Antibiotic resistance free
View Patent ↗The present invention provides Listeria strains that express a heterologous antigen and a metabolic enzyme, and methods of generating same.
1. A method of inducing an immune response against a protein antigen of interest in a subject, comprising the step of administering to said subject a recombinant Listeria strain, comprising an integrated nucleic acid molecule, wherein said integrated nucleic acid molecule does not comprise an antibiotic resistance gene, wherein said integrated nucleic acid molecule comprises a first open reading frame encoding a polypeptide and a second open reading frame encoding a metabolic enzyme, wherein said polypeptide comprises said protein antigen of interest, and wherein said metabolic enzyme complements a metabolic deficiency of said recombinant Listeria strain, thereby inducing an immune response against a protein antigen of interest in a subject.
2. The method of claim 1 , wherein said integrated nucleic acid molecule further comprises a gene encoding a transcription factor.
3. The method of claim 2 , wherein said gene encoding a transcription factor is lacking in the remainder of the chromosome of said recombinant Listeria strain.
4. The method of claim 1 , whereby said integrated nucleic acid molecule does not contain a replication region that functions in said recombinant Listeria strain.
5. The method of claim 1 , wherein said polypeptide is a fusion protein comprising said protein antigen and an additional polypeptide, wherein said additional peptide enhances the immunogenicity of said protein antigen.
6. The method of claim 5 , wherein said additional polypeptide is a non-hemolytic LLO protein or fragment thereof, a PEST amino acid sequence, or an ActA fragment.
7. The method of claim 1 , wherein said integrated nucleic acid molecule is a phage integration vector.
8. The method of claim 1 , wherein said metabolic enzyme is an amino acid metabolism enzyme.
9. The method of claim 1 , wherein said metabolic enzyme catalyzes a formation of an amino acid used for a cell wall synthesis in said recombinant Listeria strain.
10. The method of claim 1 , wherein said metabolic enzyme is an alanine racemase enzyme.
11. The method of claim 1 , wherein said metabolic enzyme is a D-amino acid transferase enzyme.
12. The method of claim 1 , wherein said recombinant Listeria strain has been passaged through an animal host.