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WO1993002103A1 - Epitopes de la proteine env du virus de l'hepatite c - Google Patents

Epitopes de la proteine env du virus de l'hepatite c Download PDF

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Publication number
WO1993002103A1
WO1993002103A1 PCT/IT1992/000081 IT9200081W WO9302103A1 WO 1993002103 A1 WO1993002103 A1 WO 1993002103A1 IT 9200081 W IT9200081 W IT 9200081W WO 9302103 A1 WO9302103 A1 WO 9302103A1
Authority
WO
WIPO (PCT)
Prior art keywords
amino acid
seq
env
sequence according
sequence
Prior art date
Application number
PCT/IT1992/000081
Other languages
English (en)
Inventor
Carlo Rosa
Silvia Griva
Fabrizio Bonelli
Original Assignee
Sorin Biomedica S.P.A.
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Sorin Biomedica S.P.A. filed Critical Sorin Biomedica S.P.A.
Publication of WO1993002103A1 publication Critical patent/WO1993002103A1/fr

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/24011—Flaviviridae
    • C12N2770/24211—Hepacivirus, e.g. hepatitis C virus, hepatitis G virus
    • C12N2770/24222—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • This invention relates to epitopes of the env protein of the hepatitis C virus.
  • this invention relates to peptides comprising epitopes of the hepatitis C virus (HCV) localized in the envelope surface viral protein (env), which are capable of reacting with antisera and/or with monoclonal antibodies and it also relates to the amino acids sequence of said epitopes, as well as to the nucleotide sequence coding for the sames.
  • HCV hepatitis C virus
  • env envelope surface viral protein
  • the virus HCV is believed to be responsible for the hepatites classified as non-A/non-B (PT-NANB) (1).
  • HCV hepatitis A
  • the preparation of immunological tests requires the availability of synthetic peptides capable of mimicking the immunological activity of viral antigens.
  • synthetic peptides capable of mimicking the immunological activity of viral antigens.
  • the identification of specific protein portions, denominated epitopes, capable of reacting with antibodies is necessary due to the short length of synthetic peptides.
  • tests which employ just the epitope of the protein are more sensitive and more accurate.
  • RNA viruses are characterized by a high frequency of spontaneous mutation.
  • variable and hypervariable domains have been identified in the sequences corresponding to the surface proteins (5 and EP 004191.82A1), possibly related to viral mechanisms of escaping of the immune response.
  • NANB hepatitis becomes a chronic disease in about 50 % of patients. It is therefore very useful to identify epitopes of surface proteins both for diagnosis and for prognosis purposes.
  • the Authors of this invention have identified variable regions with a high antigenic activity of the amino acid sequence of the env protein, and they have found that such regions correspond to epitopes of said protein.
  • the Authors also have identified some variants of such regions by means of amplification of nucleic acids from serum samples; among such regions, one is coded by a HCV variant not disclosed before.
  • the endemic distribution of the different viral variants of HCV virus makes it necessary to prepare assays able to detect epitopes of the different variants.
  • the Authors have synthesized such epitopes in vitro for immunological assays on serum samples.
  • an amino acid sequence comprising an epitope of the env protein of the HCV virus, preferably in the portion from the amino acid 209 to the amino acid 259, according to the numeration as given in (3).
  • said sequences are included in the following group of sequences: SEQ ID N1, SEQ ID N2 and SEQ ID N3; preferably from the amino acid 13 to the amino acid 46 of SEQ ID N1, more preferably from the amino acid 21 to the amino acid 30 of SEQ ID N1; alternatively from the amino acid 13 to the amino acid 46 of SEQ ID N2, preferably from the amino acid 21 to the amino acid 30 of SEQ ID N2; alternatively from the amino acid 13 to the amino acid 47 of SEQ ID N3, preferably from the amino acid 21 to the amino acid 31 of SEQ ID N3.
  • nucleotide sequence coding for an epitope of the env protein preferably comprised in one of the sequences of the following group: SEQ ID N1, SEQ ID N2, and SEQ ID N3; preferably comprising at least one fragment of 10 nucleotides of SEQ ID N3, more preferably the entire sequence of SEQ ID N3.
  • the variant env 1 and env 2 are comprised in viral variants respectively known by those skilled in the art as HCV A1 (american isolate) and HCV J1 (japan isolate).
