trials indicate that it is also immunogenic in humans but with a response almost exclusively composed of CD4 cells [15,16]. Replication-deficient recombinant adenovirus for comparison are widely accepted as one of the most efficient delivery platforms for the induction of a CD8 T cell dominated response and adeno vectors have been evaluated in a number of studies of TB antigens. The H1 molecule (Ag85B-ESAT-6) expressed in Ad5 was demonstrated to promote a strong response consisting exclusively of CD8 cells directed to a few immunodominant epitopes but unable to protect against TB [17]. Another virally-vectored vaccine is Aeras-402 Ad35, expressing a fusion of antigen 85A, antigen 85B, and TB10.4. All three antigens are present in M.tb and BCG, and all are highly immunogenic. The characterization of this vaccine in animal models has so far been limited but also this vaccine is clearly capable of inducing a strong CD8 T cell responses [18]. The antigens Ag85B and TB10.4 have been extensively characterized [11,19–22] and a fusion protein consisting of both Ag85B-TB10.4 adjuvanted with cationic liposomes induced a strong protection against infection with M.tb [23]. In the present study we have compared CD4 and CD8 responses against the H4 molecule delivered as a subunit vaccine in cationic liposomes (CAF01), expressed in Ad5 (Ad-H4) or as a heterologous prime boost vaccination. Our data demonstrate that the heterologous prime boost vaccination gives higher levels of both polyfunctional CD4 T cells, cytotoxic CD8 T cells and protection against M.tb challenge than Ad-H4 or rH4 on their own. When we monitored cellular infiltrate in the lung post M.tb challenge in mice vaccinated by the heterologous prime boost protocol we observed that whereas high numbers of both H4 specific CD4 and CD8 T cells were promoted by the vaccine, the CD4 T cells showed accelerated recruitment to the site of infection compared to CD8 cells and importantly CD4 cells were the main subset proliferating in the lung in response to M.tb infection in the vaccinated animals. Results Induction and characterization of the vaccine induced antigen specific T cell responses We first compared the immune response promoted by H4 delivered in cationic liposomes (CAF01) expressed in an adeno5 vector or as a hetrologous pirme boost. In particular, we were interested in whether we could induce both CD4 and CD8 T cells, as well as in the quantity and phenotype of these cells. CB6F1 mice were vaccinated once with rH4 followed by a booster Ad-H4 vaccination. Non-vaccinated mice were also included but had no detectable T cells responses to the vaccine components (data not shown). One week after the last vaccination, mice were sacrificed and splenocytes were stimulated in vitro with Ag85B and TB10.4, and the production of IFN-c, IL-2, and TNF-a by CD4 and CD8 T cells was analyzed by intracellular FACS. We found that all three vaccines, Ad-H4, rH4, and rH4/Ad-H4, elicited Ag85B specific CD4 T cells producing all three cytokines (Fig. 1A) and a significant number of these cells were moreover triple positive (35, 33, and 42%, respectively) (Fig. 1B). However, only rH4 and rH4/ Ad-H4 vaccinated mice contained TNF-a+IL-2+ double positive CD4 T cells (Fig. 1B and D). The triple positive and TNF-a+IL-2+ double positive T cell phenotypes generally represent effector memory- and central memory T cells, respectively [24], indicating the induction of a more memory like CD4 T cell immune response by the vaccines rH4 and rH4/Ad-H4, as compared to Ad-H4. Most importantly, the highest number of antigen specific T cells was seen in mice vaccinated with rH4/Ad-H4. This vaccine A New TB Vaccine Strategy induced a higher frequency of both IFN-c (1.1%60.3) and TNF-a (1.3%60.3) producing Ag85B specific CD4 T cells compared to