SEQ ID Nc3 ; ainsi que des séquences peptidiques homologues n'induisant pas de modification des propriétés biologiques et immunologiques. De préférence la séquence peptidique est choisie parmi les séquences d'aminoacides suivants : aa5g à aagg ; aa g à aa-j_Qi représentés sur la séquence peptidique SEQ ID N°2, aa4g à aa7g ; aagg à aa-m ; aa123 a aa133 et aa140 aa149 représentés sur la séquence peptidique SEQ ID N°3.The invention also relates to a peptide sequence of HCV E1 comprising a peptide sequence chosen from the sequences of at least 7 amino acids comprised between the following amino acids (aa): aa 5 g to aagg; aa 7 g to represented on the peptide sequence SEQ ID No. 2, aa 4 g to aa 7 g; aag to aa lll ' aa 123 a aa 133' aa 140 to aa 149 represented on the peptide sequence SEQ ID N c 3; as well as homologous peptide sequences which do not induce any modification of the biological and immunological properties. Preferably the peptide sequence is chosen from the following amino acid sequences: aa5g to aagg; aa g to aa- j _ Q i represented on the peptide sequence SEQ ID No. 2, aa 4 g to aa 7 g; aagg to aa-m; aa 123 a aa 133 and aa 140 aa 149 represented on the SEQ ID N ° 3 peptide sequence.
La séquence peptidique est en outre avanta¬ geusement choisie parmi les séquences peptidiques SEQ ID N°2, SEQ ID N°3 et SEQ ID N°4. L'invention a également pour objet une séquence nucléotidique codant par une séquence pepti¬ dique telle que définie ci-dessus.The peptide sequence is also advantageously chosen from the peptide sequences SEQ ID No 2, SEQ ID No 3 and SEQ ID No 4. The invention also relates to a nucleotide sequence coding for a peptide sequence as defined above.
L'invention a en outre pour objet une sonde polynucléotidique comprenant une séquence d'ADN telle que définie précédemment.The invention further relates to a polynucleotide probe comprising a DNA sequence as defined above.
L'invention a également pour objet un pepti- de immunogène comportant une séquence peptidique telle que définie précédemment.The invention also relates to an immunogenic peptide comprising a peptide sequence as defined above.
Les séquences peptidiques selon l'invention peuvent être obtenues par des méthodes de synthèse classique ou par .application de techniques de génie génétique comprenant 1'insertion d'une séquence d'ADN codant pour une séquence peptidique selon l'invention dans un vecteur d'expression tel qu'un plasmide et la transformation de cellules avec ce vecteur d'expres¬ sion et culture de ces cellules.The peptide sequences according to the invention can be obtained by conventional synthetic methods or by application of genetic engineering techniques comprising the insertion of a DNA sequence coding for a peptide sequence according to the invention in a vector. expression such as a plasmid and the transformation of cells with this expression vector and culture of these cells.
L'invention a également pour objet des plasmides ou des vecteurs d'expression comprenant une séquence d'ADN codant pour une séquence peptidique telle que définie précédemment ainsi que des hôtes tranformes avec ce vecteur.The invention also relates to plasmids or expression vectors comprising a DNA sequence coding for a peptide sequence as defined above as well as hosts transformed with this vector.
Les plasmides préférés sont ceux déposés à la CNCM le 5 juin 1991 sous les numéros 1-1105, 1-1106 et 1-1107.The preferred plasmids are those deposited at the CNCM on June 5, 1991 under the numbers 1-1105, 1-1106 and 1-1107.
L'invention a également pour objet des anti¬ corps monoclonaux dirigés contre une séquence peptidi¬ que selon l'invention ou une séquence immunogène d'un tel polypeptide. Les anticorps monoclonaux selon 1'invention peuvent être préparés selon une technique classique. A cet effet, les polypeptides peuvent être couplés si nécessaire à un agent immunogène, tel que l'anatoxine tétanique, par un agent de couplage tel que le glu- taraldéhyde, un carbodiimide ou une benzidine bis diazotée.The invention also relates to monoclonal antibodies directed against a peptide sequence according to the invention or an immunogenic sequence of such a polypeptide. The monoclonal antibodies according to the invention can be prepared according to a conventional technique. For this purpose, the polypeptides can be coupled if necessary to an immunogenic agent, such as tetanus toxoid, by a coupling agent such as glutaraldehyde, a carbodiimide or a bis diazotized benzidine.
La présente invention englobe également les fragments et les dérivés d'anticorps monoclonaux selon l'invention. Ces fragments sont notamment les frag¬ ments F(ab')2 qui peuvent être obtenus par clivage enzymatique des molécules d'anticorps avec la pepsine, les fragments Fab' qui peuvent être obtenus par réduction des ponts disulfure des fragments F(ab' )2 et les fragments Fab qui peuvent être obtenus par clivage enzymatique des molécules d'anticorps avec la papaîne en présence d'un agent réducteur. Ces fragments, ainsi que des fragments Fc, peuvent être également obtenus par génie génétique. Les dérivés d'anticorps monoclonaux sont par exemple des anticorps ou des fragments de ces anti¬ corps auxquels sont liés des marqueurs tels qu'un radioisotope. Les dérivés d'anticorps monoclonaux sont également des anticorps ou des fragments de ces anti- corps auxquels sont liées des molécules thérapeuti- quement actives.The present invention also encompasses the fragments and derivatives of monoclonal antibodies according to the invention. These fragments are in particular the fragments F (ab ') 2 which can be obtained by enzymatic cleavage of the antibody molecules with pepsin, the Fab' fragments which can be obtained by reduction of the disulfide bridges of the fragments F (ab ') 2 and the Fab fragments which can be obtained by enzymatic cleavage of the antibody molecules with papain in the presence of a reducing agent. These fragments, as well as Fc fragments, can also be obtained by genetic engineering. The derivatives of monoclonal antibodies are for example antibodies or fragments of these antibodies, to which markers such as a radioisotope are linked. Monoclonal antibody derivatives are also antibodies or fragments of these antibodies to which therapeutically active molecules are linked.
L'invention a également pour objet une trousse d'analyse pour la détection de séquences nucléotidiques spécifiques de la souche HVC El compor- tant une ou plusieurs sondes telles que définies précédemment.The subject of the invention is also an analysis kit for the detection of nucleotide sequences specific for the strain HVC El comprising one or more probes as defined above.
L'invention a également pour objet un pro¬ cédé de diagnostic in vitro par mise en évidence des antigènes spécifiques de HCVEl dans un prélèvement biologique pouvant contenir lesdits antigènes, dans lequel on met en présence le prélèvement biologique avec un anticorps ou un fragement d'anticorps, tels que définis précédemment; ainsi qu'une trousse de diagnostic pour la mise en oeuvre du procédé.The subject of the invention is also an in vitro diagnostic procedure by highlighting specific antigens of HCVEl in a sample. a biological which may contain said antigens, in which the biological sample is brought into contact with an antibody or an antibody frage, as defined above; as well as a diagnostic kit for implementing the process.
L'invention a également pour objet un procédé de diagnostic in vitro par mise en évidence des anticorps spécifiques de HCVEl dans un prélèvement biologique pouvant contenir lesdits anticorps, dans lequel on met en présence un prélèvement biologique avec un antigène comportant un épitope correspondant à une séquence peptidique ainsi qu'une trousse de diagnostic pour la détection d'anticorps spécifiques, comprenant un antigène comportant un épitope corres- pondant à une séquence peptidique telle que définie précédemment.The subject of the invention is also a method of in vitro diagnosis by detecting antibodies specific for HCVEl in a biological sample which may contain said antibodies, in which a biological sample is brought into contact with an antigen comprising an epitope corresponding to a sequence peptide as well as a diagnostic kit for the detection of specific antibodies, comprising an antigen comprising an epitope corresponding to a peptide sequence as defined above.
Ces procédés peuvent être basés sur une méthode radioimmunologique de type RIA, RIPA ou IRMA ou une méthode immunoenzymatique de type ESTERN-BLOT sur bandelettes ou de type ELISA.These methods can be based on a radioimmunological method of the RIA, RIPA or IRMA type or an immunoenzymatic method of the ESTERN-BLOT type on strips or of the ELISA type.
L'invention a également pour objet une com¬ position thérapeutique comprenant des anticorps monoclonaux ou des fragments d'anticorps monoclonaux ou des dérivés d'anticorps monoclonaux tels que définis précédemment.The invention also relates to a therapeutic composition comprising monoclonal antibodies or fragments of monoclonal antibodies or derivatives of monoclonal antibodies as defined above.
Avantageusement, les dérivés d'anticorps monoclonaux sont des anticorps monoclonaux ou des fragments de ces anticorps liés à une molécule thérapeutiquement active. L'invention a également pour objet une composition immunogène comportant une séquence immunogène telle que définie précédemment éven¬ tuellement liée à une protéine porteuse, ladite séquence immunogène étant susceptible d'induire des anticorps protecteurs ou des lymphocytes T cytoto- xiques. Comme protéines porteuses on peut utiliser des anatoxines telles que l'anatoxine tétanique. En va¬ riante on peut également utiliser des immunogènes constitués selon la technique du MAP (Multiple Anti- genic Peptide).Advantageously, the derivatives of monoclonal antibodies are monoclonal antibodies or fragments of these antibodies linked to a therapeutically active molecule. The subject of the invention is also an immunogenic composition comprising an immunogenic sequence as defined above possibly linked to a carrier protein, said immunogenic sequence being capable of inducing protective antibodies or cytotoxic T cells. As carrier proteins, toxoids such as tetanus toxoid can be used. Alternatively, it is also possible to use immunogens constituted according to the MAP (Multiple Antigenic Peptide) technique.
