Construction and Characterization of the Mycobacterium tuberculosis sigE fadD26 Unmarked Double Mutant as a Vaccine Candidate.

Despite the great increase in the understanding of the biology and pathogenesis of Mycobacterium tuberculosis achieved by the scientific community in recent decades, tuberculosis (TB) still represents one of the major threats to global human health. The only available vaccine (Mycobacterium bovis BCG) protects children from disseminated forms of TB but does not effectively protect adults from the respiratory form of the disease, making the development of new and more-efficacious vaccines against the pulmonary forms of TB a major goal for the improvement of global health. Among the different strategies being developed to reach this goal is the construction of attenuated strains more efficacious and safer than BCG. We recently showed that a sigE mutant of M. tuberculosis was more attenuated and more efficacious than BCG in a mouse model of infection. In this paper, we describe the construction and characterization of an M. tuberculosissigE fadD26 unmarked double mutant fulfilling the criteria of the Geneva Consensus for entering human clinical trials. The data presented suggest that this mutant is even more attenuated and slightly more efficacious than the previous sigE mutant in different mouse models of infection and is equivalent to BCG in a guinea pig model of infection.

Diverging biological roles among human monocyte subsets in the context of tuberculosis infection.

Circulating monocytes (Mo) play an essential role in the host immune response to chronic infections. We previously demonstrated that CD16(pos) Mo were expanded in TB (tuberculosis) patients, correlated with disease severity and were refractory to dendritic cell differentiation. In the present study, we investigated whether human Mo subsets (CD16(neg) and CD16(pos)) differed in their ability to influence the early inflammatory response against Mycobacterium tuberculosis. We first evaluated the capacity of the Mo subsets to migrate and engage a microbicidal response in vitro. Accordingly, CD16(neg) Mo were more prone to migrate in response to different mycobacteria-derived gradients, were more resistant to M. tuberculosis intracellular growth and produced higher reactive oxygen species than their CD16(pos) counterpart. To assess further the functional dichotomy among the human Mo subsets, we carried out an in vivo analysis by adapting a hybrid mouse model (SCID/Beige, where SCID is severe combined immunodeficient) to transfer each Mo subset, track their migratory fate during M. tuberculosis infection, and determine their impact on the host immune response. In M. tuberculosis-infected mice, the adoptively transferred CD16(neg) Mo displayed a higher lung migration index, induced a stronger pulmonary infiltration of murine leucocytes expressing pro- and anti-inflammatory cytokines, and significantly decreased the bacterial burden, in comparison with CD16(pos) Mo. Collectively, our results indicate that human Mo subsets display divergent biological roles in the context of M. tuberculosis infection, a scenario in which CD16(neg) Mo may contribute to the anti-mycobacterial immune response, whereas CD16(pos) Mo might promote microbial resilience, shedding light on a key aspect of the physiopathology of TB disease.

Expression kinetics of metalloproteinases and their tissue inhibitors in experimental murine pulmonary tuberculosis.

AIM: Explore the temporal expression of metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) during experimental tuberculosis induced by virulent Mycobacterium tuberculosis strain H37Rv. METHODS: BALB/c mice were infected via endotracheal instillation with H37Rv. Groups of mice were euthanized at different time points during infection. RNA was isolated from the lungs, and the expression of MMP-3, 8, 9, 10, 12, 13 and TIMP-1-4 was determined by quantitative PCR. Immunohistochemical detection of MMP-3, MMP-9, and MMP-10 was done to determine the cell source. RESULTS: The infection with H37Rv-induced inflammation resulted in maximal up-regulation of MMP-3, 8, 9, 10, 12 and 13 at day 21 postinfection. Additionally, MMP-13 showed another expression peak during late disease at day 60. Airway epithelium and macrophages were the most common MMP-3 and MMP-9 immunopositive cells, while for MMP-10, macrophages and endothelial cells were the most common, particularly at days 14 and 21 in well-formed granulomas. During late disease, vacuolated macrophages in pneumonic areas and bronchial epithelium showed mild MMP immunostaining. CONCLUSIONS: MMP-3, 8, 9, 10, 12, and 13 are maximally expressed at the peak of granuloma formation in the mouse tuberculosis model, with no compensation in levels or timing of TIMP expression. This data opens the possibility of participation of these molecules in the granuloma process.

Virulence and immune response induced by Mycobacterium avium complex strains in a model of progressive pulmonary tuberculosis and subcutaneous infection in BALB/c mice.

