Construction, characterization and preclinical evaluation of MTBVAC, the first live-attenuated M. tuberculosis-based vaccine to enter clinical trials

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 ofthe 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

Macrophage susceptibility to infection by Ghanaian Mycobacterium tuberculosis complex lineages 4 and 5 varies with self-reported ethnicity

BackgroundThe epidemiology of Mycobacterium tuberculosis complex (MTBC) lineage 5 (L5) infections in Ghana revealed a significantly increased prevalence in Ewes compared to other self-reported ethnic groups. In that context, we sought to investigate the early phase of tuberculosis (TB) infection using ex vivo infection of macrophages derived from the blood of Ewe and Akan ethnic group volunteers with MTBC L4 and L5 strains.MethodsThe study participants consisted of 16 controls, among which self-reported Akan and Ewe ethnicity was equally represented, as well as 20 cured TB cases consisting of 11 Akans and 9 Ewes. Peripheral blood mononuclear cells were isolated from both healthy controls and cured TB cases. CD14+ monocytes were isolated and differentiated into monocyte-derived macrophages (MDMs) before infection with L4 or L5 endemic strains. The bacterial load was assessed after 2 hours (uptake) as well as 3 and 7 days post-infection.ResultsWe observed a higher capacity of MDMs from Ewes to phagocytose L4 strains (p < 0.001), translating into a higher bacillary load on day 7 (p < 0.001) compared to L5, despite the higher replication rate of L5 in Ewe MDMs (fold change: 1.4 vs. 1.2, p = 0.03) among the controls. On the contrary, within macrophages from Akans, we observed a significantly higher phagocytic uptake of L5 (p < 0.001) compared to L4, also translating into a higher load on day 7 (p = 0.04). However, the replication rate of L4 in Akan MDMs was higher than that of L5 (fold change: L4 = 1.2, L4 = 1.1, p = 0.04). Although there was no significant difference in the uptake of L4 and L5 among cured TB cases, there was a higher bacterial load of both L4 (p = 0.02) and L5 (p = 0.02) on day 7 in Ewe MDMs.ConclusionOur results suggest that host ethnicity (driven by host genetic diversity), MTBC genetic diversity, and individual TB infection history are all acting together to modulate the outcome of macrophage infections by MTBC

Generating three-dimensional human granulomas in vitro to study Mycobacterium tuberculosis-host interaction

Granulomas are organized multicellular structures that constitute the hallmark of an infection by the human pathogen Mycobacterium tuberculosis (Mtb). A better understanding of the complex host-Mtb interactions within the granuloma’s environment may lead to new therapeutic or preventive tools to improve the control of the tuberculosis pandemic. To date, several in vitro models that are able to mimic human nascent granulomas have been reported. Here we describe a protocol in which Mtb-infected human peripheral blood mononuclear cells (PBMCs) are embedded within a collagen matrix leading to the formation of three-dimensional micro-granulomas. Subsequently, PBMCs and Mtb can be retrieved allowing multiparametric readouts from both the host and the pathogen. In addition to the incorporation of a physiological extracellular matrix, this model has the advantage of recapitulating dormant-like Mtb features, as well as reproducing Mtb resuscitation observed under immunomodulatory treatments

Playing hide-and-seek with host macrophages through the use of mycobacterial cell envelope phthiocerol dimycocerosates and phenolic glycolipids

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<em>Mycobacterium leprae</em> PGL-1 expression by BCG increases CR3 mediated entry into mouse myeloid cells and triggers the Syk/NFAT pathway to induce a specific cytokine program

