A New TB Vaccine Strategy Figure 1. Cytokine frequencies and phenotypic profiles of specific CD4 T cells in Ad-H4, rH4, or rH4/Ad-H4 vaccinated mice one week after final vaccination. CB6F1 mice were vaccinated once with Ad-H4, twice with rH4 in cationic liposomes, or once with rH4 in cationic liposomes followed two weeks later by one Ad-H4 vaccination. Mice were sacrificed one week after the final vaccination and splenocytes were stimulated with Ag85B or TB10.4 peptides prior to staining with anti-CD4, -CD44, -IFN-c, -IL-2, and –TNF-a. (A and C) Frequencies represent IFN-c, IL- 2, or TNF-a producing CD44+CD4+ T cells out of total CD4 T cells specific for Ag85B (A) or TB10.4 (C). Background staining from cells stimulated with medium alone has been subtracted. Data represent the mean+SEM of a minimum of five mice per group with ***, p,0.001, **, p,0.01, and *, p,0.05 (2-way ANOVA with Bonferroni posttest). (B and D) IFN-c, IL-2, and TNF-a cytokine profiles of Ag85B (B) or TB10.4 (D) specific CD4 T cells shown as triple, double, or single positive CD4 T cells. The color code for the pies is shown in (E). Dot plots representative of five mice are also shown in (B) and (D), depicting IFN-c expressed by CD4+CD44+ cells after stimulation with Ag85B (B) or TB10.4 (D). doi:10.1371/journal.pone.0005139.g001 TB10.470-88, and the rH4 group did not demonstrate any cytotoxic killing of the transferred target cells (Fig. 3B). Thus, both Ad-H4 and rH4/Ad-H4 induced CD8 T cells with a cytotoxic potential. The protective efficacy of the vaccine induced T cell responses We next analyzed the vaccine induced protection. Vaccinated CB6F1 mice were challenged six weeks after final vaccination, and six weeks post challenge, the mice were sacrificed and the bacterial numbers were determined in the lungs. Ad-H4, rH4, the hetrologous prime boost combination, non-vaccinated, and BCG vaccinated mice were included. All the H4 based vaccines induced significant protection against M.tb challenge but as observed with the T cell response, combining the two vaccines, rH4 and Ad-H4, showed an additive effect in terms of protection. Thus, mice vaccinated with the heterologous prime boost combination of rH4/Ad-H4 showed a bacterial reduction of 1.2 Log1060.1 CFU, which was significantly higher than the protection in Ad-H4 and rH4 vaccinated mice (p,0.01 and p,0.001, respectively). The BCG-vaccinated mice showed a protection of 1.42 Log1060.1 CFU, which was not significantly different from rH4/Ad-H4 vaccinated mice (Fig. 4A). Interestingly, the CFU levels at six weeks post infection in the vaccine groups directly correlated with the ESAT-6 CD4 T cell response (R2 = 0.78) (Fig. 4B), in agreement with previous work indicating that the ESAT-6 specific response can be used as a predictor for bacterial load [23]. In the spleen, the highest protection was seen in BCG vaccinated mice, and in the groups vaccinated with either rH4 or rH4/Ad-H4. Recruitment of vaccine induced antigen specific CD4 and CD8 T cells to the site of infection As we had observed an increase in both CD4 and CD8 T cells in the group receiving the heterologous prime boost combination of rH4/Ad-H4, and increased protection, it was important to compare how the vaccine primed CD4/CD8 T cells responded to an M.tb infection (in the group where they were both induced, e.g. the rH4/Ad-H4 group). As controls, non-vaccinated mice were included. Mice vaccinated with rH4/Ad-H4 (or non-vaccinated) PLoS ONE | www.plosone.org 3 April 2009 | Volume 4 | Issue 4 | e5139