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
1995-01-30
2001-05-22
Schwadron, Ronald B. (Department: 1644)
Drug, bio-affecting and body treating compositions
Antigen, epitope, or other immunospecific immunoeffector
Amino acid sequence disclosed in whole or in part; or...
C424S186100, C424S204100, C424S228100, C530S300000, C530S324000, C530S810000, C530S388300, C530S389400, C436S820000, C436S518000
Reexamination Certificate
active
06235284
ABSTRACT:
BACKGROUND
1. Field of the Invention
The present invention generally relates to synthetic polypeptides, that is to say which are obtained by preparative routes such as chemical synthesis, composed of consecutive amino acids which are together identical to any fragment, sequence or region of the structural protein of the nucleocapsid called (SEQ ID NO:3) CORE protein (SEQ ID NO:3) of the human hepatitis C virus (HCV). These polypeptides can be used as synthetic antigens in various applications arising from their immunogenicity and which are specified below; at the forefront of these applications is the detection HCV in various body fluids such as for example a blood sample.
2. Description of Related Art
It has been established that the nucleocapsid protein or CORE protein (SEQ ID NO:3) of HCV, which is composed as established by
FIG. 1
(SEQ ID NO:3) of 191 amino acids, is that which exhibits the greatest homology, on the one hand, between the sequences of the same group of viral isolates, and, on the other hand, between the different groups of viruses. Moreover, this CORE protein (SEQ ID NO:3) is encoded by a structural part of the HCV genome and therefore constitutes a structural protein. The high conservation of the structure of this protein makes it a particularly suitable candidate for the immunological detection of HCV.
Thus, the work of Hosein B, Fang C T, Popovsky M A, Ye J, Zhang M, Wang C Y, published in Improved serodiagnosis of hepatitis C virus infection with synthetic peptide antigen from capsid protein, Proc Natl Acad Sci USA 1991; 88: 3647-51, made it possible to determine an immunodominant region in the CORE protein (SEQ ID NO:3).
In conformity with a publication already mentioned, namely Hosein B, Fang C T et al., Improved serodiagnosis of hepatitis C virus infection with synthetic peptide antigen from capsid protein, Proc Natl Acad Sci USA 1991; 88: 3647-51, various svnthetic peptides corresponding to certain sequences of the CORE protein (SEQ ID NO:3) can be used as antigen in detection tests in a solid phase, for example on immunoabsorbent supports.
With the same objective of immunological detection of HCV, the document EP-0,442,394 describes several polypeptides comprising a polypeptide sequence belonging to the abovementioned immunodominant region of the CORE protein.
Among the said polypeptides, the one called VIIIE, was tested in an ELISA test with respect to its immunoreactivity towards the anti-HCV antibodies contained in sera from individuals infected with HCV. This polypeptide demonstrated a good immunoreactivity towards the HCV-infected sera tested.
The substitution of such known polypeptides of the prior art, obtained by chemical synthesis, for the fusion protein corresponding to the CORE protein (SEQ ID NO:3) itself in tests of detection is advantageous since it makes it possible to reduce the risks of immunoreaction with antibodies which may be present in a sample and which are different from those directed against HCV.
However, it appeared essential to the Applicant to be able to determine a minimal and sufficient sequence for a polypeptide which, from the point of view of its antigenic properties, is equivalent to the protein in its entirety.
Indeed, the longer the peptide, the higher the risks capable of interfering with the antigenicity of the said peptide because of the higher frequency of the following events:
interference between the peptide and antibodies different from those directed against HCV by cross-reactions or between the peptide and other biological molecules present in the medium,
conformational modifications relative to the structure of the native protein which may result in a disappearance of secondary and/or tertiary conformations corresponding to epitopic sites, or appearance of secondary and/or tertiary conformations different from those which the whole protein adopts, which are capable of interacting with antibodies other than the anti-HCV antibodies.
According to EP-0,442,394, the inventors have tried to shorten the length of the polypeptide VIIIE by respectively amputating 9, 19, 29 and 39 amino acids from its N-terminal part.
The immunoreactivity of each of these peptides was evaluated in ELISA tests and it is observed that the higher the number of amino acids amputated, the lower the immunoreactivity.
