NEW HUMAN PARVOVIRUS PEPTIDES WITH DISULFIDE BRIDGE FOR IMMUNIZATION OR DIAGNOSIS

Abstract: 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, is described. Furthermore, an artificial antigen which reacts with antibodies induced by a human parvovirus is described. Said antigen mainly consists of an artificial peptide according to the invention. Additionally, a method of detecting antibodies induced by a human parvovirus in a sample of body fluid, wherein said sample is subjected to an immunoassay, especially ELISA, and wherein an artificial antigen according to the invention is used as a diagnostic antigen, is described. A diagnostic immunoassay kit for said method is also described. Finally, a vaccine composition comprising, as an immunizing component, at least one antigen according to the invention, is described.

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-NH 9 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: 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-NH 2 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-NH 2 . 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.

Inventors: FRIDELL EVA, TROJNAR JERZY, WAHREN BRITTA, FRIDELL, EVA, TROJNAR, JERZY, WAHREN, BRITTA
Applicants: FERRING DIAGNOSTICA AB
Application year: 1990
Main classification: G01N33
IPC codes: A61K39, C07K14