3A–C). However, antigen-specific selleck kinase inhibitor proliferation responses were observed in response to a given protein only with spleen cells of mice immunized with the recombinant DNA vaccine construct expressing that protein (Figs. 4 and 5A,C–E; SI > 5.0), except

for PPE68, which failed to induce proliferation responses in animals immunized with pUMVC6/PPE68 or pUMVC7/PPE68 (Figs. 4B and 5B, respectively). The failure of BCG vaccine in humans has prompted the research to develop alternative vaccines against TB. Among the novel vaccine candidates, plasmid DNA–based TB vaccines have drawn close attention because of their unique features compared to conventional live or subunit vaccines, including induction of strong Th1-based CD4+ responses as well as CTL responses [21]. Therefore, the potency of plasmid DNA expressing a variety of immunogenic M. tuberculosis antigens has been intensively evaluated [22]. Unfortunately, their performance is generally not superior to BCG, especially in large animals. However, the licensure of a DNA vaccine in horses highlights the potential of DNA vaccine technology in the prevention of TB infection [23]. Besides, it is generally believed that novel TB vaccines will

be tested in the context of the widely used BCG and perhaps different kinds of vaccines are needed for the eradication of TB [24, 25]. As a result, enhancement of TB DNA vaccine efficacy has become the active field of current research [26]. In this context, Baldwin et al. have shown that inclusion of Paclitaxel cell line the secretion signal peptide from tissue plasminogen activator (tPA) into a DNA vaccine construct resulted in stronger immune responses to Ag85A, and provided sustained protection upon M. tuberculosis challenge in mice, when compared to the DNA vaccine construct based on the parent plasmid lacking tPA [27]. Furthermore, BCKDHA a plasmid DNA vaccine expressing heat shock protein 65 (HSP65-DNA vaccine) provided improved protective and therapeutic effects in mice when fused with human interleukin-2 [28]. The plasmid vectors used in this

study, i.e. pUMVC6 and pUMVC7, are eukaryotic expression vectors, which have been prepared by University of Michigan Vector Core (UMVC) and provided by Aldevron, USA. Both vectors have CMV promoter at 5′ end of the cloning site. pUMVC6 is characterized by having a secretion signal peptide from human interleukin-2 (hIL2 secretory peptide) as an immuno-stimulatory sequence, whereas pUMVC7 has a signal peptide for targeting peptides to a secretory pathway by fusion to the tPA signal peptide. To our knowledge, these DNA plasmid vectors have not been previously used to determine the expression and immunogenicity of M. tuberculosis-specific genes. Furthermore, this study provided direct comparison of tPA and hIL-2 in determining their immunopotentiating effects in DNA vaccine constructs. We cloned genes encoding five major antigenic proteins of RD1 and RD9 of M.