Live attenuated Mycobacterium bovis strains combined with the encapsulated H65 antigen as a vaccine strategy against bovine tuberculosis in a mouse model.

Mycobacterium bovis is an etiological agent of bovine tuberculosis (bTB) that also infects other mammals, including humans. The lack of an effective vaccine for the control of bTB highlights the need for developing new vaccines. In this study, we developed and evaluated an M. bovis strain deleted in the virulence genes phoP, esxA and esxB as a vaccine candidate against bTB in BALBc mice. The evaluated strains were the new live vaccine and BCG, alone or in combination with ncH65vD. The immunogen ncH65vD is a fusion protein H65, encapsulated together with vitamin D3, within the oily body of a nanocapsule composed of an antigen-loading polymeric shell. All vaccines conferred protection against the M. bovis challenge. However, no significant differences were detected among the vaccinated groups regarding bacterial loads in lungs and spleen. Mice vaccinated with the mutant strain plus ncH65vD showed negative Ziehl Neelsen staining of mycobacteria in their lungs, which suggests better control of bacteria replication according to this protection parameter. Consistently, this vaccination scheme showed the highest proportion of CD4 + T cells expressing the protection markers PD-1 and CXCR3 among the vaccinated groups. Correlation studies showed that PD-1 and CXCR3 expression levels in lung-resident CD4 T cells negatively correlated with the number of colony forming units of M. bovis in the lungs of mice. Therefore, the results suggest a link between the presence of PD-1 + and CXCR3 + cells at the site of the immune response against mycobacteria and the level of mycobacterial loads.

An update on Mycobacterium tuberculosis lipoproteins.

Almost 3% of the proteins of Mycobacterium tuberculosis (M. tuberculosis), the main causative agent of human tuberculosis, are lipoproteins. These lipoproteins are characteristic of the mycobacterial cell envelope and participate in many mechanisms involved in the pathogenesis of M. tuberculosis. In this review, the authors provide an updated analysis of M. tuberculosis lipoproteins and categorize them according to their demonstrated or predicted functions, including transport of compounds to and from the cytoplasm, biosynthesis of the mycobacterial cell envelope, defense and resistance mechanisms, enzymatic activities and signaling pathways. In addition, this updated analysis revealed that at least 40% of M. tuberculosis lipoproteins are glycosylated.

A Transcriptional Analysis of Cattle Immune Cells Reveals a Central Role of Type 1 Interferon in the In Vitro Innate Immune Response against Mycobacterium bovis.

Bovine tuberculosis is a chronic infectious disease primarily caused by Mycobacterium bovis, a bacterium that affects cattle and other mammals, including humans. Despite the availability of vast research about the immune response mechanisms of human tuberculosis caused by Mycobacterium tuberculosis, the knowledge of bovine tuberculosis's immunology, particularly regarding the innate immune response, still remains scarce. In this study, we compared the transcriptome of cell cultures containing lymphocytes and M. bovis infected-macrophages with two strains of variable virulence, the virulent Mb04-303 strain and the attenuated Mb534. To that end, we infected bovine macrophages at a multiplicity of infection of one, and co-cultured the infections with autologous lymphocytes. RNA obtained from the co-cultures was sequenced to identify differentially expressed gene pathways by using the database Reactome. The RNA-seq analysis showed that the Mb04-303 infection upregulated the type 1 interferon signalling pathway, while it downregulated the KEAP1-NFE2L2 pathway. According to the literature, this last pathway is involved in the activation of antioxidant genes and inflammasome. In addition, the macrophages infected with Mb04-303 recruited more Galectin 8 than those infected with Mb534. This result indicates that Mb04-303 induced higher phagosome membrane damage, with the possible concomitant release of bacterial compounds into the cytoplasm that activates the type I signalling pathway. Altogether, Mb04-303 repressed the antioxidant and anti-inflammatory responses, likely impairing interleukin-1ß activation, and trigged the canonical type 1 interferon signalling. Although these responses led to the control of bacterial replication during early infection, the virulent strain eventually managed to establish a successful infection.

Assessment of Tuberculosis Biomarkers in Paratuberculosis-infected Cattle.

