Influence of ESAT-6 secretion system 1 (RD1) of Mycobacterium tuberculosis on the interaction between mycobacteria and the host immune system

The chromosomal locus encoding the early secreted antigenic target, 6 kDa (ESAT-6) secretion system 1 of Mycobacterium tuberculosis, also referred to as "region of difference 1 (RD1)," is absent from Mycobacterium bovis bacillus Calmette-Guerin (BCG). In this study, using low-dose aerosol infection in mice, we demonstrate that BCG complemented with RD1 (BCG::RD1) displays markedly increased virulence which albeit does not attain that of M. tuberculosis H37Rv. Nevertheless, phenotypic and functional analyses of immune cells at the site of infection show that the capacity of BCG::RD1 to initiate recruitment/activation of immune cells is comparable to that of fully virulent H37Rv. Indeed, in contrast to the parental BCG, BCG::RD1 mimics H37Rv and induces substantial influx of activated (CD44highCD45RB(-)CD62L(-)) or effector (CD45RB(-)CD27(-)) T cells and of activated CD11c(+)CD11bhigh cells to the lungs of aerosol-infected mice. For the first time, using in vivo analysis of transcriptome of inflammatory cytokines and chemokines of lung interstitial CD11c+ cells, we show that in a low-dose aerosol infection model, BCG::RD1 triggered an activation/inflammation program comparable to that induced by H37Rv while parental BCG, due to its overattenuation, did not initiate the activation program in lung interstitial CD11c+ cells. Thus, products encoded by the ESAT-6 secretion system 1 of M. tuberculosis profoundly modify the interaction between mycobacteria and the host innate and adaptive immune system. These modifications can explain the previously described improved protective capacity of BCG::RD1 vaccine candidate against M. tuberculosis challenge

An Increase in Antimycobacterial Th1-Cell Responses by Prime-Boost Protocols of Immunization Does Not Enhance Protection against Tuberculosis

Bordetella pertussis adenylate cyclase (CyaA) toxoid is a powerful nonreplicative immunization vector targeting dendritic cells, which has already been used successfully in prophylactic and therapeutic vaccination in various preclinical animal models. Here, we investigated the potential of CyaA, harboring strong mycobacterial immunogens, i.e., the immunodominant regions of antigen 85A or the complete sequence of the 6-kDa early secreted antigenic target (ESAT-6) protein, to induce antimycobacterial immunity. By generating T-cell hybridomas or by using T cells from mice infected with mycobacteria, we first demonstrated that the in vitro delivery of 85A or ESAT-6 to antigen-presenting cells by CyaA leads to processing and presentation, by major histocompatibility complex class II molecules, of the same epitopes as those displayed upon mycobacterial infection. Importantly, compared to the recombinant protein alone, the presentation of ESAT-6 in vitro was 100 times more efficient upon its delivery to antigen-presenting cells in fusion to CyaA. Immunization with CyaA-85A or CyaA-ESAT-6 in the absence of any adjuvant induced strong antigen-specific lymphoproliferative, interleukin-2 (IL-2) and gamma interferon (IFN-γ) cytokine responses, in the absence of any IL-4 or IL-5 production. When used as boosters after priming with a BCG expressing ESAT-6, the CyaA-85A and CyaA-ESAT-6 proteins were able to strikingly increase the sensitivity and intensity of proliferative and Th1-polarized responses and notably the frequency of antigen-specific IFN-γ-producing CD4(+) T cells. However, immunization with these CyaA constructs as subunit vaccines alone or as boosters did not allow induction or improvement of protection against Mycobacterium tuberculosis infection. These results question the broadly admitted correlation between the frequency of IFN-γ-producing CD4(+) T cells and the level of protection against tuberculosis