Compositions and methods of using capsid protein from flaviviruses and pestiviruses

Abstract

This invention provides methods of inducing cell death with Flavivirus or Pestivirus capsid protein, such as West Nile virus (WNV) capsid protein, and functional fragments thereof. The invention also provides methods of treating patients suffering from diseases characterized by hyperproliferating cells by administering pharmaceutical compositions comprising WNV or other virus including Flavivirus or Pestivirus capsid or other protein or a nucleic acid molecule encoding the same. Methods of identifying compounds which have anti-viral and/or anti-WNV and/or anti-Flavivirus and/or anti-Pestivirus capsid or other protein activity are disclosed. The invention also provides vaccine compositions comprising capsid or other proteins, or fragments thereof, or nucleic acids encoding same, from WNV or other virus including Flavivirus or Pestivirus and a pharmaceutically acceptable carrier. The invention also provides diagnostic methods and kits for identifying individuals exposed to WNV or other viruses including Flavivirus or Pestivirus.

1 . A method of inducing cell death comprising the step of 
       contacting a cell with an amount of isolated Flavivirus or Pestivirus capsid protein, or a functional fragment thereof, effective to induce cell death; or 
       introducing into said cell a nucleic acid molecule comprising a nucleotide sequence encoding a Flavivirus or Pestivirus capsid protein, or a functional fragment thereof, said nucleic acid being free from an entire Flavivirus or Pestivirus virus genome, wherein said nucleotide sequence is expressed in said cell at a level effective to induce cell death. 
     
     
         2 . The method of  claim 1 , wherein the isolated capsid protein, or functional fragment thereof, or the nucleic acid molecule is from a virus selected from the Japanese encephalitis virus group subgenus. 
     
     
         3 . The method of  claim 1 , wherein the isolated capsid protein, or functional fragment thereof, or the nucleic acid molecule is from West Nile virus (WNV). 
     
     
         4 . The method of  claim 3 , wherein the functional fragment comprises SEQ ID NO:8. 
     
     
         5 . The method of  claim 3 , wherein the nucleic acid molecule encodes SEQ ID NO:8. 
     
     
         6 . The method of  claim 1 , wherein the cell is a tumor cell. 
     
     
         7 . The method of  claim 1 , wherein the cell is contacted with the Flavivirus or Pestivirus capsid protein, or a functional gent thereof. 
     
     
         8 . The method of  claim 1 , wherein the nucleic acid molecule is introduced into said cell. 
     
     
         9 . A method of identifying compounds that inhibit Flavivirus or Pestivirus capsid protein, or a functional fragment thereof, from inducing apoptosis in cells comprising the steps of
 a) contacting the 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 detectable level of apoptosis in the cells; and   b) comparing the level of apoptosis detected in step (a) with the level of apoptosis that occurs when cells are contacted with Flavivirus or Pestivirus capsid protein, or a functional fragment thereof, in the absence of said test compound.   
     
     
         10 . The method of  claim 9 , wherein the cells are contacted with Flavivirus or Pestivirus capsid protein. 
     
     
         11 . The method of  claim 9 , wherein the cells are contacted with a functional fragment of Flavivirus or Pestivirus capsid protein. 
     
     
         12 . The method of  claim 11 , wherein the functional fragment comprises SEQ ID NO:8. 
     
     
         13 . The method of  claim 9 , wherein the cells are selected from the group consisting of Hela cells, RD cells, and 293 cells. 
     
     
         14 . The method of  claim 9 , wherein the detecting step is an assay that detects a marker of apoptosis. 
     
     
         15 . The method of  claim 14 , wherein the marker is phosphatidylserine (PS) or free 3′-hydroxy DNA termini. 
     
     
         16 . The method of  claim 15 , wherein the assay is GEL analysis or annexin V flow cytometry. 
     
     
         17 . A kit for performing the method of  claim 9  comprising
 a) a container comprising Flavivirus or Pestivirus capsid protein or function fragment thereof; and   b) at least one additional component selected from the group consisting of: instructions, positive controls, negative controls, photos depicting data, and figures depicting data.   
     
     
         18 . An injectable pharmaceutical composition comprising
 a) a Flavivirus or Pestivirus capsid protein, or a functional fragment thereof, or a nucleic acid molecule that comprises a nucleotide sequence that encodes a Flavivirus or Pestivirus capsid protein or a functional fragment thereof; and   b) a pharmaceutically acceptable carrier.   
     
     
         19 . The injectable pharmaceutical composition of  claim 18  comprising
 a) a nucleic acid molecule that comprises a nucleotide sequence that encodes a Flavivirus or Pestivirus capsid protein or a functional fragment thereof; and   b) a pharmaceutically acceptable carrier.   
     
     
         20 . The injectable pharmaceutical composition of  claim 18  comprising
 a) a Flavivirus or Pestivirus capsid protein, or a functional fragment thereof; and   b) a pharmaceutically acceptable carrier.   
     
     
         21 . The injectable pharmaceutical composition of  claim 18  comprising
 a) a WNV capsid protein, or a functional fragment thereof; and   b) a pharmaceutically acceptable carrier.   
     