  • the variant env 3 is coded by a viral variant which is not included in any HCV isolate disclosed up to the present invention, denominated HCV 3.
  • Such variant differentiates mainly by the insertion of a histidine residue into a region delimited by 2 cyste- ines, which modifies the hydrophilic profile of the genie product ( Figures 1A, 1B and 1C).
  • Such modification is of particular relevance for the analogy with the transmembrane region of the HIV1 surface protein (8, 9).
  • Oligopeptides comprising respectively the sequence of the env protein from the amino acid 13 to the amino acid 32 of the SEQ ID N1 (the env 1 variant); the sequence of the env protein from the amino acid 13 to the amino acid 32 of SEQ ID N2 (the env 2 variant); the sequence of the env protein from the amino acid 13 to the amino acid 33 of SEQ ID N3 (the env 3 variant) are synthesized according to Merrifield's method (10), employing as the solid phase a polyamide resin "Pepsin K polyamide Kieselguliz" (Milligen, Novato, California), which had been previously functionalized with ethilendiamine and with 4-(alpha-Fmoc-amino- 2',4'-dimethoxybenzyl)phenoxyacetic acid.
  • Merrifield's method 10
  • the amino acids employed for the synthesis are protected on the side chains by tert-butyl groups and on the alpha-amino position with the F-moc group (9-fluoro-methyloxycarbonyl group).
  • the guanidinium group of arginine and the imidazole group of histidine is respectively protected with the substituents consisting of the 2,2,5,7,8-pentamethylchroman- 6-sulfonyl and trityl groups.
  • the carboxy group of the amino acids employed is activated by the formation of an ester-type bond with the pentafluorophenyl group.
  • the synthesis is performed with the Milligen 9050 synthesizer (Novato, California) employing the continuous flow method.
  • the removal of protection and the separation of the peptides from the resin are carried out by treatment with trifluoroacetic acid.
  • the peptide sequence is checked with an automatic microsequencer (Portan Instruments).
  • Oligopeptides comprising respectively the sequence of the env protein from the amino acid 21 to the amino acid 30 of the SEQ ID N1 (the env 1 variant); the sequence of the env protein from the amino acid 21 to the amino acid 30 of SEQ ID N2 (the env 2 variant); the sequence of the env protein from the amino acid 21 to the amino acid 31 of SEQ ID N3 (the env 3 variant) are synthesized according to the Example 1.
  • the cyclization of a fraction of the peptides is carried out in the following way: the peptide is dissolved in water to a concentration of 0.1 mg/ml. The pH value is adjusted to 7 with 1M NH 4 OH. Potassium ferricyanide is then added slowly to the solution (400 mg K 3 Fe(CN) 6 in 200 ml of water) till persistence of the yellow colour. The disappearance of the free SH groups is obtained employing the method of Edman (11).
  • the peptide is dissolved at 0.2 mg/ml in distilled/deionized water (Milliq) and the pH is adjusted to pH 8 using a solution of 3M NH.Cl. The solution is allowed to stir for four days and the loss of the free sulphide groups is monitored using the Edman titration. Briefly, 24 mg of 5-5'dithio-bis (2-nitrobenzoic acid) is dissolved in 5 ml of phosphate buffer pH 7. 20 ⁇ l of this solution is mixed with 1 ml of the peptide solution and the absorbance is read at 412 nm. After four days 96% of the free sulphide groups are disappeared.
  • the ELISA assay is performed by dispensing 200 ⁇ l/well of sera, previously diluted, using a HCV negative serum, as sample diluent. The samples are incubated for 1 hr at 37°C. The plates are then washed five times with a solution containing 0.05% Tween-20, 0.1% BSA in 50mM phosphate buffer pH 7.4 (washing buffer) and incubated for 1 hr at 37°C with 200 ⁇ l of a solution containing goat IgG anti-human IgGs, conjugated with horse radish peroxidase (HRP).
  • HRP horse radish peroxidase
  • the serum utilized (21) belongs to the panel BBI mixed HCV (Boston Biomedica Inc.).
  • the control HCV negative serum gives constantly values lower than 0.04.
  • MOLECULAR TYPE cDNA from. genomic RNA
  • CHARACTERISTICS coding for a portion of env protein variant env 1
  • PROPERTY coding sequence
  • SEQUENCE TYPE Nucleotide with corresponding protein LENGTH OF THE SEQUENCE: 153 base pairs
  • MOLECULAR TYPE cDNA from genomic RNA
  • CHARACTERISTICS coding for a portion of env protein env 2 variant
  • PROPERTY coding sequence
  • MOLECULAR TYPE cDNA from genomic RNA
  • CHARACTERISTICS coding for a portion of the env protein env 3 variant
  • PROPERTY coding sequence

