Compositions and methods of using capsid protein from flaviviruses and pestiviruses

This application claims benefit under 35 U.S.C. §119(e) of U.S. Provisional Patent Application Ser. No. 60/237,885, filed Oct. 4, 2000, incorporated herein by reference.

The invention relates to the use of the capsid protein from West Nile virus, and capsid and other proteins from other viruses including viruses of the Flavivirus and Pestivirus genuses, to induce the death of cells by apoptosis, and to vaccines and diagnostics for West Nile virus and other viruses including Flavivirus and Pestivirus infections. The invention also relates to methods of screening for antiviral compounds by identifying compounds that selectively inhibit the ability of capsid protein to induce apoptosis.

West Nile virus (infection has recently emerged in temperate regions of Europe and North America, presenting a threat to humans, horses, and birds. The most serious manifestations of WNV infection is fatal encephalitis. WNV, originally isolated in the West Nile District of Uganda in 1937, is a Flavivirus of the Flaviviridae family, having a size of 40-60 nm, an enveloped, icosahedral nucleocapsid, and a positive-sense, single-stranded RNA genome of 10,000-1,000 bases. For a recent review of WNV, see Holloway, 2000, Outbreak not contained. West Nile virus triggers a reevaluation of public health surveillance, Sci. Am., 282: 20,22, which is incorporated herein by reference. Reviews of the viruses in the Flaviviridae family are provided in the following references: Neyts et al., 1999, Infections with Flaviviridae, Verh. K. Acad. Geneeskd. Belg., 61: 661-697, discussion 697-699; Leyssen, et al, 2000, Perspectives for the treatment of infections with Flaviviridae, Clin. Microbiol. Rev., 13: 67-82; Sherlock, 1999, The hepatic Flaviviridae: summary, J. Viral. Hepat., 6 Suppl. 1: 1-5; and Fields, Knipe, & Howley, eds., Fields Virology (3^rd ed.) Vols. I & II, Lippincott Williams & Wilkins Pubs. (1996), each of which is incorporated herein, in its entirety, by reference.

There is a need for improved methods of prophylactic and therapeutic treatment of Flavivirus and Pestivirus infection. There is a need for improved methods of inducing cell death and of treating diseases characterized by hyperproliferating cells.

The present invention provides methods of inducing the death of cells. The methods of the invention comprise the step of contacting cells with an amount of a Flavivirus or Pestivirus capsid protein, or functional fragment thereof; effective to induce cell death. According to some embodiments of the invention, the Flavivirus capsid protein, or functional fragment thereof, is the capsid protein, or functional fragment thereof, of West Mile virus V). According to some embodiments of the present invention, cells are contacted with Flavivirus or Pestivirus capsid protein, or a functional fragment thereof, in order to induce the cells to die. According to some embodiments of the present invention, a nucleic acid molecule that comprises a sequence which encodes a Flavivirus or Pestivirus capsid protein, or a functional fragment thereof, is introduced into the cells. Expression of the sequence that encodes the Flavivirus or Pestivirus capsid protein, or functional fragment thereof, results in the production of the Flavivirus or Pestivirus capsid protein, or functional fragment thereof, within the cell, causing the cell to die. According to some embodiments of the present invention, the sequence which encodes the Flavivirus or Pestivirus capsid protein, or fractional fragment thereof, is operably linked to regulatory elements which are necessary for expression of the sequence in the cell. According to some embodiments of the present invention, the nucleic acid molecule is DNA. According to some embodiments of the invention, the cells are tumor cells.

The present invention provides methods of identifying compounds that inhibit the ability of Flavivirus or Pestivirus capsid protein, or functional fragments thereof, to induce apoptosis, Method of the invention comprise the steps of (a) contacting cells, in the presence of a test compound, with an amount of Flavivirus or Pestivirus capsid protein, or a functional fragment thereof, sufficient to induce a measurable amount of apoptosis in the cells, and (b) comparing the amount of apoptosis that occurs when the test compound is present with the amount of apoptosis that occurs when the test compound is absent. The present invention relates to a method of identifying compounds that inhibit capsid protein, or functional fragments thereof, from inducing apoptosis in cells that comprises the steps of (a) contacting cells, in the presence of a test compound, with an amount of WNV capsid protein, or a functional fragment thereof, sufficient to induce a measurable amount of apoptosis in the cells, and (b) comparing the amount of apoptosis that occurs when the test compound is present with the amount of apoptosis that occurs when the test compound is absent. According to some embodiments, the measuring step of the method is accomplished by detecting the presence of apoptosis-related markers, including phosphatidylserine (PS) of the cellular membrane, and free 3′-hydroxy termini in DNA.

The present invention relates to pharmaceutical compositions that comprise a Flavivirus or Pestivirus capsid protein, or functional fragment thereof, and a pharmaceutically acceptable carrier. According to some embodiments of the present invention, the pharmaceutical composition comprises WNV capsid protein, or a functional fragment thereof, and a pharmaceutically acceptable carrier.

The present invention relates to pharmaceutical compositions that comprise a nucleic acid molecule that comprises a sequence which encodes a Flavivirus or Pestivirus capsid protein, or functional fragment thereof, and a pharmaceutically acceptable carrier. According to some embodiments of the present invention, the pharmaceutical composition comprises a nucleic acid molecule that comprises a sequence which encodes a Flavivirus or Pestivirus capsid protein, or a functional fragment thereof that is operably linked to regulatory elements which are necessary for expression of the sequence in the cell. The present invention relates to pharmaceutical compositions that comprise a nucleic acid molecule that comprises a sequence which encodes WNV capsid protein, or a functional fragment thereof and a pharmaceutically acceptable carrier. According to some embodiments of the present invention, the pharmaceutical composition comprises a nucleic acid molecule that comprises a sequence which encodes WNV capsid protein, or a functional fragment thereof, that is operably lined to regulatory elements which are necessary for expression of the sequence in the cell. According to some embodiments of the present invention, a pharmaceutical composition comprises a nucleic acid molecule that is DNA.