Outre la séquence peptidique immunogène, la composition immunogène peut contenir un adjuvant doué de propriétés immunostimulantes. Parmi les adjuvants que l'on peut utiliser figurent les sels minéraux tels que l'hydroxyde d'aluminium, des composés hydrophobes du des surfac- tants tels que l'adjuvant incomplet de Freund, le squalane ou des liposomes, des polynucléotides syn--* thétiques, des microorganismes ou composants de microorganismes tels que le marabutide, des molécules artificielles synthétiques telles qe l'imuthiol ou le levamisole, ou encore des cytokines telles que les interférons A, p, >' ou des interleukines. L'invention a également pour objet un pro¬ cédé de dosage d'une séquence peptidique telle que définie précédemment comprenant 1'utilisation d'anticorps monoclonaux dirigés contre cette séquence peptidique. L'invention a également pour objet un procédé de préparation d'une séquence peptidique telle que définie précédemment, comprenant 1'insertion d'une séquence d'ADN codant pour la séquence peptidique dans un vecteur d'expression, la transformation de cellules avec ce vecteur d'expression et la culture des cellules.In addition to the immunogenic peptide sequence, the immunogenic composition can contain an adjuvant endowed with immunostimulatory properties. Among the adjuvants that can be used are mineral salts such as aluminum hydroxide, hydrophobic compounds of surfactants such as incomplete Freund's adjuvant, squalane or liposomes, polynucleotides syn-- * thetics, microorganisms or components of microorganisms such as marabutide, synthetic artificial molecules such as imuthiol or levamisole, or even cytokines such as interferons A, p,>' or interleukins. The invention also relates to a process for assaying a peptide sequence as defined above comprising the use of monoclonal antibodies directed against this peptide sequence. The subject of the invention is also a method for preparing a peptide sequence as defined above, comprising the insertion of a DNA sequence coding for the peptide sequence into an expression vector, the transformation of cells with this expression vector and cell culture.
On décrira ci-après plus en détail l'obten¬ tion de l'ADN des séquences de la souche HCV El en se reportant aux figures annexées sur lesquelles : - La figure 1 représente la localisation des régions du HCVEl amplifiées et séquencées ;The obtaining of the DNA of the sequences of the HCV El strain will be described below in more detail with reference to the appended figures in which: - Figure 1 shows the location of the regions of HCVEl amplified and sequenced;
- La figure 2 représente la comparaison de la séquence nucléotidique du HCVEl (1), dans la région non codan- te, avec les séquences d'un isolât américain (2) et de deux isolats japonais : HCJ1 (3) et HCJ4 (4) décrits respectivement dans WO-A-90/14436 et par Okamoto et coll. (12) ;- Figure 2 represents the comparison of the nucleotide sequence of HCVEl (1), in the non-coding region, with the sequences of an American isolate (2) and two Japanese isolates: HCJ1 (3) and HCJ4 (4 ) described respectively in WO-A-90/14436 and by Okamoto et al. (12);
- La figure 3 représente la comparaison de la séquence nucléotidique du HCVEl (1), dans la région El, avec les séquences d'un isolât américain (HCVpt) (2) décrit dans WO 90/14436 et de trois isolats japonais : HCVJ-1 (3), HCJ1 (4) et HCJ4 (5) décrits dans Takeuchi et coll. (15) ; Okamoto et coll., (12) ; - La figure 4 représente la comparaison de la séquence en aminoacides, dans la région El, du HCVEl (1) avec l'isolât américain ; HCVpt (2) et les isolats japonais : HCVJ1 (3), HCJ1 (4) et HCJ4 (5) ; les régions variables sont encadrées ; - La figure 5 représente la comparaison de la séquence nucléotidique, dans la région E2/NS1, du HCVEl (1) avec l'isolât américain HCVpt (2) décrit dans W0-A- 90/14436 et les isolats japonais HCJ1 (3), HCJ4 (4) et HCVJ1 (5) décrits par Okamoto et coll., (12) ; Takeu- chi et coll., (15) ;FIG. 3 represents the comparison of the nucleotide sequence of HCVEl (1), in the El region, with the sequences of an American isolate (HCVpt) (2) described in WO 90/14436 and three Japanese isolates: HCVJ- 1 (3), HCJ1 (4) and HCJ4 (5) described in Takeuchi et al. (15); Okamoto et al., (12); - Figure 4 shows the comparison of the amino acid sequence in the E1 region of HCVEl (1) with the American isolate; HCVpt (2) and Japanese isolates: HCVJ1 (3), HCJ1 (4) and HCJ4 (5); the variable regions are framed; - Figure 5 shows the comparison of the nucleotide sequence, in the E2 / NS1 region, of HCVEl (1) with the American isolate HCVpt (2) described in W0-A- 90/14436 and the Japanese isolates HCJ1 (3) , HCJ4 (4) and HCVJ1 (5) described by Okamoto et al., (12); Takeuchi et al., (15);
- La figure 6 représente la comparaison de la séquence en aminoacides, dans la région E2/NS1, du HCVEl (1) avec l'isolât américain HCVpt (2) et les isolats japo¬ nais HCJ1 (3), HCJ4 (4) et HCVJ1 (5) ; les régions variables sont encadrées ;FIG. 6 represents the comparison of the amino acid sequence, in the E2 / NS1 region, of HCVEl (1) with the American isolate HCVpt (2) and the Japanese isolates HCJ1 (3), HCJ4 (4) and HCVJ1 (5); the variable regions are framed;
- La figure 7 représente le profil d'hydrophilicité du HCVEl dans la région E2/NS1 ; les régions hydrophobe sont localisées sous la ligne médiane ;- Figure 7 shows the hydrophilicity profile of HCVEl in the E2 / NS1 region; the hydrophobic regions are located below the midline;
- La figure 8 représente la comparaison de la séquence nucléotidique, dans la région NS3/NS4, du HCVEl (1) avec l'isolât américain HCVpt (2) décrit dans WO-A-- Figure 8 shows the comparison of the sequence nucleotide, in the NS3 / NS4 region, of HCVEl (1) with the American isolate HCVpt (2) described in WO-A-
90/14436 et de l'isolât japonais HCVJl (3) décrit par90/14436 and the Japanese isolate HCVJl (3) described by
Kubo et coll. , (7) ;Kubo et al. , (7);
- La figure 9 représente la comparaison de la séquence en aminoacides, dans la région NS3/NS4, du HCVEl (1) avec l'isolât américain HCVpt (2) et l'isolât japonais- Figure 9 shows the comparison of the amino acid sequence, in the NS3 / NS4 region, of HCVEl (1) with the American isolate HCVpt (2) and the Japanese isolate
HCVJl (3).HCVJl (3).
I - Obtention des séquences nucléotidiquesI - Obtaining the nucleotide sequences
1) Préparation de l'ARN HCVEl1) Preparation of HCVEl RNA
L'ARN d'HCVEl a été préparé comme précé¬ demment décrit dans EP-A-0 318 216 à partir du sérum d'un donneur de sang français atteint d'une hépatite chronique, anti-HCV positif (anti-ClOO) (Kubo et coll., (7).The HCVE1 RNA was prepared as previously described in EP-A-0 318 216 from the serum of a French blood donor suffering from chronic hepatitis, anti-HCV positive (anti-ClOO) ( Kubo et al., (7).
100 ul de sérum ont été dilués dans un volume final de 1 ml, dans le tampon d'extraction suivant : 50 mM tris-HCl, pH.8, 1 mM EDTA, 100 mM NaCl, 1 mg/ml de protéinase K, et 0,5% de SDS. Après digestion par la protéinase K pendant lh à 37°C, les protéines ont été extraites avec un volume de phénol saturé avec du TE (10 mM Tris-HCl, pH.8, 1 mM EDTA). La phase aqueuse a été ensuite extraite 2 fois avec 1 volume de phénol/chloroforme (1:1) et 1 fois avec 1 volume de chloroforme. La phase aqueuse a été alors amenée à une concentration finale de 0,2 M d'acétate de sodium et les acides nucléiques ont été précipités par addition de deux volumes d'éthanol. Après centri- fugation les acides nucléiques ont été suspendus dans 30 ul d'eau distillé stérile traité au DEPC.100 μl of serum was diluted in a final volume of 1 ml, in the following extraction buffer: 50 mM tris-HCl, pH.8, 1 mM EDTA, 100 mM NaCl, 1 mg / ml of proteinase K, and 0.5% SDS. After digestion with proteinase K for 1 h at 37 ° C., the proteins were extracted with a volume of phenol saturated with TE (10 mM Tris-HCl, pH.8, 1 mM EDTA). The aqueous phase was then extracted 2 times with 1 volume of phenol / chloroform (1: 1) and 1 time with 1 volume of chloroform. The aqueous phase was then brought to a final concentration of 0.2 M sodium acetate and the nucleic acids were precipitated by the addition of two volumes of ethanol. After centrifugation the nucleic acids were suspended in 30 µl of sterile distilled water treated with DEPC.
2) Transcription reverse et amplification2) Reverse transcription and amplification
Un ADN complémentaire (ADNc) a été syn¬ thétisé en utilisant comme amorce soit des oligonu- cléotides spécifiques de l'HCV représenté au tableau I ci-après, soit un mélange d'hexanucléotides non spé¬ cifiques du HCV et la transcriptase reverse murine. Une PCR (Polymerase Chain Reaction) a été effectuée sur 40 cycles aux températures suivantes : 94°CA complementary DNA (cDNA) was synthesized using as oligonucleotide primer HCV-specific cleotides shown in Table I below, ie a mixture of non-specific hexanucleotides of HCV and the murine reverse transcriptase. A PCR (Polymerase Chain Reaction) was carried out over 40 cycles at the following temperatures: 94 ° C
(1 mn), 55°C (1 mn), 72°C (1 mn), sur l'ADNc ainsi obtenu en utilisant des couples d'amorces spécifiques du HCV (Tableau I ci- dessous). Les différentes amor¬ ces HCV ont été fabriquées à partir de la séquence du HCV prototype (HCVpt), isolé d'un chimpanzé chroni- quement infecté (Bradley et coll., (2) ; Alter et coll., (1), EP-A-0 318 216). La séquence nucléotidique de la région 5' du gène E2/NS1 a été obtenue en utili¬ sant une stratégie dérivée de la technique d'amplifi- cation à partir d'une seule amorce indépendante de la séquence "Séquence Independent Single Primer Amplifi¬ cation" (SISPA) décrite par Reyes et coll., (13). Elle consiste à ligaturer des adaptateurs doubles brins aux extrémités du DNA synthétisé avec une amorce HCV-spé- cifique localisée en 5' de la séquence du HCVpt (amor¬ ce NS1A du Tableau I). Ensuite, une amplification semi-spécifique est effectuée en utilisant une amorce HCV-spécifique ainsi qu'une amorce correspondant à l'adaptateur. Cette approche permet d'obtenir des produits d'amplification s'étendant en 5' de 1'amorce utilisée pour la synthèse de 1'ADNc. (1 min), 55 ° C (1 min), 72 ° C (1 min), on the cDNA thus obtained using pairs of primers specific for HCV (Table I below). The different HCV primers were made from the sequence of the prototype HCV (HCVpt), isolated from a chronically infected chimpanzee (Bradley et al., (2); Alter et al., (1), EP -A-0 318 216). The nucleotide sequence of the 5 ′ region of the E2 / NS1 gene was obtained using a strategy derived from the amplification technique from a single primer independent of the sequence "Sequence Independent Single Primer Amplifi¬ cation "(SISPA) described by Reyes et al., (13). It consists in ligating double stranded adapters at the ends of the DNA synthesized with an HCV-specific primer located 5 ′ of the HCVpt sequence (primer NS1A in Table I). Next, semi-specific amplification is performed using an HCV-specific primer as well as a primer corresponding to the adapter. This approach provides amplification products extending 5 'to the primer used for the synthesis of cDNA.
