Methods for constructing antibiotic resistance free vaccines
The present invention provides Listeria vaccine strains that express a heterologous antigen and a metabolic enzyme, and methods of generating same.
1. A composition comprising an antibiotic resistance gene-free plasmid, said plasmid comprising: i) a first nucleic acid sequence encoding a polypeptide, wherein said polypeptide is a fusion protein comprising a heterologous antigen and a non-hemolytic Listeriolysin (LLO) protein or N-terminal fragment thereof, and ii) a second nucleic acid sequence encoding a metabolic enzyme.
2. The composition of claim 1 , wherein said heterologous antigen is a human papilloma virus E7 antigen (HPV-E7) or a human papilloma virus E6 antigen (HPV-E6).
3. The composition of claim 1 , wherein said first nucleic acid sequence is operably linked to a promoter/regulatory sequence and said second nucleic acid sequence is operably linked to a promoter/regulatory sequence.
4. The composition of claim 3 , wherein said promoter/regulatory sequence linked to said first nucleic acid sequence comprises a hly promoter.
5. The composition of claim 3 , wherein said promoter/regulatory sequence linked to said second nucleic acid sequence comprises a truncated P60 promoter region.
6. The composition of claim 5 , wherein the nucleic acid sequence of said truncated P60 promoter region is set forth in SEQ ID NO: 7.
7. The composition of claim 1 , wherein said plasmid comprises a gram positive and a gram negative origin of replication region.
8. The composition of claim 7 , wherein said gram negative origin of replication is an E. coli origin of replication.
9. The composition of claim 1 , wherein said metabolic enzyme is a D-alanine racemase enzyme or D-amino acid transferase enzyme.
10. The composition of claim 1 , wherein said plasmid does not confer antibiotic resistance upon said recombinant Listeria strain.
11. The composition of claim 1 , wherein said antibiotic resistance gene-free plasmid further comprises a nucleic acid sequence encoding a transcription factor.
12. The composition of claim 11 , wherein said transcription factor is a prfA gene.
13. The composition of claim 1 , further comprising a recombinant, auxotrophic Listeria strain, wherein said plasmid is incorporated into the genome of said Listeria strain, and wherein the metabolic enzyme in said plasmid complements a mutation or deletion in said Listeria strain's chromosome.
14. The composition of claim 13 , wherein said first nucleic acid sequence is operably linked to a promoter/regulatory sequence comprising a hly promoter.
15. The composition of claim 13 , wherein said heterologous antigen is a human papilloma virus E7 antigen (HPV-E7) or a human papilloma virus E6 antigen (HPV-E6).
16. The composition of claim 13 , wherein said second nucleic acid sequence is operably linked to a promoter/regulatory sequence comprising a truncated P60 promoter region.
17. The composition of claim 13 , wherein said metabolic enzyme is a D-alanine racemase enzyme or D-amino acid transferase enzyme.
18. The composition of claim 13 , wherein the chromosome of said recombinant Listeria strain comprises a mutation or deletion of a gene encoding a transcription factor or wherein the chromosome lacks a gene encoding a transcription factor.