HCV peptide antigens and methods for the determination of HCV

This application is a divisional of U.S. application Ser. No. 08/604,365, filed on Feb. 2, 1996, now U.S. Pat. No. 6,183,949, issued on Feb. 6, 2001, which in turn is a continuation application of U.S. application Ser. No. 07/977,398, filed on Mar. 11, 1993 and now abandoned. Priority is also claimed, under 35 U.S.C. § 119 and or 35 U.S.C. § 365(b), to German applications: No; P 41 41 304.0, filed on Dec. 14, 1991; P 41 22 160.5, filed on Jul. 4, 1991; P 42 09 215.9, filed on Mar. 21, 1992; and PCT/EP92/01468, filed on Jun. 30, 1992. The disclosures of the priority applications are incorporated by reference herein in their entireties.

The invention concerns new HCV peptide antigens, a process for the production of these peptide antigens as well as a method for the determination of HCV using the peptide antigens.

The occurrence of viral hepatitis in the absence of serologic markers of previously known hepatotropic agents (e.g. hepatitis A virus, hepatitis B virus, hepatitis Δ virus, cytomegalovirus and Epstein-Barr virus) is termed non-A, non-B hepatitis (NANB hepatitis). NANB hepatitis is in turn subdivided into parenterally and sporadically transmitted non-a, non-B hepatitis and non-A, non-B hepatitis transmitted by the intestinal route. The causative agent for the parenterally and sporadically transmitted NANB hepatitis, the hepatitis C virus (HCV), has recently been isolated (Choo Q. -L. et al., Science 244 (1989) 359-362 and Kuo, G. et al., Science 244 (1989) 362-364).

HCV is worldwide an important cause of NANB hepatitis and is transmitted by contaminated blood or blood products, by blood transfusions or close personal contact.

The amino acid sequence of the HCV viral proteins is known from EP-A 0 318 216, EP-A 0 363 025, EPA 388 232 and EP-A 0 396 748. The genome of the HCV has a length of 10862 nucleotides. The proteins arising from translation have a total length of ca. 3000 amino acids. The proteins can be divided into structural proteins (envelope and core proteins) and non-structural proteins (NS1-NS5).

It is expedient to carry out the determination of HCV by detecting antibodies against HCV in body fluids using immunological tests. Therefore binding partners for anti-HCV antibodies are necessary for such immunological tests.

Thus it is known that for example the non-structural C 100-3-HCV protein can be used as a binding partner in immunological tests (tests from ABBOTT LABORATORIES, USA and ORTHO DIAGNOSTIC SYSTEMS INC., USA; Science 244 (1989) 359-364; Van der Poel C. L. et al. Lancet 337 (1991) 317; Alter H. J. J. Gastroent. Hepatol. (suppl.) 1990, 78).

A disadvantage of these tests is that a recombinant protein is used as antigen. Proteins are difficult to handle in diagnostic tests because of their susceptibility to denaturation and consequent reduced solubility and function. As a result of the low epitope density on a protein the magnitude of the measurement signal is also less than in a test in which a short-chained peptide antigen is used as the binding partner of the antibody. In addition, when proteins or long-chained peptides are used as antigens in an immunological test there can be an increase in cross-reactivities and unspecific bindings of antibodies. Moreover, reactions with proteins are often diffusion controlled which is an impediment to achieving the desired short times for immunological tests. In addition the production of protein which can be used for diagnostics in sufficient quantity and quality is time-consuming and expensive. Peptides are easily accessible by synthesis and are defined molecules.

Accordingly it is advantageous in an immunological test for anti-HCV antibodies to use peptide antigens which are as short-chained as possible and only represent sections of the total proteins. Such an immunological method is described by Okamoto (Japan J. Exp. Met. 60 (1990) 223 -234). However, it has been shown that the short-chained peptide antigen (sequence 9) described in this publication which is derived from the core region is not sufficiently sensitive to HCV.

The object of the present invention is therefore to provide peptide antigens which are specific for anti-HCV antibodies and are suitable for immunological tests for anti-HCV antibodies.

