Dual delivery system for heterologous antigens

Abstract

Provided herein are recombinant Listeria strains expressing a tumor-specific antigenic polypeptide and, optionally, an angiogenic polypeptide wherein a nucleic acid molecule encoding at least one of the polypeptides is operably integrated into the Listeria genome in an open reading frame with a nucleic acid sequence encoding a PEST-containing polypeptide, methods of preparing same, and methods of inducing an immune response, and treating, inhibiting, or suppressing cancer or tumors comprising administering same.

1 . A recombinant  Listeria  strain comprising a first and second nucleic acid molecule, each said nucleic acid molecule encoding a heterologous antigenic polypeptide or an immunogenic fragment thereof, wherein said first nucleic acid molecule is integrated into the  Listeria  genome in an open reading frame with an endogenous PEST-containing gene, and wherein said recombinant  Listeria  strain comprises an episomal expression vector comprising said second nucleic acid molecule encoding a heterologous antigen. 
     
     
         2 . (canceled) 
     
     
         3 . (canceled) 
     
     
         4 . (canceled) 
     
     
         5 . The recombinant  Listeria  strain of  claim 1 , wherein said second nucleic acid molecule encoding a heterologous antigen is present in said episomal expression vector in an open reading frame with a nucleic acid sequence encoding a PEST-containing polypeptide. 
     
     
         6 . The recombinant  Listeria  strain of  claim 1 , wherein said PEST-containing gene is LLO or ActA. 
     
     
         7 . (canceled) 
     
     
         8 . The recombinant  Listeria  strain of  claim 1 , wherein said first or second nucleic acid molecule encodes a angiogenic polypeptide expressed by a tumor cell. 
     
     
         9 . (canceled) 
     
     
         10 . The recombinant  Listeria  strain of  claim 1 , wherein said first or second nucleic acid molecule encodes a prostate specific antigen (PSA) or a High Molecular Weight-Melanoma Associated Antigen (HMW-MAA). 
     
     
         11 . (canceled) 
     
     
         12 . The recombinant  Listeria  strain of  claim 1 , wherein said first nucleic acid molecule is a vector designed for site-specific homologous recombination into the  Listeria  genome. 
     
     
         13 . (canceled) 
     
     
         14 . The recombinant  Listeria  strain of  claim 1 , wherein said recombinant  Listeria  strain is an auxotrophic  Listeria  strain. 
     
     
         15 . The recombinant  Listeria  strain of  claim 14 , wherein said auxotrophic  Listeria  strain is a dal/dat mutant. 
     
     
         16 . (canceled) 
     
     
         17 . (canceled) 
     
     
         18 . (canceled) 
     
     
         19 . (canceled) 
     
     
         20 . (canceled) 
     
     
         21 . The recombinant  Listeria  strain of  claim 1 , wherein said recombinant  Listeria  strain has been passaged through an animal host. 
     
     
         22 . The recombinant  Listeria  strain of  claim 1 , wherein said recombinant  Listeria  strain is a recombinant  Listeria  monocytogenes strain. 
     
     
         23 . (canceled) 
     
     
         24 . A method of inducing an immune response to an antigen in a subject comprising administering a recombinant  Listeria  strain to said subject, wherein said recombinant  Listeria  strain comprises a first and second nucleic acid molecule, each said nucleic acid molecule encoding a heterologous antigenic polypeptide or an immunogenic fragment thereof, wherein said first nucleic acid molecule is operably integrated into the  Listeria  genome in an open reading frame with an endogenous PEST-containing gene, and wherein said recombinant  Listeria  strain comprises an episomal expression vector comprising said second nucleic acid molecule encoding a heterologous antigen. 
     
     
         25 . (canceled) 
     
     
         26 . (canceled) 
     
     
         27 . (canceled) 
     
     
         28 . The method of  claim 24 , wherein said second nucleic acid molecule encoding a heterologous antigen is present in said episomal expression vector in an open reading frame with a nucleic acid sequence encoding a PEST-containing polypeptide. 
     
     
         29 . The method of  claim 24 , wherein said PEST-containing gene is LLO or ActA. 
     
     
         30 . (canceled) 
     
     
         31 . The method of  claim 24 , wherein said first or second nucleic acid molecule encodes a angiogenic polypeptide expressed by a tumor cell. 
     
     
         32 . (canceled) 
     
     
         33 . The method of  claim 24 , wherein said first or second nucleic acid molecule encodes a prostate specific antigen (PSA) or a High Molecular Weight-Melanoma Associated Antigen (HMW-MAA). 
     