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  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Biophysics (AREA)
  • Biochemistry (AREA)
  • Gastroenterology & Hepatology (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Virology (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)

Abstract

Séquences d'acides aminés présentant une activité antigénique et comprises dans la séquence de la protéine env du virus de l'hépatite C (HCV), et peptides synthétiques possédant lesdites séquences et présentant une réactivité accrue vis-à-vis des sérums anti-HCV lorsqu'ils sont cyclisés. On décrit également diverses variantes desdites séquences, ainsi que des séquences nucléotidiques codant pour celles-ci.
PCT/IT1992/000081 1991-07-19 1992-07-16 Epitopes de la proteine env du virus de l'hepatite c WO1993002103A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
ITRM91/A000546 1991-07-19
ITRM910546A IT1249684B (it) 1991-07-19 1991-07-19 Epitopi della proteina env del virus dell'epatite c.

Publications (1)

Publication Number Publication Date
WO1993002103A1 true WO1993002103A1 (fr) 1993-02-04

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AU (1) AU2370592A (fr)
IT (1) IT1249684B (fr)
WO (1) WO1993002103A1 (fr)

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995018382A1 (fr) * 1993-12-27 1995-07-06 Euro-Diagnostica Ab Antigene diagnostique et procede pour diagnostiquer in vitro une infection active causee par le virus de l'hepatite c
WO1995001442A3 (fr) * 1993-06-29 1995-08-10 Us Health Sequences de nucleotides et sequences d'acides amines du gene d'enveloppe 1 de 51 isolats du virus de l'hepatite c et utilisation de reactifs derives desdites sequences en tant que reactifs de diagnostic et vaccins
WO1996004385A3 (fr) * 1994-07-29 1996-03-07 Innogenetics Nv Proteines purifiees d'enveloppe de virus de l'hepatite c a usage diagnostic et therapeutique
WO1999024466A3 (fr) * 1997-11-06 1999-07-15 Innogenetics Nv Peptides multimeres derives de proteines d'enveloppe de virus d'hepatite c (hcv) a des fins de diagnostic et de vaccination
US6521403B1 (en) 1998-03-27 2003-02-18 Innogenetics N.V. Epitopes in viral envelope proteins and specific antibodies directed against these epitopes: use for detection of HCV viral antigen in host tissue
EP1201770A3 (fr) * 2000-10-30 2004-02-25 Tosoh Corporation Oligonucléotide pour la détéction ultrasensible du virus de l'hépatite C et méthode de détection
US6743941B2 (en) 2001-06-15 2004-06-01 Aventis Pharma Deutschland Gmbh Process for the production of piperidine derivatives
US7101561B2 (en) 2000-12-01 2006-09-05 Innogenetics N.V. Purified hepatitis C virus envelope proteins for diagnostic and therapeutic use
US7108855B2 (en) 1998-06-24 2006-09-19 Innogenetics N.V. Purified hepatitis C virus envelope proteins for diagnostic and therapeutic use