This object is achieved by the peptide antigens of the sequences

1: SerGlyLysProAlallelleProAspArgGluValLeuTyrArgGluPheAsp (SEQ ID NO. 1)

2: GluCysSerGlnHisLeuProTyrIleGluGInGlyMetMetLeuAlaGluGlnPheLysGInLysGInLysAlaLeuGlyLeuLeuGInThrAlaSerArg- Gln (SEQ ID NO. 2)

3: AlaValGInThrAsnTrpGlnLysLeuGluThrPheTrpAlaLysHisMetTrpAsn (SEQ ID NO. 11) 4: AsnProLysProGlnLysLysAsnLysArgAsnThrAsnArgArg (SEQ ID NO. 12)

5: AsnProLysProGlnArgLysThrLysArgAsnThrAsnArgArg (SEQ ID NO. 15)

6: ProGlnAspValLysPheProGlyGlyGlyGlnlleValGlyGlyVal (SEQ ID NO. 16)

7: ProArgGlySerArgProSerTrpGlyProThrAspProArgArg (SEQ ID NO. 22)

8: GlnLeuPheThrPheSerProArgArgHisTrpThrThrGlnGly CysAsnCysSerlleTyrProGlyHisileThrGlyHisArgMetAlaTrp Asp-MetMetMetAsnTrpSerProThrThrAlaLeuValMetAla (SEQ ID NO. 23)

10: GlnLysLysAlaAlaArgAsnThrAsnArgArg (SEQ ID NO. 29)

11: HisTrpThrThrGlnGlySerAsnSerSerIleTyrProGlyHis (SEQ ID NO. 30)

12: SerSerIleTyrProGlyHisIleThrGlyH isArgMetAlaTrpAspMetMet (SEQ ID NO. 31)

13: ProGluGlyArgThrTrpAlaGlnProGlyTyrProTrpProLeuTyr (SEQ ID NO. 32)

or peptide antigens which represent partial sequences of these peptide antigens with a length of at least four, preferably of at least seven amino acids.

Suitable partial sequences are shown in the sequence protocols and are indicated by letters/number combinations (e.g. 6 a, 2 b).

Particularly preferred partial sequences are:

From sequence 2:

GluCysSerGlnHisLeuProTyrlleGluGlnGlyMetMetLeu (sequence 2a) (SEQ ID NO. 3)

MetMetLeuAlaGluGlnPheLysGlnLysAlaLeuGlyLeuLeuGlnThrAla (sequence 2b) (SEQ ID NO. 4)

MetMetLeuAlaGluGlnPheLysGlnLysAlaLeuGlyLeuLeuGlnThrAlaSerArgGln (sequence 2c) (SEQ ID NO. 5)

HisLeuProTyrIleGlu (sequence 2d) (SEQ ID NO. 6)

Ser Gln His Leu Pro Tyr Ile Glu Gln (sequence 2e) (SEQ ID NO. 7)

Lys Ala Leu Gly Leu Leu Gln (sequence 2f) (SEQ ID NO. 8)

Gln Lys Ala Leu Gly Leu Leu Gln Thr (sequence 2g) (SEQ ID NO. 9)

from sequence 4: Lys Asn Lys Arg Asn Thr Asn Arg Arg (sequence 4a) (SEQ ID NO. 13)

from sequence 6;

ProGlnAspValLysPheProGlyGlyGlyGlnIle (sequence 6a) (SEQ ID NO. 17) Lys Phe Pro Gly Gly Gly Gln Ile Phe (sequence 6b) (SEQ ID NO. 18)

Lys Phe Pro Gly Gly Gly Gln Ile Val (sequence 6d) (SEQ ID NO. 20)

Gln Asp Val Lys Phe Pro Gly Gly Gly (sequence 6e) (SEQ ID NO. 21)

Partial sequences are particularly preferred which have a maximum length of 9 amino acids. These are in particular the sequences 6b, 6d, 6e, 2e, 2f, 2d, 2g, 4a.