Introduction: Mycobacterium bovis and Mycobacterium avium subsp. paratuberculosis, respectively the causative agents of bovine tuberculosis (bTB) and bovine paratuberculosis (PTB), share a high number of antigenic proteins. This characteristics makes the differential diagnosis of the diseases difficult. The interferon gamma (IFN-γ), C-X-C motif chemokine ligand 10 (CXCL10), matrix metallopeptidase 9 (MMP9), interleukin 22 (IL-22) and thrombospondin 1 (THBS1) bovine genes have already been shown to be accurate transcriptional biomarkers of bTB. In order to improve the diagnosis of bTB and PTB, in the present study we evaluated the risk of false positivity of these bTB biomarkers in cattle with PTB. Material and Methods: The transcription of these genes was studied in 13 PTB-infected cattle, using Mycobacterium avium subsp. paratuberculosis (MAP)-stimulated peripheral blood mononuclear cells (PBMC). Results: Overall, the levels of IFN-γ, CXCL10, MMP9 and IL-22 transcripts in MAP-stimulated PBMC failed to differentiate animals with PTB from healthy animals. However, as bTB-afflicted cattle do, the MAP-infected group also displayed a lower level of THBS1 transcription than the non-infected animals. Conclusion: The results of this study add new specificity attributes to the levels of transcription of IFN-γ, CXCL10, MMP9 and IL-22 as biomarkers for bTB.

Replication and transmission features of two experimental vaccine candidates against bovine tuberculosis subcutaneously administrated in a murine model.

Cattle vaccination is an attractive approach in compliance with control and eradication programs against Bovine Tuberculosis (bTB). Today, there is no anti bTB vaccine licensed. Two vaccine candidates, MbΔmce2 and MbΔmce2-phoP previously designed were evaluated in BALB/c mice, including the parental M. bovis NCTC10772 and a M. bovis hypervirulent Mb04-303 strains as controls. Sentinel mice (non-inoculated) cohoused with subcutaneous inoculated mice. Persistence, visible tuberculosis lesions (VTL) in lungs and spleens and bacillary load were investigated subcutaneously delivered at 60 and 90 days after inoculation (dpi) as well as their potential transmission to naïve mice. While a 100% survival was observed at 90 dpi without VTL in all groups, transmission was not evidenced in the sentinels mice. Vaccine candidates and control strains were isolated from the spleen of all inoculated mice, while Mb04-303 was isolated from the lungs of one inoculated mouse. Vaccine candidate's attenuation considering survival, lung bacillary load and VTL was confirmed, administrated by the subcutaneous route. Future experiments are necessary to demonstrate whether the persistence of both mutants in the spleen, with low CFU, remains over time to increase the potential increasing risk of dissemination to organs and subsequent transmission to other animals by airborne or other routes.

Semi-stable Production of Bovine IL-4 and GM-CSF in The Mammalian Episomal Expression System.

INTRODUCTION: Granulocyte-macrophage colony stimulating factor (GM-CSF) and interleukin-4 (IL-4) are cytokines widely used in ex vivo monocyte differentiation experiments, vaccine formulations and disease treatment. The aim of this study was to produce recombinant bovine GM-CSF and IL-4 in an episomal expression system that conserves the postransductional modification of the native proteins and to use the products to differentiate bovine monocytes into dendritic cells. MATERIAL AND METHODS: The recombinant proteins rGM-CSF and rIL-4 were expressed in PEAKrapid CRL-2828 human kidney cells, ATCC CRL-2828. The functional activity of the recombinant cytokines was monitored by registering morphological changes in bovine monocytes and assessing the expression of CD14 upon incubation with them. RESULTS: Both recombinant proteins were detected in the cell culture supernatant of transfected cells. Culture supernatants of transfected cells induced in bovine monocytes morphological changes that resemble macrophages or dendritic cells. In addition, bovine cells treated with rGM-CSF and rIL-4 showed reduced expression of the macrophage surface marker CD14 compared with untreated cells. This effect indicates the expected differentiation. The expression of the cytokines was stable after many successive cell passages and a freeze/thaw cycle. CONCLUSIONS: The semi-stable mammalian episomal expression system used in this study allowed us to easily produce functional bovine rGM-CSF and rIL-4 without the need for protein purification steps.

H65 fusion protein fails to improve the protection of a rationally attenuated live vaccine candidate against bovine tuberculosis in a mouse model of tuberculosis.