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
UA39944C2 (uk) * 1992-07-07 2001-07-16 Чірон Корпорейшн Спосіб визначення ранньої сероконверсії у ссавця-хазяїна до вірусу гепатиту с і набір для використання в способі

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1990011089A1 (fr) * 1989-03-17 1990-10-04 Chiron Corporation Diagnostics et vaccins de nanbv (virus de l'hepatite non a, non b)
EP0398748A2 (fr) * 1989-05-18 1990-11-22 Chiron Corporation Diagnostics de NANBV: polynucléotides utiles pour le criblage du virus de l'hépatite C
EP0463848A2 (fr) * 1990-06-25 1992-01-02 The Research Foundation for Microbial Diseases of Osaka University Particules du virus de l'hépatite non-A non-B
WO1992011370A2 (fr) * 1990-12-21 1992-07-09 Boehringer Mannheim Gmbh Polypeptides derives de proteines du virus de l'hepatite c

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1990011089A1 (fr) * 1989-03-17 1990-10-04 Chiron Corporation Diagnostics et vaccins de nanbv (virus de l'hepatite non a, non b)
EP0398748A2 (fr) * 1989-05-18 1990-11-22 Chiron Corporation Diagnostics de NANBV: polynucléotides utiles pour le criblage du virus de l'hépatite C
EP0463848A2 (fr) * 1990-06-25 1992-01-02 The Research Foundation for Microbial Diseases of Osaka University Particules du virus de l'hépatite non-A non-B
WO1992011370A2 (fr) * 1990-12-21 1992-07-09 Boehringer Mannheim Gmbh Polypeptides derives de proteines du virus de l'hepatite c

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS. vol. 175, no. 1, 28 February 1991, DULUTH, MINNESOTA US pages 220 - 228 HIJIKATA, MAKOTO; KATO, NOBUYUKI; OOTSUYAMA, YUKO; NAKAGAWA, MASANORI; OHKOSHI, SHOGO; SHIMOTOHNO, KUNITADA 'Hypervariable regions in the putative glycoprotein of hepatitis C virus .' *
JAPAN JOURNAL OF EXPERIMENTAL MEDECINE vol. 60, no. 3, 1990, pages 167 - 177 OKAMOTO, H. ET AL. 'The 5'-terminal sequence of the hepatitis c virus genome' *
PEPTIDE CHEMISTRY, 1991, OSAKA, JP *
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF USA. vol. 87, no. 24, 1990, WASHINGTON US pages 9524 - 9528 KATO, NOBUYUKI; HIJIKATA, MAKOTO; OOTSUYAMA, YUKO; NAKAGAWA, MASANORI; OHKOSHI, SHOWGO; SUGIMURA, TAKASHI; SHIMOTOHNO, KUNITADA 'Molecular cloning of the human hepatitis C virus genome from Japanese patients with non-A, non-B hepatitis.' *
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF USA. vol. 88, April 1991, WASHINGTON US pages 3392 - 3396 OGATA, NORIO; ALTER, HARVEY J.; MILLER, ROGER H.; PURCELL, ROBERT H. 'Nucleotide sequence and mutation rate of the H strain of hepatitis C virus' *
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF USA. vol. 88, no. 9, 1 May 1991, WASHINGTON US pages 3647 - 3651 HOSSEIN, B. ET AL. 'Improved serodiagnosis of hepatitis C virus infection with synthetic antigen from capsid protein' See Paragraph "Synthetic peptides"; Results, Discussion *

