WO1993002363A1

WO1993002363A1 - Method to detect antibodies against hepatitis c virus and kits for the use thereof

Info

Publication number: WO1993002363A1
Application number: PCT/IT1992/000082
Authority: WO
Inventors: Carlo Rosa; Silvia Griva; Fabrizio Bonelli
Original assignee: Sorin Biomedica S.P.A.
Priority date: 1991-07-19
Filing date: 1992-07-16
Publication date: 1993-02-04
Also published as: AU2370692A; ITRM910547A1; ITRM910547A0; IT1249685B

Abstract

An immunological method to detect antibodies that react with epitopes of the HCV virus env protein is disclosed. Said epitopes are preferentially cyclized.

Description

METHOD TO DETECT ANTIBODIES AGAINST HEPATITIS C VIRUS AND KITS FOR THE USE THEREOF

This invention relates to a method to detect antibodies against the env protein of the hepatitis C virus and to a kit for the use thereof.

More particularly, this invention relates to a method to detect antibodies against any variant of the envelope (env) protein of the hepatitis C virus (HCV) for employment in the preparation of in vitro diagnostic assays either as marker of HCV infection, or of viral activity in chemical or pharmaceutical preparations, or for identifying new serotypes.

The documents cited with a numeral reference are listed at the end of this disclosure.

The virus HCV is believed to be responsible for the hepatites classified as non-A/non-B (PT-NANB) (1). The existence of an etiological agent for NANB hepatitis has been also proved by Alter et al. (2). The virus has been identified as an RNA virus, of positive polarity, and the genome, in the form of cDNA, has been wholly cloned and sequenced. From an analysis of the sequence it turned out that the sequence in question consists of about 10,000 ribonucleotides and forms a single reading frame that potentially codes for a single amino acid chain. This same organization is also present in other viral families such as those of flavivirus and of Pestivirus; however, other structural characteristics make it uncertain to set forth a precise taxonomic position of HCV.

The cloning of a first portion of the genome has been disclosed by Choo, Q.L. et al. (3), and the sequence has been published in the European Patent EP 88310922.5. The regions identified correspond to the so-called nonstructural regions which, in a way similar to that of flavivirus, have been called NS1, NS2, NS3, NS4 and NS5.

More recently, structural regions, coding for capsid and for surface proteins, have been cloned and sequenced. Such sequences have been published by

Okamoto, H. et al. (4) and in the European Patent application EP 90302866.0.

In order to identify immunological markers of HCV infection, large amounts of viral antigens are needed. However, differently from other hepatotropic viruses, such as HBV and HDV, the concentration of HCV in the liver and in the blood is very low and, differently from the virus of hepatitis A (HAV), HCV cannot be grown in vitro. Therefore it is not available a good natural source of viral antigens.

Accordingly, the preparation of immunological tests requires the availability of synthetic peptides capable of mimicking the immunological activity of viral antigens. To that aim, the identification of specific protein portions, denominated epitopes, capable of reacting with antibodies is necessary due to the short length of synthetic peptides. Moreover, it is well known that tests which employ just the epitope of the protein are more sensitive and more accurate.

Up to the present time it has been impossible to identify portions with antigenic activity of HCV env protein, capable of reacting with antibodies and, therefore, the env protein or portions thereof has never been employed for immunological tests.

It is well known that RNA viruses are characterized by a high frequency of spontaneous mutation. In the case of HCV, 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. Moreover 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.

The availability of an anti-env marker with serological characteristics such as those of the object of this invention, can lead to more specific tests, which can be particularly employed for anti-HCV screening of blood samples. Indeed, an analysis, carried out by Contreras et al. (6) just employing the test based on the c100 protein gives rise to a remarkable number of false positive results, with no precise identification of the infected samples.

Finally, as tests which employ amplification procedures such as the PCR (polymerase chain reaction) are not exploitable for massive screenings, it is useful to correlate the positive results obtained with the assay realized by the Authors and the results obtained with the PCR.

The immunological assays that are already known in the present state of the art exploit recombinant polypeptides which are expressed in different organisms

(S.cerevisiae or E. coli). More particularly, a so-called "first-generation" assay makes use of a fusion protein of 363 amino acids (protein c100), which is expressed in S.cerevisiae, coded by the genome portion which has been called NS4. The employment of such protein in an indirect ELISA assay allows anti-HCV antibodies to be identified in 80-85 % of chronic PT-NANB hepatites and in 15 X of acute PT-NANB (6).

