Istograms) to assess PZ-51 specific uptake. These data presented are from one of three separate experiments. doi:10.1371/journal.pone.0047724.gstrategies, with no significant difference between the two. The strong response of the two-inoculation schedule at low dose is particularly notable, as this was a more robust performance than found in other adjuvants similarly tested in our laboratory previously. However, high dosages of PA-MSHA were ineffective and sometimes even detrimental for promoting immune responses. 1676428 Within the three-inoculation strategy, high dose PA-MSHA (108 CFU) promoted specific cellular responses up to the same strength as low-dose groups, while humoral responses were augmented only for the lowest-dose group (Fig. 4 and Fig. 5B). Similarly, within thetwo-inoculation regimen, cellular responses were attenuated for the high dose group, while humoral responses were not enhanced for any adjuvant dose (Fig. 5A). The dose dependency of both cellular and humoral responses may be due to certain immunosuppressive or tolerance-eliciting properties of PA-MSHA. Previous studies have shown that PAMSHA to inhibits cellular proliferation and induces apoptosis 
in human breast cancer cell lines in a dose-dependent manner [24] through EGFR pathway signaling [36]. High concentrations of PA-MSHA (3.6?6108 CFU) induced high levels of apoptosis andP. aeruginosa Enhanced 25837696 DNA Vaccine ImmunoreactivityFigure 4. HIV-1 Env specific cellular immune responses as order SC-66 detected by IFN-c and IL-2 ELISPOT. Mice were immunized for either two or three times at a 3 week interval with an HIV DNA plasmid co-formulated with different concentrations of PA-MSHA adjuvant. * indicates p,0.05 when compared with 50 mg DNA alone in 2 administrations; # indicates p,0.05 when compared with 50 mg DNA alone in 3 administrations; and ** indicates p,0.05 when comparing between 2 and 3 inoculations at 108 CFU dose PA-MSHA. doi:10.1371/journal.pone.0047724.gproliferation inhibition [24]. Therefore, it is possible that high dose PA-MSHA also inhibited normal immunological cell proliferation in our assays here through EGFR signaling. And our other data also demonstrated the immunosuppressive in other different immunization strategies (shown in Fig. S2). Meanwhile, the enhancement of cellular immune responses (compared to DNA vaccine alone) in the high dose, three-inoculation group may be due to a yet unknown mechanism. In addition, IgG isotype assay also suggests that high dose PAMSHA induces immune tolerance or immunosuppression responses. Our results showed that a dose range of PA-MSHA from102 to 106 CFU induced Th1/Th2 type immune responses in balance (Fig. 5C/5D). However, a typical Th2 type response was induced by 108 CFU of PA-MSHA in the three inoculations regimen, which would be expected to elicit an antibody biased response; yet in reality, the 108 dose could not enhance a humoral response. This therefore raises the question of why the Th2-type immune response resulted in a low humoral response. As inactivated bacterium, PA-MSHA is a powerful pathogen and is ?involved in the activation of naive immunity, which may also contribute to negative immunological regulation at high doseFigure 5. The HIV-1 Env 
specific humoral immune response as detected by ELISA. (A) and (B) show that the HIV-Env specific antibody titer detected by ELISA after two and three inoculations, respectively. We analyzed IgG isotypes (IgG1/IgG2a) in the humoral response after two (C) and three (D) inoculati.Istograms) to assess specific uptake. These data presented are from one of three separate experiments. doi:10.1371/journal.pone.0047724.gstrategies, with no significant difference between the two. The strong response of the two-inoculation schedule at low dose is particularly notable, as this was a more robust performance than found in other adjuvants similarly tested in our laboratory previously. However, high dosages of PA-MSHA were ineffective and sometimes even detrimental for promoting immune responses. 1676428 Within the three-inoculation strategy, high dose PA-MSHA (108 CFU) promoted specific cellular responses up to the same strength as low-dose groups, while humoral responses were augmented only for the lowest-dose group (Fig. 4 and Fig. 5B). Similarly, within thetwo-inoculation regimen, cellular responses were attenuated for the high dose group, while humoral responses were not enhanced for any adjuvant dose (Fig. 5A). The dose dependency of both cellular and humoral responses may be due to certain immunosuppressive or tolerance-eliciting properties of PA-MSHA. Previous studies have shown that PAMSHA to inhibits cellular proliferation and induces apoptosis in human breast cancer cell lines in a dose-dependent manner [24] through EGFR pathway signaling [36]. High concentrations of PA-MSHA (3.6?6108 CFU) induced high levels of apoptosis andP. aeruginosa Enhanced 25837696 DNA Vaccine ImmunoreactivityFigure 4. HIV-1 Env specific cellular immune responses as detected by IFN-c and IL-2 ELISPOT. Mice were immunized for either two or three times at a 3 week interval with an HIV DNA plasmid co-formulated with different concentrations of PA-MSHA adjuvant. * indicates p,0.05 when compared with 50 mg DNA alone in 2 administrations; # indicates p,0.05 when compared with 50 mg DNA alone in 3 administrations; and ** indicates p,0.05 when comparing between 2 and 3 inoculations at 108 CFU dose PA-MSHA. doi:10.1371/journal.pone.0047724.gproliferation inhibition [24]. Therefore, it is possible that high dose PA-MSHA also inhibited normal immunological cell proliferation in our assays here through EGFR signaling. And our other data also demonstrated the immunosuppressive in other different immunization strategies (shown in Fig. S2). Meanwhile, the enhancement of cellular immune responses (compared to DNA vaccine alone) in the high dose, three-inoculation group may be due to a yet unknown mechanism. In addition, IgG isotype assay also suggests that high dose PAMSHA induces immune tolerance or immunosuppression responses. Our results showed that a dose range of PA-MSHA from102 to 106 CFU induced Th1/Th2 type immune responses in balance (Fig. 5C/5D). However, a typical Th2 type response was induced by 108 CFU of PA-MSHA in the three inoculations regimen, which would be expected to elicit an antibody biased response; yet in reality, the 108 dose could not enhance a humoral response. This therefore raises the question of why the Th2-type immune response resulted in a low humoral response. As inactivated bacterium, PA-MSHA is a powerful pathogen and is ?involved in the activation of naive immunity, which may also contribute to negative immunological regulation at high doseFigure 5. The HIV-1 Env specific humoral immune response as detected by ELISA. (A) and (B) show that the HIV-Env specific antibody titer detected by ELISA after two and three inoculations, respectively. We analyzed IgG isotypes (IgG1/IgG2a) in the humoral response after two (C) and three (D) inoculati.