Methods for constructing antibiotic resistance free vaccines

A method of producing a recombinant Listeria strain, said method comprising the steps of:

• (a) introducing a mutation in a D-alanine racemase gene and in a D-amino acid transferase gene in said Listeria strain's chromosome, (b) transforming said Listeria strain with a plasmid comprising:
• i) a first nucleic acid sequence encoding a polypeptide, wherein said polypeptide comprises a heterologous antigen, ii) a second nucleic acid sequence encoding a metabolic enzyme, wherein said metabolic enzyme complements the mutation in said recombinant Listeria strain's chromosome, wherein said metabolic enzyme encoded by said second nucleic acid is a D-alanine racemase enzyme, wherein said polypeptide is a fusion protein comprising said heterologous antigen and a non-hemolytic fragment of a listeriolysin (LLO) protein, wherein the chromosome of said recombinant Listeria strain further lacks a prfA gene, wherein said first and said second nucleic acids are operably linked to a promoter/regulatory sequence, wherein said promoter/regulatory sequence linked to said second nucleic acid sequence comprises a truncated p60 promoter region, wherein said plasmid comprises a gram positive and a gram negative origin of replication region, and wherein said plasmid does not confer antibiotic resistance upon said recombinant Listeria strain, thereby producing a recombinant Listeria strain comprising a mutation in a D-alanine racemase gene and in a D-amino acid transferase gene in said Listeria's chromosome.