We also evaluated TNF-α levels because TNF-α is known to play a key role in granuloma formation and induction of

macrophage activation [29]. The adenoviral vector expressing the CRT-ESAT-6 fusion protein demonstrated an enhanced ability to induce both of these cytokines in comparison with ESAT-6, which generated levels of cytokines similar to those induced by control vector, Lac. These data support calreticulin being able to enhance immunity against M. tuberculosis antigens. The fact that ESAT-6 alone did not show a better cytokine Talazoparib response than the control may be because the C57BL/6 mice do not recognize AdESAT6 epitopes or that the immune response generated in these mice is relatively small and cannot be

observed above background levels. It is important to determine whether the increased response is caused by CD8 and/or CD4 T cells, and whether it is a short- or long-term response. Even though it has been demonstrated previously that intranasal vaccination with adenovirus gives rise to a better immune response in the lung versus parenteral vaccination [10, 12], it would be interesting to demonstrate how this will work in our system. Our results support those of others that also showed that calreticulin increased the production of cytokines important in the control of TB [28]. Other studies have demonstrated see more that using a fusion of different M. tuberculosis antigens provides better control of infection in a mouse model of TB [30]. Thus, we also investigated

whether multivalent or mixture-based adenoviral TB vaccines expressing an ESAT-6–CFP10 fusion protein could perform better than ESAT-6 alone when both were linked to CRT. Our result demonstrated that there was no difference between the ESAT-6 and ESAT-6–CFP10 constructs when the cells were stimulated with the ESAT-6 protein. Thus, the fusion did not increase the T cell response to ESAT-6 nor did the ESAT-6 protein stimulate Mannose-binding protein-associated serine protease the T cell response to CFP10. Accordingly, with this result, none of these adenoviruses gave protection against a challenge infection, even those constructs that induce increased IFN-γ and TFN-α antigen-specific cytokine levels. Others have reported similar data: Bennekov et al. [31], using a recombinant adenovirus expressing Ag85B–ESAT, found no protection after vaccination with adenoviral vaccine and also demonstrated that the adenovirus vaccine induced a non-protective, CD8 T cell-targeted response. Recent evidence also demonstrated differences in the types of protective immune response between the C57BL/6 and BALB/c mouse strains. In BALB/c (H-2d) mice, a dominant CD8 T cell response has been reported [32], whereas in C57BL/6 (H-2b) mice, more balanced CD4/CD8 T cell responses, with a more pronounced CD4 response in the lungs, has been reported [33].