ESAT-6 and HspX improve the effectiveness of BCG to induce human Dendritic Cells-dependent T cells and NK cells activation

The limited efficacy of Bacillus Calmette-Guerin (BCG) vaccination against some forms of tuberculosis is partly due to a missing expression of critical immunogenic proteins. We hypothesized that addition of ESAT-6 and HspX Mycobacterium Tuberculosis (Mtb) antigens could ameliorate the BCG ability to activate human dendritic cells (DC), that play an essential role in immune response. Here we report that BCG showed a weak ability to induce DC maturation, cytokine release and the subsequent CD4+ lymphocytes and NK cells activation. Addition of single ESAT-6 or HspX to BCG-stimulated DC did not significantly improve these processes. However, simultaneous addition of ESAT-6 and HspX enhanced BCG-dependent DC maturation and IL-12, IL-1β, IL-23, IL-6 and TNFα release. Moreover, DC incubated with BCG in presence of both ESAT-6 and HspX elicited IFN-γ release by CD4+ lymphocytes, and increased IFN-γ secretion and CD69 cytolysis marker expression in NK cells. These effects were inhibited by IL-12-blocking antibodies. A specific TLR2-blocking antibody decreased IL-12 release by BCG-stimulated DC incubated with ESAT-6 and HspX, as well as IFN-γ secretion by CD4+ lymphocytes co-cultured with these cells. Moreover, HspX and ESAT-6 improved the capacity of BCG-treated DC to induce the expression of the memory phenotype marker CD45RO in naïve CD4+ T cells. Our results indicate that ESAT-6 and HspX cooperation enhances the ability of BCG to stimulate human DC, that become able to induce T lymphocytes and NK cells-mediated immune responses through TLR2-dependent IL-12 secretion. Therefore ESAT-6 and HspX represent good candidates for improving the effectiveness of vaccination with BCG.