Tableau I. Séquence des amorces et des sondes.Table I. Sequence of primers and probes.
a) amorties3- • 'a) amortized 3 - • '
NS3 (+) 5' ACAATÀCGTGTGTCACC (3013-3029)NS3 (+) 5 'ACAATÀCGTGTGTCACC (3013-3029)
NS4 (-) 5' AAGTTCCACATATGCTTCGC (3955-3935)NS4 (-) 5 'AAGTTCCACATATGCTTCGC (3955-3935)
NS1A (-) 5' TCCGTTGGCATAACTGATAG (83-64)NS1A (-) 5 'TCCGTTGGCATAACTGATAG (83-64)
NS1B (+) 5' CTATCAGTTATGCCAACGGA (64-83)NS1B (+) 5 'CTATCAGTTATGCCAACGGA (64-83)
NS1C (-) 5' GTTGCCCGCCCCTCCGATGT (380-361)NS1C (-) 5 'GTTGCCCGCCCCTCCGATGT (380-361)
NS1D (+) 5' CCCAGCCCCGTGGTGGTGGG (183-202)NS1D (+) 5 'CCCAGCCCCGTGGTGGTGGG (183-202)
NS1E (-) 5' CCACAAGCAGGAGCAGACGC (860-841)NS1E (-) 5 'CCACAAGCAGGAGCAGACGC (860-841)
NCA (+) 5' CCATGGCGTTAGTATGAGT (-259- -239)NCA (+) 5 'CCATGGCGTTAGTATGAGT (-259- -239)
NCB (-) 5' GCAGGTCTACGAGACCTC (-4- -23)NCB (-) 5 'GCAGGTCTACGAGACCTC (-4- -23)
E1A (+) 5' TTCTGGAAGACGGCGTGAAC (470-489)E1A (+) 5 'TTCTGGAAGACGGCGTGAAC (470-489)
E1B (-) 5' TCATCATATCCCATGCCATG (973-954) b) sondes3 : .E1B (-) 5 'TCATCATATCCCATGCCATG (973-954) b) probes 3 :.
NS3/NS4 (+) 5'CCTTCACCATTGAGACAATCACGCTCCCCCAGGATGCTGT (3058-3097)NS3 / NS4 (+) 5'CCTTCACCATTGAGACAATCACGCTCCCCCAGGATGCTGT (3058-3097)
NSI (+) 5'CTGTCCTGAGAGGCTAGCCAGCTGCCGACCCCTTACCGAT (5-44)NSI (+) 5'CTGTCCTGAGAGGCTAGCCAGCTGCCGACCCCTTACCGAT (5-44)
NSIB/C (+) 5'AGGTCGGGCGCGCCCACCTACAGCTGGGGTGAAAATGATA (210-248)NSIB / C (+) 5'AGGTCGGGCGCGCCCACCTACAGCTGGGGTGAAAATGATA (210-248)
NC (+) 5' GTGCAGCCTCCAGGACCCCC (-235- -216)NC (+) 5 'GTGCAGCCTCCAGGACCCCC (-235- -216)
El (-) 5' CTCGTACACAATACTCGAGT (646-627)El (-) 5 'CTCGTACACAATACTCGAGT (646-627)
a. Les séquences nucléotidiques et leurs localisations correspondent au HCV prototype (HCV pt) (EP-A-0 318 216 εt WO-A-90/14436)- 3) Clonage et séquençageat. The nucleotide sequences and their locations correspond to the prototype HCV (HCV pt) (EP-A-0 318 216 εt WO-A-90/14436) - 3) Cloning and sequencing
Les produits d'amplification ont été clones dans le M13 mpl9 ou dans le bacteriophage lambda gt 10 comme décrit par Thiers et coll. (17). Les sondes utilisées pour le criblage des séquences d'ADN sont représentées au tableau I précédent. La séquence nucléotidique des inserts a été déterminée par la méthode utilisant des dideoxy-nucléotides décrite par Sanger et coll., (14). II - Etude des séquences nucléotidiques de l'isolât français (HCVEl)The amplification products were cloned into M13 mpl9 or into bacteriophage lambda gt 10 as described by Thiers et al. (17). The probes used for screening the DNA sequences are shown in Table I above. The nucleotide sequence of the inserts was determined by the method using dideoxy-nucleotides described by Sanger et al., (14). II - Study of the nucleotide sequences of the French isolate (HCVEl)
La localisation des différents produits d'amplification ayant permis l'obtention de la séquence nucléotidique de 1'isolât HCV El dans des régions non structurales et structurales ainsi que dans la région non codante du virus, est schématisée sur la figure 1.The location of the various amplification products which have made it possible to obtain the nucleotide sequence of the HCV El isolate in non-structural and structural regions as well as in the non-coding region of the virus, is shown diagrammatically in FIG. 1.
1) Séquence nucléotidique du HCV El dans la région 5' non codante La région 5' non codante du HCV El ampli¬ fiée et séquencée est appelée SEQ ID N°l. Elle corres¬ pond à un fragment de 256 paires de bases (pb) locali¬ sée en position -259 à -4 sur 1'HCV pt tel que décrit dans WO-A-90/14436. La comparaison de la séquence du HCV El avec celles préalablement publiées montre une très forte conservation en acides nucléiques (Fig.2).1) Nucleotide sequence of HCV El in the 5 ′ non-coding region The 5 ′ non-coding region of HCV El amplified and sequenced is called SEQ ID No. 1. It corresponds to a fragment of 256 base pairs (bp) localized in position -259 to -4 on the HCV pt as described in WO-A-90/14436. The comparison of the HCV E1 sequence with those previously published shows a very high conservation in nucleic acids (Fig. 2).
2) Séquences nucléotidique et peptidique du HCV El dans la région structurale2) Nucleotide and peptide sequences of HCV El in the structural region
Les séquences nucléotidiques correspon- dent vraisemblablement à deux régions codant pour les protéines d'enveloppe du virus (actuellement dési¬ gnées, comme les régions El et E2/NS1).The nucleotide sequences probably correspond to two regions coding for the envelope proteins of the virus (currently designated, such as the El and E2 / NS1 regions).
Pour la région El, la séquence obtenue pour le HCV El correspond à la moitié 3' du gène. Elle a été dénommée SEQ ID N°2. Cette séquence de 501 pb est localisée en position 470 et 973 sur la séquence du HCV pt tel que décrit dans WO-A-90/14436. La com¬ paraison de cette séquence avec celles préalablement décrites montre une importante variabilité génétique (Fig.3). En effet, selon les isolats étudiés on peut observer une divergence de 10 à 27 % en acides nucléiques et de 7 à 20 % en acides aminés comme représenté au Tableau II ci-après. De plus, la com- paraison de la séquence peptidique met en évidence 1'existence de deux régions hyper variables encadrées sur la figure 4.For the E1 region, the sequence obtained for the HCV E1 corresponds to the 3 'half of the gene. She was designated SEQ ID N ° 2. This 501 bp sequence is located at position 470 and 973 on the HCV pt sequence as described in WO-A-90/14436. The comparison of this sequence with those previously described shows significant genetic variability (Fig. 3). Indeed, depending on the isolates studied, a divergence of 10 to 27% in nucleic acids and 7 to 20% in amino acids can be observed, as shown in Table II below. In addition, the comparison of the peptide sequence highlights the existence of two hyper-variable regions framed in FIG. 4.
Pour la région E2/NS1, les données de séquence du HCV El ont été obtenues à partir de trois produits d'amplification se chevauchant (Fig.l). La séquence consensus ainsi obtenue (1210 pb) contient la totalité du gène E2/NS1, et a été dénomée SEQ ID N°3. La séquence de la région E2/NS1 du HCVEl est située en position 999 et 2209 par rapport à la séquence du HCV Pt décrite dans WO-A-90/14436. La comparaison des sé¬ quences du HCVEl avec les isolats précédemment décrits montre une divergence de 13 à 33 % en ce qui concerne les acides nucléiques et de 11 à 30 % en ce qui con¬ cerne les acides aminés (Fig.5 et 6, Tableau II). La variabilité la plus importante est observé en 5' du gène E2/NS1 (Fig.5). La comparaison en acides aminés montre l'existence de quatre régions hyper variables encadrées sur la Fig.6. Le profil d'hydrophilicité de la région E2/NS1 (Kyte et Dolittle, (9)) est donné sur la figure 7. Une région hydrophile entourée de deux régions hydrophobes sont observées. Les deux régions hydrophobes correspondent vraisemblablement à la séquence signal ainsi qu'au segment transmembranaire. Enfin, la région centrale possède dix sites potentiels de glycolisation (N-X-T/S), conservés sur les diffé¬ rents isolats (Fig.6).For the E2 / NS1 region, the HCV E1 sequence data were obtained from three overlapping amplification products (Fig.l). The consensus sequence thus obtained (1210 bp) contains the entire E2 / NS1 gene, and has been designated SEQ ID No. 3. The sequence of the E2 / NS1 region of HCVE1 is located at position 999 and 2209 relative to the sequence of HCV Pt described in WO-A-90/14436. The comparison of the sequences of HCVEl with the isolates previously described shows a divergence of 13 to 33% as regards the nucleic acids and of 11 to 30% as regards the amino acids (FIGS. 5 and 6, Table II). The greatest variability is observed in 5 'of the E2 / NS1 gene (Fig. 5). The amino acid comparison shows the existence of four hyper variable regions framed in Fig. 6. The hydrophilicity profile of the E2 / NS1 region (Kyte and Dolittle, (9)) is given in Figure 7. A hydrophilic region surrounded by two hydrophobic regions are observed. The two hydrophobic regions probably correspond to the signal sequence as well as to the transmembrane segment. Finally, the central region has ten potential sites glycolization (NXT / S), preserved on the different isolates (Fig. 6).