SUMMARY OF THE INVENTION
In contrast to these results, the present invention provides a polypeptide, or its fragments, which although consisting of an amino acid sequence much shorter than that of the VIIIE polypeptide structure manifests an immunoreactivity with all the sera of individuals or samples infected with HCV and which carry antibodies directed against the nucleocapsid protein.
The origin of the present invention is the following completely unexpected discoveries, which result from the experimental procedure outlined below:
1) an immunodominant region represented by at most the first 45 amino acids exists in the CORE protein (SEQ ID NO:3) of HCV;
2) this immunodominant region is sufficient by itself in order to obtain the same sensitivity as the total CORE protein (SEQ ID NO:3) regarding the detection of anti-HCV antibodies;
3) this immunodominant region must be continuous if it is desired to react with all the sera of individuals or blood samples infected with HCV and which possess antibodies directed against the CORE protein (SEQ ID NO:3);
4) this immunodominant region clearly contains conformational type epitopes and linear type epitopes.
Consequently, the polypeptide used in conformity with the invention comprises an isolated peptide sequence which is composed of about the 45 N-terminal amino acids of the HCV virus CORE protein (SEQ ID NO1).
Preferably, the polypeptide of the invention consists of only or of an isolated peptide sequence composed of the 45 N-terminal amino acids of the said protein or alternatively of any homologous polypeptide comprising about 45 amino acids and exhibiting an antigenic reactivity towards HCV.
Still preferably, the polypeptide of the invention consists of a peptide sequence which is composed of the N-terminal amino acids 2 to 45 of the CORE protein (SEQ ID NO:2).
REFERENCES:
patent: 5106726 (1992-04-01), Wang
patent: 0442394 (1991-02-01), None
patent: 0484787 (1992-05-01), None
patent: 0525910 (1992-07-01), None
patent: WO 92/22571 (1992-12-01), None
Ishida et al., J. Clin. Microbiol., 31:936-940, 1993 Harlow, “Antibodies: A Laboratory Manual”, Cold Spring Harbor Laboratories, 1988, pp. 75, 76.*
Ngo et al., “Chapter 14”, From: The Protein Folding Problem and Tertiary Structure Prediction, Eds Merz et al., Birkhauser, 1994, pp. 492-495.*
Rudinger, Chapter 1, From “Peptide Hormones”, Ed. J.A. Parsons, pp. 1-7, University Park Press, 1976.*
M. Nasoff et al., “Identification of an Immunodominant Epitope Within the Capsid Protein of Hepatitis C Virus”,Proc. Natl. Acad. Sci. USA, vol. 88, No. 12, pp. 5462-5466, Jun. 1991.
B. Hosein et al., “Improved Serodiagnosis of Hepatitis C Virus Infection With Synthetic Peptide Antigen from Capsid Protein,”Proc. Natl. Acad. Sci. USA, vol. 88, No. 9, pp. 3647-3651, May 1991.
E. Munekata et al., “Epitope-Mapping of Hepatitis C Virus Constituting Protein”,Peptide Chemistry 1990, pp. 211-214 (1991).
J. Chiba et al., “Serodiagnosis of Hepatitis C Virus (HCV) Infection With an HCV Core Protein Molecularly Expressed By A Recombinant Baculovirus”,Proc. Natl. Acad. Sci. USA, vol. 88, pp. 4641-4645, Jun. 1991.
G. Barany et al.,The Peptides, vol. 2, pp. 1-284 (1979).
C.L. Van Der Poel et al., “Confirmation of Hepatitis C Virus Infection by New Four-Antigen Recombinant Immunoblot Assay”,The Lancet, vol. 337, Feb. 1991.
Okamoto et al., “Enzyme-Linked Immunosorbent Assay for Antibodies Against the Capsid Protein of Hepatitis C Virus with A Synthetic Oligopeptide”,Japan, J. Exp. Med., vol. 60, pp. 223-233, (1990).
Dalbon Pascal
Jolivet Michel C.
Bio Merieux
Oliff & Berridg,e PLC
Schwadron Ronald B.
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