Intracellular Delivery Vehicles

Abstract

The invention provides methods and compositions relating to intracellular delivering of agents to eukaryotic cells. The compositions include microbial delivery vehicles such as nonvirulent bacteria comprising a first gene encoding a nonsecreted foreign cytolysin operably linked to a heterologous promoter and a second gene encoding a different foreign agent. The foreign agent may be a nucleic acid or protein, and is frequently bioactive in and therapeutic to the target eukaryote. In addition, the invention provides eukaryotic cells comprising the subject nonvirulent bacteria and nonhuman eukaryotic host organisms comprising such cells.

1 . A method of determining whether a library contains or encodes an immunogenic polypeptide, the method comprising:
 (a) providing a cell member library, which cell member library comprises a plurality of cell members, each cell member comprising a first polynucleotide encoding a polypeptide encoded by the genome of a pathogenic organism, the first polynucleotide operably linked to a promoter such that each cell member of the cell member library produces its respective polypeptide;   (b) individually contacting each cell member of the cell member library with a second cell capable of (i) endocytosing the contacted cell member and (ii) displaying the polypeptide produced by the endocytosed cell member on its surface through the MHC class I pathway;   (c) individually contacting each second cell of step (b) with a sample containing a least one cytotoxic T-lymphocyte derived from a mammal previously infected with the pathogenic organism; and   (d) detecting whether a cytotoxic T-lymphocyte within the sample is activated, wherein activation of the cytotoxic T-lymphocyte indicates that the polypeptide is immunogenic, thereby determining whether the cell member library contains or encodes an immunogenic polypeptide.   
     
     
         2 . The method of  claim 1 , wherein the step of providing a cell member library comprises:
 providing a plurality of first polynucleotides, which plurality represents a genomic library of the pathogenic organism; and   contacting the plurality of first polynucleotides with a plurality of cell members so that first polynucleotides are introduced into cell members and the provided cell member library is thereby generated.   
     
     
         3 . The method of  claim 1  or  2 , wherein the each cell member of the cell member library further comprises a second polynucleotide encoding a pore-forming protein. 
     
     
         4 . The method of  claim 3 , wherein the pore-forming protein is listeriolysin O. 
     
     
         5 . The method of  claim 1  or  2 , wherein the second cell is a macrophage. 
     
     
         6 . The method of  claim 1  or  2 , wherein the each cell member of the library is killed prior to the contacting step (b). 
     
     
         7 . The method of  claim 1  or  2 , wherein prior to the contacting step (c), the second cell is killed. 
     
     
         8 . The method of  claim 1  or  2 , wherein, prior to contacting step (b), a replica of the library is made. 
     
     
         9 . The method of  claim 8 , further comprising a step of:
 (e) recovering the first polynucleotide encoding the polypeptide identified in step (d) from a replica copy of the cell member library.   
     
     
         10 . The method of  claim 1  or  2 , further comprising a step of:
 (e) identifying an epitope sufficient for cytotoxic T-lymphocyte activation within the polypeptide determined to be immunogenic in step (d).   
     
     
         11 . A vaccine comprising at least one epitope identified by the method of  claim 10  and a pharmaceutically acceptable carrier. 
     
     
         12 . A vaccine comprising at least one immunogenic polypeptide identified by the method of  claim 1  or  2  and a pharmaceutically acceptable carrier. 
     
     
         13 . The method of  claim 1  or  2 , wherein the contacting step (c) is performed using a sample containing a plurality of cytotoxic T-lymphocytes. 
     
     
         14 . The method of  claim 1  or  2 , further comprising performing the method steps (c) and (d) at least one further time using the cell member library. 
     
     
         15 . The method of  claim 1  or  2 , wherein a different sample containing a different cytotoxic T-lymphocyte is contacted in step (c) each time the method is performed. 
     
     
         16 . The method of  claim 1  or  2 , wherein the cell member library comprises a plurality of bacterial cell members. 
     
     
         17 . The method of  claim 16 , wherein the cell member library comprises a plurality of  E. coli  cell members. 
     
     
         18 . The method of  claim 1  or  2 , wherein the pathogenic organism is a bacterium. 
     
     
         19 . The method of  claim 1  or  2 , wherein the pathogenic organism is a virus. 
     
     
         20 . The method of  claim 1  or  2 , wherein the promoter is an inducible promoter. 
     
     
         21 . The method of  claim 1  or  2 , wherein each cell member of the cell member library comprises at least one of a plurality of different first polynucleotides. 
     
     
         22 . The method of  claim 21 , wherein each cell member of the cell member library comprises a different first polynucleotide. 
     
     
         23 . The method of  claim 21 , wherein the plurality of first polynucleotides comprises a genomic library of the pathogenic organism. 
     
     
         24 . The method of  claim 23 , wherein the genomic library comprises polynucleotides encoding each polypeptide encoded by the genome of the pathogenic organism. 
     
     
         25 . The method of  claim 24 , wherein the step of detecting comprises detecting specific antigens of the pathogenic organism.