Papillomavirus infected epithelial cells and methods of producing infectious papillomavirus in epithelial cells and uses thereof

Abstract

The present invention is discloses an improved method of producing infectious papillomavirus in vitro, with a method comprising (a) introducing papillomavirus or papillomavirus DNA or portions thereof necessary for replication, into an epithelial cell; and (b) providing conditions that produce papillomavirus, wherein the conditions comprise not contacting the epithelial cell a fibroblast and does not comprise an organotypic raft culture or a dermal equivalent. The present invention also discloses a papillomavirus infected non-keratinocyte epithelial cell produced by the methods of the present invention. Further, uses of the disclosed method includes detection methods, methods for screening anti-papillomavirus drugs, methods of making recombinant papillomavirus for vaccines and studying the life cycle. Additionally, a method of reducing and assessing the risk of spontaneous abortion is disclosed.

We claim:  
     
         1 . A method of producing infectious papillomavirus in vitro, wherein said method comprises: 
 (a) introducing papillomavirus or papillomavirus DNA or portions thereof necessary for replication, into an epithelial cell; and    (b) providing conditions that produce papillomavirus, wherein said conditions comprise said epithelial cell not contacting a fibroblast.    
     
     
         2 . The method of  claim 1 , wherein said epithelial cell is not a keratinocyte.  
     
     
         3 . The method of  claim 1 , wherein said epithelial cell is a mammary epithelial cell.  
     
     
         4 . The method of  claim 3 , wherein said mammary epithelial cell is a breast ductal epithelial cell or a breast lobular epithelial cell.  
     
     
         5 . The method of  claim 1 , wherein said introducing of said papillomavirus or said papillomavirus DNA is performed by one of the methods selected from the group consisting of transfecting into, lipofecting into, electroporating into and contacting with said papillomavirus or papillomavirus DNA into said epithelial cell.  
     
     
         6 . The method of  claim 1 , wherein said papillomavirus DNA is papillomavirus genomic DNA.  
     
     
         7 . The method of  claim 1 , wherein said papillomavirus DNA is mutant DNA, and when expressed in said epithelial cell, results in an attentuated or defective infectious papillomavirus.  
     
     
         8 . The method of claims  1 , wherein said papillomavirus is a human papillomavirus.  
     
     
         9 . A papillomavirus infected non-keratinocyte epithelial cell produced by the method of  claim 1 .  
     
     
         10 . A papillomavirus infected epithelial cell produced by the method of  claim 9 , wherein said epithelial cell is a mammary epithelial cell.  
     
     
         11 . The papillomavirus infected epithelial cell of  claim 10 , wherein said mammary epithelial cell is a breast ductal epithelial cell or a breast lobular epithelial cell.  
     
     
         12 . The paillomavirus infected epithelial cell of  claim 9 , wherein said papillomavirus is a human papillomavirus.  
     
     
         13 . A method of producing infectious papillomavirus in vitro, wherein said method comprises: 
 (a) introducing papillomavirus or papillomavirus DNA or portions thereof necessary for replication, into an epithelial cell; and    (b) providing conditions that produce infectious papillomavirus, wherein said conditions comprise said epithelial cell not contacting an organotypic raft culture or a dermal equivalent.    
     
     
         14 . The method of  claim 13 , wherein said epithelial cell is a mammary epithelial cell.  
     
     
         15 . The method of  claim 14 , wherein said mammary epithelial cell is a breast ductal epithelial cell or a breast lobular epithelial cell.  
     
     
         16 . A method of producing infectious papillomavirus in vitro, wherein said method consists essentially of: 
 (a) introducing papillomavirus or papillomavirus DNA or portions thereof necessary for replication, into an epithelial cell; and    (b) providing conditions that produce infectious papillomavirus.    
     
     
         17 . The method of  claim 16 , wherein said epithelial cell is a mammary epithelial cell.  
     
     
         18 . The method of  claim 17 , wherein said mammary epithelial cell is a breast ductal epithelial cell or a breast lobular epithelial cell.  
     
     
         19 . A method of detecting the presence of papillomavirus in a subject comprising: 
 (a) introducing papillomavirus or papillomavirus DNA or portions thereof necessary for replication, into an epithelial cell;    (b) providing conditions that produce infectious papillomavirus in said epithelial cell, wherein said conditions comprise said epithelial cell not contacting a fibroblast; and    (c) detecting the presence of papillomavirus in said epithelial cell.    
     
     
         20 . The method of  claim 19 , wherein said epithelial cell is not a keratinocyte.  
     
     
         21 . The method of  claim 19 , wherein said epithelial cell is a mammary epithelial cell.  
     
     
         22 . The method of  claim 21 , wherein said mammary epithelial cell is a breast ductal epithelial cell or a breast lobular epithelial cell.  
     
     
         23 . The method of  claim 19 , wherein said papillomavirus is a human papillomavirus.  
     
     
         24 . A method of evaluating the inhibition of a papillomavirus comprising: 
 (a) introducing papillomavirus or papillomavirus DNA or portions thereof necessary for replication, into an epithelial cell;    (b) providing conditions that produce infectious papillomavirus in said epithelial cell, wherein said conditions comprise said epithelial cell not contacting a fibroblast;    (c) contacting a potential inhibitor of papillomavirus with said infectious papillomavirus; and    (d) evaluating the presence or absence of said papillomavirus.    
     
     
         25 . The method of  claim 24 , wherein said epithelial cell is not a keratinocyte.  
     
     
         26 . The method of  claim 24 , wherein said epithelial cell is a mammary epithelial cell.  
     
     
         27 . The method of  claim 26 , wherein said mammary epithelial cell is a breast ductal epithelial cell or a breast lobular epithelial cell.  
     
     
         28 . The method of  claim 24 , wherein said papillomavirus is a human papillomavirus.  
     
     
         29 . A method of producing a mutant or recombinant papillomavirus, wherein said method comprises: 
 (a) introducing mutant or recombinant papillomavirus DNA or portions thereof necessary for replication, into an epithelial cell; and    (b) providing conditions that produce infectious mutant or recombinant papillomavirus in said epithelial cell, wherein said conditions comprise said epithelial cell not contacting a fibroblast;    
     
     
         30 . The method of  claim 29 , wherein said epithelial cell is not a keratinocyte.  
     
     
         31 . The method of  claim 29 , wherein said epithelial cell is a mammary epithelial cell.  
     
     
         32 . The method of  claim 31 , wherein said mammary epithelial cell is a breast ductal epithelial cell or a breast lobular epithelial cell.  
     
     
         33 . The method of  claim 29 , wherein said introducing of said papillomavirus DNA is performed by transfecting, lipofecting, or electroporating said papillomavirus DNA into said epithelial cell.  
     
     
         34 . The method of  claim 29 , wherein said papillomavirus is a human papillomavirus.  
     
     
         35 . A method of producing a non-keratinocyte epithelial helper cell for production of mutant or recombinant papillomavirus comprising: 
 (a) introducing papillomavirus DNA or portions thereof, into a non-keratinocyte epithelial cell, wherein said DNA or portions thereof, supplements the replication of said mutant or recombinant papillomavirus; and    (b) providing conditions that produce said non-keratinocyte epithelial helper cell containing said papillomavirus DNA or portions thereof, that supplements the replication of said mutant or recombinant papillomavirus, wherein said conditions comprise said epithelial cell not contacting a fibroblast.    
     
     
         36 . A method of reducing the risk of spontaneous abortion caused by papillomavirus comprising: 
 administering to a subject at risk of spontaneous abortion a drug that inhibits papillomavirus.