3) Séquence nucléotidique et peptidique du HCV El dans la région non structurale Les données de séquence du HCV El dans la région non structurale correspondent aux parties 3' et 5' terminales des gènes NS3 et NS4, respectivement (Fig.l). La séquence obtenue pour le HCV El (943 pb) est localisée en position 4361 à 5303 sur la séquence du HCV pt et a été dénomée SEQ ID N°4. L'homologie de séquences est de 95 % avec l'isolât HCV pt et de 78,6 % avec un isolât japonais (Fig.8, Tableau II précédent). En ce qui concerne la comparaison en acides aminés, une homologie de 98 % et 93 % a été observée avec les isolats HCV pt et japonais, respectivement (Fig.8, Tableau II précédent).3) Nucleotide and peptide sequence of HCV E1 in the non-structural region The sequence data of HCV E1 in the non-structural region correspond to the 3 ′ and 5 ′ terminal parts of the NS3 and NS4 genes, respectively (FIG. 1). The sequence obtained for the HCV El (943 bp) is located in position 4361 to 5303 on the sequence of the HCV pt and has been designated SEQ ID No. 4. The sequence homology is 95% with the HCV pt isolate and 78.6% with a Japanese isolate (Fig. 8, Table II above). Regarding the amino acid comparison, 98% and 93% homology was observed with the HCV pt and Japanese isolates, respectively (Fig. 8, Table II above).
Ainsi, la comparaison de la séquence nucléotidique de 1'isolât HCV El avec celle des isolats américain et japonais montre que l'isolât français est distinct des isolats précédemment décrits. Elle met en évidence l'existence de régions fortement variables dans les protéines d'enveloppe. La variabilité de la région non structurale étudiée est plus faible. Enfin, la région 5' non codante montre une forte conservation.Thus, the comparison of the nucleotide sequence of the HCV EI isolate with that of the American and Japanese isolates shows that the French isolate is distinct from the previously described isolates. It highlights the existence of highly variable regions in the envelope proteins. The variability of the nonstructural region studied is lower. Finally, the 5 'non-coding region shows strong conservation.
- Ces résultats ont des implications à la fois pour le diagnostic et la prévention contre le HVC.- These results have implications for both diagnosis and prevention of HCV.
En ce qui concerne le diagnostic, la définition de régions hyper variables et de régions conservées peut conduire à :With regard to the diagnosis, the definition of hyper variable regions and conserved regions can lead to:
- la définition de peptides synthétiques permettant l'expression d'épitopes spécifiques des différents groupes d'HCV. Pour la protéine d'enveloppe El, des peptides pour la détermination d'épitopes spécifiques de types sont avantageusement définis dans une région comprise entre les acides aminés 75 à 100 (Fig.4). De même, pour la protéine E2/NS1 des peptides permettent la caractérisation d'épitopes spécifiques sont syn¬ thétisés dans des régions comprises de préférence entre les acides aminés 50 à 149, (Fig.6).- the definition of synthetic peptides allowing the expression of specific epitopes of the different groups of HCV. For the envelope protein E1, peptides for determining type-specific epitopes are advantageously defined in a region comprised between amino acids 75 to 100 (FIG. 4). Likewise, for the E2 / NS1 protein, peptides allow the characterization of specific epitopes are synthesized in regions preferably between amino acids 50 to 149, (FIG. 6).
L'expression de la totalité ou d'une partie des séquences clonées, en particulier des clones correspondants aux régions de 1'enveloppe du virus, permet l'obtention de nouveaux antigènes pour la mise au point de réactifs diagnostiques ainsi que pour la réalisation de compositions immunogènes. Enfin l'obtention d'une bonne partie de la séquence en nucléotides de cet isolât permet la réalisation d'ADN complémentaire de longueur complète pouvant être uti¬ lisé pour une meilleure compréhension des mécanismes de 1'infection virale et également dans des buts de diagnostic et de prévention. The expression of all or part of the cloned sequences, in particular of the clones corresponding to the regions of the envelope of the virus, makes it possible to obtain new antigens for the development of diagnostic reagents as well as for the production of immunogenic compositions. Finally, obtaining a good part of the nucleotide sequence of this isolate allows the production of complementary DNA of full length which can be used for a better understanding of the mechanisms of viral infection and also for diagnostic purposes. and prevention.
Tableau II, Divergence en acidesnucléiques (a.n.) et acidesaminés (a.a.) entre l'isolat français (HCVEl) et les isolats Américain (HCVpt) et Japonais (HCVJl, HCJl, HCJ4) .Table II, Divergence in nucleic acids (a.n.) and amino acids (a.a.) between the French isolate (HCVEl) and the American isolates (HCVpt) and Japanese (HCVJl, HCJl, HCJ4).
REFERENCESREFERENCES
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13. Reyes, G.R., Purdy, M.A., Kim, J.P., Luk, K. -C, Young, L.M., Fry, K.E. & Bradley, D.W. (1990). Isolation of a cDNA from the virus responsible for enterically transmitted non-A, non-B hepatitis. Science 247, 1335-1339.13. Reyes, G.R., Purdy, M.A., Kim, J.P., Luk, K. -C, Young, L.M., Fry, K.E. & Bradley, D.W. (1990). Isolation of a cDNA from the virus responsible for enterically transmitted non-A, non-B hepatitis. Science 247, 1335-1339.
14. Sanger, F.S., Nicklen, S. & Coulsen, A.R. (1977). DNA sequencing with chain terminating inhibition. Proceedings of the National Academy of Sciences, U.S.A. 74, 5463-5467.14. Sanger, F.S., Nicklen, S. & Coulsen, A.R. (1977). DNA sequencing with chain terminating inhibition. Proceedings of the National Academy of Sciences, U.S.A. 74, 5463-5467.
15. Takeuchi, K., Boonmar, S., Kubo, Y., Katayama, T., Harada, H., Ohbayashi, A., Choo, Q. -L., Houghton, M., Saito, I. & Miyamura, T. (1990a). Hepatitis C viral cDNA clones isolated from a healthy carrier donor implicated in post-transfusion non-A, non-B hepatitis. Gène 91 (2), 287-291. 16. Takeuchi, K. , Kubo, Y., Boonmar, S., Watanabe, Y., Katayama, T., Choo, Q. -L., Kuo, G., Houghton, M., Saito, I. & Miyamura, T. (1990b). Nucleotide séquence of core and envelope gènes of the hepatitis C virus génome derived directly from human healthy carriers. Nucleic Acids Research 18, 4626.15. Takeuchi, K., Boonmar, S., Kubo, Y., Katayama, T., Harada, H., Ohbayashi, A., Choo, Q. -L., Houghton, M., Saito, I. & Miyamura, T. (1990a). Hepatitis C viral cDNA clones isolated from a healthy carrier donor implicated in post-transfusion non-A, non-B hepatitis. Gene 91 (2), 287-291. 16. Takeuchi, K., Kubo, Y., Boonmar, S., Watanabe, Y., Katayama, T., Choo, Q. -L., Kuo, G., Houghton, M., Saito, I. & Miyamura, T. (1990b). Nucleotide sequence of core and envelope genes of the hepatitis C virus genome derived directly from human healthy carriers. Nucleic Acids Research 18, 4626.
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Symboles des acides aminésAmino acid symbols
LISTE DES SEQUENCES LIST OF SEQUENCES
I - INFORMATION GENERALEI - GENERAL INFORMATION
(1) DEMANDEUR : INSTITUT PASTEUR(1) APPLICANT: INSTITUT PASTEUR
(2) TITRE DE L'INVENTION :(2) TITLE OF THE INVENTION:
Nouvelles séquences nucléotidiques et peptidiques du virus de l'hépatite C. Applications et diagnos¬ tiques et thérapeutiques.New nucleotide and peptide sequences of the hepatitis C virus. Applications and diagnostics and therapeutics.