Cited By (17)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6572864B1 (en) 1993-06-29 2003-06-03 The United States Of America As Represented By The Department Of Health And Human Services Nucleotide and deduced amino acid sequences of the envelope 1 gene of 51 isolates of hepatitis C virus and the use of reagents derived from these sequences in diagnostic methods and vaccines
US5514539A (en) * 1993-06-29 1996-05-07 The United States Of America As Represented By The Department Of Health And Human Services Nucleotide and deduced amino acid sequences of the envelope 1 gene of 51 isolates of hepatitis C virus and the use of reagents derived from these sequences in diagnostic methods and vaccines
US5871962A (en) * 1993-06-29 1999-02-16 The United States Of America As Represented By The Department Of Health And Human Services Nucleotide and deduced amino acid sequences of the envelope 1 gene of 51 isolates of hepatitis C virus and the use of reagents derived from these sequences in diagnostic methods
WO1995001442A3 (fr) * 1993-06-29 1995-08-10 Us Health Sequences de nucleotides et sequences d'acides amines du gene d'enveloppe 1 de 51 isolats du virus de l'hepatite c et utilisation de reactifs derives desdites sequences en tant que reactifs de diagnostic et vaccins
WO1995018382A1 (fr) * 1993-12-27 1995-07-06 Euro-Diagnostica Ab Antigene diagnostique et procede pour diagnostiquer in vitro une infection active causee par le virus de l'hepatite c
WO1996004385A3 (fr) * 1994-07-29 1996-03-07 Innogenetics Nv Proteines purifiees d'enveloppe de virus de l'hepatite c a usage diagnostic et therapeutique
US7026457B2 (en) 1994-07-29 2006-04-11 Innogenetics N.V. Purified hepatitis C virus envelope proteins for diagnostic and therapeutic use
US6245503B1 (en) 1994-07-29 2001-06-12 N.V. Innogenetics S.A. Purified hepatitis C virus envelope proteins for diagnostic and therapeutic use
US6890737B1 (en) 1994-07-29 2005-05-10 Innogenetics N.V. Purified hepatitis C virus envelope proteins for diagnostic and therapeutic use
WO1999024466A3 (fr) * 1997-11-06 1999-07-15 Innogenetics Nv Peptides multimeres derives de proteines d'enveloppe de virus d'hepatite c (hcv) a des fins de diagnostic et de vaccination
US6855318B1 (en) 1997-11-06 2005-02-15 N.V. Innogenetics S.A. Multi-mer peptides derived from hepatitis C virus envelope proteins for diagnostic use and vaccination purposes
US6841353B2 (en) 1998-03-27 2005-01-11 Innogenetics N.V. Epitopes in viral envelope proteins and specific antibodies directed against these epitopes: use for detection of HCV viral antigen in host tissue
US6521403B1 (en) 1998-03-27 2003-02-18 Innogenetics N.V. Epitopes in viral envelope proteins and specific antibodies directed against these epitopes: use for detection of HCV viral antigen in host tissue
US7108855B2 (en) 1998-06-24 2006-09-19 Innogenetics N.V. Purified hepatitis C virus envelope proteins for diagnostic and therapeutic use
EP1201770A3 (fr) * 2000-10-30 2004-02-25 Tosoh Corporation Oligonucléotide pour la détéction ultrasensible du virus de l'hépatite C et méthode de détection
US7101561B2 (en) 2000-12-01 2006-09-05 Innogenetics N.V. Purified hepatitis C virus envelope proteins for diagnostic and therapeutic use
US6743941B2 (en) 2001-06-15 2004-06-01 Aventis Pharma Deutschland Gmbh Process for the production of piperidine derivatives

Also Published As

Publication number Publication date
IT1249684B (it) 1995-03-09
ITRM910546A0 (it) 1991-07-19
AU2370592A (en) 1993-02-23
ITRM910546A1 (it) 1993-01-19

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