Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
1999-04-21
2002-05-14
Wortman, Donna C. (Department: 1648)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S005000, C435S236000, C435S239000, C424S196110, C424S228100, C530S350000
Reexamination Certificate
active
06387662
ABSTRACT:
BACKGROUND OF THE INVENTION
Hepatitis C virus (HCV) is a major causative agent of post-transfusion and community-acquired non-A, non-B hepatitis world-wide (Kuo, G. et al.,
Science
244:362-364, 1989; Choo O. L. et al.,
Science
244:359-362, 1989; Alter H. J. et al.,
N. Engl. J. Med.
321:1494-1500, 1989; Kato N. et al.,
Proc. Natl. Acad. Sci. USA
87: 9524-9528, 1990). The majority of HCV infected individuals develop chronic hepatitis which progresses eventually to liver cirrhosis and hepatocellular carcinoma (Tong M. J. et al., N. Engl. J. Med. 332:1463-1466, 1995). Currently, no effective vaccine to prevent HCV infection or treatment for chronic HCV infection exists (Lemon, S. M. & Thomas, D. L.,
New Engl. J. Med.
336:177-203, 1997; Hoofuagle, J. & DiBisceclie,
New Engl. J. Med.
336:347-356, 1997). Development of an effective vaccine and/or treatment has been hampered by the inability to propagate HCV efficiently in cultured cells and the lack of a small animal model.
HCV is a member of the flavivirus family (; Francki R. I. B. et al.,
Arch. Virol.,
Suppl. 2.223-233, 1991). The HCV virion contains a positive-strand RNA genome of 9.5 kilobases (kb) including a highly conserved 5′ noncoding region followed by a long open reading frame of 9030 to 9099 nucleotides (nt) that is translated into a single polyprotein of about 3,010 to 3,030 amino acids (Matsuura Y. & Miyamura T.,
Seminars in Virol.
4:297-304, 1993; Hijikata M. et al.,
Proc. Natl. Acad. Sci. USA
88:5547-5551, 1991). Initiation of translation occurs by a mechanism of internal ribosomal entry requiring the 5′ untranslated region (UTR) and a short stretch of HCV coding sequences (Reynolds J. E. et al,
EMBO J.
14:6010-6020, 1995). Processing of the polyprotein occurs by a combination of host and viral proteases to produce at least ten putative viral structural and nonstructural (NS) proteins. The HCV structural proteins comprise the nucleocapsid or core protein (C) and the two putative virion envelope glycoproteins E1 and E2 (Miyamura T. & Matsuura Y.,
Trends Microbiol.
1(6):229-231, 1993). The cleavage of structural proteins from the polyprotein is catalyzed by a host signal peptidase (Hijikata M. et al.,
Proc. Natl. Acad. Sci. USA
88:5547-5551, 1991; Lin C. et al.,
J. Virol.
68(8):5063-5073, 1994), whereas polyprotein cleavage in the nonstructural region requires the presence of HCV-encoded proteinases encoded by the nonstructural region (Grakoui A. et al.,
Proc. Natl. Acad. Sci. USA
90:10583-10587, 1993).
Although the viral genomic organization has been characterized in detail, morphologic analysis of hepatitis C virus has been hampered by low levels of HCV particles in infected patients and the inability to propagate efficiently the virus in cultured cells. The levels of the viral particles present in infected patient plasma and/or liver tissues are very low, making it difficult to visualize the virus. By analogy to other members of the Flaviviridae, the HCV genomic organization suggests a virus consisting of a nucleocapsid comprising a viral genome and core protein coated by a lipid envelope containing the envelope glycoproteins E1 and E2. Studies of HCV infection in chimpanzees, a reliable animal model for hepatitis C, have provided evidence that HCV is inactivated by chloroform, indicating that it contains essential lipids and therefore is probably enveloped (Feinstone, S. M. et al.,
Infect. Immun.
41:816-821, 1983). Filtration studies have estimated the virion particle size to be about 30-60 nm in diameter (He et al.,
J. Infect. Dis.
156:636-640, 1987).
