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BCG turns 100 this year and while it might not be the perfect vaccine, it has certainly contributed significantly towards eradication and prevention of spread of tuberculosis (TB). The search for newer and better vaccines for TB is an ongoing endeavor and latest results from trials of candidate TB vaccines such as M72AS01 look promising. However, recent encouraging data from BCG revaccination trials in adults combined with studies on mucosal and intravenous routes of BCG vaccination in non-human primate models have renewed interest in BCG for TB prevention. In addition, several well-demonstrated non-specific effects of BCG, for example, prevention of viral and respiratory infections, give BCG an added advantage. Also, BCG vaccination is currently being widely tested in human clinical trials to determine whether it protects against SARS-CoV-2 infection and/or death with detailed analyses and outcomes from several ongoing trials across the world awaited. Through this review, we attempt to bring together information on various aspects of the BCG-induced immune response, its efficacy in TB control, comparison with other candidate TB vaccines and strategies to improve its efficiency including revaccination and alternate routes of administration. Finally, we discuss the future relevance of BCG use especially in light of its several heterologous benefits.

BACKGROUND: Bacille Calmette-Guérin (BCG) vaccine is protective against Tuberculosis (TB) in children, but its efficacy wanes with age. Consequently, determining if BCG revaccination augments anti-TB immunity in young adults in TB endemic regions is vital. METHODS: Two hundred healthy adults, BCG vaccinated at birth, were tested for their IFN-γ release assay (IGRA) status. Of these, 28 IGRA(+) and 30 IGRA(–) were BCG revaccinated, and 24 IGRA(+) and 23 IGRA(–) subjects served as unvaccinated controls. T and innate cell responses to mycobacterial antigens were analyzed by 14-color flow cytometry over 34 weeks. RESULTS: IFN-γ and/or IL-2 Ag85A- and BCG-specific CD4(+) and CD8(+) T cell responses were boosted by revacciantion at 4 and 34 weeks, respectively, and were > 2-fold higher in IGRA(+) compared with IGRA(–) vaccinees. Polyfunctional Ag85A, BCG, and mycobacterium tuberculosis (Mtb) latency Ag–specific (LTAg-specific) CD4(+) T cells expressing up to 8 cytokines were also significantly enhanced in both IGRA(+) and IGRA(–) vaccinees relative to unvaccinated controls, most markedly in IGRA(+) vaccinees. A focused analysis of Th17 responses revealed expansion of Ag85A-, BCG-, and LTAg-specific total IL-17A(+),IL-17F(+),IL-22(+), and IL-10(+) CD4(+) T cell effectors in both IGRA(+) and IGRA(–) subjects. Also, innate IFN-γ(+) NK/γδ/NKT cell responses were higher in both IGRA(+) and IGRA(–) vaccinees compared with controls. This is the first evidence to our knowledge that BCG revaccination significantly boosts antimycobacterial Th1/Th17 responses in IGRA(+) and IGRA(–) subjects. CONCLUSION: These data show that BCG revaccination is immunogenic in IGRA(–) and IGRA(+) subjects, implying that Mtb preinfection in IGRA(+) subjects does not impact immunogenicity. This has implications for public health and vaccine development strategies. FUNDING: This work was funded principally by DBT-NIH (BT/MB/Indo-US/HIPC/2013).

The capacity of Mycobacterium tuberculosis (Mtb) to tolerate multiple antibiotics represents a major problem in tuberculosis (TB) management. Heterogeneity in Mtb populations is one of the factors that drives antibiotic tolerance during infection. However, the mechanisms underpinning this variation in bacterial population remain poorly understood. Here, we show that phagosomal acidification alters the redox physiology of Mtb to generate a population of replicating bacteria that display drug tolerance during infection. RNA sequencing of this redox-altered population revealed the involvement of iron-sulfur (Fe-S) cluster biogenesis, hydrogen sulfide (H(2)S) gas, and drug efflux pumps in antibiotic tolerance. The fraction of the pH- and redox-dependent tolerant population increased when Mtb infected macrophages with actively replicating HIV-1, suggesting that redox heterogeneity could contribute to high rates of TB therapy failure during HIV-TB coinfection. Pharmacological inhibition of phagosomal acidification by the antimalarial drug chloroquine (CQ) eradicated drug-tolerant Mtb, ameliorated lung pathology, and reduced postchemotherapeutic relapse in in vivo models. The pharmacological profile of CQ (C(max) and AUC(last)) exhibited no major drug-drug interaction when coadministered with first line anti-TB drugs in mice. Our data establish a link between phagosomal pH, redox metabolism, and drug tolerance in replicating Mtb and suggest repositioning of CQ to shorten TB therapy and achieve a relapse-free cure.

