LIVE BACTERIAL VACCINES RESISTANT TO CARBON DIOXIDE (CO2), ACIDIC PH AND/OR OSMOLARITY FOR VIRAL INFECTION PROPHYLAXIS OR TREATMENT
The present invention relates to gram-negative bacterial mutants resistant to one or more stress conditions, including, but not limited to, CO 2 , acid pH, and high osmolarity. The present invention also relates 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. The present invention provides compositions comprising one or more stress-resistant gram-negative bacterial mutants, preferably attenuated stress-resistant gram-negative bacterial mutants. In particular, the present invention relates to methods for prophylaxis or treatment of a virally induced disease in a subject comprising administering to said subject one or more stress-resistant gram-negative bacterial mutants, preferably attenuated stress-resistant gram-negative bacterial mutants. The present invention further relates to methods for prophylaxis or treatment of a virally induced disease in a subject comprising 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 of the invention provide more efficient delivery of therapeutic molecules by stress-resistant gram-negative bacterial mutants engineered to express said therapeutic molecules.
1 . A mutant Salmonella sp. comprising a genetically modified msbB gene and a mutation characterized by an increased growth when grown under at least one of high CO 2 conditions, low pH media, and high osmolarity, compared to the msbB- mutant Salmonella designated YS1646.
2 . The mutant Salmonella sp. according to claim 1 , further comprising a genetically modified zwf gene.
3 . The mutant Salmonella sp. according to claim 2 , capable of being inhibited or killed by the addition of gluconate at a level which does not kill or inhibit wild type Salmonella.
4 . The mutant Salmonella sp. for viral prophylaxis or treatment of claim 1 which further comprises one or more genetically modified genes to auxotrophy.
5 . The mutant Salmonella sp. for viral prophylaxis or treatment of claim 4 , wherein at least one of the genetically modified genes to auxotrophy is a gene 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.
6 . The mutant Salmonella sp. for viral prophylaxis or treatment of claim 4 , wherein at least one of the genetically modified genes to auxotrophy is a genetically modified AroA gene.
7 . The mutant Salmonella sp. for viral prophylaxis or treatment of claim 4 , wherein at least one of the genetically modified genes to auxotrophy is a gene in a uracil or purine biosynthesis pathway.
8 . The mutant Salmonella sp. for viral prophylaxis or treatment of claim 1 of which is engineered to contain one or more nucleic acid molecules encoding one or more therapeutic agents.
9 . A pharmaceutical composition comprising an amount of the mutant Salmonella sp. of claim 1 effective to prevent or treat a virally induced disease and a pharmaceutically acceptable carrier.
10 . A method for viral prophylaxis or treatment in a subject, said method comprising administering to a subject in need thereof an effective amount of the mutant Salmonella sp. of claim 1 .
11 . A method for limiting the growth of a bacterial strain comprising the addition of gluconate to a media for the bacterial strain.
12 . A live Salmonella bacteria, that is genetically engineered to express one or more viral antigens capable of viral prophylaxis or treatment having a first attenuating mutation that reduces TNF-α induction but confers sensitivity to CO 2 , osmolarity and/or acidic pH and a second mutation that confers resistance to CO 2 , osmolarity and/or acidic pH that maintains reduced TNF-α induction.
13 . The live Salmonella bacterium according to claim 12 , having improved ability to establish a population (infection) in the gut and, if properly modified they could provide a desirable source of immunogenic avian influenza antigen polypeptide(s) to elicit an immune response in the mucosal tissue of the individual.