Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
1998-03-04
2002-05-21
Caputa, Anthony C. (Department: 1642)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C435S363000, C435S364000, C435S366000, C435S370000
Reexamination Certificate
active
06392028
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to the determination of functional HCV virus genomic RNA sequences, to construction of infectious HCV DNA clones, and to use of the clones, or their derivatives, in therapeutic, vaccine, and diagnostic applications. The invention is also directed to HCV vectors, e.g., for gene therapy or gene vaccines.
BACKGROUND OF THE INVENTION
Brief General Overview of Hepatitis C Virus
After the development of diagnostic tests for hepatitis A virus and hepatitis B virus, an additional agent, which could be experimentally transmitted to chimpanzees [Alter et al.,
Lancer
1, 459-463 (1978); Hollinger et al.,
Intervirology
10, 60-68 (1978): Tabor et al.,
Lancet
1, 463-466 (1978)], became recognized as the major cause of transfusion-acquired hepatitis. cDNA clones corresponding to the causative non-A non-B (NANB) hepatitis agent, called hepatitis C virus (HCV), were reported in 1989 [Choo et al.,
Science
244, 359-362 (1989)]. This breakthrough has led to rapid advances in diagnostics, and in our understanding of the epidemiology, pathogenesis and molecular virology of HCV (see Houghton et al.,
Curr Stud Hematol Blood Transfus
61, 1-11 (1994) for review). Evidence of HCV infection is found throughout the world, and the prevalence of HCV-specific antibodies ranges from 0.4-2% in most countries to more than 14% in Egypt [Hibbs et al.,
J. Inf. Dis
. 168, 789-790 (1993)]. Besides transmission via blood or blood products, or less frequently by sexual and congenital routes, sporadic cases, not associated with known risk factors, occur and account for more than 40% of HCV cases [Alter et al.,
J. Am. Med. Assoc
. 264, 2231-2235 (1990); Mast and Alter,
Semin. Virol
. 4, 273-283 (1993)]. Infections are usually chronic [Alter et al.,
N. Eng. J. Med.
327, 1899-1905 (1992)], and clinical outcomes range from an in apparent carrier state to acute hepatitis, chronic active hepatitis, and cirrhosis which is strongly associated with the development of hepatocellular carcinoma.
Although interferon (IFN)-&agr; has been shown to be useful for tile treatment of a minority of patients with chronic HCV infections (Davis et al.,
N. Engl. J. Med
321, 1501-1506 (1989); DiBisceglie et al.,
New Engl. J. Med
321, 1506-1510 (1989)) and subunit vaccines show some promise in the chimpanzee model [Choo et al.,
Proc. Natl. Acad. Sci. USA
91, 1294-1298 (1994)], future efforts are needed to develop more effective therapies and vaccines. The considerable diversity observed among different HCV isolates [for review, see Bukh et al.,
Sem. Liver Dis
. 15, 41-63 (1995)], the emergence of genetic variants in chronically infected individuals [Enomoto et al.,
J. Hepatol
. 17, 415-416 (1993); Hijikata et al.,
Biochem. Biophys. Res. Comm
. 175, 220-228 (1991); Kato et al.,
Biochem. Biophys. Res. Comm
. 189, 119-127 (1992); Kato et al.,
J. Virol
. 67, 3923-3930 (1993); Kurosaki et al.,
Hepatology
18, 1293-1299 (1993); Lesniewski et al.,
J. Med. Virol
. 40, 150-156 (1993): Ogata et al.,
Proc. Natl. Acad. Sci. USA
88, 3392-3396 (1991); Weiner et al.,
Virology
180, 842-848 (1991); Weiner et al.,
Proc. Natl Acad. Sci. USA
89, 3468-3472 (1992)], and the lack of protective immunity elicited after HCV infection [Farci et al.,
Science
258, 135-140 (1992); Prince et al.,
J. Infect. Dis
. 165, 438-443 (1992)] present major challenges towards these goals.
Molecular Biology of HCV
Classification. Based on its genome structure and virion properties, HCV has been classified as a separate genus in the flavivirus family, which includes two other genera: the flaviviruses (e.g., yellow fever (YF) virus) and the animal pestiviruses (e.g., bovine viral diarrhea virus (BVDV) and classical swine fever virus (CSFV)) [Francki et al.,
Arch. Virol
. Suppl. 2, 223 (1991)]. All members of this family have enveloped virions that contain a positive-strand RNA genome encoding all known virus-specific proteins via translation of a single long open reading frame (ORF).