The genus Mycobacterium comprises more than 150 species, including important pathogens for humans which cause major public health problems. The vast majority of efforts to understand the genus have been addressed in studies with Mycobacterium tuberculosis. The biological differentiation between M. tuberculosis and nontuberculous mycobacteria (NTM) is important because there are distinctions in the sources of infection, treatments, and the course of disease. Likewise, the importance of studying NTM is not only due to its clinical significance but also due to the mechanisms by which some species are pathogenic while others are not. Mycobacterium avium complex (MAC) is the most important group of NTM opportunistic pathogens, since it is the second largest medical complex in the genus after the M. tuberculosis complex. Here, we evaluated the virulence and immune response of M. avium subsp. avium and Mycobacterium colombiense, using experimental models of progressive pulmonary tuberculosis and subcutaneous infection in BALB/c mice. Mice infected intratracheally with a high dose of MAC strains showed high expression of tumor necrosis factor alpha (TNF-α) and inducible nitric oxide synthase with rapid bacillus elimination and numerous granulomas, but without lung consolidation during late infection in coexistence with high expression of anti-inflammatory cytokines. In contrast, subcutaneous infection showed high production of the proinflammatory cytokines TNF-α and gamma interferon with relatively low production of anti-inflammatory cytokines such as interleukin-10 (IL-10) or IL-4, which efficiently eliminate the bacilli but maintain extensive inflammation and fibrosis. Thus, MAC infection evokes different immune and inflammatory responses depending on the MAC species and affected tissue.

Immune response elicited by two rBCG strains devoid of genes involved in c-di-GMP metabolism affect protection versus challenge with M. tuberculosis strains of different virulence.

Pellicles, a type of biofilm, have gathered a renewed interest in the field of tuberculosis as a structure that mimics some characteristics occurring during M. tuberculosis infection, such as antibiotic recalcitrance and chronicity of infection, and as a source of antigens for humoral response in infected guinea pigs. In other bacteria, it has been well documented that the second messenger c-di-GMP modulates the transition from planktonic cells to biofilm formation. In this work, we used the live vaccine Mycobacterium bovis BCG to determine whether deletion of genes involved in c-di-GMP metabolism would affect interaction with macrophages, capacity to induce immune response in a murine cell line and mice, and how the protein profile was modified when grown as surface pellicles. We found that deletion of the BCG1419c (Delta c-di-GMP phosphodiesterase, ΔPDE) gene, or deletion of the BCG1416c (Delta c-di-GMP diguanylate cyclase, ΔDGC) gene, altered production of TNF-α, IL-6, and IL-1ß, in murine macrophages, and resulted in attenuation in intra-macrophage replication. Moreover, in addition to the improved immunogenicity of the BCGΔBCG1419c mutant already reported, deletion of the BCG1416c gene leads to increased T CD4+ and T CD8+ activation. This correlated with protection versus lethality in mice infected with the highly virulent M. tuberculosis 5186 afforded by vaccination with all the tested BCG strains, and controlled the growth of the mildly virulent M. tuberculosis H37Rv in lungs by vaccination with BCGΔBCG1419c during chronic late infection from 4 to 6 months after challenge. Furthermore, when grown as surface pellicles, a condition used to manufacture BCG vaccine, in comparison to BCG wild type, both rBCGs changed expression of antigenic proteins such as DnaK, HbhA, PstS2, 35KDa antigen, GroEL2, as well as AcpM, a protein involved in synthesis of mycolic acids, molecules relevant to modulate inflammatory responses.

Mycobacterium smegmatis proteoliposome induce protection in a murine progressive pulmonary tuberculosis model.

Tuberculosis (TB) remains an important cause of mortality and morbidity. The TB vaccine, BCG, is not fully protective against the adult form of the disease and is unable to prevent its transmission although it is still useful against severe childhood TB. Hence, the search for new vaccines is of great interest. In a previous study, we have shown that proteoliposomes obtained from Mycobacterium smegmatis (PLMs) induced cross reactive humoral and cellular response against Mycobacterium tuberculosis (Mtb) antigens. With the objective to evaluate the protective capability of PLMs, a murine model of progressive pulmonary TB was used. Animals immunized with PLMs with and without alum (PLMs/PLMsAL respectively) showed protection compared to non-immunized animals. Mice immunized with PLMsAL induced similar protection as that of BCG. Animals immunized with BCG, PLMs and PLMsAL showed a significant decrease in tissue damage (percentage of pneumonic area/lung) compared to non-immunized animals, with a more prominent effect in BCG vaccinated mice. The protective effect of the administration of PLMs in mice supports its future evaluation as experimental vaccine candidate against Mtb.

