Rare Variable M. tuberculosis Antigens induce predominant Th17 responses in human infection.

CD4 T cells are essential for immunity to M. tuberculosis (Mtb), and emerging evidence indicates that IL-17-producing Th17 cells contribute to immunity to Mtb. While identifying protective T cell effector functions is important for TB vaccine design, T cell antigen specificity is also likely to be important. To identify antigens that induce protective immunity, we reasoned that as in other pathogens, effective immune recognition drives sequence diversity in individual Mtb antigens. We previously identified Mtb genes under evolutionary diversifying selection pressure whose products we term Rare Variable Mtb Antigens (RVMA). Here, in two distinct human cohorts with recent exposure to TB, we found that RVMA preferentially induce CD4 T cells that express RoRγt and produce IL-17, in contrast to 'classical' Mtb antigens that induce T cells that produce IFNγ. Our results suggest that RVMA can be valuable antigens in vaccines for those already infected with Mtb to amplify existing antigen-specific Th17 responses to prevent TB disease.

Developing tuberculosis vaccines for people with HIV: consensus statements from an international expert panel.

New tuberculosis vaccine candidates that are in the development pipeline need to be studied in people with HIV, who are at high risk of acquiring Mycobacterium tuberculosis infection and tuberculosis disease and tend to develop less robust vaccine-induced immune responses. To address the gaps in developing tuberculosis vaccines for people with HIV, a series of symposia was held that posed six framing questions to a panel of international experts: What is the use case or rationale for developing tuberculosis vaccines? What is the landscape of tuberculosis vaccines? Which vaccine candidates should be prioritised? What are the tuberculosis vaccine trial design considerations? What is the role of immunological correlates of protection? What are the gaps in preclinical models for studying tuberculosis vaccines? The international expert panel formulated consensus statements to each of the framing questions, with the intention of informing tuberculosis vaccine development and the prioritisation of clinical trials for inclusion of people with HIV.

Persistent expression of activation markers on Mycobacterium tuberculosis-specific CD4 T cells in smear negative TB patients.

BACKGROUND: T cell activation (HLA-DR, CD-38), proliferation (KI-67), and functional (IFN-γ, TNF-α) markers have recently been shown to be useful in predicting and monitoring anti-TB responses in smear positive TB, but previous research did not characterize the activation and proliferation profiles after therapy of smear negative TB. METHODOLOGY: In this study, we used polychromatic flow cytometry to assess selected PPD-specific T cell markers using fresh PBMC of smear negative and positive pulmonary tuberculosis (PTB) patients, recruited from health facilities in Addis Ababa. RESULT: Levels of activation (HLA-DR, CD38) and proliferation (Ki-67) among total unstimulated CD4 T cells decreased significantly after therapy, particularly at month 6. Similarly, levels of PPD-specific T cell activation markers (HLA-DR, CD-38) were significantly lower in smear positive PTB patients following treatment, whereas a consistent decline in these markers was less apparent among smear negative PTB patients at the sixth month. CONCLUSION: After six months of standard anti-TB therapy, persistent levels of activation of HLA-DR and CD-38 from PPD specific CD4+T cells in this study could indicate that those markers have little value in monitoring and predicting anti-TB treatment response in smear negative pulmonary TB patients in Ethiopian context.

Isoniazid and rifapentine treatment effectively reduces persistent M. tuberculosis infection in macaque lungs.

A once-weekly oral dose of isoniazid and rifapentine for 3 months (3HP) is recommended by the CDC for treatment of latent tuberculosis infection (LTBI). The aim of this study is to assess 3HP-mediated clearance of M. tuberculosis bacteria in macaques with asymptomatic LTBI. Twelve Indian-origin rhesus macaques were infected with a low dose (~10 CFU) of M. tuberculosis CDC1551 via aerosol. Six animals were treated with 3HP and 6 were left untreated. The animals were imaged via PET/CT at frequent intervals. Upon treatment completion, all animals except 1 were coinfected with SIV to assess reactivation of LTBI to active tuberculosis (ATB). Four of 6 treated macaques showed no evidence of persistent bacilli or extrapulmonary spread until the study end point. PET/CT demonstrated the presence of significantly more granulomas in untreated animals relative to the treated group. The untreated animals harbored persistent bacilli and demonstrated tuberculosis (TB) reactivation following SIV coinfection, while none of the treated animals reactivated to ATB. 3HP treatment effectively reduced persistent infection with M. tuberculosis and prevented reactivation of TB in latently infected macaques.