Structure and physical properties of the virion. Little information is available on the structure and replication of HCV. Studies have been hampered by the lack of a cell culture system able to support efficient virus replication and the typically low titers of infectious virus present in serum. The size of infectious virus, based on filtration experiments, is between 30-80 nm [Bradley et al.,
Gastroenterology
88, 773-779 (1985); He et al.,
J. Infect. Dis
. 156, 636-640 (1987); Yuasa et al.,
J. Gen. Virol
. 72, 2021-2024 (1991)]. Initial measurements of the buoyant density of infectious material in sucrose yielded a range of values, with the majority present in a low density pool of <1.1 g/ml [Bradley et al.,
J. Med. Virol
. 34, 206-208 (1991)]. Subsequent studies have used RT/PCR to detect HCV-specific RNA as an indirect measure of potentially infectious virus present in sera from chronically infected humans or experimentally infected chimpanzees. From these studies, it has become increasingly clear that considerable heterogeneity exists between different clinical samples, and that many factors can affect the behavior of particles containing HCV RNA [Hijikata et al.,
J. Virol
. 67, 1953-1 958 (1993); Thomssen et al.,
Med. Microbiol. Immunol
. 181, 293-300 (1992)]. Such factors include association with immunoglobulins [Hijikata et al., (1993) supra] or low density lipoprotein [Thomssen et al., 1992, supra; Thomssen et al.,
Med. Microbiol. Immunol
. 182, 329-334 (1993)]. In highly infectious acute phase chimpanzee serum, HCV-specific RNA is usually detected in fractions of low buoyant density (1.03-1.1 g/ml) [Carrick et al.,
J. Virol. Meth
. 39, 279-289 (1992); Hijikata et al., (1993) supra]. In other samples, the presence of HCV antibodies and formation of immune complexes correlate with particles of higher density and lower infectivity [Hijikata et al., (1993) supra]. Treatment of particles with chloroform, which destroys infectivity [Bradley et al.,
J. Infect. Dis
. 148, 254-265 (1983); Feinstone et al.,
Infect Immun
. 41, 816-821 (1983)], or with nonionic detergents, produced RNA containing particles of higher density (1.17-1.25 g/ml) believed to represent HCV nucleocapsids [Hijikata et al., (1993) supra; Kanto et al.,
Hepatology
19, 296-302 (1994); Miyamoto et al.,
J. Gen. Virol
. 73, 715-718 (1992)].
There have been reports of negative-sense HCV-specific RNAs in sera and plasma [see Fong et al.,
Journal of Clinical Investigation
88:1058-60 (1991)]. However, it seems unlikely that such RNAs are essential components of infectious particles since some sera with high infectivity can have low or undetectable levels of negative-strand RNA [Shimizu et al.,
Proc. Natl. Acad. Sci. USA
90: 6037-6041 (1993)].
The virion protein composition has not been rigorously determined, but putative HCV structural proteins include a basic C protein and two membrane glycoproteins,E1 and E2.
HCV replication. Early events in HCV replication are poorly understood. Cellular receptors for the HCV glycoproteins have not been identified. The association of some HCV particles with beta-lipoprotein and immunoglobulins raises the possibility that these host molecules may modulate virus uptake and tissue tropism. Studies examining HCV replication have been largely restricted to human patients or experimentally inoculated chimpanzees. In the chimpanzee model, HCV RNA is detected in the serum as early as three days post-inoculation and persists through the peak of serum alanine aminotransferase (ALT) levels (an indicator of liver damage) [Shimizu et al.,
Proc. Natl. Acad. Sci. USA
87: 6441-6444 (1990)]. The onset of viremia is followed by the appearance of indirect hallmarks of HCV infection of the liver. These include the appearance of a cytoplasmic antigen [Shimizu et al., (1990) supra] and ultrastruc
Kolykhalov Alexander A.
Rice, III Charles Moen
Caputa Anthony C.
Howell & Haferkamp LC
Washington University
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