Background: The fusion protein H65, composed of Mycobacterium tuberculosis (TB) ESX-secreted antigens, has improved the bacillus Calmette-Guerin-induced immune protection in a mouse model of bovine TB when formulated in the liposomal adjuvant CAF01. In this study, we aimed to evaluate the protective efficacy of an attenuated Mycobacterium bovis strain - a mutant in mce2 and phoP genes - combined with H65+CAF01 immunization. We evaluated the protection of MbΔmce2-phoP alone or combined with H65+CAF01 against M. bovis challenge in mice. Methods: Groups of BALBc mice were inoculated with the vaccine candidates or phosphate buffered saline (PBS), and 6 weeks after the last immunization, the animals were aerogenically challenged with virulent M. bovis. Bacterial load in organs was counted after 45 days of the challenge. One-way analysis of variance and Bonferroni's posttest were used for statistical analysis. Results: All vaccinated mice showed reduced bacterial loads in lungs compared to unvaccinated animals. However, the protection level was similar between vaccinated groups. Conclusions: The MbΔmce2-phoP strain combined with three doses of H65+CAF01 induced equivalent protection than the MbΔmce2-phoP strain alone. Thus, the use of combined vaccination strategies requires a careful analysis of the potential interactions of each of their components with the host's immune system.

Identifying Bacterial and Host Factors Involved in the Interaction of Mycobacterium bovis with the Bovine Innate Immune Cells.

Bovine tuberculosis is an important animal and zoonotic disease caused by Mycobacterium bovis. The innate immune response is the first line of defense against pathogens and is also crucial for the development of an efficient adaptive immune response. In this study we used an in vitro co-culture model of antigen presenting cells (APC) and autologous lymphocytes derived from peripheral blood mononuclear cells to identify the cell populations and immune mediators that participate in the development of an efficient innate response capable of controlling the intracellular replication of M. bovis. After M. bovis infection, bovine immune cell cultures displayed upregulated levels of iNOS, IL-22 and IFN-γ and the induction of the innate immune response was dependent on the presence of differentiated APC. Among the analyzed M. bovis isolates, only a live virulent M. bovis isolate induced an efficient innate immune response, which was increased upon stimulation of cell co-cultures with the M. bovis culture supernatant. Moreover, we demonstrated that an allelic variation of the early secreted protein ESAT-6 (ESAT6 T63A) expressed in the virulent strain is involved in this increased innate immune response. These results highlight the relevance of the compounds secreted by live M. bovis as well as the variability among the assessed M. bovis strains to induce an efficient innate immune response.

The subunit vaccine H65 + CAF01 increased the BCG- protection against Mycobacterium bovis infection in a mouse model of bovine tuberculosis.

H65, a fusion protein of three pairs of ESX-secreted antigens of Mycobacterium tuberculosis and Mycobacterium bovis, formulated with the liposomal adjuvant CAF01 has been shown to confer protection against M. tuberculosis infection in mice. In this study, we evaluated the impact of combining BCG with H65 + CAF01 immunization in a M. bovis mouse model of infection. We found that a BCG-H65 + CAF01/ H65 + CAF01 prime-boost scheme induced higher protection than BCG and H65 + CAF01 alone. Altogether, H65 antigen formulated in liposomal adjuvant improved the BCG-induced immune protection, thus making this vaccine strategy a promising tool to control bovine tuberculosis.

Elimination of ESAT-6 and CFP-10 from a candidate vaccine against bovine tuberculosis impaired its protection efficacy in the BALBc mouse model.

Background: Bovine tuberculosis (bTB) is a zoonotic disease caused by Mycobacterium bovis that mainly affects cattle. Although vaccination is the most effective strategy to control bTB, it may interfere with the diagnosis of the infection. Therefore, ancillary tests to differentiate vaccinated from infected animals (DIVA) are essential in a cattle vaccination scenario. ESAT-6 and CFP-10 are the most promissory DIVA antigens. Method: In this study, we deleted esat6 and cfp10 genes from the M. bovis Δ mce2 live-attenuated vaccine candidate and evaluated its protection level against bTB in BALBc mice. Results: We found that the M. bovis strain mutant in mce2, esat-6 and cfp-10 failed to confer protection against virulent M. bovis challenge in a mouse model of tuberculosis. Conclusions: This result highlights the relevant role of ESAT-6 and CFP-10 in the induction of protective immune response against M. bovis infection and reveals the need of evaluating different strategies to compensate for the lack of these DIVA antigens in new vaccine formulations.

Rv2577 of Mycobacterium tuberculosis Is a Virulence Factor With Dual Phosphatase and Phosphodiesterase Functions.

