EP0470205A1 - Nouveaux peptides de parvovirus humain dotes d'un pont de bisulfure pour l'immunisation ou le diagnostic - Google Patents

Nouveaux peptides de parvovirus humain dotes d'un pont de bisulfure pour l'immunisation ou le diagnostic

Info

Publication number
EP0470205A1
EP0470205A1 EP90908131A EP90908131A EP0470205A1 EP 0470205 A1 EP0470205 A1 EP 0470205A1 EP 90908131 A EP90908131 A EP 90908131A EP 90908131 A EP90908131 A EP 90908131A EP 0470205 A1 EP0470205 A1 EP 0470205A1
Authority
EP
European Patent Office
Prior art keywords
ser
cys
ala
antigen
ile
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.)
Withdrawn
Application number
EP90908131A
Other languages
German (de)
English (en)
Inventor
Jerzy Trojnar
Britta Wahren
Eva Fridell
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
FERRING DIAGNOSTICA AB
Original Assignee
FERRING DIAGNOSTICA AB
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 FERRING DIAGNOSTICA AB filed Critical FERRING DIAGNOSTICA AB
Publication of EP0470205A1 publication Critical patent/EP0470205A1/fr
Withdrawn legal-status Critical Current

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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
    • 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
    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14211—Erythrovirus, e.g. B19 virus
    • C12N2750/14222—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • the present invention relates to artificial peptides having an amino acid sequence which corresponds to a naturally occurring amino acid sequence of a human parvovirus comprising an epitope and which further has two cysteine residues located on each side of said epitope, and further having a sulphur bridge between said two cysteine residues which has been formed by a chemical oxidation step.
  • the invention also relates to artificial antigens, which react with antibodies induced by a human parvovirus, a method of detecting antibodies induced by a human parvovirus in a sample of body fluid, a diagnostic immunoassay kit for said method, and a vaccine composition compri ⁇ sing, as an immunizing component, at least one antigen of the invention.
  • a human parvovirus, B19 gives rise to erythema infectiosum in children. It is also associated with aplastic crisis in patients with chronic hemolytic (e.g. sickle cell) anemia and with spontaneous abortion intrauterine death or hydrops fetalis when the woman aquires the infection during pregnancy. The fetus infection might be cured if the diagnosis is made early. (J Virol 58: 921 - 936, 1986; Shade et al).
  • the virus can only be cultivated in human bone arrow, which hampers isolation for serology. Peptide-based serology would therefore be a means of diagnosis.
  • ELISA enzyme- -linked immunosorbent assay
  • the present invention provides i.a. a rapid, sensitive and specific assay for detection of anti ⁇ bodies induced by a human parvovirus.
  • an artificial peptide having an amino acid sequence which corresponds to a naturally occurring amino acid sequence of a human parvovirus comprising an epitope and which further has two cysteine residues located on each side of said epitope, and further having a sulphur bridge between said two cysteine residues which has been formed by a chemical oxidation step. It is believed that this stabilization of the peptide by a sulphur bridge between two cysteine residues is responsible for the useful properties of the peptide, such as an enhancement of the antibody binding activity, as well as the chemical stability of the final product.
  • the artificial peptide includes at least two cysteine residues, which are cyclized to form a sul ⁇ phur bridge.
  • the two cysteine residues which are linked together may have one or more amino acid residues comprising an epitope between themselves, such as 2 to 10 residues.
  • the artificial peptide according to the invention includes more than two cysteine resi ⁇ dues, still only one sulphur bridge between two cysteine residues is formed by a chemical oxidation step.
  • an artificial peptide which is chosen from the group consisting of the peptide having the modified amino acid sequence
  • an artificial antigen which reacts with antibodies induced by a human parvovirus, which antigen mainly consists of an artificial peptide having an amino acid sequence which corresponds to a naturally occurring amino acid sequence of a human parvovirus comprising an epitope and which further has two cysteine residues located on each side of said epitope, and further having a sulphur bridge between said two cysteine residues which has been formed by a chemical oxidation step.
  • the expression "antigen mainly consists of an artificial peptide” indicates that the ability of the antigen to react with antibodies derives from the artificial peptide.
  • an artificial antigen which reacts with antibodies induced by a human parvo ⁇ virus, which antigen mainly consists of a preferred artificial peptide according to the invention, exem ⁇ plified above.
  • the artificial antigens according to the invention can be immobilized or coupled to a carrier, such as mineral carriers, e.g. aluminium hydroxide, calcium phosphate, etc., plastic surfaces, e.g. microplates, beads, etc., proteins, such as bovine serum albumin or an immunizing component, such as keyhole limpet haemocyanin.