The peptide antigens with the sequences 1-3 are contained in the C 100-3 region of the HCV proteins and the peptide antigens with the sequences 4-8, 10-13 are contained in the env/core region of the HCV proteins. The peptide antigens with the sequences 1-8, 10-13 according to the present invention and the peptide antigen 9 of sequence (ArgGlyProArgLeuGlyValArgAlaThrArg LysThrSerGluArgSerGInProArgGlyArgArgGln ProlleProLysAlaArgArgProGluGlyArgThrTrpAlaGlnProGlyTyrProTrpPro, (SEQ ID NO. 25), Okamoto loc. cit) are specified in the sequence protocols SEQ ID NO: 1-32.

An anti-HCV antibody test is carried out according to methods known to one skilled in the art. The invention therefore also concerns a method for the determination of HCV antibodies which is characterized in that the sample is incubated with a combination of at least two peptide antigens from the group of sequences 1-13 or peptide antigens which represent partial sequences of these peptide antigens which have a length of at least 4, preferably of at least 7 amino acids and the amount of anti-HCV antibodies bound to the peptide antigen is determined under conditions which allow the formation of an antibody-antigen complex.

According to the present invention a combination of at least two of the peptide antigens or partial sequences thereof according to the present invention are used. It is particularly preferred that the peptide antigens of sequences 1-3 or partial sequences thereof be combined with at least one peptide antigen from the group of the sequences 4-13 or partial sequences thereof.

Suitable partial sequences of sequence 9 are:

ArgGlyProArgLeuGlyValArgAlaThrArg LysThrSerGluArgSerGlnProArgGly (sequence 9a) (SEQ ID NO. 26)

SerGlnProArgGlyArgArgGlnProlleProLysAlaArgArgProGluGlyArgThr (sequence 9b) (SEQ ID NO. 27)

LysAlaArgArgProGluGlyArgThrTrpAlaGln ProGlyTyr (sequence 9c) (SEQ ID NO. 28)

The combination of the antigens can for example be carried out by using several individual peptide antigens or in that peptide antigens are covalently bound to one another, appropriately by means of an amino acid bridge which differs from the amino acid sequences that naturally occur in HCV proteins or by means of a peptide linker.

The following combinations of antigens are particularly preferred:

Sequence 2b (SEQ ID NO. 4), 4 (SEQ ID NO. 12), and 6 (SEQ ID NO. 16)

Sequence 2b (SEQ ID NO. 4), 2c (SEQ ID NO. 5),4 (SEQ ID NO. 12), and 6 (SEQ ID NO. 16)

Sequence 2a (SEQ ID NO. 3), 2b (SEQ ID NO. 4), 2c (SEQ ID NO. 5), 4 (SEQ ID NO. 12), and 6 (SEQ ID NO. 16)

Sequence 2a (SEQ ID NO. 3), 2b (SEQ ID NO. 4), 2c (SEQ ID NO. 5), 4 (SEQ ID NO. 12), 6 (SEQ ID NO. 16), 9a (SEQ ID NO. 26), and 9b (SEQ ID NO. 27)

Sequence 2a (SEQ ID NO. 3), 2b (SEQ ID NO. 4), 4 (SEQ ID NO. 12), 6 (SEQ ID NO. 16), 9a (SEQ ID NO. 26), and 3 (SEQ ID NO. 11)

Sequence 2a (SEQ ID NO. 3), 2b (SEQ ID NO. 4), 4 (SEQ ID NO. 12), 6 (SEQ ID NO. 16), and 9a (SEQ ID NO. 26)

Sequence 2e (SEQ ID NO. 7), 2g (SEQ ID NO. 9), 4a (SEQ ID NO. 13), 6d (SEQ ID NO. 20), and 6e (SEQ ID NO. 21)

Sequence 2d (SEQ ID NO. 6), 2f (SEQ ID NO. 8), 4a (SEQ ID NO. 13), 6c (SEQ ID NO. 19), and 9c (SEQ ID NO. 28)

Sequence 11 (SEQ ID NO. 30), 12 (SEQ ID NO31), and 8a (SEQ ID NO. 24)

The antigens in the combinations are preferably used in approximately equimolar amounts.

