Vaccines comprising heat-sensitive transgenes

Abstract

The present disclosure provides temperature sensitive essential nucleic acid molecules from a psychrophilic bacterium, proteins encoded by the nucleic acid molecules, as well as recombinant cells into which have been introduced such nucleic acid molecules. The disclosed recombinant cells containing one or more essential nucleic acid molecules from a psychrophilic bacterium are thereby made temperature sensitive, and can be administered to a mammal to induce an immune response in the mammal.

What is claimed is: 
     
         1 . A method of stimulating an immune response in an animal, comprising:
 administering to the animal a therapeutically effective amount of a recombinant mesophilic bacterium that is temperature-sensitive (TS), wherein the recombinant mesophilic bacterium that is TS is made by a method comprising:
 introducing into the genome of a mesophilic bacterium a nucleic acid construct comprising a TS essential nucleic acid molecule from a psychrophilic bacterium, and 
 functionally replacing the mesophilic bacterium's homolog of the TS essential nucleic acid molecule, thereby making the recombinant mesophilic bacterium that is TS, 
 wherein a protein encoded by the TS essential nucleic acid molecule is operable at a temperature less than 30° C. and inoperable at a temperature greater than 30° C., and 
   wherein the recombinant mesophilic bacterium that is TS has a restrictive temperature between 33° C. and 44° C.,   thereby stimulating the immune response in the animal.   
     
     
         2 . The method of  claim 1 , wherein the psychrophilic bacterium is  Colwellia  sp.,  Pseudoalteromonas  sp., or  Shewanella  sp. 
     
     
         3 . The method of  claim 1 , wherein the recombinant mesophilic bacterium that is TS is  Salmonella  sp.,  Mycobacterium  sp. or  Escherichia  sp. 
     
     
         4 . The method of  claim 1 , wherein the TS essential nucleic acid molecule comprises the nucleotide sequence shown in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25 or 27, wherein expression of the TS essential nucleic acid molecule confers temperature sensitivity. 
     
     
         5 . The method of  claim 1 , wherein the protein encoded by the TS essential nucleic acid molecule comprises the amino acid sequence shown in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, or 28. 
     
     
         6 . The method of  claim 1 , wherein the nucleic acid construct comprising the TS essential nucleic acid molecule comprises a vector. 
     
     
         7 . The method of  claim 1 , wherein the recombinant mesophilic bacterium that is TS is present in a composition comprising an adjuvant. 
     
     
         8 . The method of  claim 1 , wherein the recombinant mesophilic bacterium that is TS is present in a composition comprising a pharmaceutically acceptable carrier. 
     
     
         9 . The method of  claim 1 , wherein the animal is a bird. 
     
     
         10 . The method of  claim 1 , wherein the animal is a mammal. 
     
     
         11 . The method of  claim 1 , wherein at least 10 5  CFU of the recombinant mesophilic bacterium that is TS are administered to the animal. 
     
     
         12 . The method of  claim 1 , wherein administering comprises intramuscular injection, subcutaneous injection, oral administration, intranasal administration or inhalation. 
     
     
         13 . The method of  claim 1 , wherein the immune response comprises a cytotoxic T lymphocyte (CTL) response. 
     
     
         14 . The method of  claim 1 , wherein the immune response comprises a B cell response. 
     
     
         15 . The method of  claim 1 , wherein the method treats a  Francisella novicida, Mycobacterium tuberculosis, Salmonella enteritidis , or  Escherichia coli  infection in the animal. 
     
     
         16 . The method of  claim 1 , wherein the method prevents a  Francisella novicida, Mycobacterium tuberculosis, Salmonella enteritidis , or  Escherichia coli  infection in the animal. 
     
     
         17 . The method of  claim 1 , wherein more than one type of recombinant mesophilic bacterium that is TS is administered to the animal. 
     
     
         18 . The method of  claim 1 , further comprising:
 culturing the recombinant mesophilic bacterium that is TS at a temperature wherein a protein encoded by the TS essential nucleic acid molecule is operable, whereby the recombinant mesophilic bacterium that is TS produces a plurality of peptides;   increasing the culturing temperature to a temperature at which a protein encoded by the TS nucleic acid molecule is inoperable;   maintaining said culturing for a period of time sufficient to kill the recombinant mesophilic bacterium that is TS; and   harvesting the killed recombinant mesophilic bacterium that is TS.   
     
     
         19 . A method of stimulating an immune response in an animal, comprising:
 administering to the animal a therapeutically effective amount of a recombinant mesophilic bacterium that is temperature-sensitive (TS), wherein the recombinant mesophilic bacterium that is TS is made by a method comprising:   introducing into the genome of a mesophilic bacterium by homologous recombination a nucleic acid construct comprising a TS essential nucleic acid molecule from a psychrophilic bacterium flanked on both sides by a nucleic acid molecule homologous to a region of the mesophilic bacterium genome where the TS essential nucleic acid molecule from the psychrophilic bacterium will be inserted into the mesophilic bacterium genome, and   functionally replacing the mesophilic bacterium's homolog of the TS essential nucleic acid molecule, thereby making a recombinant mesophilic bacterium that is TS,   wherein a protein encoded by the TS essential nucleic acid molecule is operable at a temperature less than 30° C. and inoperable at a temperature greater than 30° C., and   wherein the recombinant mesophilic bacterium that is TS has a restrictive temperature between 33° C. and 44° C.,   thereby stimulating the immune response in the animal.   
     
     
         20 . A method of stimulating an immune response in an animal, comprising:
 administering to the animal a therapeutically effective amount of a recombinant mesophilic bacterium that is temperature-sensitive (TS), wherein the recombinant mesophilic bacterium that is TS is made by a method comprising:   introducing into the genome of a mesophilic bacterium a nucleic acid construct comprising a TS essential nucleic acid molecule from a psychrophilic bacterium flanked on both sides by a nucleic acid molecule homologous to a region of the mesophilic bacterium genome where the TS essential nucleic acid molecule from the psychrophilic bacterium will be inserted into the mesophilic bacterium genome,
 wherein a protein encoded by the TS essential nucleic acid molecule is operable at a temperature less than 30° C. and inoperable at a temperature greater than 30° C., and 
 wherein the recombinant mesophilic bacterium that is TS is viable at a temperature of 0° C. to 30° C., nonviable at a temperature greater than 30° C., and has a restrictive temperature between 33° C. and 44° C.; 
   culturing the recombinant mesophilic bacterium that is TS at a temperature wherein a protein encoded by the TS essential nucleic acid molecule is operable, whereby the recombinant mesophilic bacterium that is TS produces a plurality of peptides;   increasing the culturing temperature to a temperature at which a protein encoded by the TS nucleic acid molecule is inoperable;   maintaining said culturing for a period of time sufficient to kill the recombinant mesophilic bacterium that is TS; and   harvesting the killed recombinant mesophilic bacterium that is TS,   thereby stimulating the immune response in the animal.