(3) NOMBRE DE SEQUENCES : 4(3) NUMBER OF SEQUENCES: 4
II - INFORMATION POUR SEQ ID N°lII - INFORMATION FOR SEQ ID N ° l
CARACTERISTIQUES DES SEQUENCESCHARACTERISTICS OF THE SEQUENCES
TYPE : Séquence nucléotidiqueTYPE: Nucleotide sequence
LONGUEUR : 256LENGTH: 256
NOMBRE DE BRINS : simpleNUMBER OF STRANDS: simple
CONFIGURATION : linéaireCONFIGURATION: linear
TYPE DE MOLECULE : ADNc pour ARN viralTYPE OF MOLECULE: cDNA for viral RNA
ORIGINEORIGIN
ORGANISME : virus hépatite C CARACTERISTIQUEORGANISM: hepatitis C virus CHARACTERISTICS
LOCALISATION DE L'ADN : région 5' non codante DESCRIPTION DE LA SEQUENCEDNA LOCATION: 5 'non-coding region DESCRIPTION OF THE SEQUENCE
CCATGGCGTTAGTATGAGTGTCGTACAGCCTCCAGGACCCCCCCTCCCGGGAGAGCCATA 60 GTGGTCTGCGGAGCCGGTGAGTACACCGGAATTGCCAGGACGACCGGGTCCTTTCTTGGA 12 TCAACCCGCTCAATGCCTGGAGATTTGGGCGTGCCCCCGCAAGACTGCTAGCCGAGTAGT 18 GTTGGGTCGCGAAAGGCCTTGTGGTACTGCCTGATAGGGTGCTTGCGAGTGCCCCGGGAG 24 GTCTCGTAGACCGTGC 25 I - INFORMATION POUR SEQ ID N°2CCATGGCGTTAGTATGAGTGTCGTACAGCCTCCAGGACCCCCCCTCCCGGGAGAGCCATA 60 GTGGTCTGCGGAGCCGGTGAGTACACCGGAATTGCCAGGACGACCGGGTCCTTTCTTGGA 12 TCAACCCGCTCAATGCCTGGAGATTTGGGCGTGCCCCCGCAAGACTGCTAGCCGAGTAGT GTTGGGTCGCGAAAGGCCTTGTGGTACTGCCTGATAGGGTGCTTGCGAGTGCCCCGGGAG 18 24 25 GTCTCGTAGACCGTGC I - INFORMATION FOR SEQ ID N ° 2
TYPE : Séquence nucléotidique et sa protéine correspondanteTYPE: Nucleotide sequence and its corresponding protein
LONGUEUR : 501 paires de basesLENGTH: 501 base pairs
166 acides aminés166 amino acids
NOMBRE DE BRINS : simpleNUMBER OF STRANDS: simple
CARACTERISTIQUE : ADN codant pour une séquence peptidique appartenant à la protéine d'enveloppe El. (moitié 3' du gène de El)CHARACTERISTIC: DNA coding for a peptide sequence belonging to the El envelope protein (3 'half of the El gene)
DESCRIPTION DE LA SEQUENCEDESCRIPTION OF THE SEQUENCE
TT CTG GAA GAC GGC GTG AAC TAT GCA ACA GGG AAC* CTT 38TT CTG GAA GAC GGC GTG AAC TAT GCA ACA GGG AAC * CTT 38
Leu Glu Asp Gly Val Asn Tyr Ala Thr Gly Asn Leu 12Leu Glu Asp Gly Val Asn Tyr Ala Thr Gly Asn Leu 12
CCT GGT TGC TCT TTC TCT ATC CTC CTC CTG GCC CTG CTC 77CCT GGT TGC TCT TTC TCT ATC CTC CTC CTG GCC CTG CTC 77
Pro Gly Cys Ser Phe Ser Ile Leu Leu Leu Ala Leu Leu 25Pro Gly Cys Ser Phe Ser Ile Leu Leu Leu Ala Leu Leu 25
TCT TGC CTG ACT GTG CCC GCG TCA GCC TAC CAA GTA CGC 116TCT TGC CTG ACT GTG CCC GCG TCA GCC TAC CAA GTA CGC 116
Ser Cys Leu Thr Val Pro Ala Ser Ala Tyr Gin Val Arg 38Ser Cys Leu Thr Val Pro Ala Ser Ala Tyr Gin Val Arg 38
AAT TCT CGC GGC CTT TAC CAT GTC ACC AAT GAT TGC CCT 155AAT TCT CGC GGC CTT TAC CAT GTC ACC AAT GAT TGC CCT 155
Asn Ser Arg Gly Leu Tyr His Val Thr Asn Asp Cys Pro 51Asn Ser Arg Gly Leu Tyr His Val Thr Asn Asp Cys Pro 51
AAC TCG AGT ATT GTG TAC GAG ACG GCC GAT AGC ATT CTA 194AAC TCG AGT ATT GTG TAC GAG ACG GCC GAT AGC ATT CTA 194
Asn Ser Ser Ile Val Tyr Glu Thr Ala Asp Ser Ile Leu 64Asn Ser Ser Ile Val Tyr Glu Thr Ala Asp Ser Ile Leu 64
CAC TCT CCG GGG TGT GTC CCT TGC GTT CGC GAG GGT AAC 233CAC TCT CCG GGG TGT GTC CCT TGC GTT CGC GAG GGT AAC 233
Kis Ser Pro Gly Cys Val Pro Cys Val Arg Glu Gly Asn 77Kis Ser Pro Gly Cys Val Pro Cys Val Arg Glu Gly Asn 77
ACC TCG AAA TGT TGG GTG GCG GTG GCC CCT ACA GTC GCC 272ACC TCG AAA TGT TGG GTG GCG GTG GCC CCT ACA GTC GCC 272
Thr Ser Lys Cys Trp Val Ala Val Ala Pro Thr Val Ala 90Thr Ser Lys Cys Trp Val Ala Val Ala Pro Thr Val Ala 90
ACC AGA GAC GGC AGA CTC CCC ACA ACG CAG CTT CGA CGT 311ACC AGA GAC GGC AGA CTC CCC ACA ACG CAG CTT CGA CGT 311
Thr Arg Asp Gly Arg Leu Pro Thr Thr Gin Leu Arg Arg 103Thr Arg Asp Gly Arg Leu Pro Thr Thr Gin Leu Arg Arg 103
CAT ATC GAT CTG CTC GTC GGG AGC GCC ACC CTC TGC TCG 350CAT ATC GAT CTG CTC GTC GGG AGC GCC ACC CTC TGC TCG 350
His Ile Asp Leu Leu Val Gly Ser Ala Thr Leu Cys Ser 116His Ile Asp Leu Leu Val Gly Ser Ala Thr Leu Cys Ser 116
GCC CTC TAT GTG GGG GAC TTG TGC GGG TCC GTC TTC CTC 389GCC CTC TAT GTG GGG GAC TTG TGC GGG TCC GTC TTC CTC 389
Ala Leu Tyr Val Gly Asp Leu Cys Gly Ser Val Phe Leu 129Ala Leu Tyr Val Gly Asp Leu Cys Gly Ser Val Phe Leu 129
GTC GGT CAA TTG TTC ACC TTC TCC CCC AGG CGC CAC TGG 428GTC GGT CAA TTG TTC ACC TTC TCC CCC AGG CGC CAC TGG 428
Val Gly Gin Leu Phe Thr Phe Ser Pro Arg Arg His Trp 142Val Gly Gin Leu Phe Thr Phe Ser Pro Arg Arg His Trp 142
ACA ACG CAA GAC TGC AAC TGT TCC ATC TAC CCC GGC CAC 467ACA ACG CAA GAC TGC AAC TGT TCC ATC TAC CCC GGC CAC 467
Thr Thr Gin Asp Cys Asn Cys Ser Ile Tyr Pro Gly His 155 IV - INF0RATI0N POUR SEQ ID N°3 TYPE Séquence nucléotidique et sa protéine correspondante.Thr Thr Gin Asp Cys Asn Cys Ser Ile Tyr Pro Gly His 155 IV - INF0RATI0N FOR SEQ ID N ° 3 TYPE Nucleotide sequence and its corresponding protein.
LONGUEUR 1210 paires de bases 403 acides aminésLENGTH 1210 base pairs 403 amino acids
NOMBRE DE BRINS simple CARACTERISTIQUES ADN codant pour la protéine E2/NS1 (totalité du gène E2/NS1 )NUMBER OF STRANDS simple CHARACTERISTICS DNA coding for the protein E2 / NS1 (entire gene E2 / NS1)
DESCRIPTION DE LA SEQUENCEDESCRIPTION OF THE SEQUENCE
A ATG GCT CAA CTG CTC AGG GTC CCG CAA GCC ATC TTG 37A ATG GCT CAA CTG CTC AGG GTC CCG CAA GCC ATC TTG 37
Met Ala Gin Leu Leu Arg Val Pro Gin Ala Ile Leu 12Met Ala Gin Leu Leu Arg Val Pro Gin Ala Ile Leu 12
GAC ATG ATC GCT GGT GCC CAC TGG GGA GTC CTA GCG GGC 76GAC ATG ATC GCT GGT GCC CAC TGG GGA GTC CTA GCG GGC 76
Asp Met Ile Ala Gly Ala His Trp Gly Val Leu Ala Gly 25Asp Met Ile Ala Gly Ala His Trp Gly Val Leu Ala Gly 25
ATA GCG TAT TTC TCC ATG GTG GGG AAC TGG GCG AAG GTC 115ATA GCG TAT TTC TCC ATG GTG GGG AAC TGG GCG AAG GTC 115
Ile Ala Tyr Phe Ser Met Val Gly Asn Trp Ala Lys Val 38Ile Ala Tyr Phe Ser Met Val Gly Asn Trp Ala Lys Val 38
CTG CTA GTG CTG TTG CTG TTC GCC GGC GTC GAT GCG GAA 154CTG CTA GTG CTG TTG CTG TTC GCC GGC GTC GAT GCG GAA 154
Leu Leu Val Leu Leu Leu Phe Ala Gly Val Asp Ala Glu 51Leu Leu Val Leu Leu Leu Phe Ala Gly Val Asp Ala Glu 51
ACC TAC ACC ACC GGG GGG AGT ACT GCC AGG ACC ACG CAA 193ACC TAC ACC ACC GGG GGG AGT ACT GCC AGG ACC ACG CAA 193
Thr Tyr Thr Thr Gly Gly Ser Thr Ala Arg Thr Thr Gin 64Thr Tyr Thr Thr Gly Gly Ser Thr Ala Arg Thr Thr Gin 64