The present invention relates to methods of treating individuals diagnosed with or suspected of suffering from diseases characterized by hyperproliferating cells which comprise the step of administering to an individual an amount of a Flavivirus or Pestivirus capsid protein, or functional fragment thereof, sufficient to kill the hyperproliferating cells. The present invention relates to methods of treating individuals diagnosed with or suspected of suffering from diseases characterized by hyperproliferating cells which comprise the step of administering to an individual an amount of WNV capsid protein, or a functional fragment thereof, sufficient to kin the hyperproliferating cells. According to some embodiments, methods comprise the steps of administering to such individuals, an effective amount of WNV capsid protein, or a functional fragment of WNV capsid protein. According to some embodiments of the present invention, the sequence that encodes the Flavivirus or Pestivirus capsid protein, or functional fragment thereof, is operably linked to regulatory elements which are necessary for expression of the sequence in cells. According to some embodiments of the present invention, methods comprise the steps of administering to such individuals, an effective amount of a nucleic acid molecule that comprises a sequence which encodes WNV capsid protein, or a functional fragment thereof. According to some embodiments of the present invention, the sequence that encodes the WNV capsid protein, or functional fragment thereof, is operably linked to regulatory elements which are necessary for expression of the sequence in cells. According to some embodiments of the present invention, the nucleic acid molecule is DNA. According to some embodiments of the present invention, the disease characterized by hyperproliferating cells is cancer or psoriasis.

The present invention relates to vaccine compositions that comprise an immunologically effective amount of capsid protein from WNV or a related member of the Flaviviruses or Pestiviruses and a pharmaceutically acceptable carrier. According to some embodiments of the present invention, the vaccine composition comprises an immunologically effective amount of an immunogenic fragment of capsid protein from WNV or a related member of the Flaviviruses or Pestiviruses and a pharmaceutically acceptable carrier.

The present invention relates to vaccine compositions that comprise a nucleic acid molecule that comprises a sequence which encodes capsid protein from WNV or a related member of the Flaviviruses or Pestiviruses and a pharmaceutically acceptable carrier. According to some embodiments of the present invention the vaccine composition comprises a nucleic acid molecule that comprises a sequence which encodes an immunogenic fragment of capsid protein from WNV or a related member of the Flaviviruses or Pestiviruses and a pharmaceutically acceptable carrier. According to some embodiments of the present invention, the vaccine composition comprises a nucleic acid molecule that comprises a sequence which encodes and immunogenic fragment of WNV or related Flavivirus or Pestivirus capsid protein, operably linked to regulatory elements necessary for expression of the sequence in a cell. According to some embodiments of the present invention, a vaccine composition comprises a nucleic acid molecule that is DNA. According to some embodiments of the present invention, a vaccine composition comprises a plasmid.

The present invention relates to methods of immunizing individuals against WNV or a related member of the Flaviviruses or Pestiviruses. The immune responses generated may be prophylactic or therapeutic. The methods comprise the steps of administering to the individual an immunologically effective amount of capsid protein, or immunogenic fragment thereof, from WNV or a related member of the Flaviviruses or Pestiviruses, or a nucleic acid molecule that encodes capsid protein, or an immunogenic fragment thereof, from WNV or a related member of the Flaviviruses or Pestiviruses.

The present invention relates to methods of identifying individuals exposed to capsid protein from WNV or a related Flavivirus or Pestivirus by detecting the presence of capsid protein from WNV or a related Flavivirus or Pestivirus in a sample using antibodies which specifically bind to capsid protein from WNV or a related Flavivirus or Pestivirus. The antibodies are preferably monoclonal antibodies. Quantification of the amount of capsid protein from WNV or a related Flavivirus or Pestivirus present in a sample of an individual may be used in determining the prognosis of an infected individual.

The present invention relates to kits for identifying individuals exposed to WNV or a related Flavivirus or Pestivirus and reagents used in such kits. The kits comprise a first container which contains antibodies which specifically bind to capsid protein from WNV or a related Flavivirus or Pestivirus and a second container which contains capsid protein from WNV or a related Flavivirus or Pestivirus. The antibodies are preferably monoclonal antibodies. The kits may be adapted for quantifying of the amount of capsid protein from WNV or a related Flavivirus or Pestivirus present in a sample of an individual. Such information may be used in determining the prognosis of an infected individual.

The present invention relates to methods of identifying individuals exposed to WNV or a related Flavivirus or Pestivirus by detecting the presence of antibodies against capsid protein from WNV or a related Flavivirus or Pestivirus in a sample using capsid protein from or a related Flavivirus or Pestivirus. Quantification of the amount of anti-capsid protein from WNV or a related Flavivirus or Pestivirus antibodies present in a sample of an individual may be used in determining the prognosis of an infected individual.

The present invention relates to kits for identifying individuals exposed to WNV or a related Flavivirus or Pestivirus and reagents used therein. The kits comprise a first container which contains antibodies which were produced in response to exposure to capsid protein from WNV or a related Flavivirus or Pestivirus and a second container which contains capsid protein from WNV or a related Flavivirus or Pestivirus. The kits may be adapted for quantifying the amount of anti-capsid protein from W or a related Flavivirus or Pestivirus antibodies present in a sample of an individual. Such information may be used in determining the prognosis of an infected individual.