DUAL DELIVERY SYSTEM FOR HETEROLOGOUS ANTIGENS
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.