GGA CTC GTC AGC CTT TTC AGT CGA GGC GCC AAG CAG GAC 232GGA CTC GTC AGC CTT TTC AGT CGA GGC GCC AAG CAG GAC 232
Gly Leu Val Ser Leu Phe Ser Arg Gly Ala Lys Gin Asp 77Gly Leu Val Ser Leu Phe Ser Arg Gly Ala Lys Gin Asp 77
ATC CAG CTG ATC AAC ACC AAC GGC AGC TGG CAC ATT AAT 271ATC CAG CTG ATC AAC ACC AAC GGC AGC TGG CAC ATT AAT 271
Ile Gin Leu Ile Asn Thr Asn Gly Ser Trp His Ile Asn 90Ile Gin Leu Ile Asn Thr Asn Gly Ser Trp His Ile Asn 90
CGC ACA GCT TTG AAC TGT AAT GAG AGC CTC GAC ACC GGC 310CGC ACA GCT TTG AAC TGT AAT GAG AGC CTC GAC ACC GGC 310
Arç Thr Ala Leu Asn Cys Asn Glu Ser Leu Asp Thr Gly 103Arç Thr Ala Leu Asn Cys Asn Glu Ser Leu Asp Thr Gly 103
TGG GTA GCG GGG CTC TTC TAT TAC CAC AAA TTC AAC TCT 349TGG GTA GCG GGG CTC TTC TAT TAC CAC AAA TTC AAC TCT 349
Trp Val Ala Gly Leu Phe Tyr Tyr His Lys Phe Asn Ser 116Trp Val Ala Gly Leu Phe Tyr Tyr His Lys Phe Asn Ser 116
TCA GGC TGC CCC GAG AGG ATG GCC AGC TGC AGA CCC CTT 388TCA GGC TGC CCC GAG AGG ATG GCC AGC TGC AGA CCC CTT 388
Ser Gly Cys Pro Glu Arg Met Ala Ser Cys Arg Pro Leu 129Ser Gly Cys Pro Glu Arg Met Ala Ser Cys Arg Pro Leu 129
GCC GAT TTC GAC CAG GGC TGG GGC CCT ATC AGT TAT GCC 427GCC GAT TTC GAC CAG GGC TGG GGC CCT ATC AGT TAT GCC 427
Ala Asp Phe Asp Gin Gly Trp Gly Pro Ile Ser Tyr Ala 142Ala Asp Phe Asp Gin Gly Trp Gly Pro Ile Ser Tyr Ala 142
AAC GGA ACC GGC CCT GAA CAC CGC CCC TAC TGC TGG CAC 466AAC GGA ACC GGC CCT GAA CAC CGC CCC TAC TGC TGG CAC 466
Asn Gly Thr Gly Pro Glu His Arg Pro Tyr Cys Trp His 155 TAC CCC CCA AAG CCT TGT GGT ATC GTG CCA GCA CAG ACC 505 Tyr Pro Pro Lys Pro Cys Gly Ile Val Pro Ala Gin Thr 168 Asn Gly Thr Gly Pro Glu His Arg Pro Tyr Cys Trp His 155 TAC CCC CCA AAG CCT TGT GGT ATC GTG CCA GCA C A G ACC 505 Tyr Pro Pro Lys Pro Cys Gly Ile Val Pro Ala Gin Thr 1 68
GTA TGT GGC CCA GTG TAT TGC TTC ACT CCT AGC CCC GTG 544 Val Cys Gly Pro Val Tyr Cys Phe Thr Pro Ser Pro Val ιsi GTG GTG GGG ACG ACC AAT AAG TTG GGC GCA CCC ACT TAC 583 Val Val Gly Thr Thr Asn Lys Leu Gly Ala Pro Thr Tyr 194GTA TGT GGC CCA GTG TAT TGC TTC ACT CCT AGC CCC G T G 5 44 Val Cys Gly Pro Val Tyr Cys Phe Thr Pro Ser Pro Val ιs i G T GG TG GGG ACG ACC AAT AAG TTG GGC GCA CCC ACT TAC 583 Val Val Gly Thr Thr Asn Lys Leu Gly Ala Pro Thr Tyr 194
AAC TGG GGT TGT AAT GAT ACG GAC GTC TTC GTC CTT AAT 622 Asn Trp Gly Cys Asn Asp Thr Asp Val Phe Val Leu Asn 207AA C TGG GGT TGT AAT GAT ACG GAC GTC TTC GTC CTT AAT 622 Asn Trp Gly Cys Asn Asp Thr Asp Val Phe Val Leu Asn 207
AAC ACC AGG CCA CCG CTG GGC AAT TGG TTC GGC TGC ACC 661 Asn Thr Arg Pro Pro Leu Gly Asn Trp Phe Gly Cys Thr 220AA C ACC AGG CCA CCG CTG GGC AAT TGG TTC GGC TGC ACC 661 Asn Thr Arg Pro Pro Leu Gly Asn Trp Phe Gly Cys Thr 220
TGG GTG AAC TCA TCT GGA TTT ACT AAA GTG TGC GGA GCG 700T GG G TG AAC TCA TCT GGA TTT ACT AAA GTG TGC GGA GCG 700
Trp Val Asn Ser Ser Gly Phe Thr Lys Val Cys Gly Ala 233Trp Val Asn Ser Ser Gly Phe Thr Lys Val Cys Gly Ala 233
CCT CCC TGT GTC ATC GGA GGA GCG GGC AAT AAC ACC TTG 739 Pro Pro Cys Val Ile Gly Gly Ala Gly Asn Asn Thr Leu 246CCT CCC TGT GTC ATC GGA GGA GCG GGC AAT AAC ACC TTG 739 Pro Pro Cys Val Ile Gly Gly Ala Gly Asn Asn Thr Leu 246
TAC TGC CCC ACT GAC TGT TTC CGC AAG CAT CCG GAA GCT 778 Tyr Cys Pro Thr Asp Cys Phe Arg Lys His Pro Glu Ala 259TAC TGC CCC ACT GAC TGT TTC CGC AAG CAT CCG GAA GCT 778 Tyr Cys Pro Thr Asp Cys Phe Arg Lys His Pro Glu Ala 259
ACA TAC TCC CGA TGT GGC TCC GGT CCT TGG ATC ACG CCC 817 Thr Tyr Ser Arg Cys Gly Ser Gly Pro Trp Ile Thr Pro 272ACA TAC TCC CGA TGT GGC TCC GGT CCT TGG ATC ACG CCC 817 Thr Tyr Ser Arg Cys Gly Ser Gly Pro Trp Ile Thr Pro 272
AGG TGC CTG GTT GGC TAT CCT TAT AGG CTC TGG CAT TAT 856 Arg Cys Leu Val Gly Tyr Pro Tyr Arg Leu Trp His Tyr 285AGG TGC CTG GTT GGC TAT CCT TAT AGG CTC TGG CAT TAT 856 Arg Cys Leu Val Gly Tyr Pro Tyr Arg Leu Trp His Tyr 285
CCC TGT ACT GTC AAC TAC ACC CTG TTC AAG GTC AGG ATG 895 Pro Cys Thr Val Asn Tyr Thr Leu Phe Lys Val Arg Met 298CCC TGT ACT GTC AAC TAC ACC CTG TTC AAG GTC AGG ATG 895 Pro Cys Thr Val Asn Tyr Thr Leu Phe Lys Val Arg Met 298
TAC GTG GGA GGG GTC GAG CAC AGG CTG CAA GTC GCT TGC 934 Tyr Val Gly Gly Val Glu His Arg Leu Gin Val Ala Cys 311TAC GTG GGA GGG GTC GAG CAC AGG CTG CAA GTC GCT TGC 934 Tyr Val Gly Gly Val Glu His Arg Leu Gin Val Ala Cys 311
AAC TGG ACG CGG GGC GAG CGT TGT AAT CTG GAC GAC AGG 973 Asn Trp Thr Arg Gly Glu Arg Cys Asn Leu Asp Asp Arg 324AAC TGG ACG CGG GGC GAG CGT TGT AAT CTG GAC GAC AGG 973 Asn Trp Thr Arg Gly Glu Arg Cys Asn Leu Asp Asp Arg 324
GAC AGG TCC GAG CTC AGT CCG CTG CTG CTG TCT ACC ACA 1012 Asp Arg Ser Glu Leu Ser Pro Leu Leu Leu Ser Thr Thr 337GAC AGG TCC GAG CTC AGT CCG CTG CTG CTG TCT ACC ACA 1012 Asp Arg Ser Glu Leu Ser Pro Leu Leu Leu Ser Thr Thr 337
CAG TGG CAG GTC CTC CCG TGT TCC TTT ACG ACC TTG CCA 1051 Gin Trp Gin Val Leu Pro Cys Ser Phe Thr Thr Leu Pro 350CAG TGG CAG GTC CTC CCG TGT TCC TTT ACG ACC TTG CCA 1051 Gin Trp Gin Val Leu Pro Cys Ser Phe Thr Thr Leu Pro 350
GCC TTG ACT ACC GGC CTC ATC CAC CTC CAC CAG AAC ATC 1090 Ala Leu Thr Thr Gly Leu Ile His Leu His Gin Asn Ile 363GCC TTG ACT ACC GGC CTC ATC CAC CTC CAC CAG AAC ATC 1090 Ala Leu Thr Thr Gly Leu Ile His Leu His Gin Asn Ile 363
GTG GAC GTG CAA TAT TTG TAC GGG GTG GGG TCA AGC ATT 1129 Val Asp Val Gin Tyr Leu Tyr Gly Val Gly Ser Ser Ile 376GTG GAC GTG CAA TAT TTG TAC GGG GTG GGG TCA AGC ATT 1129 Val Asp Val Gin Tyr Leu Tyr Gly Val Gly Ser Ser Ile 376
GTG TCC TGG GCC ATC AAG TGG GAG TAC GTC ATT CTC CTG 1168 Val Ser Trp Ala Ile Lys Trp Glu Tyr Val Ile Leu Leu 389GTG TCC TGG GCC ATC AAG TGG GAG TAC GTC ATT CTC CTG 1168 Val Ser Trp Ala Ile Lys Trp Glu Tyr Val Ile Leu Leu 389
TTT CTC CTG CTT GCA GAC GCG CGC GTC TGC TCC TGC TTG 1207 Phe Leu Leu Leu Ala Asp Ala Arg Val Cys Ser Cys Leu 402TTT CTC CTG CTT GCA GAC GCG CGC GTC TGC TCC TGC TTG 1207 Phe Leu Leu Leu Ala Asp Ala Arg Val Cys Ser Cys Leu 402
TGG 1210 V - INFORMATION POUR SEQ ID N°4 TYPE Séquence nucléotidique et sa protéine correspondante.TGG 1210 V - INFORMATION FOR SEQ ID N ° 4 TYPE Nucleotide sequence and its corresponding protein.
