A New TB Vaccine Strategy Figure 5. Cytokine frequencies and phenotypic profiles of specific CD4 and CD8 T cells in non-vaccinated or rH4/Ad-H4 vaccinated mice two weeks after challenge. CB6F1 mice were vaccinated once with rH4 in cationic liposomes followed two weeks later by one Ad-H4 vaccination, and challenged by the aerosol route with virulent M.tb six weeks after the final vaccination. Two weeks after challenge the mice were sacrificed and lung lymphocytes were stimulated with Ag85B or TB10.4 peptides prior to staining with anti-CD4, -CD8, -CD44, -IFN-c, -IL-2, and –TNF- a. Frequencies represent IFN-c, IL-2, or TNF-a producing CD44+CD4+ or CD44+CD8+ T cells specific for Ag85B (A and B) or TB10.4 (C and D). Background staining from cells stimulated with medium alone has been subtracted. Data represent the mean+SEM of four mice per group with **, p,0.01 (2-way ANOVA with Bonferroni posttest). IFN-c, IL-2, and TNF-a cytokine profiles of Ag85B or TB10.4 specific CD4 and CD8 T cells are shown in pies as triple, double, or single positive CD4 or CD8 T cells. The color code for the pies is the same as shown in Fig. 1E. doi:10.1371/journal.pone.0005139.g005 protection against acute M.tb infection in a manner that is not strictly dependent upon simultaneous induction of CD4 T cells [5,41]. In the present study we wanted to study the potential of a heterologous prime boost vaccine strategy that induce both CD4 and CD8 T cells, and to compare the phenotype of vaccine primed CD4 and CD8 T cells, as well as the recruitment and proliferation of these cells following infection with M.tb. We combined vaccination of a CD4 T cell inducer, rH4/CAF01, with a strong CD8 T cell inducer, Ad-H4. Mice vaccinated with the rH4/Ad-H4 combination showed an increase in CD4 T cell activity indicating that rH4 primed CD4 T cells can be boosted by Ad-H4 immunization. Compared to Ad-H4, rH4/Ad-H4 also induced significant higher numbers of cytokine producing CD8 T cells. In the rH4/Ad-H4 group approximately 27% of all CD8 T cells were specific for the vaccine antigens, with 7% being specific for Ag85B and 20% for TB10.4. Most of these (45–80%) CD8 T cells expressed IFN-c and TNF-a (Fig. 1 and 2) which suggests an effector phenotype. This clearly demonstrated that combining rH4 and Ad-H4 had a strong additive effect on priming for both the CD4 and CD8 T cell response. It can be speculated that the boosting of the CD4 T cells, which occurred in parallel with the priming of CD8 T cells, provided increased CD4 help for the CD8 T cells, thereby further promoting the activation and expansion of these cells. We also observed a slightly increased cytolytic function of the CD8 T cells in the rH4/Ad-H4 group, in particular against TB10.4 20-28 loaded target cells (17% increase in the specific killing of these cells in the rH4/Ad-H4 group cpmpared to the Ad-H4 group) (Fig. 3). This may be due to increased CD8 T cell numbers, increased cytolytic capacity of the individual T cells, or that the CD8 T cells in the rH4/Ad-H4 group were primed in the presence of CD4 T cells, which have been suggested to increase both the persistence of CD8 T cells and the ability of the CD8 cells to respond to a secondary encounter with their antigen [34–38]. In terms of protection against infection with M.tb, we found, as observed with the numbers of vaccine specific T cells (Fig. 1 and 2), that combining the two vaccine strategies also had a significant additive effect in the lung. Thus, mice vaccinated with rH4/Ad- H4 showed a protection of 1.2 Log1060.1 CFU, which was significantly higher than the protection observed in the Ad-H4 and rH4 mice (Fig. 4A) and not significantly different from that observed in BCG vaccinated mice. The Ad-H4 group showed a protection of approximately 0.4 Log10 CFU, which is in agreement with a recent study using Ad-35-Ag85A-Ag85B- TB10.4 [18]. Interestingly, in the spleen, the lowest protection was observed in the Ad-H4 group, which may indicate that prevention of dissemination from the lung to the spleen require CD4 T cells more than CD8 T cells (Fig. 4B). In the rH4/Ad-H4 group two weeks after infection we observed that 14% of all CD8 T cells in the lungs were specific for TB10.4 PLoS ONE | www.plosone.org 7 April 2009 | Volume 4 | Issue 4 | e5139