     
         34 . (canceled) 
     
     
         35 . The method of  claim 24 , wherein said first nucleic acid molecule is a vector designed for site-specific homologous recombination into the  Listeria  genome. 
     
     
         36 . (canceled) 
     
     
         37 . The method of  claim 24 , wherein said recombinant  Listeria  strain is an auxotrophic  Listeria  strain. 
     
     
         38 . The method of  claim 37 , wherein said auxotrophic  Listeria  strain is a dal/dat mutant. 
     
     
         39 . (canceled) 
     
     
         40 . (canceled) 
     
     
         41 . (canceled) 
     
     
         42 . (canceled) 
     
     
         43 . (canceled) 
     
     
         44 . The method of  claim 24 , wherein said recombinant  Listeria  strain has been passaged through an animal host. 
     
     
         45 . The method of  claim 24 , wherein said recombinant  Listeria  strain is a recombinant  Listeria monocytogenes  strain. 
     
     
         46 . (canceled) 
     
     
         47 . The method of  claim 24 , wherein said recombinant  Listeria  strain is administered orally or parenterally. 
     
     
         48 . A method of treating, suppressing, or inhibiting a cancer in a subject comprising administering a recombinant  Listeria  strain of  claim 1  to said subject. 
     
     
         49 . The method of  claim 48 , wherein said first or second nucleic acid molecule encodes a prostate specific antigen (PSA) or a High Molecular Weight-Melanoma Associated Antigen (HMW-MAA). 
     
     
         50 . (canceled) 
     
     
         51 . The method of  claim 49 , wherein said cancer is prostate cancer, ovarian cancer, breast cancer, brain cancer, lung cancer, gastrointestinal cancer, melanoma, pancreatic cancer, sarcoma, or a combination thereof. 
     
     
         52 . (canceled) 
     
     
         53 . (canceled) 
     
     
         54 . (canceled) 
     
     
         55 . A method of treating, suppressing, or inhibiting at least one tumor in a subject, comprising administering a recombinant  Listeria  strain of  claim 1  to said subject. 
     
     
         56 . The method of  claim 55 , wherein said first or second nucleic acid molecule encodes a prostate specific antigen (PSA) or a High Molecular Weight-Melanoma Associated Antigen (HMW-MAA). 
     
     
         57 . (canceled) 
     
     
         58 . The method of  claim 56 , wherein said tumor is an ovarian tumor, prostate tumor, brain tumor, lung tumor, gastrointestinal tumor, pancreatic tumor, breast tumor, or a combination thereof. 
     
     
         59 . (canceled) 
     
     
         60 . (canceled) 
     
     
         61 . (canceled) 
     
     
         62 . (canceled) 
     
     
         63 . (canceled) 
     
     
         64 . (canceled) 
     
     
         65 . (canceled) 
     
     
         66 . (canceled) 
     
     
         67 . (canceled) 
     
     
         68 . (canceled) 
     
     
         69 . (canceled) 
     
     
         70 . (canceled) 
     
     
         71 . (canceled) 
     
     
         72 . (canceled) 
     
     
         73 . (canceled) 
     
     
         74 . (canceled) 
     
     
         75 . (canceled) 
     
     
         76 . (canceled) 
     
     
         77 . (canceled) 
     
     
         78 . (canceled) 
     
     
         79 . (canceled) 
     
     
         80 . (canceled) 
     
     
         81 . A method of producing a recombinant  Listeria  strain expressing two antigens, the method comprising:
 (a) Genetically fusing a first nucleic acid encoding a first antigen into the  Listeria  genome in an open reading frame with an endogenous PEST-containing gene;   (b) Transforming said recombinant Listeria with an episomal expression vector comprising a second nucleic acid encoding a second antigen; and   (c) Expressing said first and second antigens under conditions conducive to antigenic expression in said recombinant  Listeria  strain.   
     
     
         82 . (canceled) 
     
     
         83 . The recombinant  Listeria  of  claim 12 , wherein said first integrated nucleic acid molecule comprises a site-specific integrase gene. 
     
     
         84 . The recombinant  Listeria  of  claim 83 , wherein said site-specific integrase gene is A118 or U153. 
     
     
         85 . The recombinant  Listeria  of  claim 12 , wherein said vector is a phage integration vector. 
     
     
         86 . The method of  claim 35 , wherein said first integrated nucleic acid molecule comprises a site-specific integrase gene. 
     
     
         87 . The method of  claim 86 , wherein said site-specific integrase gene is A118 or U153. 
     
     
         88 . The recombinant  Listeria  of  claim 35 , wherein said vector is a phage integration vector.