The combination of the antigens of sequences 11, 12, 8a is particularly suitable for detecting patient sera in which a HCV infection has been cured (convalescent sera).

The antigens are preferably used separately without being covalently bound to one another or bound together using a peptide linker.

Since a high sensitivity is necessary for the infection parameter HCV, heterogeneous immunoassays are preferably used for the detection. These heterogeneous tests allow washing steps which considerably reduce the background measurement signal resulting in an increase in sensitivity.

The determination can for example be carried out by means of a radioimmunoassay, enzyme immunoassay or by immunofluorescence. For this the peptide antigen is usually immobilized. The sample which is to be examined for anti-HCV antibodies is added and the antibodies bound to the antigen are determined by means of a labelled anti-human immunoglobulin antibody. The immobilization of the peptide antigen according to the present invention can be carried out adsorptively, covalently or by means of a biological binding pair such as biotin/streptavidin, antibody/antigen or sugar/lectin. In this process the peptide antigen is covalently bound to this partner.

The peptide antigens according to the present invention can preferably be immobilized according to methods familiar to one skilled in the art such as on beads, plastic tubes or microtitre plates (preferably polystyrene or copolymers of polystyrene). This is preferably carried out by adsorbing the peptide antigens unspecifically onto the surface or covalently binding the peptide antigen to functionalized or activated surfaces. The unspecific adsorption can be improved by linking the peptide antigen to a protein to form a conjugate and using this conjugate for the adsorption (cf. e.g. EP-A 0 269 092). The binding can also be carried out via an immobilized antibody. For this the peptide antigen should be modified in such a way that the epitope is not blocked by the antibody binding e.g. by formation of a peptide-protein conjugate.

The conjugation of the peptide antigen to the binding partner is preferably carried out via a spacer. This spacer appropriately contains 10-50, preferably 10-30 atoms and is also preferably an essentially linear molecule. Examples for this are spacers made of alkyl chains, polyether chains or polyamide chains. In a particularly preferred embodiment a peptide antigen with a length of 4 -9 amino acids is bound to the carrier via a linear spacer of 10-30 atoms. If a spacer made of amino acids is to be used, it is appropriate that it consists of amino acids which do not correspond to the sequence in the direct vicinity of the peptide antigen in the HCV gene.

In a preferred embodiment the peptide antigen according to the present invention is covalently bound to biotin whereby the immobilization is carried out by means of an avidin/streptavidin solid phase.

Methods of determination are also suitable in which the detection is not via a labelled antibody but via a labelled additional peptide antigen sequences 1 -13 or partial sequences thereof.

The peptide antigens according to the present invention can be produced according to methods for peptide synthesis familiar to one skilled in the art. The invention therefore also concerns a process for the production of the peptide antigen according to the present invention which comprises binding the amino acid forming the C-terminal end to a carrier, assembling stepwise the peptide antigen starting at the C-terminal end and subsequently cleaving it from the carrier.

The details of this process are that an amino acid is linked, for example via its carboxyl group, to an insoluble polymer which can be easily filtered and then the peptide chain is assembled stepwise starting at the C-terminal end. For this purpose a N-protected amino acid is reacted with a reactive group of the artificial resin. The Nα-protective group is removed from the amino acid which is covalently anchored to the carrier particle and the resulting amino acyl polymer is reacted with the next N-protected amino acid. The Nα-protective group is removed from the dipeptide covalently bound to the carrier resin and the resulting amino acyl polymer is reacted with the next N-protected amino acid. All excess reagents and by-products are removed by simple filtration. As soon as the desired peptide sequence has been prepared in this way, the covalent binding between the C-terminal amino acid and the anchor group of the polymeric carrier is cleaved. The insoluble carrier is removed from the peptide which is now in solution by simple filtration. The peptide is purified by chromatographic methods.

