EP0470205A1 - New human parvovirus peptides with disulfide bridge for immunization or diagnosis - Google Patents
New human parvovirus peptides with disulfide bridge for immunization or diagnosisInfo
- 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- Withdrawn
Links
- 108090000765 processed proteins & peptides Proteins 0.000 title claims abstract description 53
- 241000484121 Human parvovirus Species 0.000 title claims abstract description 24
- 230000003053 immunization Effects 0.000 title claims abstract description 8
- 102000004196 processed proteins & peptides Human genes 0.000 title claims description 15
- 238000002649 immunization Methods 0.000 title abstract description 3
- 238000003745 diagnosis Methods 0.000 title description 5
- 239000000427 antigen Substances 0.000 claims abstract description 43
- 102000036639 antigens Human genes 0.000 claims abstract description 43
- 108091007433 antigens Proteins 0.000 claims abstract description 43
- 125000000151 cysteine group Chemical group N[C@@H](CS)C(=O)* 0.000 claims abstract description 19
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract description 15
- 238000002965 ELISA Methods 0.000 claims abstract description 12
- 239000000126 substance Substances 0.000 claims abstract description 11
- 238000000034 method Methods 0.000 claims abstract description 10
- 210000001124 body fluid Anatomy 0.000 claims abstract description 9
- 239000010839 body fluid Substances 0.000 claims abstract description 9
- 230000003647 oxidation Effects 0.000 claims abstract description 9
- 238000007254 oxidation reaction Methods 0.000 claims abstract description 9
- 239000000203 mixture Substances 0.000 claims abstract description 7
- 229960005486 vaccine Drugs 0.000 claims abstract description 5
- 210000002966 serum Anatomy 0.000 claims description 13
- 235000001014 amino acid Nutrition 0.000 claims description 11
- 150000001413 amino acids Chemical class 0.000 claims description 11
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- 239000005864 Sulphur Substances 0.000 claims description 9
- 102000004190 Enzymes Human genes 0.000 claims description 8
- 108090000790 Enzymes Proteins 0.000 claims description 8
- 238000001514 detection method Methods 0.000 claims description 8
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- 231100000252 nontoxic Toxicity 0.000 claims description 2
- 230000003000 nontoxic effect Effects 0.000 claims description 2
- 238000004458 analytical method Methods 0.000 abstract description 5
- 230000000890 antigenic effect Effects 0.000 abstract 2
- 230000001900 immune effect Effects 0.000 abstract 2
- NINIDFKCEFEMDL-UHFFFAOYSA-N Sulfur Chemical compound [S] NINIDFKCEFEMDL-UHFFFAOYSA-N 0.000 abstract 1
- 239000000470 constituent Substances 0.000 abstract 1
- 229910052717 sulfur Inorganic materials 0.000 abstract 1
- 239000011593 sulfur Substances 0.000 abstract 1
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- 238000003786 synthesis reaction Methods 0.000 description 7
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- 239000011347 resin Substances 0.000 description 5
- 229920005989 resin Polymers 0.000 description 5
- 241000702617 Human parvovirus B19 Species 0.000 description 4
- 238000002835 absorbance Methods 0.000 description 4
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- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 241000700605 Viruses Species 0.000 description 3
- 238000003556 assay Methods 0.000 description 3
- 229940098773 bovine serum albumin Drugs 0.000 description 3
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- 239000000047 product Substances 0.000 description 3
- 102000002260 Alkaline Phosphatase Human genes 0.000 description 2
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- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 2
- BVKZGUZCCUSVTD-UHFFFAOYSA-L Carbonate Chemical compound [O-]C([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-L 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- KRHYYFGTRYWZRS-UHFFFAOYSA-N Fluorane Chemical compound F KRHYYFGTRYWZRS-UHFFFAOYSA-N 0.000 description 2
- 229920001213 Polysorbate 20 Polymers 0.000 description 2
- 241000125945 Protoparvovirus Species 0.000 description 2
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- 125000000539 amino acid group Chemical group 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
