The development of a new tuberculosis vaccine is an urgent need due to the failure of the current vaccine, BCG, to protect against the respiratory form of the disease. MTBVAC is an attenuated Mycobacterium tuberculosis vaccine candidate genetically engineered to fulfil the Geneva consensus requirements to enter human clinical trials. We selected a M. tuberculosis clinical isolate to generate two independent deletions without antibiotic-resistance markers in the genes phoP, coding for a transcription factor key for the regulation of M. tuberculosis virulence, and fadD26, essential for the synthesis of the complex lipids phthiocerol dimycocerosates (DIM), one of the major mycobacterial virulence factors. The resultant strain MTBVAC exhibits safety and biodistribution profiles similar to BCG and confers superior protection in preclinical studies. These features have enabled MTBVAC to be the first live attenuated M. tuberculosis vaccine to enter clinical evaluation.

Some of the most promising novel tuberculosis vaccine strategies currently under development are based on respiratory vaccination, mimicking the natural route of infection. In this work, we have compared pulmonary and subcutaneous delivery of BCG vaccine in the tuberculosis-susceptible DBA/2 mouse strain, a model in which parenterally administered BCG vaccine does not protect against tuberculosis. Our data show that intranasally but not subcutaneously administered BCG confers robust protection against pulmonary tuberculosis challenge. In addition, our results indicate that pulmonary vaccination triggers a Mycobacterium tuberculosis-specific mucosal immune response orchestrated by interleukin 17A (IL-17A). Thus, IL-17A neutralization in vivo reduces protection and abrogates M. tuberculosis-specific immunoglobulin A (IgA) secretion to respiratory airways and lung expression of polymeric immunoglobulin receptor induced following intranasal vaccination. Together, our results demonstrate that pulmonary delivery of BCG can overcome the lack of protection observed when BCG is given parenterally, suggesting that respiratory tuberculosis vaccines could have an advantage in tuberculosis-endemic countries, where intradermally administered BCG has inefficient effectiveness against pulmonary tuberculosis.

MTBVAC is a live-attenuated Mycobacterium tuberculosis vaccine, currently under clinical development, that contains the major antigens ESAT6 and CFP10. These antigens are absent from the current tuberculosis vaccine, BCG. Here we compare the protection induced by BCG and MTBVAC in several mouse strains that naturally express different MHC haplotypes differentially recognizing ESAT6 and CFP10. MTBVAC induces improved protection in C3H mice, the only of the three tested strains reactive to both ESAT6 and CFP10. Deletion of both antigens in MTBVAC reduces its efficacy to BCG levels, supporting a link between greater efficacy and CFP10- and ESAT6-specific reactogenicity. In addition, MTBVAC (but not BCG) triggers a specific response in human vaccinees against ESAT6 and CFP10. Our results warrant further exploration of this response as potential biomarker of protection in MTBVAC clinical trials.

SummaryApoptosis modulation is a procedure amply utilized by intracellular pathogens to favour the outcome of the infection. Nevertheless, the role of apoptosis during infection with Mycobacterium tuberculosis, the causative agent of human tuberculosis, is subject of an intense debate and still remains unclear. In this work, we describe that apoptosis induction in host cells is clearly restricted to virulent M. tuberculosis strains, and is associated with the capacity of the mycobacteria to secrete the 6 kDa early secreted antigenic target ESAT-6 both under in vitro and in vivo conditions. Remarkably, only apoptosis-inducing strains are able to propagate infection into new cells, suggesting that apoptosis is used by M. tuberculosis as a colonization mechanism. Finally, we demonstrate that in vitro modulation of apoptosis affects mycobacterial cell-to-cell spread capacity, establishing an unambiguous relationship between apoptosis and propagation of M. tuberculosis. Our data further indicate that BCG and MTBVAC vaccines are inefficient in inducing apoptosis and colonizing new cells, correlating with the strong attenuation profile of these strains previously observed in vitro and in vivo.