The introduction of new markers such as the capsid antigen (a protein of 120 amino acids, c22) and of a polypeptide coded by the region NS3 (the protein c33 of 210 amino acids) has contributed to a high improvement on the quality of immunological assays. Indeed, quite recently, Van der Poel et al. (7), in a comparison between the performance of the ELISA assay based on the polypeptide clOO and the confirmation assay which is called RIBA-4, containing the proteins c100, c22 and c33, have shown that, with the addition of the polypeptides c22 and c33, both the clinical sensitivity and the specificity are increased, because the presence of more precocious antibodies (anti-c22 and c33) becomes detectable, and the falsely positive results are reduced.

Accordingly, it is the object of this invention a method to detect antibodies against the HCV virus, said method comprising the following steps:

- to react epitopes of the env protein of HCV with said antibodies; - to evidentiate said reaction.

Preferably, said reaction step of the HCV env protein epitopes, with said antibodies comprises the adhesion of env synthetic peptides to a solid phase, the incubation of said peptides in the sample; and said detection step of said reaction comprises the incubation with a detecting system which is selected from the following group: an enzymatic tracer with incubation with chromogen and measurement of the optical density, a radioisotopic system.

According to a preferred embodiment of this invention, said sample is a serum sample, alternatively said sample is a chemical product, and alternatively said sample is a pharmaceutical product of natural origin or recombinant.

It is an object of this invention a method to detect antibodies against the HCV virus, wherein said epitopes are comprised in the portion from the amino acid 209 to the amino acid 259 of the env protein, according to the numbering of (3), preferably in the sequences of the following group: SEQ ID N1, SEQ ID N2 and SEQ ID N3; preferably comprising from the amino acid 13 to the amino acid 46 of SEQ ID N1, alternatively from the amino acid 21 to the amino acid 30 of SEQ ID N1; alternatively comprising from the amino acid 13 to the amino acid 46 of SEQ ID N2, alternatively from the amino acid 21 to the amino acid 30 of SEQ ID N2; alternatively comprising from the amino acid 13 to the amino acid 47 of SEQ ID N3, and alternatively from the amino acid 21 to the amino acid

31 of SEQ ID N3.

In a particular embodiment of this invention, said epitopes are synthetic peptides which are preferably cyclized between 2 residues od cysteine.

This invention will be now disclosed in some working examples of the same, with reference to the following figures, wherein:

- Figures 1A, 1B, and 1C represent the hydrophilic profiles respectively of the env 1, env 2 and env 3 variants.

EXAMPLE 1 Identification of 3 variants in the region of the eny protein and synthesis of the corresponding peptides

An investigation carried out by means of nucleic acid amplification procedures from serum samples (PCR, 7) allowed the identification of 3 main variants of the env surface protein to be carried out, said variants being called respectively env 1, env 2, env 3, and comprising the sequences disclosed respectively as SEQ ID N1, SEQ ID N2 and 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 cysteines, 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. 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).

EXAMPLE 2 Cyclization of peptides

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 l 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 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 NH₄OH. Potassium ferricyanide is then added slowly to the solution (400 mg K₃Fe(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).

Alternatively 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 NH₄Cl. 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 μl 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 disappear.

EXAMPLE 3 Immunolocrical assay

In order to determine the immunogenicity of linear and cyclized peptides described in EXAMPLE 2, an ELISA assay is carried out.

The cyclic and linear peptides are dissolved in 50 mM carbonate buffer, pH 9.6 at a concentration of 5μg/ml. 200μl/well of a microtitration plate is dispensed and incubated for 1 hr at 37°C. The overcoating of the wells is performed by coating to the empty wells 300 μl of a solution containing 50 mM Tris-HCl pH 7.4 and 0.2% bovine serum albumin (BSA, Sigma, Fraction V). The plates are incubated for 2 hrs at room temperature. Finally 300 μl/well of a solution containing 10% sucrose, 4% polyvinylpirrolidone and 9% NaCl is added and left for 1 hr at room temperature.

The ELISA assay is performed by dispensing 200 μl/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 μl of a solution containing goat IgG anti-human IgGs, conjugated with horse radish peroxidase (HRP).

After five washings with washing buffer the plates are incubated for 30 min with a chromogen-substrate solution (tetramethylbenzidine and

3% hydrogenperoxide). The reaction is stopped with IN sulphuric acid and the absorbance is read at 450nm.

The serum utilized (21) belongs to the panel BBI mixed HCV (Boston Biomedica Inc.). The control HCV negative serum gives constantly values lower than 0.002.

