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

Abstract

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.