Tuberculosis, a lung disease caused by Mycobacterium tuberculosis (Mtb), is one of the ten leading causes of death worldwide affecting mainly developing countries. Mtb can persist and survive inside infected cells through modulation of host antibacterial attack, i.e., by avoiding the maturation of phagosome containing mycobacteria to more acidic endosomal compartment. In addition, bacterial phosphatases play a central role in the interplay between host cells and Mtb. In this study, we characterized the Rv2577 of Mtb as a potential alkaline phosphatase/phosphodiesterase enzyme. By an in vitro kinetic assay, we demonstrated that purified Rv2577 expressed in Mycobacterium smegmatis displays both enzyme activities, as evidenced by using the artificial substrates p-NPP and bis-(p-NPP). In addition, a three-dimensional model of Rv2577 allowed us to define the catalytic amino acid residues of the active site, which were confirmed by site-directed mutagenesis and enzyme activity analysis, being characteristic of a member of the metallophosphatase superfamily. Finally, a mutation introduced in Rv2577 reduced the replication of Mtb in mouse organs and impaired the arrest of phagosomes containing mycobacteria in early endosomes; which indicates Rv2577 plays a role in Mtb virulence.

FasR Regulates Fatty Acid Biosynthesis and Is Essential for Virulence of Mycobacterium tuberculosis.

Mycobacterium tuberculosis, the etiologic agent of human tuberculosis, is the world's leading cause of death from an infectious disease. One of the main features of this pathogen is the complex and dynamic lipid composition of the cell envelope, which adapts to the variable host environment and defines the fate of infection by actively interacting with and modulating immune responses. However, while much has been learned about the enzymes of the numerous lipid pathways, little knowledge is available regarding the proteins and metabolic signals regulating lipid metabolism during M. tuberculosis infection. In this work, we constructed and characterized a FasR-deficient mutant in M. tuberculosis and demonstrated that FasR positively regulates fas and acpS expression. Lipidomic analysis of the wild type and mutant strains revealed complete rearrangement of most lipid components of the cell envelope, with phospholipids, mycolic acids, sulfolipids, and phthiocerol dimycocerosates relative abundance severely altered. As a consequence, replication of the mutant strain was impaired in macrophages leading to reduced virulence in a mouse model of infection. Moreover, we show that the fasR mutant resides in acidified cellular compartments, suggesting that the lipid perturbation caused by the mutation prevented M. tuberculosis inhibition of phagolysosome maturation. This study identified FasR as a novel factor involved in regulation of mycobacterial virulence and provides evidence for the essential role that modulation of lipid homeostasis plays in the outcome of M. tuberculosis infection.

A Phenotypic Characterization of Two Isolates of a Multidrug-Resistant Outbreak Strain of Mycobacterium tuberculosis with Opposite Epidemiological Fitness.

Tuberculosis (TB) is an infectious disease, caused by Mycobacterium tuberculosis, primarily affecting the lungs. The M. tuberculosis strain of the Haarlem family named M was responsible for a large multidrug-resistant TB (MDR-TB) outbreak in Buenos Aires. This outbreak started in the early 1990s and in the mid 2000s still accounted for 29% of all MDR-TB cases in Argentina. By contrast, a clonal variant of strain M, named 410, has caused a single tuberculosis case since the onset of the outbreak. The molecular bases of the high epidemiological fitness of the M strain remain unclear. To assess its unique molecular properties, herein, we performed a comparative protein and lipid analysis of a representative clone of the M strain (Mp) and the nonprosperous M variant 410. We also evaluated their growth in low pH. The variant 410 had higher levels of latency proteins under standard conditions and delayed growth at low pH, suggesting that it is more sensitive to stress stimuli than Mp. Moreover, Mp showed higher levels of mycolic acids covalently attached to the cell wall and lower accumulation of free mycolic acids in the outer layer than the 410 strain. The low expression of latency proteins together with the reduced content of surface mycolic acids may facilitate Mp to evade the host immune responses.

Klebsiella pneumoniae ST258 Negatively Regulates the Oxidative Burst in Human Neutrophils.