The results are shown in the following Table 1.

The results show that env 1, env 2 and env 3 peptides are able to react with anti HCV sera. The reactivity is greatly increased when such peptides are made cyclic and therefore have a conformational structure similar to the corresponding region of the whole env protein. The reactivity decreases proportionally with serum diluitions, thus indicating that the reaction is specific.

This invention has been disclosed with specific reference to some preferred embodiments of the same. but it is to be understood that modifications and/or changes can be introduced by those who are skilled in the art without departing from the spirit and scope of the invention for which a priority right is claimed.

LIST OF THE SEQUENCE CHARACTERISTICS

SEQ ID N1

SEQUENCE TYPE: Nucleotide with corresponding peptide LENGTH OF THE SEQUENCE: 153 base pairs

CONFORMATION: single helix

TOPOLOGY: linear

MOLECULAR TYPE: cDNA from genomic RNA

HYPOTHETIC SEQUENCE: no

ANTI-SENSE: no

ORIGINAL SOURCE: HCV virus variant A1

EXPERIMENTAL SOURCE: genic library from viral isolate CHARACTERISTICS: coding for a portion of env protein variant env 1

IDENTIFICATION METHOD: experimental

PROPERTY: coding sequence

AAC TCG ABC ATT GTB TAG GAB GCT GCC GAC 30

Asn Ser Ser lle Val Tyr Glu Ala Ala Asp

1 5 10

GCC ATC CTB CAC ACT CCG GGG TGC GTC CCT 60

Ala Ile Leu His Thr Pro Gly Cys Val Pro

11 15 20

TGC GTT CGC GAB GGT AAC GCC TCG AGG TGT 90

Cys Val Arg Glu Gly Asn Ala Ser Arg Cys

21 25 30

TGG GTG GCG ATC ACC CCC ACG GTG GCC ACC 120

Trp Val Ala lie Thr Pro Thr Val Ala Thr

31 35 40

AGG GAT GGC AAA CTC CCC ACA GCG CAC GTT 150

Arg Asp Gly Lys Leu Pro Thr Ala His Val

41 45 50

CGA

Arg

51 SEQ ID N2

SEQUENCE TYPE: Nucleotide with corresponding protein LENGTH OF THE SEQUENCE: 153 base pairs

