Signal peptide fusion partners facilitating listerial expression of antigenic sequences and methods of preparation and use thereof

Abstract

The present invention provides nucleic acids, expression systems, and vaccine strains which provide efficient expression and secretion of antigens of interest into the cytosol of host cells, and elicit effective CD4 and CD8 T cell responses by functionally linking Listerial or other bacterial signal peptides/secretion chaperones as N-terminal fusion partners in translational reading frame with selected recombinant encoded protein antigens. These N-terminal fusion partners are deleted (either by actual deletion, by mutation, or by a combination of these approaches) for any PEST sequences native to the sequence, and/or for certain hydrophobic residues.

We claim: 
     
         1 . A method for stimulating an immune response to a non-Listerial antigen in a human comprising:
 administering an effective amount of the  Listeria  bacterium to the human, the  Listeria  bacterium expressing the non-Listerial antigen as a fusion protein comprising:
 a polypeptide derived by recombinant modification of a secreted Listerial protein sequence, the secreted Listerial protein sequence in its unmodified form comprising a signal sequence and one or more PEST motifs, the modification comprising removal of each of the PEST motifs by deletion or substitution by one or more residues such that the polypeptide lacks any PEST motif; and 
 a non-Listerial antigen 
   wherein the non-Listerial antigen is expressed in one or more cells of the human.   
     
     
         2 . The method of  claim 1 , wherein the secreted Listerial protein sequence is ActA or LLO. 
     
     
         3 . The method of  claim 2 , wherein the modification further comprises truncation of the secreted Listerial protein sequence at about residue 100. 
     
     
         4 . The method of  claim 1 , wherein the polypeptide retains the signal sequence of the secreted Listerial protein sequence in unmodified form. 
     
     
         5 . The method of  claim 1 , wherein the modification further comprises:
 removal of one or more hydrophobic domains which are not part of the signal sequence of the secreted Listerial protein sequence; and/or   substitution of one or more residues within one or more hydrophobic domains which are not part of the signal sequence of the secreted Listerial protein sequence with amino acids which are not hydrophobic.   
     
     
         6 . The method of  claim 1 , wherein the secreted Listerial protein sequence is an ActA sequence and wherein at least 75% of the sequence KTEEQPSEVNTGP is deleted. 
     
     
         7 . The method of  claim 1 , wherein the sequence KTEEQPSEVNTGP or KTEEQPSEVNTGPR is deleted. 
     
     
         8 . The method of  claim 1 , wherein the secreted Listerial protein sequence is an ActA sequence and wherein one or more P, E, S, and T residues in the sequence KTEEQPSEVNTGPR is substituted with a residue other than P, E, S, and T. 
     
     
         9 . The method of  claim 8 , wherein each P, E, S, and T residue in the sequence KTEEQPSEVNTGPR is substituted with K or R. 
     
     
         10 . The method of  claim 1 , wherein the secreted Listerial protein sequence is an ActA sequence and wherein one or more hydrophobic residues within the sequence LIAML are substituted with amino acids which are not hydrophobic. 
     
     
         11 . The method of  claim 10 , wherein the sequence LIAML is replaced with the sequence QDNKR. 
     
     
         12 . The method of  claim 1 , wherein the polypeptide comprises at least the first 95 residues of one of the sequences referred to as dlPEST and dlPEST qdnkr in  FIG. 2 . 
     
     
         13 . The method of  claim 1 , wherein the secreted Listerial protein sequence is an LLO sequence and wherein at least 75% of the sequence SISSMAPPASPPASPKTPIE is deleted. 
     
     
         14 . The method of  claim 1 , wherein the sequence KENSISSMAPPASPPASPK or NSISSMAPPASPPASPKTPIEKKHAD is deleted. 
     
     
         15 . The method of  claim 1 , wherein the secreted Listerial protein sequence is an LLO sequence and wherein one or more P, E, S, and T residues in the sequence SISSMAPPASPPASPKTPIEKKHAD is substituted with a residue other than P, E, S, and T. 
     
     
         16 . The method of  claim 15 , wherein each P, E, S, and T residue in the sequence SISSMAPPASPPASPKTPIEKKHAD is substituted with K or R. 
     
     
         17 . The method of  claim 1 , wherein the polypeptide comprises at least the first 95 residues of one of the sequences referred to as LLO dlPEST and LLO dl26 in  FIG. 2 . 
     
     
         18 . The method of  claim 1 , wherein the non-Listerial antigen is a cancer cell, tumor, or infectious agent antigen.