The development of immunocompetent T cells entails a complex pathway of differentiation in the thymus. Thymic atrophy occurs with ageing and during conditions such as malnutrition, infections and cancer chemotherapy. The comparative changes in thymic subsets under different modes of thymic atrophy and the mechanisms involved are not well characterized. These aspects were investigated, using mice infected with Salmonella Typhimurium, injection with lipopolysaccharide (LPS), an inflammatory but non‐infectious stimulus, etoposide (Eto), a drug used to treat some cancers, and dexamethasone (Dex), a steroid used in some inflammatory diseases. The effects on the major subpopulations of thymocytes based on multicolour flow cytometry studies were, first, the CD4(−) CD8(−) double‐negative (DN) cells, mainly DN2–4, were reduced with infection, LPS and Eto treatment, but not with Dex. Second, the CD8(+) CD3(lo) immature single‐positive cells (ISPs) were highly sensitive to infection, LPS and Eto, but not Dex. Third, treatment with LPS, Eto and Dex reduced all three subpopulations of CD4(+) CD8(+) double‐positive (DP) thymocytes, i.e. DP1, DP2 and DP3, but the DP3 subset was relatively more resistant during infection. Fourth, both CD4(+) and CD8(+) single‐positive (SP) thymocytes were lowered by Eto and Dex, but not during infection. Notably, LPS lowered CD4(+) SP subsets, whereas the CD8(+) SP subsets were relatively more resistant. Interestingly, the reactive oxygen species quencher, N‐acetyl cysteine, greatly improved the survival of thymocytes, especially DNs, ISPs and DPs, during infection and LPS treatment. The implications of these observations for the development of potential thymopoietic drugs are discussed.

Chronic T cell activation is a hallmark of pulmonary tuberculosis (PTB). The mechanisms underpinning this important phenomenon are however, poorly elucidated, though known to rely on control of T effector cells (Teff) by regulatory T cells (Treg). Our studies show that circulating natural Treg cells in adults with PTB preserve their suppressive potential but Teff cells from such subjects are resistant to Treg-mediated suppression. We found this to be due to expansion of an activated Teff subset identified by Human Leukocyte Antigen (HLA)-DR expression. Sensitivity to suppression was restored to control levels by depletion of this subset. Comparative transcriptome analysis of Teff cells that contain HLA-DR+ cells versus the fraction depleted of this population identified putative resistance mechanisms linked to IFNG, IL17A, IL22, PD-L1 and β-chemokines CCL3L3, CCL4 expression. Antibody blocking experiments confirmed HLA-DR+ Teff cells, but not the fraction depleted of HLA-DR+ effectors, to be resistant to Treg suppression mediated via CCR5 and PD-L1 associated pathways. In the presence of HLA-DR+ Teff cells, activation of NFκB downstream of CCR5 and PD-L1 was perturbed. In addition, HLA-DR+ Teff cells expressed significantly higher levels of Th1/Th17 cytokines that may regulate Treg function through a reciprocal counter-balancing relationship. Taken together, our study provides novel insight on how activated HLA-DR+CD4+ T cells may contribute to disease associated inflammation by compromising Treg-mediated suppression in PTB., Author summary An important marker of progression to PTB following Mycobacterium tuberculosis (Mtb) infection in humans is elevated frequencies of HLA-DR+CD4+ T cells, reflecting chronic T cell activation. However, the mechanisms by which activated HLA-DR+CD4+ T cells contribute to disease process is not known. We show that CD25- HLA-DR+CD4+ memory Teff from PTB patients are resistant to suppression mediated by Treg cells. An unbiased transcriptome analysis identified several key pathways that contribute to this resistance. Specifically, presence of HLA-DR+CD4+ T cells renders the effector population resistant to CCR5 and PD-L1 mediated suppression by Treg cells. In addition, the HLA-DR+CD4+ memory Teff cells express elevated levels of Th1/Th17 cytokines known to counter-regulate and dampen Treg suppression. These findings provide fresh insight to disease process in TB and identify HLA-DR+ Teff resistant to Treg suppression as a potential functional marker of disease.