The epidemic clone of Klebsiella pneumoniae (Kpn), sequence type 258 (ST258), carbapenamase producer (KPC), commonly infects hospitalized patients that are left with scarce therapeutic option since carbapenems are last resort antibiotics for life-threatening bacterial infections. To improve prevention and treatment, we should better understand the biology of Kpn KPC ST258 infections. Our hypothesis was that Kpn KPC ST258 evade the first line of defense of innate immunity, the polymorphonuclear neutrophil (PMN), by decreasing its functional response. Therefore, our aim was to evaluate how the ST258 Kpn clone affects PMN responses, focusing on the respiratory burst, compared to another opportunistic pathogen, Escherichia coli (Eco). We found that Kpn KPC ST258 was unable to trigger bactericidal responses as reactive oxygen species (ROS) generation and NETosis, compared to the high induction observed with Eco, but both bacterial strains were similarly phagocytized and cause increases in cell size and CD11b expression. The absence of ROS induction was also observed with other Kpn ST258 strains negative for KPC. These results reflect certain selectivity in terms of the functions that are triggered in PMN by Kpn, which seems to evade specifically those responses critical for bacterial survival. In this sense, bactericidal mechanisms evasion was associated with a higher survival of Kpn KPC ST258 compared to Eco. To investigate the mechanisms and molecules involved in ROS inhibition, we used bacterial extracts (BE) and found that BE were able to inhibit ROS generation triggered by the well-known ROS inducer, fMLP. A sequence of experiments led us to elucidate that the polysaccharide part of LPS was responsible for this inhibition, whereas lipid A mediated the other responses that were not affected by bacteria, such as cell size increase and CD11b up-regulation. In conclusion, we unraveled a mechanism of immune evasion of Kpn KPC ST258, which may contribute to design more effective strategies for the treatment of these multi-resistant bacterial infections.

ERAP1 and PDE8A Are Downregulated in Cattle Protected against Bovine Tuberculosis.

Bovine tuberculosis (bTB) is a zoonotic disease caused by Mycobacterium bovis that is responsible for significant economic losses worldwide. In spite of its relevance, the limited knowledge about the host immune responses that provide effective protection against the disease has long hampered the development of an effective vaccine. The identification of host proteins with an expression that correlates with protection against bTB would contribute to the understanding of the cattle defence mechanisms against M. bovis infection. In this study, we found that ERAP1 and PDE8A were downregulated in vaccinated cattle that were protected from experimental M. bovis challenge. Remarkably, both genes encode proteins that have been negatively associated with immune protection against bTB.

Mycobacterium bovis Requires P27 (LprG) To Arrest Phagosome Maturation and Replicate within Bovine Macrophages.

Mycobacterium bovis causes tuberculosis in a wide variety of mammals, with strong tropism for cattle and eventually humans. P27, also called LprG, is among the proteins involved in the mechanisms of the virulence and persistence of M. bovis and Mycobacterium tuberculosis Here, we describe a novel function of P27 in the interaction of M. bovis with its natural host cell, the bovine macrophage. We found that a deletion in the p27-p55 operon impairs the replication of M. bovis in bovine macrophages. Importantly, we show for the first time that M. bovis arrests phagosome maturation in a process that depends on P27. This effect is P27 specific since complementation with wild-type p27 but not p55 fully restored the wild-type phenotype of the mutant strain; this indicates that P55 plays no important role during the early events of M. bovis infection. In addition, we also showed that the presence of P27 from M. smegmatis decreases the association of LAMP-3 with bead phagosomes, indicating that P27 itself blocks phagosome-lysosome fusion by modulating the traffic machinery in the cell host.

Polymorphisms of 20 regulatory proteins between Mycobacterium tuberculosis and Mycobacterium bovis.

Mycobacterium tuberculosis and Mycobacterium bovis are responsible for tuberculosis in humans and animals, respectively. Both species are closely related and belong to the Mycobacterium tuberculosis complex (MTC). M. tuberculosis is the most ancient species from which M. bovis and other members of the MTC evolved. The genome of M. bovis is over >99.95% identical to that of M. tuberculosis but with seven deletions ranging in size from 1 to 12.7 kb. In addition, 1200 single nucleotide mutations in coding regions distinguish M. bovis from M. tuberculosis. In the present study, we assessed 75 M. tuberculosis genomes and 23 M. bovis genomes to identify non-synonymous mutations in 202 coding sequences of regulatory genes between both species. We identified species-specific variants in 20 regulatory proteins and confirmed differential expression of hypoxia-related genes between M. bovis and M. tuberculosis.

Evaluation of Mycobacterium bovis double knockout mce2-phoP as candidate vaccine against bovine tuberculosis.

In this study, a Mycobacterium bovis knockout strain in phoP-phoR and mce2 operons was tested as an antituberculosis experimental vaccine in animal models. The double mutant strain was significantly more attenuated than the wild type strain in inmunocompetent and inmunodeficient mice. Vaccination with the double mutant protected mice against challenge with a virulent M. bovis strain.