Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving virus or bacteriophage
Reexamination Certificate
2000-06-12
2001-08-07
Mosher, Mary E. (Department: 1648)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving virus or bacteriophage
C435S007100, C435S007200, C435S007920, C435S069300, C435S069700, C435S069800, C436S501000, C530S300000, C530S350000, C424S189100, C424S228100
Reexamination Certificate
active
06270960
ABSTRACT:
DESCRIPTION
The invention concerns polypeptides from the non-structural protein 3 (NS3) region of the hepatitis C virus (HCV), a nucleic acid coding for such polypeptides as well as the use of the polypeptide as an antigen in an immunological test procedure or as helicase protein.
The disease denoted non-A-non-B hepatitis is caused in many cases by the hepatitis C virus (HCV). HCV is a single-stranded encapsulated RNA virus whose genome is composed of about 9 to 10,000 bases. This genome codes for structural proteins (core and envelope proteins) as well as for non-structural proteins. The non-structural protein 3 (NS3) region of HCV contains a protease and a helicase. The protease activity is localized in the amino terminal third of the NS3 region.
A partial nucleotide sequence of HCV is disclosed in the European Patent Application EP-A-0 318 216. It claims the use of nucleic acid fragments or polypeptide sections from HCV for a diagnostic test and for therapeutic treatment.
EP-A-0 450 931 incorporated by reference discloses the complete nucleotide and amino acid sequence of HCV. In addition a combination of synthetic HCV antigens is described comprising a first HCV antigen from the C domain and at least one further HCV antigen from one of the non-structural domains NS3, NS4 or NS5 and the envelope domain S. A preferred antigen from the domain NS3 is an antigen denoted C33c which comprises the amino acids 1192 to 1457 encoded by the HCV genome shown in FIG. 1 of EP-A-0 450 931.
The international Patent Application WO92/11370 concerns the cloning and sequencing of various polypeptides from the genome of HCV and the use of these polypeptides without any foreign protein parts in test kits and as a vaccine. A clone NS-3 deposited in connection with this application at the “Deutsche Sammlung für Mikroorganismen und Zellkulturen GmbH, Mascheroder Weg 1b, 38124 Braunschweig, BRD (DSM)” under the number 6847 contains the genetic information for a polypeptide of 527 amino acids from the NS3 region of HCV.
Mori et al., (Jpn. J. Cancer Res. 83 (1992), 264-268) describes the diagnostic detection of a HCV infection by determining viral antibodies in blood using viral proteins as antigens. These HCV proteins are expressed in
E. coli
as a fusion protein with &bgr;-galactosidase. A protein from the NS3 region containing the amino acids 1295 to 1541 of the HCV genome showed the highest sensitivity. However, a disadvantage of such fusion proteins is that cross-reactions with the fused protein part can occur which reduce the specificity of the test reaction.
A test for HCV in blood requires a high degree of specificity and sensitivity. Furthermore the antigen used for the test should be capable of expression in high yield and be stable. Previously known antigens from the HCV genome have disadvantages since they do not fulfil one or several of the above requirements.
Thus an object of the present invention is to provide a polypeptide encoded by the HCV genome in which these disadvantages of the state of the art are at least partially eliminated and which, especially in comparison with known antigens, has a higher specificity and sensitivity and can be expressed in higher yield and is stable.
This object is achieved by a polypeptide which is composed of amino acids 1207±10 to 1488±10 of a hepatitis C virus and has less than 20, preferably less than 15 foreign amino acids. The polypeptide according to the invention preferably contains the amino acids 1207±5 to 1488±5. More preferably they contain amino acids particularly preferably 1207±2 to 1488±2 and most preferably 1207 to 1488 of hepatitis C virus in which the numbering of the amino acid residues refers to FIG. 1 of EP-A-0 450 931 incorporated by reference.
