Regulated expression of antigen and/or regulated attenuation to enhance vaccine immunogenicity and/or safety

Abstract

The invention relates to compositions and methods for making and using recombinant bacteria that are capable of regulated attenuation and/or regulated expression of one or more antigens of interest.

What is claimed is: 
     
         1 . A recombinant bacterium capable of regulated expression of at least one nucleic acid sequence encoding an antigen of interest, wherein the bacterium comprises:
 a. at least one chromosomally integrated nucleic acid sequence encoding a repressor operably linked to a regulatable promoter, wherein the nucleic acid sequence encoding the repressor and/or promoter have been modified from the wild-type nucleic acid sequence so as to optimize the expression level of the nucleic acid sequence encoding the repressor;   b. a vector comprising at least one nucleic acid sequence encoding an antigen of interest operably linked to a promoter regulated by the repressor, such that the expression of the nucleic acid sequence encoding the antigen is repressed during in vitro growth of the bacterium, but the bacterium is capable of high level expression of the nucleic acid sequence encoding the antigen in a host; and   c. a deletion in sifA, a deletion in pabA, a deletion in pabB, a deletion in both pabA and pabB, or a deletion in pmi.   
     
     
         2 . The recombinant bacterium of  claim 1 , wherein the bacterium further comprises the mutation ΔasdA::TT araC P BAD  c2. 
     
     
         3 . The recombinant bacterium of  claim 1 , wherein the bacterium further comprises the mutation ΔrelA::araC P BAD  lacI TT. 
     
     
         4 . A recombinant bacterium capable of regulated expression of at least one nucleic acid sequence encoding an antigen of interest, wherein the bacterium comprises:
 a. at least one chromosomally integrated nucleic acid sequence encoding a repressor operably linked to a regulatable promoter, wherein the nucleic acid sequence encoding the repressor and/or promoter have been modified from the wild-type nucleic acid sequence so as to optimize the expression level of the nucleic acid sequence encoding the repressor,   b. a vector comprising at least one nucleic acid sequence encoding an antigen of interest operably linked to a promoter regulated by the repressor, such that the expression of the nucleic acid sequence encoding the antigen is repressed during in vitro growth of the bacterium, but the bacterium is capable of high level expression of the nucleic acid sequence encoding the antigen in a host; and   c. the mutation ΔP murA ::TT araC P BAD  murA.   
     
     
         5 . The recombinant bacterium of  claim 4 , wherein the bacterium further comprises a deletion in asd. 
     
     
         6 . The recombinant bacterium of  claim 4 , wherein the bacterium further comprises the mutation ΔasdA::TT araC P BAD  c2. 
     
     
         7 . The recombinant bacterium of  claim 1 , wherein the repressor is selected from the group consisting of LacI, C2, and C1. 
     
     
         8 . The recombinant bacterium of  claim 1 , wherein the nucleic acid sequence encoding the repressor comprises a modified Shine-Dalgarno sequence and optimized codons so as to optimize the expression level of the nucleic acid sequence encoding the repressor. 
     
     
         9 . The recombinant bacterium of  claim 1 , wherein the vector is a plasmid. 
     
     
         10 . The recombinant bacterium of  claim 1 , wherein the nucleic acid encoding the antigen of interest is operably linked to a P trc  promoter. 
     
     
         11 . The recombinant bacterium of  claim 1 , wherein the antigen of interest is toxic. 
     
     
         12 . The recombinant bacterium of  claim 1 , wherein the vector further comprises a nucleic acid sequence encoding a secretion signal for the antigen of interest. 
     
     
         13 . The recombinant bacterium of  claim 1 , wherein the bacterium is capable of eliciting a protective immune response in the host. 
     
     
         14 . The recombinant bacterium of  claim 1 , wherein the bacterium comprises more than one means of attenuation. 
     
     
         15 . The recombinant bacterium of  claim 1 , wherein
 the repressor is LacI;   the regulatable promoter is selected from the group consisting of P rhaBAD , P xylAB , and P xylFGH ;   the vector is a plasmid; and   the nucleic acid sequence encoding the antigen of interest is operably linked to the P trc  promoter.   
     
     
         16 . A vaccine composition comprising the recombinant bacterium of  claim 1 . 
     
     
         17 . A method for eliciting an immune response in a host, the method comprising administering to the host an effective amount of a vaccine composition comprising a recombinant bacterium of  claim 1 .