     
         22 . A method of treating an individual diagnosed with or suspected of suffering from a disease characterized by hyperproliferating cells which comprises the step of administering to said individual an effective amount of the injectable pharmaceutical composition of clam  18 . 
     
     
         23 . A method of treating an individual diagnosed with or suspected of suffering from a disease characterized by hyperproliferating cells which comprises the step of administering to said individual an effective amount of the injectable pharmaceutical composition of  claim 19 . 
     
     
         24 . A method of treating an individual diagnosed with or suspected of suffering from a disease characterized by hyperproliferating cells which comprises the step of administering to said individual an effective amount of the injectable pharmaceutical composition of  claim 20 . 
     
     
         25 . A method of treating an individual diagnosed with or suspected of suffering from a disease characterized by undesirable cells comprising eliminating the undesirable cells by administering to said individual an effective amount of the injectable pharmaceutical composition of  claim 18 . 
     
     
         26 . The method of  claim 24 , wherein the capsid protein, or functional fragment thereof, is WNV capsid protein, or functional fragment thereof. 
     
     
         27 . The method of  claim 22 , wherein the disease is cancer. 
     
     
         28 . The method of  claim 22 , wherein the administration step is accomplished by intratumoral injection of the injectable pharmaceutical composition. 
     
     
         29 . A method of identifying an individual exposed to Flavivirus or Pestivirus comprising the steps of:
 a) contacting antibodies specific for Flavivirus or Pestivirus capsid protein with a sample from the individual; and   b) detecting whether said antibodies are bound to Flavivirus or Pestivirus capsid protein from the sample,   
       wherein detection of binding of the antibodies to Flavivirus or Pestivirus capsid protein is indicative of exposure of the individual to Flavivirus or Pestivirus. 
     
     
         30 . The method of  claim 24 , where the capsid protein is WNV capsid protein. 
     
     
         31 . A kit for identifying individuals exposed to a Flavivirus or Pestivirus comprising
 a) a first container comprising antibodies specific for a Flavivirus or Pestivirus capsid protein; and   b) a second container comprising Flavivirus or Pestivirus capsid protein, or a fragment thereof.   
     
     
         32 . The kit of  claim 31 , wherein the first container comprises antibodies specific for WNV capsid protein and the second container comprises WNV capsid protein, or a fragment thereof. 
     
     
         33 . A method of identifying an individual exposed to a Flavivirus or Pestivirus comprising the steps of:
 a) contacting a sample with Flavivirus or Pestivirus capsid protein; and   b) detecting whether said Flavivirus or Pestivirus capsid protein is bound to antibodies in said sample,   
       wherein detection of binding of Flavivirus or Pestivirus a capsid protein is indicative of exposure of the individual to Flavivirus or Pestivirus. 
     
     
         34 . The method of  claim 33 , wherein the virus is WNV and the capsid protein is WNV capsid protein. 
     
     
         35 . A kit for identifying individuals exposed to a Flavivirus or Pestivirus comprising
 a) a first container comprising Flavivirus or Pestivirus capsid protein; and   b) a second container which contains antibodies which specifically bind to Flavivirus or Pestivirus capsid protein.   
     
     
         36 . The kit of  claim 35 , wherein the capsid protein is WNV capsid protein. 
     
     
         37 . A vaccine composition comprising
 a) an immunologically effective amount of Flavivirus or Pestivirus capsid protein, or an immunogenic fragment thereof; and   b) a pharmaceutically acceptable carrier.   
     
     
         38 . The vaccine of  claim 37 , wherein the Flavivirus or Pestivirus capsid protein, or immunogenic fragment thereof, is WNV capsid protein, or immunogenic fragment thereof. 
     
     
         39 . A vaccine composition comprising
 a) nucleic acid encoding Flavivirus or Pestivirus capsid protein, or an immunogenic fragment thereof; and   b) a pharmaceutically acceptable carrier.   
     
     
         40 . The vaccine of  claim 39 , wherein the nucleic acid encodes WNV capsid protein, or an immunogenic fragment thereof. 
     
     
         41 . A method of treating an individual exposed to a Flavivirus or Pestivirus by administering a therapeutically effective amount of capsid protein, or an immunogenic fragment thereof, from a Flavivirus or Pestivirus, or a nucleic acid encoding capsid protein, or an immunogenic fragment thereof, from a Flavivirus or Pestivirus. 
     
     
         42 . The method of  claim 41 , wherein the virus to which the individual is exposed is WNV, and wherein the capsid protein, or fragment thereof, or the nucleic acid encoding the capsid protein, or immunogenic fragment thereof, is from WNV. 
     
     
         43 . A method of protecting an individual from Flavivirus or Pestivirus infection by administering a prophylactically effective amount of capsid protein, or an immunogenic fragment thereof, from a Flavivirus or Pestivirus, or a nucleic acid encoding capsid protein, or an immunogenic fragment thereof, from a Flavivirus or Pestivirus. 
     
     
         44 . The method of  claim 43 , wherein the virus against which the individual is to be protected is WNV, and wherein the capsid protein, or fragment thereof, or the nucleic acid encoding the capsid protein, or immunogenic fragment thereof, is from WNV.