Ad-H4 and rH4 (p,0.001 and 0.05, respectively) (Fig. 1A), and the phenotypic profile of the vaccine induced T cell response reflected the combination of Ad-H4 and rH4 vaccinations since it contained a high frequency of triple- and double positive memory CD4 T cells, as well as a significant amount of IFN-c+TNF-a+ double positive (and single positive IFN-c+) effector CD4 T cells (Fig 1B and D) [24,25]. In general, the level of TB10.4 specific CD4 T cells was found to be low in all three vaccinated groups, however, with a significant increase of IFN-c producing CD4 T cells in the rH4/Ad-H4 vaccinated mice (Fig. 1C). In comparison, BCG vaccinated mice showed low numbers of Ag85B specific CD4 T cells, but the highest numbers of TB10.4 specific T cells of which the major phenotypes were IFN-c+TNF-a+IL-2+ or IFN- c + TNF-a+ expressing CD4 T cells. In terms of CD8 T cells, we found that only the mice vaccinated with Ad-H4 or rH4/Ad-H4 had detectable numbers of antigen specific CD8 T cells (Fig. 2A and C), but by combining rH4 and Ad-H4 a larger CD8 T cell population was observed. We observed an increase in Ag85B specific IFN-c producing CD8 T cells from 1.1%60.1 in the Ad- H4 group to 6.8%61.9 in the rH4/Ad-H4 group (p,0.001), and an increase from 6.2%60.6 to 16%62.3 in the IFN-c producing CD8 T cells specific for TB10.4 (p,0.001) (Fig 2A and C). Moreover, when summing up the T cells expressing all combinations of IFN-c, TNF-a, and IL-2 we found that in the rH4/Ad-H4 group 6.9% of all CD8 T cells were specific for Ag85B and 19.6% for TB10.4. Both vaccines induced IFN- c + TNF-a+ double positive CD8 T cells specific for both Ag85B and TB10.4, and interestingly also a minor, but still detectable, population of triple positive antigen specific CD8 T cells (Fig. 2B and D). Consistent with this, stimulation of splenocytes from the vaccinated mice in vitro for 72 hours (instead of 6 hours in the FACS analysis) followed by ELISA analysis of IFN-c secretion, also revealed significantly higher IFN-c production in the rH4/ Ad-H4 vaccine group, although mice from all vaccine groups gave measurable responses (Fig. 2E). Taken together, we found that heterologous prime boost based on rH4 and Ad-H4 had a clear additive effect on the number of vaccine antigen specific CD4 and CD8 T cells, inducing higher numbers of polyfunctional CD4 and CD8 T cells compared to rH4 and Ad-H4 vaccinations. Vaccine induced cytotoxicity To further characterize the vaccine induced T cells we next analyzed the cytotoxic potential of the induced CD8 (and CD4) T cells. We used the recently published MHC class I restricted epitopes (specific for TB10.43-11 and TB10.420-28) and one MHC class II restricted epitope (TB10.470-88) [11,21,26]. CFSE labeled splenocytes from na¨ıve mice, pulsed (CFSE high) or not pulsed (CFSE low) with either of the two MHC class I restricted peptides or the MHC class II restricted epitope from TB10.4, were adoptively transferred into mice that had been vaccinated with rH4/Ad-H4 a week before. For comparison we included Ad-H4 and rH4 vaccinated mice. After 4 hours peptide specific lysis of the transferred cells was determined by FACS analysis of recipient splenocytes. We observed 78.8%60.2 and 80.6%60.2 specific killing of the TB10.43-11 loaded target cells (CFSE high) in Ad-H4 and rH4/Ad-H4 mice, respectively, compared to non-loaded target cells (CFSE low) (p,0.05). The rH4/Ad-H4 mice also demonstrated strong cytotoxic potential against TB10.420-28 loaded target cells with 70.5%62.3 specific killing, which was slightly higher than the TB10.420-28 specific killing in Ad-H4 mice (53.5%66.6, p,0.05) (Fig. 3A and C). In contrast, there was no killing of target cells loaded with the MHC II restricted epitope PLoS ONE | www.plosone.org 2 April 2009 | Volume 4 | Issue 4 | e5139