Sepsis, a leading cause of death in intensive care units, is primarily caused due to an exaggerated immune response. The hyperactive inflammatory response mediated by immune cells against infectious organisms and their toxins results in host cell death and tissue damage, the hallmarks of septic shock. Therefore, molecules that modulate inflammatory responses are attractive therapeutic targets for sepsis. Nitric oxide (NO) is a signaling molecule, which is implicated in regulating diverse immune functions. Although, the protective roles of NO in infectious diseases are well documented, its importance in sepsis is controversial. In the present study, the effects of intra-peritoneal injection of mice with Salmonella Typhimurium, a Gram-negative intracellular pathogen, were studied which leads to a rapid upregulation of serum cytokines and infiltration of neutrophils to the peritoneal cavity. Surprisingly, the induction of inflammatory cytokines and chemokines, e.g. IL6 and CCL2, and the infiltration of neutrophils into the peritoneal cavity are mitigated in mice lacking Nitric oxide synthase 2 (NOS2). The reduced inflammatory response in Nos2(-/-) mice is accompanied by greater bacterial burden in the peritoneal cavity, lower thymic atrophy, higher liver damage and cardiovascular dysfunction followed by decreased survival. However, no significant differences are observed in other responses between C57BL/6 wild type (WT) and Nos2(-/-) mice: induction of glucocorticoids, phagocytic ability and apoptosis of peritoneal cells. This study clearly highlights the NOS2-dependent and - independent responses in this mouse model of peritonitis induced sepsis. Importantly, pre-treatment of Nos2(-/-) mice with DETA-NO, a NO donor, upon infection, restores neutrophil recruitment, reduces bacterial numbers in the peritoneal cavity, improves liver and cardio-vascular function and enhances survival. Interestingly, DETA-NO treatment does not significantly increase the survival of infected WT mice. The implications of these results and the complex roles of NO as a target molecule during sepsis are discussed.

The functional heterogeneity of T cell responses to diverse antigens expressed at different stages of Mycobacterium tuberculosis (Mtb) infection, in particular early secreted versus dormancy related latency antigens expressed later, that distinguish subjects with latent (LTBI), pulmonary (PTB) or extrapulmonary (EPTB) tuberculosis remains unclear. Here we show blood central memory CD4 T-cell responses specific to Mtb dormancy related (DosR) latency, but not classical immunodominant secretory antigens, to clearly differentiate LTBI from EPTB and PTB. The polyfunctionality score integrating up to 31 DosR-specific CD4 T-cell functional profiles was significantly higher in LTBI than EPTB or PTB subjects. Further analysis of 256 DosR-specific T-cell functional profiles identified regulatory IL10 + Th17 cells (IL10+IL17A+IL17F+IL22+) to be significantly enriched in LTBI; in contrast to pro-inflammatory Th17 cells (IFNγ+IL17A+/IL10−) in the blood and lung of EPTB and PTB subjects respectively. A blood polyfunctional, Mtb DosR latency antigen specific, regulatory, central memory response is therefore a novel functional component of T-cell immunity in latent TB and potential correlate of protection.

The thymus is known to atrophy during infections; however, a systematic study of changes in thymocyte subpopulations has not been performed. This aspect was investigated, using multi-color flow cytometry, during oral infection of mice with Salmonella Typhimurium (S. Typhimurium). The major highlights are: First, a block in the developmental pathway of CD4−CD8− double negative (DN) thymocytes is observed. Second, CD4+CD8+ double positive (DP) thymocytes, mainly in the DP1 (CD5loCD3lo) and DP2 (CD5hiCD3int), but not DP3 (CD5intCD3hi), subsets are reduced. Third, single positive (SP) thymocytes are more resistant to depletion but their maturation is delayed, leading to accumulation of CD24hiCD3hi SP. Kinetic studies during infection demonstrated differences in sensitivity of thymic subpopulations: Immature single positive (ISP) > DP1, DP2 > DN3, DN4 > DN2 > CD4+ > CD8+. Upon infection, glucocorticoids (GC), inflammatory cytokines, e.g. Ifnγ, etc are induced, which enhance thymocyte death. Treatment with RU486, the GC receptor antagonist, increases the survival of most thymic subsets during infection. Studies with Ifnγ−/− mice demonstrated that endogenous Ifnγ produced during infection enhances the depletion of DN2-DN4 subsets, promotes the accumulation of DP3 and delays the maturation of SP thymocytes. The implications of these observations on host cellular responses during infections are discussed.