  • a carrier such as mineral carriers, e.g. aluminium hydroxide, calcium phosphate, etc., plastic surfaces, e.g. microplates, beads, etc., proteins, such as bovine serum albumin or an immunizing component, such as keyhole limpet haemocyanin.
  • the artificial antigens according to the invention so far only have been used as diag ⁇ nostic antigens to detect antibodies induced by a human parvovirus, in a sample of body fluid, it is believed that they can be used as immunizing components in vaccine compositions against a human parvovirus.
  • a further aspect of the invention provides a vaccine composition, which comprises as an immunizing component, at least one antigen selected from artifi ⁇ cial antigens according to the invention, together with a nontoxic pharmaceutically acceptable carrier and/or diluent.
  • a method of detecting antibodies induced by a human parvovirus in a sample of body fluid wherein said sample is subjected to an immunoassay and an artificial antigen according to the invention is used as a diagnostic antigen.
  • useful body fluids are urine, saliva, tear fluid, milk, serum, blood and cerebrospinal fluid.
  • the immunoassay in which the artificial antigens according to the invention can be used as diagnostic antigens is any immunoassay of choice, such as enzyme- linked immunosorbent assay (ELISA), radioimmunoassay (RIA), immunodiffusion or immunoelectrophoreses (IE).
  • ELISA is used as the immunoassay of choice.
  • a diagnostic immunoassay kit for the detection of antibodies induced by a human parvovirus in a sample of body fluid, wherein an artificial antigen according to the invention is included as a diagnostic antigen.
  • the kit will comprise other items, such as a positive standard serum sample, a negative standard serum sample, in the case of ELISA an enzyme conjugate and optionally a substrate for the enzyme conjugate, and also optionally buffer solution(s) and/or washing solution(s).
  • all the reagents in the kit are contained in separate sealed test tubes or vials marked with specific labels. Synthesis of the artificial peptides of the invention
  • the artificial peptides of the invention can be produced by any known method of producing a linear peptide sequence, such as cloning, degradation, coupling of one amino acid residue to the next one in liquid phase or coupling the amino acids to one another starting with a solid phase (resin) to which the C-ter inal of the first amino acid is coupled, whereupon the C-terminal of the next amino acid is coupled to the N-terminal of the first amino acid, etc., finally releasing the built-up peptide from the solid phase (so-called Merrifield synthesis).
  • a chemical oxidation step in order to cyclize two cysteine residues thereof, whereby a sulphur bridge is formed between the cysteine residues.
  • the peptide was then cleaved from the resin by treatment with liquid hydrogen fluorid in the presence of anisole and ethyl-methyl-sulfide as scavangers (HF:anisole:EMS - 10:05:05). After removal of hydrogen fluoride by evaporation the residue was suspended in ethyl acetate (100 ml) and filtered. The solid was washed on filter with additional ethyl acetate (3x100 ml) and the cleaved peptide extracted with acetic acid (100 ml).
  • the extract was promptly diluted to the volume of 2 dm with 20% acetic acid in methanol and treated with 0.1 M solution of iodine in methanol until the faint brown colour persisted. Then the Dowex 1x8 ion exchanger in acetate form was added (3 g) (Bio-Rad, Richmond, CA and the mixture was filtered. The filtrate was evaporated and the residue freeze-dried from 1% acetic acid in water. The product was then purified by reversed phase liquid chromatography on a column filled with Vydac 20-25 ⁇ (Separation Group, CA) in a suitable system containing acetonitrile in 0.1% trifluoroacetic acid water solution.
  • Amino acid derivatives were delivered by Bachem AG, Switzerland.
  • the peptide was prepared according to the general description of synthesis. The structure was confirmed by aminoacid analysis and by FAB-MS.
  • Buffer A Trypsinization buffer without
  • BSA bovine serum albumin
  • Washing solution 0.9% NaCl with 0.05% Tween 20.
  • Coating A solution of the coating antigen (test peptide), 5 ⁇ g to 20 ⁇ g per ml, is made in carbonate buffer. 100 ⁇ l of the solution is added to each well of stripes or of 96-well microplates. The adsorption takes place over night, 18 hrs, at room temperature.
  • the coated plates can be stored with their contents in 4°C until use.
  • Serum assay 1. Empty and wash the plate 4 times with washing solution.
  • This peptide gave absorbance values of 1.55 + 0.45 when the test was performed on serum samples from 10 seropositive persons and absorbance values of 0.30 +_ 0.15 when the test was performed on serum samples from 9 seronegative persons.
  • the antigen in the test was the peptide according to the invention
  • the absorbance values of seropositive sera became significant, and the peptide showed good reactivity with antibodies induced by human parvovirus B19.