The peptide antigens according to the present invention can for example be prepared according to Merrifield, JACS 85 (1964) 2146. If a biotinylation is necessary this can for example be carried out according to PNAS USA 80 (1983) 4045. A preferred biotinylation agent for this is biotinyl-aminocaproic acid-N-hydroxysuccinimide ester.

A preferred process for the production of biotinylated peptide antigens is to introduce the biotin residue at the N-terminus during a solid phase synthesis of the peptide antigen. This process is preferably used in cases in which the peptide antigen contains several ε-lysine amino groups which are not intended to be biotinylated. This is for example the case when N-α-Fmoc-N-ε-biotinyl-aminocaproyllysine, N-α-Fmoc-N-ε-biotinyllysine is used or when for the biotinylation of the N-terminal amino acids biotin, biotinyl-aminocaproic acid or dimethoxytritylbiotin is used with an activating reagent, such as for example dicyclohexylcarbodiimide, or as an active ester.

In a further preferred embodiment a detection antibody which is for example directed against the Fc part of human IgG is immobilized. A monoclonal antibody is preferably used for this. The peptide antigen is then present in solution. The antibody (analyte) to be detected and also all other antibodies in the sample liquid are then bound by the immobilized antibody. The bound antibody can then bind the analyte which can be detected with a suitable detection system e.g. competitively with a peptide antigen-enzyme conjugate.

It is also possible using the peptide antigens according to the present invention to obtain antibodies by immunization methods familiar to one skilled in the art with which the virus itself can be detected in an immunological test.

The invention therefore also concerns a process for the production of antibodies which is characterized in that a mammal is immunized with a peptide according to the present invention which, if desired, is bound to a carrier and the antibodies are obtained, for example from the serum or the spleen, according to known methods.

In a preferred embodiment B lymphocytes of the immunized animals are fused with a suitable cell line in the presence of transforming agents, the cell line which produces the desired antibodies is cloned and cultured and the monoclonal antibodies are isolated from the cells or from the culture supernatant.

Using this antibody it is possible to directly determined HCV viruses. The invention therefore also concerns a process for the determination of HCV viruses which is characterized in that the sample is incubated with an antibody according to the present invention under conditions which allow the formation of an antigen-antibody complex and the amount of antibody-antigen complex formed is determined.

The invention in addition concerns a process for the production of vaccines using the peptide antigens according to the present invention and a vaccine for treating HCV infections containing a peptide antigen of the sequences 1-8, 10-13 which is carrier-bound if desired or partial sequences thereof or at least two peptide antigens of the sequences 1 -13 or partial sequences thereof as an immunogen in a pharmacologically effective dose and in a pharmaceutically acceptable formulation.

The production of these vaccines can be carried out according to known methods. However, the peptide antigens are preferably first lyophilized and subsequently suspended, if desired with addition of auxiliary substances.

Vaccination with these vaccines or combinations of vaccines according to the present invention can be carried out according to methods familiar to one skilled in the art for example intradermally, intramuscularly, intraperitoneally, intravenously, subcutaneously or intranasally.

For the intramuscular or subcutaneous administration, the vaccine can for example be suspended in physiological saline. For an intranasal or intraoccular application, the vaccine can for example be used in the form of a spray or an aqueous solution. For a local, for example an oral administration, it is often necessary to temporarily protect the immunogens against inactivation, for example against proteolytic enzymes in the cavity of at the mouth or in the stomach. Such a temporary protection can for example be achieved by encapsulating the immunogens. This encapsulation can for example be carried out by coating with a protective agent (microencapsulation) or by embedding a multitude of immunogens according to the present invention in a protective carrier (macroencapsulation).

The encapsulation material can be semipermeable or become semipermeable when introduced into the human or animal body. A biological degradable substance is usually used as a carrier for the encapsulation.

The invention is further elucidated by the following examples and sequence protocols.

The sequence protocols denote the following: