Methods of Transforming a Listeria

Abstract

Site-specific Listeria integration vectors and methods for their use are provided. The subject vectors include a bacteriophage integrase gene and a bacteriophage attachment site, where in many embodiments the bacteriophage that is the source 0 of these elements is a listeriophage. In certain embodiments, the subject vectors further include a multiple cloning site, where the multiple cloning site may further include a polypeptide coding sequence, e.g., for a heterologous antigen. The subject vectors and methods find use in a variety of different applications, including the study of Listeria species and the preparation of Listeria vaccines.

What is claimed is: 
     
         1 . An integration vector capable of site-specific  Listeria  genome integration. 
     
     
         2 . The integration vector according to  claim 1 , wherein said integration vector is a plasmid. 
     
     
         3 . The integration vector according to  claim 2 , wherein said integration vector comprises a bacteriophage integrase gene and a bacteriophage attachment site. 
     
     
         4 . The integration vector according to  claim 3 , wherein said bacteriophage is a listeriophage. 
     
     
         5 . The integration vector according to  claim 3 , wherein said attachment site provides for integration at an integration site selected from the group consisting of: the comK integration site and the tRNA Arg  integration site. 
     
     
         6 . The integration vector according to  claim 1 , wherein said integration vector further includes a multiple cloning site. 
     
     
         7 . The integration vector according to  claim 6 , wherein said integration vector further includes a coding sequence. 
     
     
         8 . The integration vector according to  claim 7 , wherein said coding sequence encodes a polypeptide. 
     
     
         9 . The integration vector according to  claim 8 , wherein said polypeptide is an antigen. 
     
     
         10 . The integration vector according to  claim 1 , wherein said integration vector is pPL1. 
     
     
         11 . The integration vector according to  claim 1 , wherein said integration vector is pPL2. 
     
     
         12 . A method of transforming a  Listeria , said method comprising:
 contacting said  Listeria  with an integration vector according to  claim 1  under conditions sufficient for said integration vector to integrate into said  Listeria 's genome.   
     
     
         13 . A  Listeria  transformed with a vector according to  claim 1 . 
     
     
         14 . A method of eliciting or boosting a cellular immune response to an antigen in a subject, said method comprising:
 administering to said subject an effective amount of  Listeria  cells according to  claim 13 .   
     
     
         15 . The method according to  claim 14 , wherein said  Listeria  cells are attenuated. 
     
     
         16 . A vaccine comprising a strain of  Listeria  cells according to  claim 13 , wherein said  Listeria  cells express a heterologous antigen. 
     
     
         17 . The vaccine according to  claim 16 , wherein said  Listeria  cells are attenuated. 
     
     
         18 . A recombinant culture of  Listeria  cells according to  claim 13 . 
     
     
         19 . The recombinant culture according to  claim 18 , wherein said  Listeria  cells are attenuated. 
     
     
         20 . A kit for use in preparing a vector according to  claim 7 , said kit comprising:
 a vector according to  claim 1 ; and   at least one nuclease that cuts said vector at said multiple cloning site.   
     
     
         21 . The kit according to  claim 20 , wherein said kit further comprises a host cell. 
     
     
         22 . A kit for use in preparing a cell according to  claim 13 , said kit comprising:
 a vector according to  claim 1 ;   at least one nuclease that cuts said vector at said multiple cloning site; and   a  Listeria  cell.   
     
     
         24 . A system for preparing a vaccine according to  claim 16 , said system comprising:
 a vector according to  claim 1 ;   at least one nuclease that cuts said vector at said multiple cloning site;   a coding sequence for said heterologous antigen;   and     Listeria  cells.