The recombinant fusion protein CFP10–ESAT6–dIFN has protective effect against tuberculosis in guinea pigs

Development of effective vaccine candidates against tuberculosis (TB) is currently the most important challenge in the prevention of this disease since the BCG vaccine fails to guarantee a lifelong protection, while any other approved vaccine with better efficiency is still absent. The protective effect of the recombinant fusion protein CFP10–ESAT6–dIFN produced in a prokaryotic expression system (Escherichia coli) has been assessed in a guinea pig model of acute TB. The tested antigen comprises the Mycobacterium tuberculosis (Mtb) proteins ESAT6 and CFP10 as well as modified human γ-interferon (dIFN) for boosting the immune response. Double intradermal immunization of guinea pigs with the tested fusion protein (2 × 0.5 µg) induces a protective effect against subsequent Mtb infection. The immunized guinea pigs do not develop the symptoms of acute TB and their body weight gain was five times more as compared with the non-immunized infected guinea pigs. The animal group immunized with this dose of antigen displays the minimum morphological changes in the internal organs and insignificant inflammatory lesions in the liver tissue, which complies with a decrease in the bacterial load in the spleen and average Mtb counts in macrophages

Differences between Mycobacterium-Host Cell Relationships in Latent Tuberculous Infection of Mice Ex Vivo and Mycobacterial Infection of Mouse Cells In Vitro

The search for factors that account for the reproduction and survival of mycobacteria, including vaccine strains, in host cells is the priority for studies on tuberculosis. A comparison of BCG-mycobacterial loads in granuloma cells obtained from bone marrow and spleens of mice with latent tuberculous infection and cells from mouse bone marrow and peritoneal macrophage cultures infected with the BCG vaccine in vitro has demonstrated that granuloma macrophages each normally contained a single BCG-Mycobacterium, while those acutely infected in vitro had increased mycobacterial loads and death rates. Mouse granuloma cells were observed to produce the IFNγ, IL-1α, GM-CSF, CD1d, CD25, CD31, СD35, and S100 proteins. None of these activation markers were found in mouse cell cultures infected in vitro or in intact macrophages. Lack of colocalization of lipoarabinomannan-labeled BCG-mycobacteria with the lysosomotropic LysoTracker dye in activated granuloma macrophages suggests that these macrophages were unable to destroy BCG-mycobacteria. However, activated mouse granuloma macrophages could control mycobacterial reproduction in cells both in vivo and in ex vivo culture. By contrast, a considerable increase in the number of BCG-mycobacteria was observed in mouse bone marrow and peritoneal macrophages after BCG infection in vitro, when no expression of the activation-related molecules was detected in these cells

Mycobacterium-Host Cell Relationships in Granulomatous Lesions in a Mouse Model of Latent Tuberculous Infection

Tuberculosis (TB) is a dangerous infectious disease characterized by a tight interplay between mycobacteria and host cells in granulomatous lesions (granulomas) during the latent, asymptomatic stage of infection. Mycobacterium-host cell relationships were analyzed in granulomas obtained from various organs of BALB/c mice with chronic TB infection caused by in vivo exposure to the Bacillus Calmette-Guérin (BCG) vaccine. Acid-fast BCG-mycobacteria were found to be morphologically and functionally heterogeneous (in size, shape, and replication rates in colonies) in granuloma macrophages, dendritic cells, and multinucleate Langhans giant cells. Cord formation by BCG-mycobacteria in granuloma cells has been observed. Granuloma macrophages retained their ability to ingest damaged lymphocytes and thrombocytes in the phagosomes; however, their ability to destroy BCG-mycobacteria contained in these cells was compromised. No colocalization of BCG-mycobacteria and the LysoTracker dye was observed in the mouse cells. Various relationships between granuloma cells and BCG-mycobacteria were observed in different mice belonging to the same line. Several mice totally eliminated mycobacterial infection. Granulomas in the other mice had mycobacteria actively replicating in cells of different types and forming cords, which is an indicator of mycobacterial virulence and, probably, a marker of the activation of tuberculous infection in animals

Mycobacterium tuberculosis Load in Host Cells and the Antibacterial Activity of Alveolar Macrophages Are Linked and Differentially Regulated in Various Lung Lesions of Patients with Pulmonary Tuberculosis

Tuberculosis (TB) is a disease caused by Mycobacterium tuberculosis (Mtb) infection with the formation of a broad range of abnormal lung lesions within a single patient. Although host–pathogen interactions determine disease outcome, they are poorly understood within individual lesions at different stages of maturation. We compared Mtb load in a tuberculoma wall and the lung tissue distant from tuberculomas in TB patients. These data were combined with an analysis of activation and bactericidal statuses of alveolar macrophages and other cell subtypes examined both in ex vivo culture and on the histological sections obtained from the same lung lesions. The expression of pattern recognition receptors CD14, CD11b, and TLR-2, transcription factors HIF-1α, HIF-2α, and NF-κB p50 and p65, enzymes iNOS and COX-2, reactive oxygen species (ROS) biosynthesis, and lipid production were detected for various lung lesions, with individual Mtb loads in them. The walls of tuberculomas with insufficient inflammation and excessive fibrosis were identified as being the main niche for Mtb survival (single or as colonies) in non-foamy alveolar macrophages among various lung lesions examined. The identification of factors engaged in the control of Mtb infection and tissue pathology in local lung microenvironments, where host–pathogen relationships take place, is critical for the development of new therapeutic strategies

Alveolar macrophages with <i>Mtb</i> in colonies are detected both in cavity walls and in the distant parts of lung tissue.

