Chemistry: molecular biology and microbiology – Vector – per se
Reexamination Certificate
1998-11-30
2001-04-03
Scheiner, Laurie (Department: 1641)
Chemistry: molecular biology and microbiology
Vector, per se
C435S069300, C435S235100, C536S023720
Reexamination Certificate
active
06210962
ABSTRACT:
The present invention relates to nucleotide and peptide sequences of a European, more particularly French, strain of the hepatitis C virus, as well as to the diagnostic and therapeutic applications of these sequences.
The hepatitis C virus is a major causative agent of infections by viruses previously called “Non-A Non-B” viruses. Infections by the C virus in fact now represent the most frequent forms of acute hepatitides and chronic Non-A Non-B hepatitides (Alter et al. (1), Choo et al., (3); Hopf et al., (5); Kuo et al., (8); Miyamura et al., (11). Furthermore, there is a relationship (the significance of which is still poorly understood) between the presence of anti-HCV antibodies and the development of primary liver cancers. It has also been shown that the hepatitis C virus is involved in both chronic or acute Non-A Non-B hepatitides linked to transfusions of blood products or of sporadic origin.
The genome of the hepatitis C virus has been cloned and the nucleotide sequence of an American isolate has been described in EP-A-0 318 216, EP-A-0 363 025, EP-A-0 388 232 and WO-A-90/14436. Moreover, data is currently available on the nucleotide sequences of several Japanese isolates relating both to the structural region and the nonstructural region of the virus (Okamoto et al., (12), Enomoto et al., (4), Kato et al., (6); Takeuchi et al., (15 and 16)). The virus exhibits some similarities with the group comprising Flavi- and Pestiviruses; however, it appears to form a distinct class, different from viruses known up until now (Miller and Purcell, (10)).
In spite of the breakthrough which the cloning of HCV represented, several problems persist:
a substantial genetic variability exists in certain regions of the virus which has made it possible to describe the existence of two groups of viruses,
diagnosis of the viral infection remains difficult in spite of the possibility of detecting anti-HCV antibodies in the serum of patients. This is due to the existence of false positive results and to a delayed seroconversion following acute infection. Finally there are clearly cases where only the detection of the virus RNA makes it possible to detect the HCV infection while the serology remains negative.
These problems have important implications both with respect to diagnosis and protection against the virus.
The authors of the present invention have carried out the cloning and obtained the partial nucleotide sequence of a French isolate of HCV (called hereinafter HCV E1) from a blood donor who transmitted an active chronic hepatitis to a recipient. Comparison of the nucleotide sequences and the peptide sequences obtained with the respective sequences of the American and Japanese isolates showed that there was
a high conservation of nucleic acids in the noncoding region of HCV E1,
a high genetic variability in the structural regions called E1 and E2/NS1,
a smaller genetic variability in the nonstructural region.
The present invention is based on new nucleotide and polypeptide sequences of the hepatitis C virus which have not been described in the abovementioned state of the art.
The subject of the present invention is thus a DNA sequence of HCV E1 comprising a DNA sequence chosen from the nucleotide sequences of at least 10 nucleotides between the following nucleotides (n); n
118
to n
138
; n
177
to n
202
; n
233
to n
247
; n
254
to n
272
and n
272
to n
288
represented in the sequence ID SEQ No.2, and, n
156
to n
170
; n
170
to n
217
; n
267
to n
283
and n
310
to n
334
represented in the sequence ID SEQ No.3; as well as analogous nucleotide sequences resulting from degeneracy of the genetic code.
The subject of the invention is in particular the following nucleotide sequences: ID SEQ No.2, ID SEQ No.3 and ID SEQ No.4.
The oligonucleotide sequences may be advantageously synthesised by the Applied Bio System technique.
