Live bacterial vaccines resistant to carbon dioxide (co2), acidic ph and/or osmolarity for viral infection prophylaxis or treatment

Abstract

Gram-negative bacterial mutants resistant to one or more stress conditions, including CO 2 , acid pH, and high osmolarity, and more particularly to gram-negative bacterial mutants with reduced TNF-α induction having a mutation in one or more lipid biosynthesis genes, including, but not limited to msbB, that are rendered stress-resistant by a mutation in the zwf gene. Compositions are provided comprising one or more stress-resistant gram-negative bacterial mutants, preferably attenuated stress-resistant gram-negative bacterial mutants. Methods are provided for prophylaxis or treatment of a virally induced disease in a subject comprising administering to a subject a stress-resistant gram-negative bacterial mutant, preferably attenuated stress-resistant gram-negative bacterial mutants. The stress-resistant gram-negative bacterial mutants may serve as vectors for the delivery of one or more therapeutic molecules to a host. The methods of the invention provide more efficient delivery of therapeutic molecules by stress-resistant gram-negative bacterial mutants engineered to express said therapeutic molecules.

What is claimed is: 
     
         1 . A method of treating a human or animal, comprising:
 administering to the human or mammal a live genetically engineered bacteria derived from a wild type species having an MsbB gene and a zwf gene, the live genetically engineered bacteria having a knockout mutation of MsbB and a knockout mutation of zwf;   allowing the live genetically engineered bacteria to replicate within and colonize a tissue of the human or animal having a pH of pH 6.7 or below, to cause a transient maintenance of the live genetically engineered bacteria in the tissue; and   secreting, by the live genetically engineered bacteria, within the tissue, a heterologous protein.   
     
     
         2 . The method according to  claim 1 , further comprising clearing the live genetically engineered bacteria from the tissue. 
     
     
         3 . The method according to  claim 1 , wherein the heterologous protein comprises an antigen adapted act as a vaccine. 
     
     
         4 . The method according to  claim 1 , wherein the heterologous protein comprises a eukaryotic-type antigen adapted act as a vaccine. 
     
     
         5 . The method according to  claim 1 , wherein the heterologous protein comprises a fusion of a bacterial-type secretion signal and an antigenic peptide portion. 
     
     
         6 . The method according to  claim 1 , wherein the live genetically engineered bacteria are  Salmonella.    
     
     
         7 . The method according to  claim 1 , wherein the wild type species is  Salmonella enterica.    
     
     
         8 . The method according to  claim 1 , wherein the live genetically engineered bacteria are zwf  Salmonella  YS1646 ATCC Accession No. 202165. 
     
     
         9 . The method according to  claim 1 , wherein the live genetically engineered bacteria have at least one mutation in a biosynthetic pathway selected from the group consisting of the isoleucine biosynthetic pathway, valine biosynthetic pathway, phenylalanine biosynthetic pathway, tryptophan biosynthetic pathway, tyrosine biosynthetic pathway, and arginine biosynthetic pathway. 
     
     
         10 . A live genetically engineered bacteria derived from a wild type species having an MsbB gene and a zwf gene, comprising:
 a knockout mutation of MsbB; and   a knockout mutation of zwf;   at least one gene configured to cause secretion of a heterologous protein;   the live genetically engineered bacteria being adapted to replicate within and colonize a tissue of a human or animal having a pH of pH 6.7 or below, to cause a transient maintenance of the live genetically engineered bacteria in the tissue.   
     
     
         11 . The live genetically engineered bacteria according to  claim 10 , wherein the wild type species is  Salmonella , and live genetically engineered bacteria is adapted to colonize a gut of a human recipient of the live genetically engineered bacteria. 
     
     
         12 . The live genetically engineered bacteria according to  claim 10 , wherein the heterologous protein comprises an antigen adapted to induce a protective vaccination immune response of the human or animal. 
     
     
         13 . The live genetically engineered bacteria according to  claim 10 , wherein the heterologous protein comprises an antigen adapted to induce a therapeutic immune response of the human or animal. 
     
     
         14 . The live genetically engineered bacteria according to  claim 10 , wherein the live genetically engineered bacteria, are adapted after colonization, to produce a therapeutically effective amount of the heterologous protein. 
     
     
         15 . The live genetically engineered bacteria according to  claim 10 , wherein the live genetically engineered bacteria, are adapted after colonization, to produce a therapeutically effective amount of the heterologous protein to induce an immune response against an infectious organism. 
     
     
         16 . The live genetically engineered bacteria according to  claim 10 , wherein the heterologous protein comprises a fusion protein having at least a bacterial secretion signal and a eukaryotic-type antigenic peptide. 
     
     
         17 . The live genetically engineered bacteria according to  claim 10 , in combination with a pharmaceutically acceptable carrier. 
     
     
         18 . A live genetically engineered bacterium, comprising:
 a knockout mutation of MsbB; and   a knockout mutation of zwf;   the knockout mutation of MsbB and the knockout mutation of zwf together causing the live genetically engineered bacterium to have resistance to growth suppressive effects of CO 2 ≥5%, pH≤6.7, and osmolarity of ≥455 milliosmoles;   at least one gene configured to cause secretion from the live genetically engineered bacterium of a heterologous protein;   the live genetically engineered bacteria being adapted to replicate within and colonize a tissue of a human or animal having a pH of pH 6.7 or below, to cause a transient maintenance of the live genetically engineered bacteria in the tissue.   
     
     
         19 . The live genetically engineered bacterium according to  claim 18 , further comprising:
 an attenuating mutation of at least one gene to auxotrophy; and   the heterologous protein comprises a fusion of a therapeutic peptide portion and a secretion signal.   
     
     
         20 . The live genetically engineered bacteria according to  claim 19 , wherein the therapeutic peptide sequence portion comprises a eukaryotic protein antigen.