LONGUEUR 943 paires de bases 313 acides aminésLENGTH 943 base pairs 313 amino acids
NOMBRE DE BRINS simple CARACTERISTIQUE ADN codant pour une séquence de la régon non structurale (parties 3' et 5' terminales des gènes NS3 et NS4 respective¬ ment)NUMBER OF STRANDS simple CHARACTERISTIC DNA coding for a sequence of the non-structural region (3 'and 5' terminal parts of the NS3 and NS4 genes respectively)
DESCRIPTION DE LA SEQUENCE DESCRIPTION OF THE SEQUENCE
?6? 6
AC AAT ACG TGT GTC ACC CAG ACA GTC GAC TTC AGC CTT 38AC AAT ACG TGT GTC ACC CAG ACA GTC GAC TTC AGC CTT 38
Asn Thr Cys Val Thr Gin Thr Val Asp Phe Ser Leu 12Asn Thr Cys Val Thr Gin Thr Val Asp Phe Ser Leu 12
GAC CCT ACC TTC ACC ATT GAA ACA ACA ACG CTT CCC CAG 77GAC CCT ACC TTC ACC ATT GAA ACA ACA ACG CTT CCC CAG 77
Asp Pro Thr Phe Thr Ile Glu Thr Thr Thr Leu Pro Gin 25Asp Pro Thr Phe Thr Ile Glu Thr Thr Thr Leu Pro Gin 25
GAT GCT GTC TCC CGC ACT CAA CGT CGG GGC AGG ACT GGC 116GAT GCT GTC TCC CGC ACT CAA CGT CGG GGC AGG ACT GGC 116
Asp Ala Val Ser Arg Thr Gin Arg Arg Gly Arg Thr Gly 38Asp Ala Val Ser Arg Thr Gin Arg Arg Gly Arg Thr Gly 38
AGG GGG AAG CCA GGC ATT TAC AGA TTT GTG GCA CCT GGA 155AGG GGG AAG CCA GGC ATT TAC AGA TTT GTG GCA CCT GGA 155
Arg Gly Lys Pro Gly Ile Tyr Arg Phe Val Ala Pro Gly 51Arg Gly Lys Pro Gly Ile Tyr Arg Phe Val Ala Pro Gly 51
GAG CGC CCC TCC GGC ATG TTC GAC TCG TCC GTC CTC TGC 194GAG CGC CCC TCC GGC ATG TTC GAC TCG TCC GTC CTC TGC 194
Glu Arg Pro Ser Gly Met Phe Asp Ser Ser Val Leu Cys 64Glu Arg Pro Ser Gly Met Phe Asp Ser Ser Val Leu Cys 64
GAG TGC TAT GAC GCA GGC TGT GCT TGG TAT GAG CTC ACG 233GAG TGC TAT GAC GCA GGC TGT GCT TGG TAT GAG CTC ACG 233
Glu Cys Tyr Asp Ala Gly Cys Ala Trp Tyr Glu Leu Thr 77Glu Cys Tyr Asp Ala Gly Cys Ala Trp Tyr Glu Leu Thr 77
CCC GCC GAG ACC ACA GTC AGG CTA CGA GCA TAC ATG AAC 272CCC GCC GAG ACC ACA GTC AGG CTA CGA GCA TAC ATG AAC 272
Pro Ala Glu Thr Thr Val Arg Leu Arg Ala Tyr Met Asn 90Pro Ala Glu Thr Thr Val Arg Leu Arg Ala Tyr Met Asn 90
ACC CCG GGA CTT CCC GTG TGC CAA GAC CAT CTT GAG TTT 311ACC CCG GGA CTT CCC GTG TGC CAA GAC CAT CTT GAG TTT 311
Thr Pro Gly Leu Pro Val Cys Gin Asp His Leu Glu Phe 103Thr Pro Gly Leu Pro Val Cys Gin Asp His Leu Glu Phe 103
TGG GAG GGC GTC TTC ACG GGT CTC ACC CAT ATA GAC GCC 350TGG GAG GGC GTC TTC ACG GGT CTC ACC CAT ATA GAC GCC 350
Trp Glu Gly Val Phe Thr Gly Leu Thr His Ile Asp Ala 116Trp Glu Gly Val Phe Thr Gly Leu Thr His Ile Asp Ala 116
CAC TTC CTA TCC CAG ACA AAG CAG AGT GGG GAA AAC CTT 389CAC TTC CTA TCC CAG ACA AAG CAG AGT GGG GAA AAC CTT 389
His Phe Leu Ser Gin Thr Lys Gin Ser Gly Glu Asn Leu 129His Phe Leu Ser Gin Thr Lys Gin Ser Gly Glu Asn Leu 129
CCT TAC CTG GTA GCG TAC CAA GCC ACC GTG TGC GCT AGG 428CCT TAC CTG GTA GCG TAC CAA GCC ACC GTG TGC GCT AGG 428
Pro Tyr Leu Val Ala Tyr Gin Ala Thr Val Cys Ala Arg 142Pro Tyr Leu Val Ala Tyr Gin Ala Thr Val Cys Ala Arg 142
GCC CAA GCC CCT CCC CCG TCG TGG GAC CAG ATG TGG AAG 467GCC CAA GCC CCT CCC CCG TCG TGG GAC CAG ATG TGG AAG 467
Ala Gin Ala Pro Pro Pro Ser Trp Asp Gin Met Trp Lys 155Ala Gin Ala Pro Pro Pro Ser Trp Asp Gin Met Trp Lys 155
TGC TTG ATT CGT CTC AAG CCC ACC CTC CAT GGG CCA ACA 506TGC TTG ATT CGT CTC AAG CCC ACC CTC CAT GGG CCA ACA 506
Cys Leu Ile Arg Leu Lys Pro Thr Leu His Gly Pro Thr 168Cys Leu Ile Arg Leu Lys Pro Thr Leu His Gly Pro Thr 168
CCC CTG CTA TAC CGA CTG GGC GCT GTT CAG AAT GAA GTC 545CCC CTG CTA TAC CGA CTG GGC GCT GTT CAG AAT GAA GTC 545
Pro Leu Leu Tyr Arg Leu Gly Ala Val Gin Asn Glu Val 181Pro Leu Leu Tyr Arg Leu Gly Ala Val Gin Asn Glu Val 181
ACC CTG ACG CAC CCA ATC ACC AAA TAT ATC ATG ACA TGC 584ACC CTG ACG CAC CCA ATC ACC AAA TAT ATC ATG ACA TGC 584
Thr Leu Thr His Pro Ile Thr Lys Tyr Ile Met Thr Cys 194Thr Leu Thr His Pro Ile Thr Lys Tyr Ile Met Thr Cys 194
ATG TCG GCT GAC CTG GAG GTC GTC ACG AGT ACC TGG GTG 623ATG TCG GCT GAC CTG GAG GTC GTC ACG AGT ACC TGG GTG 623
Met Ser Ala Asp Leu Glu Val Val Thr Ser Thr Trp Val 207Met Ser Ala Asp Leu Glu Val Val Thr Ser Thr Trp Val 207
CTC GTG GGC GGC GTT CTG GCT GCT TTG GCC GCG TAT TGC 662CTC GTG GGC GGC GTT CTG GCT GCT TTG GCC GCG TAT TGC 662
Leu Val Gly Gly Val Leu Ala Ala Leu Ala Ala Tyr Cys 220Leu Val Gly Gly Val Leu Ala Ala Leu Ala Ala Tyr Cys 220
CTA TCC ACA GGC TGC GTG GTC ATA GTA GGC AGG GTC ATT 701CTA TCC ACA GGC TGC GTG GTC ATA GTA GGC AGG GTC ATT 701
Leu Ser Thr Gly Cys Val Val Ile Val Gly Arg Val Ile 233 TTG TCC GGG AAG CCG GCA ATC ATA CCC GAC AGG GAA GTC 740 Leu Ser Gly Lys Pro Ala Ile Ile Pro Asp Arg Glu Val 246Leu Ser Thr Gly Cys Val Val Ile Val Gly Arg Val Ile 233 TTG TCC GGG AAG CCG GCA ATC ATA CCC GAC AGG GAA GTC 740 Leu Ser Gly Lys Pro Ala Ile Ile Pro Asp Arg Glu Val 246
CTC TAC CGG GAG TTC GAT GAG ATG GAA GAG TGC TCT CAG 779CTC TAC CGG GAG TTC GAT GAG ATG GAA GAG TGC TCT CAG 779
Leu Tyr Arg Glu Phe Asp Glu Met Glu Glu Cys Ser Gin 259Leu Tyr Arg Glu Phe Asp Glu Met Glu Glu Cys Ser Gin 259
CAC TTG CCA TAC ATC GAG CAA GGG ATG ATG CTC GCC GAG 818CAC TTG CCA TAC ATC GAG CAA GGG ATG ATG CTC GCC GAG 818
His Leu Pro Tyr Ile Glu Gin Gly Met Met Leu Ala Glu 272His Leu Pro Tyr Ile Glu Gin Gly Met Met Leu Ala Glu 272
CAG TTC AAG CAG AAG GCC CTC GGC CTC CTG CAA ACA CGG 857CAG TTC AAG CAG AAG GCC CTC GGC CTC CTG CAA ACA CGG 857
Gin Phe Lys Gin Lys Ala Leu Gly Leu Leu Gin Thr Arg 285Gin Phe Lys Gin Lys Ala Leu Gly Leu Leu Gin Thr Arg 285
TCC CGC CAG GCA GAG GTC ATC ACC CCT GCT GTC CAG ACC 896TCC CGC CAG GCA GAG GTC ATC ACC CCT GCT GTC CAG ACC 896
Ser Arg Gin Ala Glu Val Ile Thr Pro Ala Val Gin Thr 298Ser Arg Gin Ala Glu Val Ile Thr Pro Ala Val Gin Thr 298
AAC TGG CAG AGA CTC GAG GCC TTC TGG GCG AAG CAT ATG 935AAC TGG CAG AGA CTC GAG GCC TTC TGG GCG AAG CAT ATG 935
Asn Trp Gin Arg Leu Glu Ala Phe Trp Ala Lys His Met 311Asn Trp Gin Arg Leu Glu Ala Phe Trp Ala Lys His Met 311
TGG AAC TT 943TGG AAC TT 943
Trp Asn 313 Trp Asn 313

Claims

REVENDICATIONS 1. Séquence d'ADN de HCV El comprenant une séquence d'ADN choisie parmi les séquences nucléoti¬ diques d'au moins 10 nucléotides comprises entre les nucléotides (n) suivants : n^g à n^g ; nι77 à n2Q2 ' n233 a n247 ; n254 a n272 et n272 a n288 représentés sur la séquence SEQ ID N°2, et, à n217 ' n267 a n283 e^ n310 a n334 rePr®sentés sur la séquence SEQ ID N°3 ; ainsi que les séquences nucléo- tidiques analogues résultant de la dégénérescence du code génétique.CLAIMS 1. DNA sequence of HCV E1 comprising a DNA sequence chosen from nucleotide sequences of at least 10 nucleotides comprised between the following nucleotides (n): n ^ g to n ^ g; nι 77 to n 2Q 2 ' n 233 year 247 ; n 254 year 272 and n 272 year 288 represented on the sequence SEQ ID N ° 2, and, at n 217 ' n 267 year 283 e ^ n 310 year 334 re P r® sent on the sequence SEQ ID N ° 3; as well as analogous nucleotide sequences resulting from the degeneracy of the genetic code.