Recombinant HCV proteins have been produced using various expression systems, but no virus-like particles have been generated in these systems (Grakoui A. et al.,
J. Virol.
67:1385-1395, 1993; Hijikata, M. et al.,
Proc. Natl. Acad. Sci. USA
88:5547-5551, 1991; LauFord, B. et al.,
Virol.
197:225-235, 1993; Miyamura, T. & Matsuura, Y.,
Trends Microbiol.
1:229-231, 1993). Production of recombinant HCV proteins suggests that some of the HCV proteins specifically interact. For example, previous results suggest that the HCV core protein interacts with the E1 envelope protein but not with the E2 envelope protein (Lo S.-Y. et al.,
J. Virol.
70(6): 5177-5182, 1996). Recombinant HCV polypeptides produced in vitro have been disclosed in PCT application WO 9604301, PCT application WO 9533053, PCT application WO 9102820 and U.S. Pat. No. 5,372,928.
Virus-like particles have been synthesized for viruses of various families other than Flaviviridae or Pestiviridae using a baculovirus-insect cell expression system (Gheysen D. et al.,
Cell
59:103-112, 1989; Kirnbauer R. et al.,
Proc. Natl. Acad. Sci. USA
89:12180-12184; 1992; Zeng C. O.-Y. et al.,
J. Virol.
70:2736-2742, 1996). The baculovirus-insect cell expression of viral proteins is advantageous because the eukaryotic insect cells can carry out a number of co- or post-translational modifications such as fatty acid acetylation and glycosylation, similar to mammalian cells (Luckow, V. A. & Summers, M. D.,
Virol.
167:56, 1988). Moreover, the baculovirus expression system allows higher levels of heterologous protein synthesis than generally is possible in many mammalian cell expression systems (Luckow, V. A. & Summers, M. D.,
Viral.
167:56, 1988).
The present invention differs from the prior art because it utilizes a recombinant construct that contains nucleic acid that includes part of the 5′ UTR, coding sequences for HCV structural proteins including p7, and produces virus-like particles when the construct is expressed in insect cells. These virus-like particles of an enveloped RNA virus are generated without other components required for viral replication and are assembled intracellularly in vitro. These virus-like particles are effective immunogens for generating HCV-specific antibodies and thus are important for development of an effective HCV vaccine.
SUMMARY OF THE INVENTION
According to one aspect of the invention, there is provided a method of producing virus-like particles in vitro comprising the steps of providing a vector comprising an expression system capable of producing proteins in insect cells, cloning a cDNA that codes for structural proteins of an enveloped RNA virus into the vector such that the cDNA is capable of being expressed in transfected or infected insect cells, transfecting or infecting insect cells with the vector containing the cloned cDNA that codes for the structural proteins of an enveloped RNA virus, maintaining the transfected or infected insect cells in culture for sufficient time to allow expression of the cDNA to produce the structural proteins of an enveloped RNA virus, and allowing the structural proteins to form intracellular virus-like particles. In one embodiment, the method further comprises the step of purifying the intracellular virus-like particles from the cultured cells. In a preferred embodiment, the purifying step comprises lysing the cells to produce a lysate and subjecting the lysate to gradient centrifugation. In another embodiment, the purifying step comprises lysing the cells to produce a lysate and subjecting the lysate to immunoadsorption using an immunoreagent that specifically recognizes a viral protein contained within the virus-like particles. In one embodiment, the method further comprises the step of generating an immune response in a mammal by introducing an effective amount of purified virus-like particles into the mammal in a pharmaceutically acceptable carrier. In a preferred embodiment, the immunizing step is performed in a mammal selected from the group consisting of a mouse, rat, rabbit, goat, sheep, horse and human. In one embodiment of the method, the cloned cDNA is produced from an enveloped RNA virus that is a member of the Sindbis-like superfamily or a member of the Flavivirus-like superfamily. Preferrably, the cloned cDNA is produced from a member of the group consisting of Togaviridae, Bromovirus, Cucumovirus, Tobavirus, Ilarvirus, Tobravirus, Potexvirus, Flaviviridae, and Pestivirus. In
Baumert Thomas F.
Liang T. Jake
Knobbe Martens Olson & Bear LLP
The United States of America as represented by the Department of
Wortman Donna C.
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