The granule exocytosis pathway of cytotoxic lymphocytes (Tc and NK cells) is critical for control of tumor development and viral infections. Granule-associated perforin and granzymes are key components in Tc cell-mediated function(s). On the basis of studies that showed granzymes A, B, C, K and M, to induce apoptosis in vitro, all granzymes were thought to also induce cell death in vivo. This review summarizes our present understanding of the biological processes elicited by purified granzyme A and granzyme as well as the processes induced by the more physiologically relevant cytotoxic cells secreting these proteases. The combined evidence supports the concept that the granule secretion pathway is not mono-specific but rather poly-functional including induction of pro-inflammatory cytokines, besides their widely appreciated apoptotic properties.

Most cancer cells use anaerobic-like glycolysis to generate energy instead of oxidative phosphorylation. They also avoid recognition by CTLs, which occurs primarily through decreasing the level of MHC class I (MHC-I) at the cell surface. We find that the two phenomena are linked; culture conditions that force respiration in leukemia cells upregulate MHC-I transcription and protein levels at the cell surface, whereas these decrease in cells forced to perform fermentation as well as in leukemia cells lacking a functional mitochondrial respiratory chain. Forced respiration leads to increased expression of the MAPK ERK5, which activates MHC-I gene promoters, and ERK5 accumulation in mitochondria. Respiration-induced MHC-I upregulation is reversed upon short hairpin RNA-mediated ERK5 downregulation and by inactive mutants of ERK5. Moreover, short hairpin RNA for ERK5 leukemia cells do not tolerate forced respiration. Thus, the expression of ERK5 and MHC-I is linked to cell metabolism and notably diminished by the metabolic adaptations found in tumor cells.

Tumor cells have a tendency to use glucose fermentation to obtain energy instead of mitochondrial oxidative phosphorylation (OXPHOS). We demonstrated that this phenotype correlated with loss of ERK5 expression and with reduced MHC class I expression. Consequently, tumor cells could evade cytotoxic T lymphocyte (CTL)-mediated immune surveillance, but also increase their sensitivity to natural killer (NK) cells. These outcomes were evaluated using two cellular models: leukemic EL4 cells and L929 transformed fibroblasts and their derived ρ° cell lines, which lack mitochondrial DNA. We have also used a L929 cell sub-line that spontaneously lost matrix attachment (L929dt), reminiscent of metastasis generation, that also downregulated MHC-I and ERK5 expression. MHC-I expression is lower in ρ° cells than in the parental cell lines, but they were equally sensitive to CTL. On the contrary, ρ° cells were more sensitive to activated NK cells than parental cells. On the other hand, L929dt cells were resistant to CTL and NK cells, showed reduced viability when forced to perform OXPHOS, and surviving cells increased MHC-I expression and became sensitive to CTL. The present results suggest that when the reduction in MHC-I levels in tumor cells due to glycolytic metabolism is partial, the increase in sensitivity to NK cells seems to predominate. However, when tumor cells completely lose MHC-I expression, the combination of treatments that increase OXPHOS with CTL-mediated immunotherapy could be a promising therapeutic approach.

Protein kinase C-θ (PKCθ) was initially isolated as an important PKC isoform expressed in T cells, although its expression is not restricted to these cells. Despite the central function of PKCθ in several immune responses, its role in the antitumor response against MHC class I (MHC-I)-negative cells has not been investigated. This is an important issue because most tumor cells growing in vivo down-regulate MHC-I expression to escape the CTL-mediated response. In the present work, we show that in vivo development of a MHC-I-deficient tumor (RMA-S) is much favored in PKCθ−/− mice compared with wild-type mice. This is associated with a reduced recruitment of NK cells to the site of tumor development and a reduced activation status of recruited NK cells. This correlates with a reduced ex vivo and in vivo cytotoxic potential of NK cells isolated from PKCθ−/− mice treated with polyinosinic:polycytidylic acid. Consistently, polinosinic:cytidilic acid treatment induces PKCθ expression and activation of its enzymatic activity in NK cells in an indirect manner. These observations underline the relevance of PKCθ as a key molecule in NK cell-mediated antitumor immune surveillance.