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
1994-02-03
2001-04-03
Allen, Marianne P. (Department: 1631)
Drug, bio-affecting and body treating compositions
Antigen, epitope, or other immunospecific immunoeffector
Amino acid sequence disclosed in whole or in part; or...
C435S005000, C435S069300, C424S186100, C424S204100, C424S228100, C530S350000, C530S826000
Reexamination Certificate
active
06210675
ABSTRACT:
The present invention relates to the isolation and characterisation of the viral agent responsible for post-transfusional non-A non-B hepatitis (PT-NANBH) and in particular to PT-NANBH viral polypeptides, DNA sequences encoding such viral polypeptides, expression vectors containing such DNA sequences, and host cells transformed by such expression vectors. The present invention also relates to the use of a DNA sequence in a nucleic acid hybridisation assay for the diagnosis of PT-NANBH. The present invention further relates to the use of PT-NANBH viral polypeptides or polyclonal or monoclonal antibodies against such polypeptides in an immunoassay for the diagnosis of PT-NANBH or in a vaccine for its prevention.
Non-A non-B hepatitis (NANBH) is by definition a diagnosis of exclusion and has generally been employed to describe cases of viral hepatitis infection in human beings that are not due to hepatitis A or B viruses. In the majority of such cases, the cause of the infection has not been identified although, on clinical and epidemiological grounds, a number of agents have been thought to be responsible as reviewed in Shih et al (
Prog.Liver Dis.,
1986, 8, 433-452). In the USA alone, up to 10% of blood transfusions can result in NANBH which makes it a significant problem. Even for PT-NANBH there may be at least several viral agents responsible for the infection and over the years many claims have been made for the identification of the agent, none of which has been substantiated.
European Patent Application 88310922.5 purports to describe the isolation and characterisation of the aetiological agent responsible for PT-NANBH which is also referred to in the application as hepatitis C virus (HCV). A cDNA library was prepared from viral nucleic acid obtained from a chimpanzee infected with PT-NANBH and was screened using human antisera. A number of positive clones were isolated and sequenced. The resulting nucleic acid and amino acid sequence data, which are described in the application, represent approximately 70% of the 10 kb viral genome and are derived entirely from its 3′-end corresponding to the non-structural coding region.
The present inventors have now isolated and characterised PT-NANBH viral polypeptides by the cloning and expression of DNA sequences encoding such viral polypeptides. Surprisingly, the nucleic acid and amino acid sequence data both show considerable differences with the corresponding data reported in European Patent Application 88310922.5. Overall these differences amount to about 20% at the nucleic acid level and about 15% at the amino acid level but some regions of the sequences show even greater differences. The overall level of difference is much larger than would be expected for two isolates of the same virus even allowing for geographical factors, and is believed to be due to one of two possible reasons.
Firstly, the present inventors and those of the aforementioned European Patent Application used different sources for the nucleic acid used in the cDNA cloning. In particular, the European Patent Application describes the use of chimpanzee plasma as the source for the viral nucleic acid starting material, with the virus having been passaged through a chimpanzee on two occasions. PT-NANBH is of course an human disease and passaging the virus through a foreign host, even if it is a close relative to humans, is likely to result in extensive mutation of the viral nucleic acid. Accordingly, the sequence data contained in European Patent Application 88310922.5 may not be truly representative of the actual viral agent responsible for PT-NANBH in humans. In contrast, the present inventors utilised viral nucleic acid from a human plasma source as the starting material for cDNA cloning. The sequence data thus obtained is much more likely to correspond to the native nucleic acid and amino acid sequences of PT-NANBH.
Secondly, it may be that the viral agent exists as more than one subtype and the sequence data described in the European Patent Application and that elucidated by the present inventors correspond to separate and distinct subtypes of the same viral agent. Alternatively, it may be that the level of difference between the two sets of sequence data is due to a combination of these two factors.
The present invention provides a PT-NANBH viral polypeptide comprising an antigen having an amino acid sequence that is at least 90% homologous with the amino acid sequence set forth in SEQ ID NO: 3,4,5, 18,19,20,21 or 22, or is an antigenic fragment thereof.
SEQ ID NO: 3,4,5,18,19,20,21 or 22 set forth the amino acid sequence as deduced from the nucleic acid sequence. Preferably, the amino acid sequence is at least 95% or even 98% homologous with the amino acid sequence set forth in SEQ ID NO: 3,4,5,18,19,20,21 or 22. Optionally, the antigen may be fused to an heterologous polypeptide.
Two or more antigens may optionally be used together either in combination or fused as a single polypeptide. The use of two or more antigens in this way in a diagnostic assay provides more reliable results in the use of the assay in blood screening for PT-NANBH virus. Preferably, one antigen is obtained from the structural coding region (the 5′-end) and one other antigen is obtained from the non-structural coding region (the 3′-end). It is particularly preferred that the antigens are fused together as a recombinant polypeptide. This latter approach offers a number of advantages in that the individual antigens can be combined in a fixed, pre-determined ratio (usually equimolar) and only a single polypeptide needs to be produced, purified and characterised.
An antigenic fragment of an antigen having an amino acid sequence that is at least 90% homologous with that set forth in SEQ ID NO: 3,4,5, 18,19,20,21 or 22 preferably contains a minimum of five, six, seven, eight, nine or ten, fifteen, twenty, thirty, forty or fifty amino acids. The antigenic sites of such antigens may be identified using standard procedures. These may involve fragmentation of the polypeptide itself using proteolytic enzymes or chemical agents and then determining the ability of each fragment to bind to antibodies or to provoke an immune response when inoculated into an animal or suitable in vitro model system (Strohmaier et al,
J.Gen.Virol.,
1982, 59, 205-306). Alternatively, the DNA encoding the polypeptide may be fragmented by restriction enzyme digestion or other well-known techniques and then introduced into an expression system to produce fragments (optionally fused to a polypeptide usually of bacterial origin). The resulting fragments are assessed as described previously (Spence et al,
J.Gen.Virol.,
1989, 70, 2843-51; Smith et al, Gene, 1984, 29, 263-9). Another approach is to synthesise chemically short peptide fragments (3-20 amino acids long; conventionally 6 amino acids long) which cover the entire sequence of the full-length polypeptide with each peptide overlapping the adjacent peptide. (This overlap can be from 1-10 amino acids but ideally is n−1 amino acids where n is the length of the peptide; Geysen et al,
Proc. Natl. Acad. Sci.,
1984, 81, 3998-4002). Each peptide is then assessed as described previously except that the peptide is usually first coupled to some carrier molecule to facilitate the induction of an immune response. Finally, there are predictive methods which involve analysis of the sequence for particular features, e.g. hydrophilicity, thought to be associated with immunologically important sites (Hopp and Woods,
Proc. Natl. Acad. Sci.,
1981, 78, 3824-8; Berzofsky,
Science,
1985, 229, 932-40). These predictions may then be tested using the recombinant polypeptide or peptide approaches described previously.
Preferably, the viral polypeptide is provided in a pure form, i.e. greater than 90% or even 95% purity.
The PT-NANBH viral polypeptide of the present invention may be obtained using an amino acid synthesiser, if it is an antigen having no more than about thirty residues, or by recombinant DNA technology.
The present invention also provides a DNA
Barbara John Anthony James
Highfield Peter Edmund
Rodgers Brian Colin
Tedder Richard Seton
Allen Marianne P.
Glaxo Wellcome Inc.
Nixon & Vanderhye P.C.
Zeman Mary K
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