The subject of the invention is also a peptide sequence of HCV E1 comprising a peptide sequence chosen from the sequences of at least 7 amino acids between the following amino acids (aa): aa
58
to aa
66
; aa
76
to aa
101
represented in the peptide sequence ID SEQ No.2; aa
49
to aa
78
; aa
98
to aa
111
; aa
123
to aa
133
; aa
140
to aa
149
represented in the peptide sequence ID SEQ No.3; as well as homologous peptide sequences which do not induce modification of biological and immunological properties.
Preferably, the peptide sequence is chosen from the following amino acid sequences: aa
58
to aa
66
; aa
76
to aa
101
represented in the peptide sequence ID SEQ No.2, aa
49
to aa
78
; aa
98
to aa
111
; aa
123
to aa
133
and aa
140
to aa
149
represented in the peptide sequence ID SEQ No.3.
Moreover, the peptide sequence is advantageously chosen from the peptide sequences ID SEQ No.2, ID SEQ No.3 and ID SEQ No.4.
The subject of the invention is also a nucleotide sequence encoding a peptide sequence as defined above.
Moreover, the subject of the invention is a polynucleotide probe comprising a DNA sequence as defined above.
The subject of the invention is also an immunogenic peptide comprising a peptide sequence as defined above.
The peptide sequences according to the invention can be obtained by conventional methods of synthesis or by the application of genetic engineering techniques comprising the insertion of a DNA sequence, encoding a peptide sequence according to the invention, into an expression vector such as a plasmid and the transformation of cells using this expression vector and the culture of these cells.
The subject of the invention is also plasmids or expression vectors comprising a DNA sequence encoding a peptide sequence as defined above as well as hosts transformed using this vector.
The preferred plasmids are those deposited with CNCM on Jun. 5, 1991 under the numbers I-1105, I-1106 and I-1107.
The subject of the invention is also monoclonal antibodies directed against a peptide sequence according to the invention or an immunogenic sequence of such a polypeptide.
The monoclonal antibodies according to the invention can be prepared according to a conventional technique. For this purpose, the polypeptides may be coupled, if necessary, to an immunogenic agent such as tetanus anatoxin using a coupling agent such as glutaraldehyde, a carbodiimide or a bisdiazotised benzidine.
The present invention also encompasses the fragments and the derivatives of monoclonal antibodies according to the invention. These fragments are especially F(ab′)
2
fragments which can be obtained by enzymatic cleavage of the antibody molecules with pepsin, the Fab′ fragments which can be obtained by reducing the disulphide bridges of the F(ab′)
2
fragments, and the Fab fragments which can be obtained by enzymatic cleavage of the antibody molecules with papain in the presence of a reducing agent. These fragments, as well as the Fc fragments, can also be obtained by genetic engineering.
The derivatives of monoclonal antibodies are for example antibodies or fragments of these antibodies to which markers, such as a radioisotopes, are attached. The derivatives of monoclonal antibodies are also antibodies or fragments of these antibodies to which therapeutically active molecules are attached.
The subject of the invention is also an analytical kit for the detection of nucleotide sequences specific to the HVC E1 strain, comprising one or more probes as defined above.
The subject of the present invention is also an in vitro diagnostic process involving the detection of antigens specific to HCV E1, in a biological sample possibly containing the said antigens, in which, the biological sample is exposed to an antibody or an antibody fragment, as defined above; as well as a diagnostic kit for carrying out the process.
The subject of the invention is also an in vitro diagnostic process involving the detection of antibodies specific to HCV E1 in a biological sample possibly containing the said antibodies, in which a biological sample is exposed to an antigen containing an epitope corresponding to a peptide sequence, as well as a diagnostic kit for the detection
Brechot Christian
Kremsdorf Dina
Porchon Colette
Finnegan Henderson Farabow Garrett & Dunner L.L.P.
Institut Pasteur
Scheiner Laurie
LandOfFree
Nucleotide and peptide sequences of an isolate of the... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Nucleotide and peptide sequences of an isolate of the..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Nucleotide and peptide sequences of an isolate of the... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2521982