The polypeptide according to the invention can be derived from an arbitrary HCV isolate, for example from a HCV isolate with a nucleotide sequence as described in EP-A-0 450 931. However, the polypeptide is preferably derived from the HCV isolate from which the clone NS3 described in WO92/11370 is derived which was deposited at the “Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH” (DSM), Mascheroder Weg 1b, 38124 Braunschweig, under the number DSM 6847. The polypeptide according to the invention is preferably obtained by recombinant expression of the vector pUC-D26.
SEQ ID NO: 1 shows the amino acid sequence of a polypeptide according to the invention. Amino acids 1-13 are foreign amino acids. Amino acids 14-295 originate from HCV. The polypeptide according to the invention preferably contains amino acids 14-295 of the amino acid sequence shown in SEQ ID NO: 1 and 2 or an amino acid sequence which is at least 90% homologous thereto.
The present invention also concerns a polypeptide defined as above which contains at least one marker group. All known marker groups come into consideration as the marker group that can be detected in a test system i.e. directly or indirectly detectable marker groups. In this connection a directly detectable marker group is understood as a group which produces a directly detectable signal e.g. a radioactive group, an enzyme group, a luminescence group, a metal complex etc. On the other hand the marker group can also be an indirectly detectable group e.g. a biotin or hapten group that is detectable by reaction with a suitable binding partner (streptavidin, avidin or anti-hapten antibody) which in turn carries a signal-generating group. The marker group can be coupled in known manner to the antigen for example via a bifunctional spacer. Such processes for coupling marker groups to peptide antigens are known to a person skilled in the area of immunology and do not need to be described in detail here.
The polypeptide according to the invention preferably contains 1 to 12 and particularly preferably 3 to 7 marker groups.
In addition, the invention concerns a polypeptide as defined above in which one or several of the sulfhydryl groups of cysteine residues are present in covalently modified form. Examples of suitable covalent modifying groups are maleimidodioxaoctylamine (MADOO), N-methyl-maleinimide (NMM), iodoacetic acid and iodoacetamide. The covalent cysteine modification results in particularly high specific immunological reactivity.
Directly or indirectly detectable marker groups are preferably covalently coupled to the sulfhydryl groups of the polypeptide. Examples of SH-reactive bifunctional linkers for coupling to sulfhydryl groups are maleimidopropylamine (MP), maleimidoethylamine (MEA) and maleimidodioxaoctylamine (MADOO).
In addition, it may be preferable to use a polypeptide according to the invention in which one or several cysteine residues are replaced by other natural or artificial amino acids. Cysteine residues are preferably replaced by structurally analogous &agr;-amino acids e.g. serine or &agr;-aminobutyric acid. Cysteine substitutions lead to particularly high stability.
The polypeptide according to the invention has surprising advantages over already known polypeptides. Compared to the antigen C33 described in EP-A-0 450 931 which contains the amino acid residues 1192 to 1457 of the HCV sequence, the polypeptides according to the invention exhibit substantially higher specificity which is manifested in a statistically significant lower number of false positive results in negative sera. Compared to the antigen NS3 described in WO92/11370 which contains the region of amino acids 1007 to 1534 of the HCV sequence, the polypeptide according to the invention has considerably higher stability under test conditions. Compared to a polypeptide which contains amino acids 1227 to 1528 from the NS3 region of HCV, the polypeptide according to the invention has the advantage of improved expression efficiency and a higher sensitivity. Due to these advantages the polypeptides according to the invention are substantially superior to all previously known HCV antigens from the NS3 region.
In addition, the invention concerns an isolate nucleic acid which codes for a polypeptide according to the invention. A pr
Motz Manfred
Schmitt Urban
Seidel Christoph
Soutschek Erwin
Upmeier Barbara
Foley Shanon
Fulbright & Jaworski LLP
Mosher Mary E.
Roche Diagnostics GmbH
LandOfFree
Recombinant antigen from the NS3 region of the hepatitis C... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Recombinant antigen from the NS3 region of the hepatitis C..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Recombinant antigen from the NS3 region of the hepatitis C... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2474482