National audienceIntroduction : Parmi les molécules lipidiques produites dans la paroi cellulaire des mycobactéries, les glycolipides phénoliques (PGL) ne sont produits que par les principales souches pathogènes pour l’Homme : Mycobacterium tuberculosis (Mtb), M. bovis (Mb) M. leprae et M. ulcerans. Les PGL de ces quatre espèces présentent un squelette lipidique et un noyau phénolique similaires et sont différenciées par leur domaine saccharidique. Grâce à la reprogrammation génétique, nous avons produit des souches de BCG synthétisant le PGL-1 de M. lepra), le PGL-bov (naturellement produit par M. bovis) ainsi qu’une souche déficiente en PGL. Nos travaux précédents ont montré, que la souche rBCG::PGL-1 exploitait le récepteur au complément CR3 pour entrer efficacement dans les macrophages humains et diminuer la réponse inflammatoire (Tabouret et al, 2010). Méthode : Nous avons étudié in vitro et in vivo dans trois types de cellules myéloïdes ; les macrophages (MP), les cellules dendritiques (CD) et neutrophiles (PMN) exprimant toutes les trois fortement le CR3 ; l’impact de PGL-1 – dans le contexte de l’enveloppe naturelle du BCG sur l’entrée de la bactérie et sur la sécrétion de cytokines. Grâce à l’utilisation de différents inhibiteurs, nous avons déterminé les voies de signalisation impliquées. Nous avons utilisé la souris de type sauvage et la souris Itgam-/- déficiente pour le CR3. Résultats : Nous avons observé que rBCG::PGL-1 est capable d’entrer plus efficacement que rBCG::PGL-bov ou rBCG::noPGL dans les MP, les CD et les PMN. Les cellules issues de souris Itgam-/- perdent cette capacité. Le CR3 coopère avec Dectin-1 pour cette entrée plus efficace et la voie Syk est requise. Outre une phagocytose plus efficace, rBCG::PGL-1 induit une forte production d’IL-2 par les CD, d’IL-1b par les MP et d’IL-10 par les PMN en comparaison des deux autres souches. A nouveau, la voie Syk est nécessaire. Au contraire, aucune différence entre les souches n’est observée pour la production de cytokines inflammatoires dépendantes de TLR et MyD88 telles que TNF, IL-6 et IL-12p40. En dessous de la voie Syk, nous montrons également que la voie de signalisation NFAT est engagée efficacement par rBCG::PGL-1 via son entrée efficace par le CR3. Conclusion : Ces résultats montrent que l’expression de PGL-1 confère au BCG la capacité d’exploiter le récepteur CR3 pour envahir les cellules myéloïdes murines dans des conditions non opsonisantes. L’engagement du CR3 active une signalisation impliquant la kinase Syk qui aboutit à l’activation du facteur de transcription NFAT pour engager un programme spécifique de sécrétion de cytokines. La molécule PGL-1 dans le contexte de l’enveloppe mycobactérienne exerce ainsi un effet immunomodulateur important qui pourrait polariser la réponse immunitaire dcifiqu

Mycobacterial Phenolic Glycolipids Selectively Disable TRIF-Dependent TLR4 Signaling in Macrophages

International audiencePGL-1 as a model, we found that PGLs dampen the toll-like receptor (TLR)4 signaling pathway, with macrophage exposure to PGLs leading to significant reduction in TIR-domain-containing adapter-inducing interferon-β (TRIF) protein level. PGL-driven decrease in TRIF operated posttranscriptionally and independently of Src-family tyrosine kinases, lysosomal and proteasomal degradation. It resulted in the defective production of TRIF-dependent IFN-β and CXCL10 in TLR4-stimulated macrophages, in addition to iNOS. Our results unravel a mechanism by which PGLs hijack both the bactericidal and inflammatory responses of host macrophages. Moreover, they identify TRIF as a critical node in the crosstalk between CR3 and TLR4

CR3 engaged by PGL-I triggers Syk-calcineurin-NFATc to rewire the innate immune response in Leprosy.