2. Séquence d'ADN de HCV El comprenant une séquence d'ADN choisie parmi la séquence SEQ ID N°2, la séquence SEQ ID N°3 et la séquence SEQ ID N°4' ; ainsi que les séquences analogues résultant de la dégénérescence du code génétique.2. DNA sequence of HCV E1 comprising a DNA sequence chosen from the sequence SEQ ID No 2, the sequence SEQ ID No 3 and the sequence SEQ ID No 4 ' ; as well as analogous sequences resulting from the degeneration of the genetic code.
3. Séquence peptidique de HCVEl comprenant une séquence peptidique choisie parmi les séquences d'au moins 7 aminoacides compris entre les aminoacides (aa) suivants : aa5g à aagg et aa7g à aaioi représen¬ tés sur la séquence peptidique SEQ ID N°2, aa4g à aa78; aag8 à aaιn ; aai23 à aaι33 et aa^Q à aaι4g représentés sur la séquence peptidique SEQ ID N°3 ; ainsi que des séquences peptidiques homologues n'induisant pas de modification des propriétés biolo¬ giques et immunologiques.3. HCVEl peptide sequence comprising a peptide sequence chosen from the sequences of at least 7 amino acids between the following amino acids (aa): aa5g to aagg and aa 7 g to aaioi shown on the peptide sequence SEQ ID N ° 2 , aa 4 g to aa 78 ; aa g8 to aa ιn ; aa i2 3 to aaι 33 and aa ^ Q to aaι 4g represented on the peptide sequence SEQ ID N ° 3; as well as homologous peptide sequences which do not induce any modification of the biological and immunological properties.
4. Séquence peptidique de HCV El comprenant une séquence peptidique choisie parmi les séquences d'aminoacides suivants : aa5g à aagg et aa7g à aaïQi représentés sur la séquence peptidique SEQ ID N°2, aa4g à aa78 ; aag8 à aaιn ; aaι23 à aaι33 et aaι40 à aa149 représentés sur la séquence peptidique SEQ ID N°3 ; ainsi que des séquences homologues n'induisant pas de modification des propriétés biologiques et immunologiques. 4. HCV E1 peptide sequence comprising a peptide sequence chosen from the following amino acid sequences: aa5g to aagg and aa 7 g to aai Q i represented on the peptide sequence SEQ ID No. 2, aa 4g to aa 78 ; aa g8 to aa ιn ; aaι 23 to aaι 33 and aaι 40 to aa 149 represented on the peptide sequence SEQ ID N ° 3; as well as homologous sequences which do not induce any modification of the biological and immunological properties.
5. Séquence peptidique de HCV El comprenant une séquence peptidique choisie parmi les séquences peptidiques SEQ ID Nβ2, SEQ ID N°3 et SEQ ID N°4 ; ainsi que des séquences homologues n'induisant pas de modification des propriétés biologiques et immunolo¬ giques.5. HCV E1 peptide sequence comprising a peptide sequence chosen from the peptide sequences SEQ ID N β 2, SEQ ID No.3 and SEQ ID No.4; as well as homologous sequences which do not induce any modification of the biological and immunological properties.
6. Séquence nucléotidique codant pour une séquence peptidique selon la revendication 3 ou la revendication 4. 6. Nucleotide sequence coding for a peptide sequence according to claim 3 or claim 4.
7. Sonde polynucléotidique caractérisée en ce qu'elle comprend une séquence d'ADN selon la revendication 1.7. Polynucleotide probe characterized in that it comprises a DNA sequence according to claim 1.
8. Séquence peptidique immunogène comportant une séquence peptidique selon la revendication 3 ou 4. 8. Immunogenic peptide sequence comprising a peptide sequence according to claim 3 or 4.
9. Vecteur d'expression comprenant une séquence d'ADN codant pour une séquence peptidique selon 1'une des revendication 3, 4 et 5.9. Expression vector comprising a DNA sequence coding for a peptide sequence according to one of claims 3, 4 and 5.
10. Plasmide comprenant une séquence d'ADN codant pour une séquence peptidique selon la revendi- cation 5, choisi parmi les plasmides déposés à la CNCM le 5 juin 1991 sous les n° 1-1105, 1-1106 et 1-1107.10. Plasmid comprising a DNA sequence coding for a peptide sequence according to claim 5, chosen from the plasmids deposited at the CNCM on June 5, 1991 under the numbers 1-1105, 1-1106 and 1-1107.
11. Hôte transformé avec un vecteur selon la revendication 9.11. Host transformed with a vector according to claim 9.
12. Anticorps monoclonaux humains ou murins dirigés contre une séquence peptidique selon l'une des revendications 3, 4 ou 5.12. Human or murine monoclonal antibodies directed against a peptide sequence according to one of claims 3, 4 or 5.
13. Fragments Fab, Fab', F(ab' )2 ou Fc d'anticorps monoclonaux selon la revendication 12.13. Fab, Fab ', F (ab') 2 or Fc fragments of monoclonal antibodies according to claim 12.
14. Dérivés d'anticorps monoclonaux com- prenant des anticorps monoclonaux selon la revendi¬ cation 12 ou des fragments selon la revendication 13 auxquels sont liés des marqueurs ou des molécules thérapeutiquement actives.14. Monoclonal antibody derivatives comprising monoclonal antibodies according to CLAIM 12 or fragments according to claim 13 to which markers or therapeutically active molecules are linked.
15. Hybridomes produisant des anticorps monoclonaux selon la revendication 12.15. Antibody producing hybridomas monoclonals according to claim 12.
16. Trousse d'analyse pour la détection de séquences nucléotidiques de virus de 1'hépatite C comportant une ou plusieurs sonde(s) polynucléo- tidique(s) selon la revendication 7.16. Analysis kit for the detection of nucleotide sequences of hepatitis C virus comprising one or more polynucleotide probe (s) according to claim 7.
17. Procédé de diagnostic in vitro par mise en évidence des antigènes spécifiques de HCVEl dans un prélèvement biologique pouvant contenir lesdits antigènes, dans lequel on met en présence le prélève- ment biologique avec un anticorps ou un fragement d'anticorps, tels que définis dans les revendications 12 et 13.17. In vitro diagnostic method by demonstrating specific antigens of HCVEl in a biological sample which may contain said antigens, in which the biological sample is brought into contact with an antibody or an antibody frage, as defined in claims 12 and 13.
18. Trousse de diagnostic pour la détection d'antigènes spécifiques de HCV El comprenant un anticorps ou un fragment d'anticorps tels que définis à la revendication 12 ou 13 qui provoque une réaction immunologique avec un antigène HCV.18. A diagnostic kit for the detection of specific antigens of HCV E1 comprising an antibody or an antibody fragment as defined in claim 12 or 13 which causes an immunological reaction with an HCV antigen.
19. Procédé de diagnostic in vitro par mise en évidence des anticorps spécifiques de HCV El dans un prélèvement biologique pouvant contenir lesdits anticorps, dans lequel on met en présence un prélè¬ vement biologique avec un antigène comportant un épitope correspondant à une séquence peptidique selon la revendication 3, 4 ou 5. 19. In vitro diagnostic method by detecting antibodies specific for HCV El in a biological sample which may contain said antibodies, in which a biological sample is brought into contact with an antigen comprising an epitope corresponding to a peptide sequence according to the claim 3, 4 or 5.
20. Trousse de diagnostic pour la détection d'anticorps spécifiques de HCV El comprenant un antigène comportant un épitope correspondant à une séquence peptidique selon la revendication 3, 4 ou 5.20. A diagnostic kit for the detection of antibodies specific to HCV E1 comprising an antigen comprising an epitope corresponding to a peptide sequence according to claim 3, 4 or 5.
21. Composition thérapeutique comprenant des anticorps monoclonaux selon la revendication 12, des fragments d'anticorps monoclonaux selon la revendica¬ tion 13 ou des dérivés d'anticorps monoclonaux selon la revendication 14.21. Therapeutic composition comprising monoclonal antibodies according to claim 12, fragments of monoclonal antibodies according to CLAIM 13 or derivatives of monoclonal antibodies according to claim 14.
22. Composition thérapeutique selon la revendication 21, dans laquelle les dérivés d'anti¬ corps monoclonaux sont des anticorps monoclonaux ou des fragments de ces anticorps liés à une molécule thérapeutiquement active. 22. Therapeutic composition according to the claim 21, in which the derivatives of monoclonal antibodies are monoclonal antibodies or fragments of these antibodies linked to a therapeutically active molecule.
23. Composition immunogène comportant une séquence immunogène selon la revendication 8 liée à une protéine porteuse, ladite séquence immunogène étant susceptible d'induire des anticorps protecteurs ou d'induire des lymphocytes T cytotoxiques. 23. An immunogenic composition comprising an immunogenic sequence according to claim 8 linked to a carrier protein, said immunogenic sequence being capable of inducing protective antibodies or of inducing cytotoxic T lymphocytes.
24. Procédé de dosage d'un polypeptide selon les revendications 3, 4 ou 5 comprenant 1'utilisation d'anticorps monoclonaux dirigés contre un polypeptide selon la revendication 3, 4 ou 5 ou une séquence immunogène d'un tel polypeptide. 24. A method of assaying a polypeptide according to claims 3, 4 or 5 comprising the use of monoclonal antibodies directed against a polypeptide according to claim 3, 4 or 5 or an immunogenic sequence of such a polypeptide.
25. Procédé de préparation d'un polypeptide selon l'une quelconque des revendications 3 à 5, com¬ prenant 1'insertion d'une séquence d'ADN codant pour le polypeptide dans un vecteur d'expression, la trans¬ formation de cellules avec ce vecteur d'expression et l culture de ces cellules. 25. A method of preparing a polypeptide according to any one of claims 3 to 5, comprising the insertion of a DNA sequence coding for the polypeptide in an expression vector, the transformation of cells with this expression vector and the culture of these cells.
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FR2677372B1 (en) 1991-06-06 1994-11-10 Pasteur Institut NUCLEOTIDE AND PEPTIDE SEQUENCES OF A HEPATITIS C VIRUS ISOLATE, DIAGNOSTIC AND THERAPEUTIC APPLICATIONS.
RO117329B1 (en) * 1991-06-24 2002-01-30 Chiron Corp Emeryville Polypeptides containing a hcv sequence
US5866139A (en) * 1992-06-04 1999-02-02 Institut Pasteur Nucleotide and peptide sequences of a hepatitis C virus isolate, diagnostic and therapeutic applications
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US6210962B1 (en) 2001-04-03

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