Compositions and methods useful for hcv infection

Abstract

The present invention provides compositions comprising cells that can effectively produce HCV after HCV infection, compositions for culturing the cells, methods for making the composition and methods for infecting the cells in the composition with HCV. The present invention also provides methods for assaying HCV production and methods for evaluating compounds that affect the production of HCV.

1 - 15 . (canceled) 
     
     
         16 . A composition comprising a serum-free media comprising calcium, free fatty acids (FFAs), high density lipoprotein (HDL), nicotinamide, trace elements, epidermal growth factor (EGF), insulin, transferrin, hydrocortisone, and not low density lipoprotein (LDL) and, optionally, any one of the ingredients selected from the group consisting of glucagon, liver growth factor, ethanolamine and thyrotropin releasing factor. 
     
     
         17 . A method for isolating and cultivating a cell mixture comprising the steps of:
 a. dissecting a liver of a human aged three months or older after conception up to 1 year after birth in a buffer comprising ethylene glycol bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetate (EGTA);   b. incubating the dissected liver in a buffer comprising collagenase to separate cells from the liver;   c. remove objects 40 micron or greater from the separated cells;   d. removing red blood cells from the separated cells;   e. resuspending the cells of step (d) in a serum-free media, comprising calcium, FFAs, HDL, nicotinamide, trace elements, EGF, insulin, transferrin, hydrocortisone, and optionally, further comprising any one of the ingredients selected from the group consisting of: glucagon, liver growth factor, ethanolamine and thyrotropin releasing factor; and   f. culturing the cells in the serum-free media of step (e).   
     
     
         18 . The method according to  claim 17 , wherein the red blood cells are removed by centrifuging the suspended cells at low speed (50×g) to pellet the larger cells for approximately 3-4 minutes, washing the cells that pellet and repeating the centrifuging and washing steps. 
     
     
         19 . The method according to  claim 17 , wherein the serum-free media does not contain low density lipoprotein (LDL). 
     
     
         20 . (canceled) 
     
     
         21 . (canceled) 
     
     
         22 . A method for assaying HCV infection comprising the steps of:
 a. incubating a composition comprising a cell mixture comprising liver cells and hematopoietic cells, the cells being released from the liver of a human aged three months or older after conception up to 1 year after birth, with a feeder cell;   b. contacting the cells in the composition with RNA898 (“RNA 898” deposited on Mar. 27, 2001, in the American Type Culture Collection, 10801 University Boulevard, Manassas, Va. 20110-2209; ATCC Deposit No.: PTA-3237); and   c. measuring the presence of the HCV RNA associated with the cells of the composition, the media in which the cells are cultured or both the cells and the media.   
     
     
         23 . The method according to  claim 22 , wherein the feeder cell is the STO(Reid-99) cell (“STO(Reid-99)”, deposited Mar. 27, 2001, in the American Type Culture Collection, 10801 University Boulevard, Manassas, Va. 20110-2209; ATCC Deposit No: PTA-3236). 
     
     
         24 . The method according to  claim 22 , wherein the quantity of HCV RNA is measured by comparing (a) the amount of HCV RNA present in or associated with the cells or media in which the cells are cultivated with (b) an amount of RNA from a second virus that is used as an internal control. 
     
     
         25 . The method according to  claim 24 , wherein the second viral RNA is from Bovine Viral Diarrhea Virus (BVDV). 
     
     
         26 . A method for evaluating the ability of a compound to affect the production of an HCV comprising the steps of:
 a. incubating a composition comprising a cell mixture comprising liver cells and hematopoietic cells, the cells being released from the liver of a human aged three months or older after conception up to 1 year after birth, with a feeder cell;   b. contacting the cells in the composition with RNA898;   c. administering the compound to the composition before or after the cells are contacted with RNA898 or its infectious equivalent;   d. measuring the HCV associated with the cells, the media in which the cells are cultured, or both the cells and the media.   
     
     
         27 . The method according to  claim 26 , wherein the compound inhibits HCV production. 
     
     
         28 . The method according to  claim 26 , wherein a plurality of compounds are screened simultaneously for their ability to inhibit HCV production. 
     
     
         29 . The method according to  claim 26 , comprising the further step of comparing the measurement of step (d) with the amount of HCV associated with control cells, the media in which the control cells are cultured or both the control cells and the media, wherein the control cells have been subjected to steps (a)-(d) except that no compound or a known inactive compound has been administered. 
     
     
         30 . The method according to  claim 26 , wherein the feeder cell is the STO(Reid-99) cell. 
     
     
         31 . The method according to  claim 26 , wherein the presence of HCV is measured by determining the quantity of HCV RNA associated with the cells or media in which the cells are cultivated. 
     
     
         32 . The method according to  claim 31 , wherein the quantity of HCV RNA is determined by reverse-transcriptase polymerase chain reaction (RT-PCR). 
     
     
         33 . The method according to  claim 32 , wherein the quantity of HCV RNA is measured by comparing (a) the amount of HCV RNA associated with the cell or the media in which the cells are cultivated with (b) an amount of RNA from a second virus that is used as an internal control. 
     
     
         34 . The method according to  claim 33 , wherein the second virus is Bovine Viral Diarrhea Virus (BVDV).