Mycobacterium bovisBCG is widely used as a vaccine against tuberculosis due toM. tuberculosis(Mtb), which kills millions of people each year. BCG variably protects children, but not adults against tuberculosis. BCG evades phagosome maturation, autophagy, and reduces MHC-II expression of antigen-presenting cells (APCs) affecting T-cell activation. To bypass these defects, an autophagy-inducing, TLR-2 activating C5 peptide from Mtb-derived CFP-10 protein was overexpressed in BCG in combination with Ag85B. Recombinant BCG^85C5induced a robust MHC-II-dependent antigen presentation to CD4 T cells in vitro, and elicited stronger T_H1 cytokines (IL-12, IL-1β, and TNFα) from APCs of C57Bl/6 mice increasing phosphorylation of p38MAPK and ERK. BCG^85C5also enhanced MHC-II surface expression of MΦs by inhibiting MARCH1 ubiquitin ligase that degrades MHC-II. BCG^85C5infected APCs from MyD88 or TLR-2 knockout mice showed decreased antigen presentation. Furthermore, BCG^85C5induced LC3-dependent autophagy in macrophages increasing antigen presentation. Consistent with in vitro effects, BCG^85C5markedly expanded both effector and central memory T cells in C57Bl/6 mice protecting them against both primary aerosol infection with Mtb and reinfection, but was less effective among TLR-2 knockout mice. Thus, BCG^85C5induces stronger and longer lasting immunity, and is better than BCG against tuberculosis of mice.

Human mesenchymal stem cells (MSCs) express scavenger receptors that internalize lipids, including oxidized low-density lipoprotein (oxLDL). We report that MSCs phagocytoseMycobacterium tuberculosis(Mtb) through two types of scavenger receptors (SRs; MARCO and SR-B1), as blockade of the receptors with antibodies or siRNA knockdown decreased the uptake of Mtb. MSCs also expressed mannose receptor (MR) that was found to endocytose rhodamine-labeled mannosylated BSA (rMBSA), though the receptor was not involved in the uptake of Mtb. Dil-oxLDL and rMBSA taken up into MSC endosomes colocalized with Mtb phagosomes, thus suggesting that the latter were fusion competent. Phagocytosed Mtb did not replicate within MSCs, thus suggesting an intrinsic control of bacterial growth. Indeed, MSCs exhibited intrinsic autophagy, which was up-regulated after activation with rapamycin. SiRNA knockdown of autophagy initiator beclin-1 enhanced Mtb survival, whereas rapamycin-induced autophagy increased intracellular killing of Mtb. In addition, MSCs secreted nitric oxide after Mtb infection, and inhibition of NO by N(G)-monomethyl-L-arginine enhanced intracellular survival of Mtb. MSCs can be grown in large numbersin vitro, and autologous MSCs transfused into tuberculosis patients have been found to be safe and improve lung immunity. Thus, MSCs are novel phagocytic cells with a potential for immunotherapy in treating multidrug-resistant tuberculosis.

Mycobacterium tuberculosis H37Rv (Mtb) excludes phagocyte oxidase (phox) and inducible nitric oxide synthase (iNOS) while preventing lysosomal fusion in macrophages (MΦs). The antigen 85A deficient (ΔfbpA) mutant of Mtb was vaccinogenic in mice and the mechanisms of attenuation were compared with MΦs infected with H37Rv and BCG. ΔfbpA contained reduced amounts of trehalose 6, 6, dimycolate and induced minimal levels of SOCS-1 in MΦs. Blockade of oxidants enhanced the growth of ΔfbpA in MΦs that correlated with increased colocalization with phox and iNOS. Green fluorescent protein-expressing strains within MΦs or purified phagosomes were analysed for endosomal traffick with immunofluorescence and Western blot. ΔfbpA phagosomes were enriched for rab5, rab11, LAMP-1 and Hck suggesting enhanced fusion with early, recycling and late endosomes in MΦs compared with BCG or H37Rv. ΔfbpA phagosomes were thus more mature than H37Rv or BCG although, they failed to acquire rab7 and CD63 preventing lysosomal fusion. Finally, ΔfbpA infected MΦs and dendritic cells (DCs) showed an enhanced MHC-II and CD1d expression and primed immune T cells to release more IFN-γ compared with those infected with BCG and H37Rv. ΔfbpA was thus more immunogenic in MΦs and DCs because of an enhanced susceptibility to oxidants and increased maturation.

Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), is the leading cause of death due to bacterial infections in mankind, and BCG, an attenuated strain of Mycobacterium bovis, is an approved vaccine. BCG sequesters in immature phagosomes of antigen presenting cells (APCs), which do not fuse with lysosomes, leading to decreased antigen processing and reduced Th1 responses. However, an Mtb derived ΔfbpA attenuated mutant underwent limited phagosome maturation, enhanced immunogenicity and was as effective as BCG in protecting mice against TB. To facilitate phagosome maturation of ΔfbpA, we disrupted an additional gene sapM, which encodes for an acid phosphatase. Compared to the wild type Mtb, the ΔfbpAΔsapM (double knock out; DKO) strain was attenuated for growth in mouse macrophages and PMA activated human THP1 macrophages. Attenuation correlated with increased oxidants in macrophages in response to DKO infection and enhanced labeling of lysosomal markers (CD63 and rab7) on DKO phagosomes. An in vitro Antigen 85B peptide presentation assay was used to determine antigen presentation to T cells by APCs infected with DKO or other mycobacterial strains. This revealed that DKO infected APCs showed the strongest ability to present Ag85B to T cells (>2500 pgs/mL in 4 hrs) as compared to APCs infected with wild type Mtb or ΔfbpA or ΔsapM strain (<1000 pgs/mL in 4 hrs), indicating that DKO strain has enhanced immunogenicity than other strains. The ability of DKO to undergo lysosomal fusion and vacuolar acidification correlated with antigen presentation since bafilomycin, that inhibits acidification in APCs, reduced antigen presentation. Finally, the DKO vaccine elicited a better Th1 response in mice after subcutaneous vaccination than either ΔfbpA or ΔsapM. Since ΔfbpA has been used in mice as a candidate vaccine and the DKO (ΔfbpAΔsapM) mutant is more immunogenic than ΔfbpA, we propose the DKO is a potential anti-tuberculosis vaccine.