Methods for constructing antibiotic resistance free vaccines

A composition comprising an antibiotic gene-free plasmid, said 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 a mutation or deletion in a recombinant Listeria strain's chromosome, wherein said polypeptide is a fusion protein comprising said heterologous antigen and a non-hemolytic Listeriolysin (LLO) protein or N-terminal fragment thereof.