Drug – bio-affecting and body treating compositions – Antigen – epitope – or other immunospecific immunoeffector – Amino acid sequence disclosed in whole or in part; or...
Reexamination Certificate
2000-10-13
2003-07-15
Housel, James (Department: 1648)
Drug, bio-affecting and body treating compositions
Antigen, epitope, or other immunospecific immunoeffector
Amino acid sequence disclosed in whole or in part; or...
C424S185100, C424S186100, C424S205100, C424S228100, C424S192100, C435S005000, C435S007100, C435S007500, C536S023400, C536S023720
Reexamination Certificate
active
06592871
ABSTRACT:
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 &Dgr; 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
umber 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 f
Bayer Hubert
Ehrlich-Weinreich Gertraud
Ihlenfeldt Hans Georg
Jung Günther Gerhard
Seidel Christoph
Brinks Hofer Gilson & Lione
Housel James
Li Bao Qun
Roche Diagnostics GmbH
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