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- 239000012467 final product Substances 0.000 description 2
- 238000004128 high performance liquid chromatography Methods 0.000 description 2
- 208000015181 infectious disease Diseases 0.000 description 2
- UZKWTJUDCOPSNM-UHFFFAOYSA-N methoxybenzene Substances CCCCOC=C UZKWTJUDCOPSNM-UHFFFAOYSA-N 0.000 description 2
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 2
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 2
- 238000003127 radioimmunoassay Methods 0.000 description 2
- 239000007790 solid phase Substances 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- BGGHCRNCRWQABU-JTQLQIEISA-N (2s)-2-amino-5-oxo-5-phenylmethoxypentanoic acid Chemical compound OC(=O)[C@@H](N)CCC(=O)OCC1=CC=CC=C1 BGGHCRNCRWQABU-JTQLQIEISA-N 0.000 description 1
- ONOURAAVVKGJNM-SCZZXKLOSA-N (2s,3r)-2-azaniumyl-3-phenylmethoxybutanoate Chemical compound [O-]C(=O)[C@@H]([NH3+])[C@@H](C)OCC1=CC=CC=C1 ONOURAAVVKGJNM-SCZZXKLOSA-N 0.000 description 1
- UHPQFNXOFFPHJW-UHFFFAOYSA-N (4-methylphenyl)-phenylmethanamine Chemical compound C1=CC(C)=CC=C1C(N)C1=CC=CC=C1 UHPQFNXOFFPHJW-UHFFFAOYSA-N 0.000 description 1
- XXMFJKNOJSDQBM-UHFFFAOYSA-N 2,2,2-trifluoroacetic acid;hydrate Chemical compound [OH3+].[O-]C(=O)C(F)(F)F XXMFJKNOJSDQBM-UHFFFAOYSA-N 0.000 description 1
- XZKIHKMTEMTJQX-UHFFFAOYSA-N 4-Nitrophenyl Phosphate Chemical compound OP(O)(=O)OC1=CC=C([N+]([O-])=O)C=C1 XZKIHKMTEMTJQX-UHFFFAOYSA-N 0.000 description 1
- ZCYVEMRRCGMTRW-UHFFFAOYSA-N 7553-56-2 Chemical compound [I] ZCYVEMRRCGMTRW-UHFFFAOYSA-N 0.000 description 1
- 206010000234 Abortion spontaneous Diseases 0.000 description 1
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- 206010055690 Foetal death Diseases 0.000 description 1
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- 206010020529 Hydrops foetalis Diseases 0.000 description 1
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 1
- 229910000831 Steel Inorganic materials 0.000 description 1
- 101710172711 Structural protein Proteins 0.000 description 1
- WNROFYMDJYEPJX-UHFFFAOYSA-K aluminium hydroxide Chemical compound [OH-].[OH-].[OH-].[Al+3] WNROFYMDJYEPJX-UHFFFAOYSA-K 0.000 description 1
- 229910021502 aluminium hydroxide Inorganic materials 0.000 description 1
- 150000003862 amino acid derivatives Chemical class 0.000 description 1
- 229910021529 ammonia Inorganic materials 0.000 description 1
- 208000007502 anemia Diseases 0.000 description 1
- 230000000840 anti-viral effect Effects 0.000 description 1
- 239000011324 bead Substances 0.000 description 1
- 230000027455 binding Effects 0.000 description 1
- 210000000988 bone and bone Anatomy 0.000 description 1
- 210000004899 c-terminal region Anatomy 0.000 description 1
- 239000011575 calcium Substances 0.000 description 1
- 229910052791 calcium Inorganic materials 0.000 description 1
- 239000001506 calcium phosphate Substances 0.000 description 1
- 229910000389 calcium phosphate Inorganic materials 0.000 description 1
- 235000011010 calcium phosphates Nutrition 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
- 210000001175 cerebrospinal fluid Anatomy 0.000 description 1
- 230000001684 chronic effect Effects 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 235000018417 cysteine Nutrition 0.000 description 1
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- ZBCBWPMODOFKDW-UHFFFAOYSA-N diethanolamine Chemical compound OCCNCCO ZBCBWPMODOFKDW-UHFFFAOYSA-N 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- -1 etc. Substances 0.000 description 1
- 238000001704 evaporation Methods 0.000 description 1
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- 238000010265 fast atom bombardment Methods 0.000 description 1
- 210000003754 fetus Anatomy 0.000 description 1
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- 239000012530 fluid Substances 0.000 description 1
- 230000002949 hemolytic effect Effects 0.000 description 1
- 229910000040 hydrogen fluoride Inorganic materials 0.000 description 1
- 230000000951 immunodiffusion Effects 0.000 description 1
- 229910052500 inorganic mineral Inorganic materials 0.000 description 1
- 231100001046 intrauterine death Toxicity 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 239000011630 iodine Substances 0.000 description 1
- 229910052740 iodine Inorganic materials 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 108010045069 keyhole-limpet hemocyanin Proteins 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 239000007791 liquid phase Substances 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- 238000003760 magnetic stirring Methods 0.000 description 1
- IOPLHGOSNCJOOO-UHFFFAOYSA-N methyl 3,4-diaminobenzoate Chemical compound COC(=O)C1=CC=C(N)C(N)=C1 IOPLHGOSNCJOOO-UHFFFAOYSA-N 0.000 description 1
- 235000013336 milk Nutrition 0.000 description 1
- 239000008267 milk Substances 0.000 description 1
- 210000004080 milk Anatomy 0.000 description 1
- 239000011707 mineral Substances 0.000 description 1
- 229920003023 plastic Polymers 0.000 description 1
- 239000004033 plastic Substances 0.000 description 1
- 229920001467 poly(styrenesulfonates) Polymers 0.000 description 1
- 230000035935 pregnancy Effects 0.000 description 1
- 230000004224 protection Effects 0.000 description 1
- 235000018102 proteins Nutrition 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 230000009257 reactivity Effects 0.000 description 1
- 238000004366 reverse phase liquid chromatography Methods 0.000 description 1
- 210000003296 saliva Anatomy 0.000 description 1
- 229920006395 saturated elastomer Polymers 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 229910000029 sodium carbonate Inorganic materials 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 208000000995 spontaneous abortion Diseases 0.000 description 1
- 230000006641 stabilisation Effects 0.000 description 1
- 238000011105 stabilization Methods 0.000 description 1
- 239000010959 steel Substances 0.000 description 1
- ZERULLAPCVRMCO-UHFFFAOYSA-N sulfure de di n-propyle Natural products CCCSCCC ZERULLAPCVRMCO-UHFFFAOYSA-N 0.000 description 1
- 210000001138 tear Anatomy 0.000 description 1
- 125000005931 tert-butyloxycarbonyl group Chemical group [H]C([H])([H])C(OC(*)=O)(C([H])([H])[H])C([H])([H])[H] 0.000 description 1
- QORWJWZARLRLPR-UHFFFAOYSA-H tricalcium bis(phosphate) Chemical compound [Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O QORWJWZARLRLPR-UHFFFAOYSA-H 0.000 description 1
- 210000002700 urine Anatomy 0.000 description 1
- 230000003612 virological effect Effects 0.000 description 1
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.Disclosed is an artificial peptide having an amino acid sequence which corresponds to an amino acid sequence of a naturally occurring human parvovirus, comprising an antigenic determinant and having two cysteine residues placed on each side of said antigenic determinant as well as a bridge of sulfur between said two cysteine residues, said bridge being driven by a chemical oxidation process. An artificial antigen is also described which reacts with antibodies induced by a human parvovirus, said antigen consisting mainly of an artificial peptide, according to the invention. Furthermore, a method is provided for detecting the antibodies induced by a human parvovirus in a sample of body fluid, during which said sample is subjected to an immunological analysis, in particular ELISA. An artificial antigen is used as a diagnostic antigen. A kit for immunological analysis is also described, as well as a vaccine composition, comprising, in the form of an immunization constituent, at least one antigen according to the invention.
Description
NEW HUMAN PARVOVIRUS PEPTIDES WITH DISULFIDE BRIDGE FOR IMMUNIZATION OR DIAGNOSIS
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. Background
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.
One common way to establish a diagnosis through antibody detection is to screen serum samples by enzyme- -linked immunosorbent assay (ELISA) (Sarngadharan, M.G., Popovic, M. , Bruch, L. , Schϋpbach, J. and Gallo, R.C.) Wells of microplates coated with viral antigens are reacted with the serum samples under investigation, washed, and antihuman Ig added. The latter reagent
is labelled with an enzyme. After washing, the enzyme labelled antihuman Ig remains only if specific antiviral Ig was present in the serum sample. It is visualized by addition of a substrate for the enzyme and the color reaction quantified in a spectrophotometer.
No assay has so far been developed for detection of antibodies induced by human parvovirus in a sample of body fluid. Description of the invention
The present invention provides i.a. a rapid, sensitive and specific assay for detection of anti¬ bodies induced by a human parvovirus.
In one aspect of the invention there is provided 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. -If 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.
In a preferred embodiment of this aspect of the invention there is provided an artificial peptide,
which is chosen from the group consisting of the peptide having the modified amino acid sequence
H-Phe-Ser-Pro-Ala-Ala-Ser-Ser-Cys-His-Asn-Ala-Ser-
-Gly-Lys-Glu-Ala-Lys-Val-Cys-Thr-Ile-Ser-Pro-Ile-NH- I
and peptides having a shorter sequence thereof including the modified sequence
-Cys-His-Asn-Ala-Ser-Gly-Lys-Glu-Ala-Lys-Val-Cys-;
I I
and the peptide having the modified amino acid sequence
H-Gly-Lys-Glu-Ala-Lys-Val-Cys-Thr-Ile-Ser-Pro-Ile-
I
-Cys-Gly-Tyr-Ser-Thr-Pro-Trp-Arg-Tyr-Leu-NH
_l
and peptides having a shorter sequence thereof including the modified sequence
-Cys-Thr-Ile-Ser-Pro-Ile-Cys-;
I I
In another aspect of the invention there is pro¬ vided 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.
In the specification and claims 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.
In a preferred embodiment of this aspect of the invention there is provided 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.
Even though 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.
Thus, 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.
In yet another aspect of the invention there is provided 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. Examples of 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).
In a preferred embodiment of this aspect of the invention ELISA is used as the immunoassay of choice.
In a further aspect of the invention there is provided 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. Depending on the immunoassay to be used, 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). Optionally 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). Once the appropriate linear peptide sequence is ready, it is subjected to a chemical oxidation step in order to cyclize two cysteine residues thereof, whereby a sulphur bridge is formed between the cysteine residues. General description of synthesis
In the examples below, all peptides were synthe¬ sized on an Applied Biosystems 430A Peptide Synthesizer
using a double coupling program with termination step after the second coupling. The resin used was of 4-methyl- benzhydrylamine type with theoretical loading of 0.66 meq/g (Peptides International, Louisville, KY, USA). The final product of the synthesis was dried in vacuo over night. After drying the peptide-resin was suspended in ethanol (70 ml) and saturated with ammonia. The mixture was placed in pressurized steel vessel and left over night with magnetic stirring. The resin was then separated by filtration, washed several times with methanol and thoroughly dried in vacuo. 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 μ (Separation Group, CA) in a suitable system containing acetonitrile in 0.1% trifluoroacetic acid water solution. The samples collected from the column were analyzed by analytical HPLC - Varian 5500 (Sunny¬ vale, CA) equipped with Bondapak C18 column (Millipore, Milford, Mass.). Fractions containing pure substance (>99,5%) were pooled, the solvent was evaporated and the product freeze-dried from 1% acetic acid in water.
The final HPLC analysis was performed on ready product and the structure of the peptide was confirmed by amino acid analysis and FAB-MS (Fast atom bombardment spectro etry) .
All amino acids used during the synthesis were protected with tert-butoxycarbonyl group at α-amino- function. The side chain protections used were as follows: Ser(BZL), Thr(BZL), Tyr(2-BrCbz) , Lys(2-ClCbz) , Glu(BZL), Arg(Tos), Cys(Mob).
Amino acid derivatives were delivered by Bachem AG, Switzerland.
The genome of human parvovirus B19 encodes two structural proteins. The sequences corresponding to VP2 were first synthesized as linear peptides and then a sulphur bridge between two cysteine residues were formed by a chemical oxidation step. EXAMPLE I
(The sequence corresponds to a sequence found in B19's (parvovirus) VP2 region).
H-Phe-Ser-Pro-Ala-Ala-Ser-Ser-Cys-His-Asn-Ala-Ser-Gly-
I
-Lys-Glu-Ala-Lys-Val-Cys-Thr-Ile-Ser-Pro-Ile-NH9 I
The peptide was prepared according to the general description of synthesis. The structure was confirmed by aminoacid analysis and by FAB-MS. EXAMPLE II
(The sequence corresponds to a sequence found in B19's (parvovirus) VP2-region).
H-Gly-Lys-Glu-Ala-Lys-Val-Cys-Thr-Ile-Ser-Pro-Ile-Cys-
I I
-Gly-Tyr-Ser-Thr-Pro-Trp-Arg-Tyr-Leu-NH-
The peptide was prepared according to the general description of synthesis. The structure was confirmed by aminoacid analysis and by FAB-MS.
Detection of antibodies induced by human parvovirus in blood samples
For the detection of antibodies induced by human parvovirus B19 in blood samples use was made of ELISA. (Engvall, E. and Perl ann, P.: Enzyme-linked immuno- sorbent assay (ELISA). Ill Quantification of specific antiboides by enzyme labelled anti-immunoglobulin in antigen-coated tubes. J. Immunol. 109.129-135,1972). Materials;
Plates: Nunc 96 F, Roskilde, Denmark Conjugate: Alkaline phosphatase labelled anti-human
Ig, Sigma Buffers: Carbonate buffer: 0.05 M sodium carbonate buffer, pH approx. 9.5.
Buffer A: Trypsinization buffer without
++ ++ Ca and Mg , and with 0.5% bovine serum albumin (BSA) and
0.05% Tween 20
Diethanolamine, Sigma.
Washing solution: 0.9% NaCl with 0.05% Tween 20.
Methods:
Coating: A solution of the coating antigen (test peptide), 5 μg to 20 μg per ml, is made in carbonate buffer. 100 μl 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.
2. Add 100 μl of serum diluted 1:100 in buffer A.
3. Add 100 μl of just buffer A to one well; this well serves as a blank.
4. Incubate 2 h at 37°C. (Dilute the conjugate during this period, see 6).
5. Wash 4 times with washing solution. Empty.
6. Add 100 μl of a conjugat y—e.g« "Alkaline phosphatase labelled anti-human IgG, Sigma diluted 1:1500 in buffer A.
7. Incubate at 37°C for 2 h. (Prepare substrate solution during this period, see 9) .
8. Wash 4 times with washing solution. Empt .
9. Add 100 μl Paranitrophenylphosphate (2 tablets/10 ml diethanola ine) . Use a clean vessel. 5 in are needed to dissolve the tablets.
10. Incubate 30 min at room temperature. Stop the reaction with 25 μl 5 M NaOH.
11. Read plate at 405 nm.
Using the above described materials and methods the following materials were tested as diagnostic antigens (coating antigens) for the detection of antibodies induced by human parvovirus B19 in serum samples from infected patients:
A. A region of linear peptides corresponding to a sequence found in the VP2-region of human parvo¬ virus B19. They gave absorbance values of 0.2-0.4 with known seropositive samples.
B. An artificial peptide according to the invention described in Example I of this specification.
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.
Thus, when 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.
Claims
1. An artificial peptide having an amino acid se¬ quence 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, c h a r a c t e r i s e d in that the peptide has a sulphur bridge between said two cysteine residues which has been formed by a chemical oxidation step.
2. An artificial peptide according to claim 1, c h a r a c t e r i s e d in that it is chosen from the group consisting of the peptide having the modified amino acid sequence
H-Phe-Ser-Pro-Ala-Ala-Ser-Ser-Cys-His-Asn-Ala-Ser-Gly-
I
-Lys-Glu-Ala-Lys-Val-Cys-Thr-Ile-Ser-Pro-Ile-NH, 1
and peptides having a shorter sequence thereof including the modified sequence
and the peptide having the modified amino acid sequence
H-Gly-Lys-Glu-Ala-Lys-Val-Cys-Thr-Ile-Ser-Pro-Ile-Cys-
I I
-Gly-Tyr-Ser-Thr-Pro-Trp-Arg-Tyr-Leu-NH2
and peptides having a shorter sequence thereof including the modified sequence -Cys-Thr-Ile-Ser-Pro-Ile-Cys-;
I L
3. An artificial antigen which reacts with anti¬ bodies induced by a human parvovirus, c h a r a c t e ¬ r i s e d in that the 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.
4. An artificial antigen according to claim 3, c h a r a c t e r i s e d in that the antigen mainly consists of an artificial peptide chosen from the group consisting of the peptide having the modified amino acid sequence
H-Phe-Ser-Pro-Ala-Ala-Ser-Ser-Cys-His-Asn-Ala-Ser-Gly-
J
-Lys-Glu-Ala-Lys-Val-Cys-Thr-Ile-Ser-Pro-Ile-NH- L
and peptides having a shorter sequence thereof including the modified sequence
-Cys-His-Asn-Ala-Ser-Gly-Lys-Glu-Ala-Lys-Val-Cys-;
1 I
and the peptide having the modified amino acid sequence
H-Gly-Lys-Glu-Ala-Lys-Val-Cys-Thr-Ile-Ser-Pro-Ile-Cys-
-Gly-Tyr-Ser-Thr-Pro-Trp-Arg-Tyr-Leu-NH2. and peptides having a shorter sequence thereof including the modified sequence
-Cys-Thr-Ile-Ser-Pro-Ile-Cys-;
5. An artificial antigen according to claim 3 or
4, c h a r a c t e r i s e d in that it has been immo¬ bilized or coupled to a carrier.
6. A method of detecting antibodies induced by a human parvovirus in a sample of body fluid, wherein said sample is subjected to an immunoassay, c h a r a σ t e r i s e d in that an artificial antigen according to any one of claims 3-5 is used as a diagnostic antigen.
7. A method according to claim 6, wherein said sample is subjected to enzyme-linked immunosorbent assay (ELISA) c h a r a c t e r i s e d in that an artifi¬ cial antigen according to any one of claims 3-5 is used as a diagnostic coating antigen.
8. A diagnostic immunoassay kit for the detection of antibodies induced by a human parvovirus in a sample of body fluid, c h a r a c t e r i s e d in that it comprises as a diagnostic antigen an artificial antigen according to any one of claims 3-5.
9. A diagnostic immunoassay kit according to claim 8, c h a r a c t e r i s e d in that it additionally comprises a positive standard serum sample, a negative standard serum sample, an enzyme conjugate, optionally a substrate for the enzyme conjugate, and optionally buffer solution(s) and/or washing solution(s) .
10. A vaccine composition, c h a r a c t e r i ¬ s e d in that it comprises as an immunizing component, at least one antigen selected from the artificial anti- gens according to any one of claims 3-5, together with a nontoxic pharmaceutically acceptable carrier and/or diluent.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| SE8901566 | 1989-04-28 | ||
| SE8901566A SE8901566D0 (en) | 1989-04-28 | 1989-04-28 | NEW PEPTIDES INCLUDING A DISULFID BOND |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| EP0470205A1 true EP0470205A1 (en) | 1992-02-12 |
Family
ID=20375840
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| EP90908131A Withdrawn EP0470205A1 (en) | 1989-04-28 | 1990-04-25 | New human parvovirus peptides with disulfide bridge for immunization or diagnosis |
Country Status (6)
| Country | Link |
|---|---|
| EP (1) | EP0470205A1 (en) |
| AU (1) | AU636145B2 (en) |
| CA (1) | CA2053240A1 (en) |
| IL (1) | IL94218A0 (en) |
| SE (1) | SE8901566D0 (en) |
| WO (1) | WO1990013567A1 (en) |
Families Citing this family (4)
| 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 (en) | 1995-06-26 | 1997-01-14 | Nippon Sekijiyuujishiya | Detection of human parvovirus and reagent therefor |
| US6238860B1 (en) | 1998-11-05 | 2001-05-29 | Dyax Corp. | Binding moieties for human parvovirus B19 |
Family Cites Families (2)
| 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 (en) * | 1986-03-20 | 1992-12-03 | Abbott Lab | RECOMBINANT PARVOVIRUS RA-1 PRODUCTS AND METHOD FOR USE THEREOF. |
-
1989
- 1989-04-28 SE SE8901566A patent/SE8901566D0/en unknown
-
1990
- 1990-04-25 EP EP90908131A patent/EP0470205A1/en not_active Withdrawn
- 1990-04-25 CA CA002053240A patent/CA2053240A1/en not_active Abandoned
- 1990-04-25 WO PCT/SE1990/000276 patent/WO1990013567A1/en not_active Application Discontinuation
- 1990-04-25 AU AU55596/90A patent/AU636145B2/en not_active Expired - Fee Related
- 1990-04-26 IL IL94218A patent/IL94218A0/en unknown
Non-Patent Citations (1)
| Title |
|---|
| See references of WO9013567A1 * |
Also Published As
| Publication number | Publication date |
|---|---|
| WO1990013567A1 (en) | 1990-11-15 |
| AU5559690A (en) | 1990-11-29 |
| SE8901566D0 (en) | 1989-04-28 |
| AU636145B2 (en) | 1993-04-22 |
| IL94218A0 (en) | 1991-01-31 |
| CA2053240A1 (en) | 1990-10-29 |
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