<p>(A-C and D-F) Lung tissues and alveolar macrophages obtained from patients 6 and 8, respectively. (A, B, D, E) Representative histological images of the cavity walls (A, D) and the distant parts of lung tissue (B, E) stained by the ZN method demonstrate a massive load of replicating <i>Mtb</i> in the cavities and single <i>Mtb</i> within alveolar macrophages in other lung regions, respectively. (B, E) Enlarged views of the parts of these images with alveolar macrophages containing single <i>Mtb</i> (black arrows) are shown in the upper right corners. (C) Alveolar macrophages obtained from the cavity wall and after <i>ex vivo</i> culture for 16 hours contain colonies of replicating <i>Mtb</i>. (F) Alveolar macrophages obtained from the distant part of lung tissue and after <i>ex vivo</i> culture for 18 hours contain replicating <i>Mtb</i> in colonies with cording morphology. Alveolar macrophages (C, F) were stained by the ZN method. The black arrows point to alveolar macrophages with acid-fast <i>Mtb</i>. The scale bars are 10 μm each.</p

<i>Ex vivo</i> expansion of alveolar macrophages with <i>Mycobacterium tuberculosis</i> from the resected lungs of patients with pulmonary tuberculosis

<div><p>Tuberculosis (TB), with the <i>Mycobacterium tuberculosis</i> (<i>Mtb</i>) as the causative agent, remains to be a serious world health problem. Traditional methods used for the study of <i>Mtb</i> in the lungs of TB patients do not provide information about the number and functional status of <i>Mtb</i>, especially if <i>Mtb</i> are located in alveolar macrophages. We have developed a technique to produce <i>ex vivo</i> cultures of cells from different parts of lung tissues surgically removed from patients with pulmonary TB and compared data on the number of cells with <i>Mtb</i> inferred by the proposed technique to the results of bacteriological and histological analyses used for examination of the resected lungs. The <i>ex vivo</i> cultures of cells obtained from the resected lungs of all patients were largely composed of CD14-positive alveolar macrophages, foamy or not, with or without <i>Mtb</i>. Lymphocytes, fibroblasts, neutrophils, and multinucleate Langhans giant cells were also observed. We found alveolar macrophages with <i>Mtb</i> in the <i>ex vivo</i> cultures of cells from the resected lungs of even those TB patients, whose sputum smears and lung tissues did not contain acid-fast <i>Mtb</i> or reveal growing <i>Mtb</i> colonies on dense medium. The detection of alveolar macrophages with <i>Mtb</i> in <i>ex vivo</i> culture as soon as 16–18 h after isolation of cells from the resected lungs of all TB patients suggests that the technique proposed for assessing the level of infection in alveolar macrophages of TB patients has higher sensitivity than do prolonged bacteriological or pathomorphological methods. The proposed technique allowed us to rapidly (in two days after surgery) determine the level of infection with <i>Mtb</i> in the cells of the resected lungs of TB patients and, by the presence or absence of <i>Mtb</i> colonies, including those with cording morphology, the functional status of the TB agent at the time of surgery.</p></div

Different cell types obtained from the cavity wall in the resected lung of patient 6 and stained by the ZN method after <i>ex vivo</i> culture for 16 hours.

<p>(A, B) A Langhans giant cell and a neutrophil containing acid-fast <i>Mtb</i> are indicated by the red and green arrows, respectively. (C) A lymphocyte interplaying with alveolar macrophages is indicated by the black snowflake. The other cells (A, B, C) are alveolar macrophages. Those with acid-fast <i>Mtb</i> are indicated by the black arrows, others are uninfected. The scale bars are 10 μm each.</p

Histological examination of the resected lung from patient 7 shows the absence of cells with <i>Mtb</i>. <i>Ex vivo</i> analysis of alveolar macrophages disagrees.

<p>(A) A surgically removed part of the lung. (B-F) Multiple TB lesions detected by histological analysis of tissue from the resected lung (A): N, caseous necrosis; M, a ring of macrophages; F, fibrous capsule; L, multinucleate Langhans giant cell. The scale bars are (B, D, E) 20 μm each, (C) 50 μm, and (F) 10 μm. (B, C, and E, F) Representative histological images show enclosed necrotizing granulomas stained by hematoxylin/eosin, a mixture of picric acid and fuchsin acid to detect collagen, and after the ZN method, respectively. (D) Multinucleate Langhans giant cells determined in an early non-necrotizing granuloma by ZN staining. (F) Scanty <i>Mtb</i> revealed in the caseous matter. Enlarged view of the part of this image with <i>Mtb</i> indicated by black arrows is shown in the upper right corner. (G) The tissue specimen of the resected lung (A) was cut into small pieces for producing alveolar macrophages. The diameter of the Petri dish is 10 cm. (H, I) Alveolar macrophages stained by the ZN method after <i>ex vivo</i> culture for 18 hours contain <i>Mtb</i> in isolation and as a colony, respectively. The black arrows point to alveolar macrophages with acid-fast <i>Mtb</i>. The scale bars are 10 μm each.</p

CD14 staining of alveolar macrophages with <i>Mtb</i> performed after <i>ex vivo</i> culture for 18 hours.

<p>(A, B, C) Representative confocal fluorescent 3D images show alveolar macrophages stained by human CD14-specific (green signal) and <i>Mtb</i> Ag38-specific antibodies (red signal). Nuclei are stained by DAPI (blue signal). Alveolar macrophages were obtained from the resected lungs of patients 16 (A, B) and 18 (C). Black or white arrows indicate a single <i>Mtb</i> (A) and mycobacterial colonies (B, C) residing within alveolar macrophages.</p