CONFORMATION: single helix

TOPOLOGY: linear

MOLECULAR TYPE: cDNA from genomic RNA

HYPOTHETIC SEQUENCE: no

ANTI-SENSE: no

ORIGINAL SOURCE: HCV virus variant J1

EXPERIMENTAL SOURCE: genic library from viral isolate

CHARACTERISTICS: coding for a portion of env protein env 2 variant

IDENTIFICATION METHOD: experimental

PROPERTY: coding sequence

AAC TCA AGC ATC GTG TAT GAG GCA GCA GAC 30

Asn Ser Ser He Val Tyr Glu Ala Ala Asp

1 5 10

TTG ATC ATG CAC ACC CCC GGB TGC GTG CCC 60

Leu lle Met His Thr Pro Gly Cys Val Pro

11 15 20

TGC GTT CGG GAG AAC AAC CTC TCC CGC TGC 90

Cys Val Arg Glu Asn Asn Leu Ser Arg Cys

21 25 30

TGB GTA GCG CTC ACT CCC ACG CTT GCG GCC 120

Trp Val Ala Leu Thr Pro Thr Leu Ala Ala

31 35 40

AGG AAT GTC AGC GTC CCC ACA GCA ACA ATA 150

Arg Asn Val Ser Val Pro Thr Ala Thr lie

41 45 50

CGA

Arg

51 SEQ ID N3

SEQUENCE TYPE: Nucleotide with corresponding protein

LENGTH OF THE SEQUENCE: 156 base pairs

CONFORMATION: single helix

TOPOLOGY: linear

MOLECULAR TYPE: cDNA from genomic RNA

HYPOTHETIC SEQUENCE: no

ANTI-SENSE: no

ORIGINAL SOURCE: HCV virus variant 3

EXPERIMENTAL SOURCE: genic library from viral isolate

CHARACTERISTICS: coding for a portion of the env protein env 3 variant

IDENTIFICATION METHOD: experimental

PROPERTY: coding sequence

AAC TCA AGT ATT GTG TAT GAG GCA GCG GAC 30

Asn Ser Ser He Val Tyr Glu Ala Ala Asp

1 5 10

CTB ATC ATG CAC ACC CCC GGG TGC GTG CCC 60

Leu Ile Met His Thr Pro Gly Cys Val Pro

11 15 20

TGC GTT CBG GAA GGA GAC AAC CAC TCC CGC 90

Cys Val Arg Glu Gly Asp Asn His Ser Arg

21 25 30

TGC TGG GTA GCG CTC ACT CCC ACT CTC GCG ISO

Cys Trp Val Ala Leu Thr Pro Thr Leu Ala

31 35 40

GCC AGG AAT AGC AGC GTC CCC ACC ACG ACA 150

Ala Arg Asn Ser Ser Val Pro Thr Thr Thr

41 45 50

ATA CGA

Ile Arg

51 BIBLIOGRAPHY

1) Prince A.M. Lancet (1974), II:241-246.

2) Alter M. at al. Lancet (1975), II:838-841

3) Choo, Q-L. et al. Science (1988), 244:359-362.

4) Okamoto, H. et al. Japan J. Exp. Med. (1990), 60:167-177.

5) Weiner A.J. et al. Virology (1999), 180:842-848.

6) Contreras et al. Lancet (1991) 337-753-757.

7) Sambrook J., Fritsch E.F. and Maniatis T. Molecular cloning: a laboratory manual (1989) II Ed. CSH Lab.

Press.

8) Norby, E. et al. Nature (1987) 329:248-250.

9) Oldstone, M. et al., J. of Virology (1991) 65:1727-1734

10) Rink, Tetrahedron Lett. (1987) 28:3787.

11) Ellman, G.L. Arch. Biochem. Biophys. (1959) 82:70.

Claims

1. A method to detect HCV virus antibodies, said method being characterized in that it comprises the following steps:

- to react epitopes of the HCV env protein with said antibodies;

- to evidentiate said reaction.

2. A method to detect HCV virus antibodies according to claim 1, said method being characterized in that said reaction step of the HCV env protein epitopes with said antibodies comprises the adhesion of said synthetic peptides to a solid phase, the incubation of said peptides in the sample; and the step of detecting said reaction comprises the incubation with a detecting system selected from the following group: an enzymatic tracer with chromogen incubation and measurement of the optical density, a radioisotopic system.

3. A method to detect HCV virus antibodies according to claim 2, characterized in that said sample is a serum sample.

4. A method to detect antibodies against the HCV virus according to claim 2, said method being characterized in that said sample is a chemical product.

5. A method to detect antibodies against the HCV virus according to claim 2, characterized In that said sample is a pharmaceutical product of natural or recombinant origin.

6. A method to detect antibodies against the HCV virus according to any one of the preceding claims, characterized in that said epitopes are included in the portion from the amino acid 209 to the amino acid 259 of the env protein according to the numbering of Choo, Q.L. et al., Science (1988), 244:359-362 (3).

7. A method to detect HCV virus antibodies according to claim 6, said method being characterized in that said sequence is comprised in the sequence SEQ ID N1.

8. A method to detect antibodies against the HCV virus according to claim 7, said method being characterized in that said sequence comprises the portion from the amino acid 13 to the amino acid 46 of the sequence SEQ ID N1.

9. A method to detect antibodies against the HCV virus according to claim 7, said method being characterized in that said sequence comprises the portion from the amino acid 21 to the amino acid 30 of the sequence SEQ ID N1.

10. A method to detect HCV virus antibodies according to claim 6, said method being characterized in that said sequence is included in the sequence SEQ ID N2.

11. A method to detect HCV virus antibodies according to claim 10, characterized in that said sequence comprises the portion from the amino acid 13 to the amino acid 46 of the sequence SEQ ID N2.

12. A method to detect HCV virus antibodies according to claim 10, characterized in that said sequence comprises the portion from the amino acid 21 to the amino acid 30 of the sequence SEQ ID N2.

13. A method to detect HCV virus antibodies according to claim 6, said method being characterized in that said sequence is included in the sequence SEQ ID N3.

14. A method to detect HCV virus antibodies according to claim 13, said method being characterized in that said sequence comprises the portion from the amino acid 13 to the amino acid 47 of the sequence SEQ ID N3.

15. A method to detect antibodies against the HCV virus according to claim 13, said method being characterized in that said sequence comprises the portion from the amino acid 21 to the amino acid 31 of the sequence SEQ ID N3.

16. A method to detect HCV virus antibodies according to any one of the preceding claims. characterized in that said epitopes are synthetic peptides.

17. A method to detect HCV virus antibodies according to claim 16 characterized in that said peptides are cyclized.