International audienceMycobacterium leprae, the causative agent of leprosy, is unique amongst human pathogens in its capacity to produce the virulence factor phenolic glycolipid (PGL)-I. In addition to mediating bacterial tropism for neurons, PGL-I interacts with Complement Receptor (CR)3 on macrophages (MPs) to promote infection. We demonstrate here that PGL-I binding to CR3 also enhances bacterial invasion of both polymorphonuclear neutrophils (PMNs) and dendritic cells (DCs). Moreover, in all cell types CR3 engagement by PGL-I activates the Syk tyrosine kinase, inducing calcineurin-dependent nuclear translocation of the transcription factor NFATc. This selectively augments the production of IL-2 by DCs, IL-10 by PMNs and IL-1β by MPs. In intranasally-infected mice PGL-I binding to CR3 heightens mycobacterial phagocytosis by lung PMNs and MPs, and stimulates NFATc-controlled production of Syk-dependent cytokines. Our study thus identifies the CR3-Syk-NFATc axis as a novel signaling pathway activated by PGL-I in innate immune cells, rewiring host cytokine responses to M. leprae

Attenuated <em>Mycobacterium tuberculosis</em> SO2 Vaccine Candidate Is Unable to Induce Cell Death

<div><p>It has been proposed that <em>Mycobacterium tuberculosis</em> virulent strains inhibit apoptosis and trigger cell death by necrosis of host macrophages to evade innate immunity, while non-virulent strains induce typical apoptosis activating a protective host response. As part of the characterization of a novel tuberculosis vaccine candidate, the <em>M. tuberculosis phoP</em> mutant SO2, we sought to evaluate its potential to induce host cell death. The parental <em>M. tuberculosis</em> MT103 strain and the current vaccine against tuberculosis Bacillus Calmette-Guérin (BCG) were used as comparators in mouse models <em>in vitro</em> and <em>in vivo</em>. Our data reveal that attenuated SO2 was unable to induce apoptotic events neither in mouse macrophages <em>in vitro</em> nor during lung infection <em>in vivo</em>. In contrast, virulent MT103 triggers typical apoptotic events with phosphatidylserine exposure, caspase-3 activation and nuclear condensation and fragmentation. BCG strain behaved like SO2 and did not induce apoptosis. A clonogenic survival assay confirmed that viability of BCG- or SO2-infected macrophages was unaffected. Our results discard apoptosis as the protective mechanism induced by SO2 vaccine and provide evidence for positive correlation between classical apoptosis induction and virulent strains, suggesting apoptosis as a possible virulence determinant during <em>M. tuberculosis</em> infection.</p> </div

Virulent MT103 strain, but not the attenuated SO2 and BCG strains, replicates in vivo, causes lung pathology and induces apoptosis in mouse lungs.

<p>Groups of five C57BL/6 mice were intratracheally infected with a low dose (100 bacteria/mouse) of MT103, BCG or SO2 strains as described in Materials and Methods. Lungs were harvested and CFU counted at 21 days post-infection (A, left panel). Lung histopathology (hematoxilin/eosin) representative images (10x magnification) of mock-treated (A, right panel, image 1) or MT103-, BCG- or SO2-infected mice (A, right panel, images 2, 3, 4, respectively) at 3 weeks post inoculation. Representative images of active caspase 3 immunohistochemical and Ziehl-Neelsen staining of mock-treated or MT103-, BCG- or SO2-infected lungs at three weeks post inoculation (B): primary antibody control of MT103-infected lung section incubated only with secondary antibody (10x magnification) (image 1); active caspase-3 staining of MT103-infected lung section (10x magnification) (image 2); active caspase-3 staining of MT103-infected lung section (100x magnification) (image 3); active caspase-3 staining of MT103-infected lung section (600x magnification) (image 4); Ziehl-Neelsen staining of MT103-infected lung section (600x magnification) (image 5); active caspase-3 staining of mock-treated lung section (10x, 100x and 600x magnification, images 6, 9 and 12, respectively); active caspase-3 staining of BCG-infected lung section (10x, 100x and 600x magnification, images 7, 10 and 13, respectively); active caspase-3 staining of SO2-infected lung section (10x, 100x and 600x magnification, images 8, 11 and 14, respectively).</p