Towards a second generation of Salmonella-mediated oral DNA vaccines

Abstract

Salmonella-mediated oral DNA vaccination has established itself as a very potent and versatile way of immunization. In this method, the use of commercially available multicopy expression plasmids based on pUC origin of replication (ori) transferred by the metabolic mutant Salmonella typhimurium aroA to mice has resulted in protective responses against pathogens and tumors. However, general applicability of this system has been hampered by a severe instability of transformants carrying these expression plasmids. Therefore new low copy number expression plasmids were constructed using different oris to stabilize the transformants. Comparative studies between transformants of the high copy number plasmid pCMVs and the different low copy number plasmids that contain the pMB1, p15A or pSC101 ori on the pCMVs backbone, revealed a dramatic increase in plasmid stability in vitro and in vivo. Analyzing the immune responses against antigens encoded by these vectors indicated that the increased stability resulted in a strong and reproducible induction of antigen specific CD4+ and CD8+ T cell as well as antibody responses even after a single application. In protection studies using listeriolysin as antigen, mice were protected against a high lethal dose of Listeria monocytogenes independent of the plasmid copy number. Thus, we consider the improved stability as a first step towards a second generation of Salmonella-based oral DNA vaccines. In addition, it was shown that Salmonella transferring two independent antigens on these compatible low copy number plasmids elicited robust responses to either antigen that is effective as Salmonella transformed with each plasmid singly.