To circumvent limitations of poor antigen presentation and immunogenicity of DNA vaccines that target induction of CD8(+) T cell immunity, we have generated single chain MHC I trimers (MHC I SCTs) composed of a single polypeptide chain with a linear composition of antigenic peptide, β2-microglobulin, and heavy chain of a MHC class I molecule connected by flexible linkers. Because of its pre-assembled nature, the SCT presents enhanced expression and presentation of the antigenic peptide/MHC complexes at the cell surface. Furthermore, DNA vaccination with a plasmid DNA encoding an SCT incorporating an immunodominant viral epitope elicited protective CD8(+) T cell responses against lethal virus infection. To extend these findings, here we tested the efficacy of SCT DNA vaccines against bacterial infections. In a mouse infection model of Listeria monocytogenes, the SCT DNA vaccine encoding H-2K(d) and the immunodominant peptide LLO 91-99 generated functional primary and memory peptide-specific CD8(+) T cells that confer partial protection against L. monocytogenes infection. DNA immunization of K(d)/LLO(91-99) SCTs generated functional memory CD8(+) T cells independently of CD4(+) T cells, although the expression of cognate or non-cognate CD4(+) helper T cell epitopes further enhanced the protective efficacy of SCTs. Our study further demonstrates that the SCT serves as a potent platform for DNA vaccines against various infectious diseases