Immunogenicity and protection conferred by Mycobacterium habana in a murine model of pulmonary tuberculosis.

Mycobacterium habana was isolated in Cuba in 1971. Later, was demonstrated its protection capacity in mycobacterial infection. Here we determined the level of virulence, immunogenicity and the efficacy of three different M. habana strains as attenuated live vaccines. Intratracheal infection of BALB/c mice with high dose M. habana TMC 5135 or IPK-337 strains permitted 100% survival and limited tissue damage. Mice infected with M. habana IPK-220 showed lower attenuation, so it was discarded for the vaccination experiments. Strains IPK-337 and TMC 5135 were used as subcutaneous vaccine and compared with BCG. Nude mice vaccinated with strain 5135 showed longer but non-significant survival than BCG vaccinated animals. Cell suspensions from M. habana vaccinated mice produced higher IFNγ after stimulation with mycobacterial antigens than BCG recipients. After four months of challenge with Mycobacterium tuberculosis strain H37Rv, mice vaccinated with BCG substrain Phipps or strain TMC 5135 showed total survival, while 60% survival was exhibited by animals vaccinated with M. habana IPK-337. Both M. habana strains do not prevent the infection with M. tuberculosis but avoided the progression of the experimental disease; strain TMC 5135 showed similar level of protection than BCG.

Protective effect of a lipid-based preparation from Mycobacterium smegmatis in a murine model of progressive pulmonary tuberculosis.

A more effective vaccine against tuberculosis (TB) is urgently needed. Based on its high genetic homology with Mycobacterium tuberculosis (Mtb), the nonpathogenic mycobacteria, Mycobacterium smegmatis (Ms), could be an attractive source of potential antigens to be included in such a vaccine. We evaluated the capability of lipid-based preparations obtained from Ms to provide a protective response in Balb/c mice after challenge with Mtb H37Rv strain. The intratracheal model of progressive pulmonary TB was used to assess the level of protection in terms of bacterial load as well as the pathological changes in the lungs of immunized Balb/c mice following challenge with Mtb. Mice immunized with the lipid-based preparation from Ms either adjuvanted with Alum (LMs-AL) or nonadjuvanted (LMs) showed significant reductions in bacterial load (P < 0.01) compared to the negative control group (animals immunized with phosphate buffered saline (PBS)). Both lipid formulations showed the same level of protection as Bacille Calmette and Guerin (BCG). Regarding the pathologic changes in the lungs, mice immunized with both lipid formulations showed less pneumonic area when compared with the PBS group (P < 0.01) and showed similar results compared with the BCG group. These findings suggest the potential of LMs as a promising vaccine candidate against TB.

The protective effect of immunoglobulin in murine tuberculosis is dependent on IgG glycosylation.

Antibodies have demonstrated having a protective effect in animal models of tuberculosis (TB). These experiments have considered the specificity of antigen recognition and the different isotypes and subclasses as significant contributors of this effect. However, the carbohydrate chain heterogeneity on the Fc region of IgG (Fc-IgG) can play an important role in modulating the immune response. Patients with TB usually have high titers of specific IgG; however, the carbohydrate associated with Fc-IgG usually lacks galactose. To assess the effect of this abnormal IgG in murine pulmonary TB, we evaluated the specificity of recognition to Mycobacterium tuberculosis antigens in vitro and protective effects in vivo comparing human intravenous immunoglobulin (IVIg) and IVIg treated with an endoglycosidase to remove the glycan residues (EndoS-treated IVIg). Our results showed similar antigen recognition. The study of distribution and kinetics of IVIg in serum and bronchial lavage after intraperitoneal (i.p.) administration in mice showed that IVIg circulates for 21 days. Finally, the protective effect of intact and EndoS-treated IVIg administered by i.p was studied in a murine model of progressive TB. IVIg treatment caused reduction in pulmonary bacilli loads, larger granulomas, and less pneumonia, while animals treated with EndoS-treated IVIg were not protected compared with control animals. Thus, IVIg has a protective activity in experimental pulmonary TB, and this effect requires intact Fc oligosaccharides.