Figure 4. The protective efficacy and correlation of protection with infection driven immune response in the vaccinated and challenged mice. (A and B) Protection in vaccinated mice (expressed as Log10 CFU reduction) compared to unvaccinated and BCG vaccinated controls challenged by the aerosol route with virulent M.tb. six weeks after the final vaccination. Six weeks post-challenge, the mice were sacrificed and the bacterial burden (CFU) was measured in the lung or spleen. Data represent the mean+SEM of a minimum of five mice per group in two individual experiments with ***, p,0.001 and **, p,0.01 (2-way ANOVA with Bonferroni posttest). (C) Relation between protection (CFU) and ESAT-6 specific IFN-c producing CD4 T cells is shown as a fitted regression line with the correlation coefficient (R2). Six weeks post challenge, the mice were sacrificed and the lung cells were A New TB Vaccine Strategy stimulated with ESAT-6 overlapping peptides prior to staining with anti- CD4, -CD44, and –IFN-c. Frequencies represent IFN-c producing ESAT-6 specific CD44+CD4+ T cells out of total CD4 T cells. Background staining from cells stimulated with medium alone has been subtracted. Data represent the mean of +SEM of a minimum of five mice per group in two individual experiments. doi:10.1371/journal.pone.0005139.g004 CD8 T cell population, the majority of the cytokine producing cells were non-proliferating (Fig. 7E). Moreover, when looking at the frequency of cytokine producing CD4 T cells, we found a clear tendency towards higher IFN-c, IL-2, and TNF-a production by the BrdU+ CD4 T cells compared to the BrdU2 CD4 T cell population (Fig. 7D). This tendency was less pronounced, however still present, in the cytokine producing antigen specific CD8 T cells (Fig. 7E). In conclusion, both the vaccine induced CD4 and CD8 T cells undergo cell division and were recruited to the site of infection, at the onset of the infection. However, the CD4 T cells showed a higher frequency of memory-like phenotypes, were recruited to the lungs at a higher rate, and proliferated more than CD8 T cells. Discussion CD4 T cells are crucial for defense against M.tb. However, it is still not clear whether the optimal response against infection with M.tb in fact involves both CD4 and CD8 T cells and if these subsets play a role in distinct and different stages of the infection [27]. In the present study we therefore compared the well characterized vaccine fusion molecule rH4 (Ag85B-TB10.4) delivered either as a subunit vaccine in cationic liposomes (rH4/ CAF01) expressed in an adeno5 vector (ad-H4) or as a heterologous prime boost combination. We clearly show that rH4/CAF01 induces primarily CD4 T cells, and Ad-H4 CD8 T cells, but the heterologous prime boost combination induces both CD4 and CD8 T cells. Although CD8 T cells are a significant part of the immune response induced by the natural infection, the role of CD8 T cells in the defense against M.tb is not fully resolved. Lately, several vaccine strategies have made use of viral vectors to deliver the vaccine antigens, and to induce vaccine specific CD8 T cells [28– 30]. These studies all point to a protective role for CD8 T cells, even though in most of these studies CD4 T cells were also primed. Studies using MVA as delivery of the vaccine antigens have indicated that this vector is particularly efficient when used as a booster vaccine, and boosting of both CD4 and CD8 T cells have been observed [15,31]. Likewise, adenoviral vectors have been found to serve as efficient boosters of existing immune responses. Thus, adenovirus expressing Ag85A was able to boost BCG immunity, which in turn increased the protection against M.tb [32]. As with MVA, adenovirus boosted both the CD4 and CD8 T cell activity, and priming of both these subsets was also observed when following immunization with adenovirus expressing myco- bacterial antigens [18,29,33]. In other recent studies, in which only CD8 T cells to immunodominant antigens (such as ESAT-6 or TB10.4) were induced, only minor or no protection was observed [17,21], indicating that the contribution of CD8 T cells to protection may depend on, or increase inthe presence of, an ongoing CD4 T cell response. This is in line with studies showing that both CD8 T cell memory induction and maintenance can be influenced by CD4 T cells [34–40]. However, it should be mentioned that in two studies using vaccination with a DNA vaccine encoding CFP10, or a mix of several TB proteins, the vaccine induced CD8 T cells were shown to induce protection, demonstrating that in some instances CD8 T cells do provide PLoS ONE | www.plosone.org 6 April 2009 | Volume 4 | Issue 4 | e5139