The difference was considered statistically significant when P ≤ 0.05. Leica Microscopy system was used to take the picture, and magnification used was 40 with numerical aperture of the objectives, at temperature room. The slides were mounted using Vectashield mounting medium (Vector laboratories), and Alexa 488 fluorochrome was used to detect the positive signal (Invitrogen). As a first step, we designed recombinant adenovirus vectors containing ESAT-6 with and without calreticulin to determine whether calreticulin increased the immune response to the antigen. AdESAT-6 and AdCRT–ESAT-6 were created as described in the Materials and methods. Expression of ESAT-6 in both constructs was under the control of a cytomegalovirus promoter (Fig. 1A–C). The capacity of these constructs to express ESAT-6 was first verified by immunoblot RGFP966 in vitro analyses of HEK293 cells transfected with one of the recombinant vectors (data not shown). ESAT-6 protein expression was also demonstrated by immunofluorescence analysis of HEK293 cells transfected with AdESAT-6, AdCRT-ESAT-6 or AdLacZ (Fig. 1D). As shown in Fig. 1D, only cells transfected with
AdESAT-6 and AdCRT-ESAT-6 express ESAT-6. Therefore, our recombinant adenovirus constructs were proven to be capable of producing ESAT-6. To test the ability of AdCRT–ESAT-6 or AdESAT-6 to generate ESAT-6-specific cellular immune responses in vivo, mice Enzalutamide concentration were immunized by the intranasal route with the adenovirus constructs.
At 4 weeks post-vaccination, splenocyte cultures were prepared Cobimetinib order and restimulated with ESAT-6, and the resultant cytokine responses were analysed. It was found that while splenocytes from mice immunized with the antigen alone (AdESAT-6) showed no differences in cytokine production compared to splenocytes from LacZ-immunized mice (controls), there were significant inductions of IFN-γ and TNF-α (measured by ELISPOT and ELISA, respectively) in splenocytes from mice immunized with the antigen ESAT-6 fused to calreticulin (AdCRT–ESAT-6) (Fig. 2A,B). Taken together, these data demonstrate that immunization with ESAT-6 linked to calreticulin is an effective approach to generate potent immune responses. It has been previously shown that fusion of ESTA-6 with CFP-10 enhances the immune response. Hence, using the same strategy, we expressed a calreticulin–ESAT-6–CFP10 fusion protein (AdCRT–ESAT-6–CFP10) and compared its ability to induce a cytokine response against AdCRT–ESAT-6. The expression of the fusion protein was demonstrated by immunoblot analysis of lysates of cells transfected with the fusion vector using an anti-CFP10 polyclonal mouse antibody (Fig. 3A). While no reaction was observed in the uninfected HEK293 cell lysates, a single antibody-reactive band of approximately 90 kDa was detected in the AdCRT–ESAT-6–CFP10 cell lysates. The size of the reactive band correlated with the predicted size of the CRT-ESAT-6–CFP10 fusion protein.