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 nucleic acid molecule encoding a fusion polypeptide, wherein said fusion polypeptide comprises a heterologous antigen fused to an N-terminal LLO, wherein said recombinant Listeria is an attenuated Listeria comprising a mutation in the dal/dat genes, wherein said Listeria comprises an episomal vector comprising a nucleic acid sequence encoding a metabolic enzyme that complements said dal/dat mutation, wherein said episomal vector is stably maintained in said recombinant Listeria in the absence of antibiotic selection, and wherein said Listeria comprises a deletion of the endogenous actA gene.
2. The recombinant Listeria of claim 1 , wherein said nucleic acid molecule is integrated into the Listeria genome.
3. The recombinant Listeria of claim 1 , wherein said nucleic acid molecule is in said episomal vector in said recombinant Listeria strain.
4. The recombinant Listeria of claim 3 , wherein said episomal vector does not confer antibiotic resistance upon said recombinant Listeria.
5. The recombinant Listeria of claim 1 , wherein said metabolic enzyme is an alanine racemase enzyme or a D-amino acid transferase enzyme.
6. The recombinant Listeria of claim 1 , wherein the heterologous antigen is Prostate Specific Antigen (PSA) or a fragment thereof, or Human Papilloma Virus E7 (HPV-E7) or a fragment thereof.
7. An immunogenic composition comprising the recombinant Listeria of claim 6 , and an adjuvant.
8. The recombinant Listeria of claim 7 , wherein said adjuvant comprises a granulocyte/macrophage colony-stimulating factor (GM-CSF) protein, a nucleic acid molecule encoding a GM-CSF protein, saponin QS21, monophosphoryl lipid A, or an unmethylated CpG-containing oligonucleotide.
9. The recombinant Listeria of claim 1 , wherein said Listeria is a Listeria monocytogenes strain.
10. A recombinant Listeria strain comprising a nucleic acid molecule encoding a fusion polypeptide, wherein said fusion polypeptide comprises a Prostate Specific Antigen (PSA) or fragment thereof fused to an N-terminal LLO, wherein said recombinant Listeria is an attenuated Listeria comprising a mutation in the dal/dat genes, wherein said Listeria comprises a metabolic enzyme that complements said dal/dat mutation, and wherein said Listeria comprises a deletion of the endogenous actA gene.