At 4 weeks post-immunization, mice were sacrificed, and their spleens were removed. Splenocytes were restimulated with ESAT-6 protein in vitro, and the number of IFN-γ-secreting cells and the concentration of TNF-α in the supernatant were measured using ELISPOT and ELISA, respectively. No significant differences in the number of IFN-γ-secreting cells or the concentration of TNF-α were observed

between the two groups (Fig. 3B,C). Thus, the addition of CFP-10 to the calreticulin–ESAT-6 fusion did not provide an enhancement of the Selleck 3-deazaneplanocin A ESAT-6-specific immune response. We next investigated the ability of the vaccine-induced immune response to reduce the mycobacterial burden after low-dose aerosol infection in the mouse model. Mice were Navitoclax supplier vaccinated with AdCRT–ESAT-6–CFP10 via the intranasal route and BCG via the subcutaneous route, only once as described in Materials and methods. At 4 weeks post-immunization, mice were infected with M. tuberculosis. Four weeks after challenge, the M. tuberculosis burden of infected animals was determined to evaluate the

protective efficacy in both lung and spleen. The trends were similar in both organs (Fig. 4A,B). BCG caused a reduction in CFU in both the lungs and spleen of infected animals. However, there was no significant difference between mice vaccinated with the adenovirus constructs and the saline-treated group for both organs. The high incidence of TB has Bay 11-7085 stimulated interest in understanding the immune response to infection, resulting in the accelerated identification of novel immunodominant mycobacterial proteins as possible vaccine candidates. Culture filtrates and RD sequences have attracted particular interest as a source of antigens. ESAT-6, TB 10.4, CFP10, MTB12, MTB39 and Ag85 A and B have all been shown to elicit protective immune responses in various animal models of TB [12, 16, 27, 28]. Even though many strategies for vaccination increase the overall immune response, this may not be the ideal solution. When multiple antigens are presented to the immune system, they will compete for

presentation, and the antigens dominating the response will not necessarily be those most relevant for protection. Thus, a targeted approach may be ideal. It has been repeatedly demonstrated that calreticulin can enhance immune responses when linked to antigens in DNA and viral vaccines [23–26]. This suggests that the use of calreticulin may be broadly applicable as a strategy to enhance vaccine efficacy. In addition, several reports have suggested the efficacious use of vaccines against TB in mice using adenoviral vectors expressing different M. tuberculosis antigens [10]. We herein demonstrate the effects of a replication-deficient adenoviral vector that contains the M. tuberculosis ESAT-6 antigen fused to calreticulin and show that there is an increased immune response to this antigen as demonstrated by increased cytokine expression.