A fourth individual observed erythema and induration at the site of the first vaccination after the 2nd vaccination (Table 1). Systemic adverse inhibitors reactions included Gemcitabine order headache, fatigue, malaise and fever in one subject given antigen only. Extensive follow-up of blood and urine parameters did not reveal any obvious trends within or differences

between the three vaccination groups, or laboratory abnormalities with respect to change from baseline that could be related to the vaccinations. In the two subjects who developed a transient fever the day after vaccination, a small rise in C-reactive protein occurred that had subsided within a week. Stimulation with H1, Ag85B and ESAT-6 gave rise to an increased number of spot forming units (SFU) in all adjuvant groups (Fig. 2A and B). The highest proportion of responders to vaccination was seen AP24534 cell line in the low CAF01 group at week 32 and in the intermediate CAF01 group at

week 32 and 52 (Fig. 2C). At this time point median responses were 301 SFU/per million PBMC (inter quartile range (IQR) 111–668 SFU) for H1; 308 SFU (IQR 108–558 SFU) for Ag85B and 39 SFU (IQR 9.5–136 SFU) for ESAT-6, p < 0.05 ( Fig. 2B). No changes from baseline were seen in the non-adjuvant group at any time points. Overall, there was a clear trend in the adjuvant groups that responses increased after the first vaccination and that a second vaccination further increased the magnitude of responses ( Fig. 2A). To assess the breadth of the vaccine-induced immune memory, we performed an exploratory multiplex

analysis of 14 cytokines and chemokines in supernatants of 24 h H1 stimulated PBMCs. We observed a broad induction of multiple cytokines and chemokines at both weeks 14 and 32 for the else three groups vaccinated with adjuvanted H1, responses in the intermediate CAF01 group are presented in Fig. 3 (all groups in supplementary Figure 1). The dominating markers were Th1 associated (IFN-γ, TNF-α, IP-10, MIG, MIP-1b and GM-CSF), but we also observed a substantial release of IL-13, but not IL-4. IL-2, IL-10 and IL-17 followed the same kinetic pattern, but levels were very low (<20 pg/ml) and failed to reach significance (Fig. 3 and data not shown). No clear pattern emerged for VEGF, IL-22 and MCP-1 (supplementary Figure 1). To further assess the long-term immunogenicity of H1:CAF01, PBMC samples at week 150 were analyzed by Intracellular flow cytometry. Compared to the non-adjuvant group, intermediate and high dose CAF01 groups had increased frequencies of Ag85B-specific CD4 T-cells producing IFN-γ and/or IL-2 and/or TNF-α (Fig. 4A). Moreover, intermediate and high dose CAF01 groups induced significant TNF-α production, but only the intermediate CAF01 group reached significant levels of IL-2 (Fig.