Live bacterial vaccines resistant to carbon dioxide (CO
Gram-negative bacterial mutants resistant stress conditions, including CO 2 , acid pH, and high osmolarity are provided, having reduced TNF-α induction having a msbB mutation, that are rendered stress-resistant by a mutation in the zwf gene. A method for prophylaxis or treatment of a virally induced disease in a subject comprising administering to said subject attenuated stress-resistant gram-negative bacterial mutants, is also provided, along with Methods for prophylaxis or treatment of a virally induced disease in a subject comprise administering to said subject one or more stress-resistant gram-negative bacterial mutants as vectors for the delivery of one or more therapeutic molecules. The methods provide efficient delivery of therapeutic molecules by stress-resistant gram-negative bacterial mutants engineered to express said therapeutic molecules.
1. A live attenuated stress-resistant Salmonella species (sp.) genetically engineered from a wild type Salmonella sp., the wild type Salmonella having an msbB gene and a zwf gene, and producing pyrogenic Lipid A, and comprising an intact pentose pathway,
the mutant Salmonella sp. comprising:
a defective msbB gene resulting in reduction of Lipid A pyrogenicity compared to the wild type Salmonella , and
a defect in the zwf gene which interrupts the pentose phosphate pathway,
the mutant Salmonella sp. growing under conditions of at least one of: (a) pH 6.7 in air, (b) 5% CO 2 , and (c) 300 milliosmoles.
2. The mutant Salmonella sp. according to claim 1 , wherein the mutant Salmonella sp. is inhibited or killed by the addition of gluconate at a level which does not kill or inhibit wild type Salmonella.
3. The mutant Salmonella sp. according to claim 1 , which is further engineered to contain one or more nucleic acid molecules encoding one or more therapeutic agents.
4. The mutant Salmonella sp. according to claim 3 , in combination with a pharmaceutically acceptable carrier.
5. The mutant Salmonella sp. according to claim 4 , wherein the pharmaceutically acceptable carrier is formulated for oral administration to a human.
6. The mutant Salmonella sp. according to claim 3 , wherein the mutant Salmonella sp. is adapted to colonize human gut, and produce a therapeutically effective amount of the one or more therapeutic agents within the human gut.
7. The mutant Salmonella sp. according to claim 3 , wherein the one or more nucleic acid molecules encode a secreted antigen.
8. The mutant Salmonella sp. according to claim 3 , wherein the one or more nucleic acid molecules encode an antigen heterologous to Salmonella.
9. The mutant Salmonella sp. according to claim 3 , wherein the one or more nucleic acid molecules encode a viral antigen.
10. The mutant Salmonella sp. according to claim 3 , wherein the one or more nucleic acid molecules encoding one or more therapeutic agents comprise one or more nucleic acid molecules encoding one or more therapeutic agents fused to a secretion promoter.
11. The mutant Salmonella sp. according to claim 3 , wherein the one or more nucleic acid molecules encoding one or more therapeutic agents encode a non-bacterial protein, wherein the nucleic acid molecules are optimized for bacterial expression by use of bacterial codons.
12. A live attenuated stress-resistant Salmonella sp. genetically engineered from a wild type Salmonella sp., said mutant Salmonella having a defective msbB gene in the lipopolysaccharide biosynthetic pathway and resulting in reduced Lipid A pyrogenicity with respect to the respective wild type Salmonella having an intact lipopolysaccharide biosynthetic pathway comprising a non-defective msbB gene, and a defective zwf gene in the pentose phosphate pathway which reduces synthesis of pentose phosphate as compared to the wild type Salmonella having the intact lipopolysaccharide biosynthetic pathway comprising the non-defective msbB gene, the mutant Salmonella sp. exhibiting growth after at least 2 hours under incubation conditions selected from the group consisting of at least one of:
a growth medium having a pH 6.7 in air;
a growth medium subjected to 5% CO 2 , and
a growth medium having an osmolarity of 300 milliosmoles.
13. The mutant Salmonella sp. according to claim 12 , further comprising a nucleic acid molecule encoding a therapeutic agent which is secreted from the mutant Salmonella sp.
14. A live attenuated stress-resistant Salmonella sp. genetically engineered from a wild type Salmonella sp. comprising an intact lipopolysaccharide biosynthetic pathway comprising a non-defective msbB gene, said mutant Salmonella comprising:
an altered lipopolysaccharide pathway comprising a defective msbB gene, resulting in reduced Lipid A pyrogenicity with respect to the wild type Salmonella ; and
an altered pentose phosphate pathway comprising a defective zwf gene, which reduces synthesis of pentose,
the mutant Salmonella sp. being characterized by exhibition of growth after at least 2 hours under incubation conditions selected from the group consisting of at least one of:
a growth medium having a pH 6.7 in air;
a growth medium subjected to 5% CO 2 , and
a growth medium having an osmolarity of 300 milliosmoles.
15. The mutant Salmonella sp. according to claim 14 , further comprising a nucleic acid molecule encoding a therapeutic agent which is secreted from the mutant Salmonella sp.
16. The mutant Salmonella sp. according to claim 15 , wherein the nucleic acid molecule encoding the therapeutic agent encodes a non-bacterial protein, wherein the nucleic acid molecule is optimized for bacterial expression by use of bacterial codons.