Landscapes

  • 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

On décrit un peptide artificiel ayant une séquence d'aminoacides qui correspond à une séquence d'aminoacides d'un parvovirus humain se présentant naturellement, comprenant un déterminant antigénique et présentant deux résidus de cystéine placés de chaque côté dudit déterminant antigénique ainsi qu'un pont de soufre entre lesdits deux résidus de cystéine, ledit pont étant entraîné par un procédé d'oxydation chimique. On décrit également un antigène artificiel qui réagit avec des anticorps induits par un parvovirus humain, ledit antigène consistant principalement en un peptide artificiel, selon l'invention. De plus, on prévoit un procédé pour détecter les anticorps induits par un parvovirus humain dans un échantillon de liquide organique, au cours duquel on soumet ledit échantillon à une analyse immunologique, en particulier ELISA. Un antigène artificiel est utilisé en tant qu'antigène diagnostic. Un kit pour l'analyse immunologique est également décrit, ainsi qu'une composition de vaccin, comprenant, sous forme de constituant d'immunisation, au moins un antigène selon l'invention.
EP90908131A 1989-04-28 1990-04-25 Nouveaux peptides de parvovirus humain dotes d'un pont de bisulfure pour l'immunisation ou le diagnostic Withdrawn EP0470205A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
SE8901566 1989-04-28
SE8901566A SE8901566D0 (sv) 1989-04-28 1989-04-28 Nya peptider inkluderande en disulfidbindning

Publications (1)

Publication Number Publication Date
EP0470205A1 true EP0470205A1 (fr) 1992-02-12

Family

ID=20375840

Family Applications (1)

Application Number Title Priority Date Filing Date
EP90908131A Withdrawn EP0470205A1 (fr) 1989-04-28 1990-04-25 Nouveaux peptides de parvovirus humain dotes d'un pont de bisulfure pour l'immunisation ou le diagnostic

Country Status (6)

Country Link
EP (1) EP0470205A1 (fr)
AU (1) AU636145B2 (fr)
CA (1) CA2053240A1 (fr)
IL (1) IL94218A0 (fr)
SE (1) SE8901566D0 (fr)
WO (1) WO1990013567A1 (fr)

Families Citing this family (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6274307B1 (en) * 1990-02-08 2001-08-14 Mikrogen Molekularbiologische Entwicklungs-Gmbh Immunologically active peptides or polypeptides from the parvovirus B19
US5244785A (en) * 1991-02-01 1993-09-14 Microgenics Corporation Determination of high molecular weight analytes using a β-galactosidase complementation assay
JPH0910000A (ja) 1995-06-26 1997-01-14 Nippon Sekijiyuujishiya ヒトパルボウイルスの検出方法及びそのための試薬
US6238860B1 (en) 1998-11-05 2001-05-29 Dyax Corp. Binding moieties for human parvovirus B19

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
IL70704A0 (en) * 1983-01-19 1984-04-30 Amgen Methods and materials for development of parvovirus vaccines
DE3782350D1 (de) * 1986-03-20 1992-12-03 Abbott Lab Rekombinante parvovirus-ra-1-produkte und verfahren zu deren anwendung.

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO9013567A1 *

Also Published As

Publication number Publication date
WO1990013567A1 (fr) 1990-11-15
AU5559690A (en) 1990-11-29
SE8901566D0 (sv) 1989-04-28
AU636145B2 (en) 1993-04-22
IL94218A0 (en) 1991-01-31
CA2053240A1 (fr) 1990-10-29

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