Using an effective TB vaccination regimen to identify immune responses associated with protection in the murine model.

A vaccine against tuberculosis (TB), a disease resulting from infection with Mycobacterium tuberculosis (M.tb), is urgently needed to prevent more than a million deaths per year. Bacillus Calmette-Guérin (BCG) is the only available vaccine against TB but its efficacy varies throughout the world. Subunit vaccine candidates, based on recombinant viral vectors expressing mycobacterial antigens, are one of the strategies being developed to boost BCG-primed host immune responses and efficacy. A promising vaccination regimen composed of intradermal (i.d.) BCG prime, followed by intranasally (i.n.) administered chimpanzee adenoviral vector (ChAdOx1) and i.n. or i.d. modified vaccinia Ankara virus (MVA), both expressing Ag85A, has been previously reported to significantly improve BCG efficacy in mice. Effector and memory immune responses induced by BCG-ChAdOx1.85A-MVA85A (B-C-M), were evaluated to identify immune correlates of protection in mice. This protective regime induced strong Ag85A-specific cytokine responses in CD4+ and CD8+ T cells, both in the systemic and pulmonary compartments. Lung parenchymal CXCR3+ KLRG1- Ag85A-specific memory CD4+ T cells were significantly increased in B-C-M compared to BCG immunised mice at 4, 8 and 20 weeks post vaccination, but the number of these cells decreased at the latter time point. This cell population was associated with the protective efficacy of this regime and may have an important protective role against M.tb infection.

Author Correction: The influence of haemoglobin and iron on in vitro mycobacterial growth inhibition assays.

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Regulation of mycobacterial infection by macrophage Gch1 and tetrahydrobiopterin.

Inducible nitric oxide synthase (iNOS) plays a crucial role in controlling growth of Mycobacterium tuberculosis (M.tb), presumably via nitric oxide (NO) mediated killing. Here we show that leukocyte-specific deficiency of NO production, through targeted loss of the iNOS cofactor tetrahydrobiopterin (BH4), results in enhanced control of M.tb infection; by contrast, loss of iNOS renders mice susceptible to M.tb. By comparing two complementary NO-deficient models, Nos2-/- mice and BH4 deficient Gch1fl/flTie2cre mice, we uncover NO-independent mechanisms of anti-mycobacterial immunity. In both murine and human leukocytes, decreased Gch1 expression correlates with enhanced cell-intrinsic control of mycobacterial infection in vitro. Gene expression analysis reveals that Gch1 deficient macrophages have altered inflammatory response, lysosomal function, cell survival and cellular metabolism, thereby enhancing the control of bacterial infection. Our data thus highlight the importance of the NO-independent functions of Nos2 and Gch1 in mycobacterial control.

Identification and Evaluation of Novel Protective Antigens for the Development of a Candidate Tuberculosis Subunit Vaccine.

The development of a vaccine against tuberculosis (TB), a disease caused by Mycobacterium tuberculosis, is urgently needed. The only currently available vaccine, M. bovis BCG, has variable efficacy. One approach in the global vaccine development effort is focused on boosting BCG using subunit vaccines. The identification of novel antigens for inclusion in subunit vaccines is a critical step in the TB vaccine development pathway. We selected four novel mycobacterial antigens recognized during the course of human infection. A replication-deficient chimpanzee adenovirus (ChAdOx1) was constructed to express each antigen individually, and these vectors were evaluated for protective efficacy in murine M. tuberculosis challenge experiments. One antigen, PPE15 (Rv1039c), conferred significant and reproducible protection when administered alone and as a boost to BCG vaccination. We identified immunodominant epitopes to define the protective immune responses using tetramers and intravascular staining. Lung parenchymal CD4+ and CD8+ CXCR3+ KLRG1- T cells, previously associated with protection against M. tuberculosis, were enriched in the vaccinated groups compared to the control groups. Further work to evaluate the protective efficacy of PPE15 in more stringent preclinical animal models, together with the identification of further novel protective antigens using this selection strategy, is now merited.

Hepcidin deficiency and iron deficiency do not alter tuberculosis susceptibility in a murine M.tb infection model.

Tuberculosis (TB), caused by the macrophage-tropic pathogen Mycobacterium tuberculosis (M.tb) is a highly prevalent infectious disease. Since an immune correlate of protection or effective vaccine have yet to be found, continued research into host-pathogen interactions is important. Previous literature reports links between host iron status and disease outcome for many infections, including TB. For some extracellular bacteria, the iron regulatory hormone hepcidin is essential for protection against infection. Here, we investigated hepcidin (encoded by Hamp1) in the context of murine M.tb infection. Female C57BL/6 mice were infected with M.tb Erdman via aerosol. Hepatic expression of iron-responsive genes was measured by qRT-PCR and bacterial burden determined in organ homogenates. We found that hepatic Hamp1 mRNA levels decreased post-infection, and correlated with a marker of BMP/SMAD signalling pathways. Next, we tested the effect of Hamp1 deletion, and low iron diets, on M.tb infection. Hamp1 knockout mice did not have a significantly altered M.tb mycobacterial load in either the lungs or spleen. Up to 10 weeks of dietary iron restriction did not robustly affect disease outcome despite causing iron deficiency anaemia. Taken together, our data indicate that unlike with many other infections, hepcidin is decreased following M.tb infection, and show that hepcidin ablation does not influence M.tb growth in vivo. Furthermore, because even severe iron deficiency did not affect M.tb mycobacterial load, we suggest that the mechanisms M.tb uses to scavenge iron from the host must be extremely efficient, and may therefore represent potential targets for drugs and vaccines.

The influence of haemoglobin and iron on in vitro mycobacterial growth inhibition assays.

The current vaccine against tuberculosis, live attenuated Mycobacterium bovis BCG, has variable efficacy, but development of an effective alternative is severely hampered by the lack of an immune correlate of protection. There has been a recent resurgence of interest in functional in vitro mycobacterial growth inhibition assays (MGIAs), which provide a measure of a range of different immune mechanisms and their interactions. We identified a positive correlation between mean corpuscular haemoglobin and in vitro growth of BCG in whole blood from healthy UK human volunteers. Mycobacterial growth in peripheral blood mononuclear cells (PBMC) from both humans and macaques was increased following the experimental addition of haemoglobin (Hb) or ferric iron, and reduced following addition of the iron chelator deferoxamine (DFO). Expression of Hb genes correlated positively with mycobacterial growth in whole blood from UK/Asian adults and, to a lesser extent, in PBMC from South African infants. Taken together our data indicate an association between Hb/iron levels and BCG growth in vitro, which may in part explain differences in findings between whole blood and PBMC MGIAs and should be considered when using such assays.