Mycobacterium tuberculosis antigen-specific T-cell responses in smear-negative pulmonary tuberculosis patients.

Despite recent improvements in microbial detection, smear-negative TB remains a diagnostic challenge. In this study, we investigated the potential discriminatory role of polychromatic flow cytometry of M. tuberculosis antigen-specific T cells to discriminate smear-negative TB from health controls with or without latent TB infection, and non-TB respiratory illnesses in an endemic setting. A cross-sectional study was conducted on HIV negative, newly diagnosed smear-positive PTB (n = 34), smear-negative/GeneXpert negative PTB (n = 29) patients, non-TB patients with respiratory illness (n = 33) and apparently healthy latent TB infected (n = 30) or non-infected (n = 23) individuals. The expression of activation (HLA-DR, CD-38), proliferation (Ki-67), and functional (IFN-γ, TNF-α) T-cell markers using polychromatic flow cytometry was defined after stimulation with PPD antigens. Sputum samples were collected and processed from all patients for Mtb detection using a concentrated microscopy, LJ/MGIT culture, and RD9 typing by PCR. Our study showed CD4 T cells specific for PPD co-expressed activation/proliferation markers together with induced cytokines IFN-γ or TNF-α were present at substantially higher levels among patients with smear-positive and smear-negative pulmonary TB than among healthy controls and to a lesser extent among patients with non-TB illness. Our study conclude that smear-negative TB can be distinguished from non-TB respiratory illness and healthy controls with a flow cytometric assay for PPD-specific T cells co-expressing activation/proliferation markers and cytokines.

Mycobacterium tuberculosis impedes CD40-dependent notch signaling to restrict Th17 polarization during infection.

Early Th17 responses are necessary to provide protection against Mycobacterium tuberculosis (Mtb). Mtb impedes Th17 polarization by restricting CD40 co-stimulatory pathway on dendritic cells (DCs). We previously demonstrated that engaging CD40 on DCs increased Th17 responses. However, the molecular mechanisms that contributed to Th17 polarization were unknown. Here, we identify the Notch ligand DLL4 as necessary for Th17 polarization and demonstrate that Mtb limits DLL4 on DCs to prevent optimal Th17 responses. Although Mtb infection induced only low levels of DLL4, engaging CD40 on DCs increased DLL4 expression. Antibody blockade of DLL4 on DCs reduced Th17 polarization in vitro and in vivo. In addition, we show that the Mtb Hip1 protease attenuates DLL4 expression on lung DCs by impeding CD40 signaling. Overall, our results demonstrate that Mtb impedes CD40-dependent DLL4 expression to restrict Th17 responses and identify the CD40-DLL4 pathways as targets for developing new Th17-inducing vaccines and adjuvants for tuberculosis.

Antiretroviral therapy timing impacts latent tuberculosis infection reactivation in a Mycobacterium tuberculosis/SIV coinfection model.

Studies using the nonhuman primate model of Mycobacterium tuberculosis/simian immunodeficiency virus coinfection have revealed protective CD4+ T cell-independent immune responses that suppress latent tuberculosis infection (LTBI) reactivation. In particular, chronic immune activation rather than the mere depletion of CD4+ T cells correlates with reactivation due to SIV coinfection. Here, we administered combinatorial antiretroviral therapy (cART) 2 weeks after SIV coinfection to study whether restoration of CD4+ T cell immunity occurred more broadly, and whether this prevented reactivation of LTBI compared to cART initiated 4 weeks after SIV. Earlier initiation of cART enhanced survival, led to better control of viral replication, and reduced immune activation in the periphery and lung vasculature, thereby reducing the rate of SIV-induced reactivation. We observed robust CD8+ T effector memory responses and significantly reduced macrophage turnover in the lung tissue. However, skewed CD4+ T effector memory responses persisted and new TB lesions formed after SIV coinfection. Thus, reactivation of LTBI is governed by very early events of SIV infection. Timing of cART is critical in mitigating chronic immune activation. The potential novelty of these findings mainly relates to the development of a robust animal model of human M. tuberculosis/HIV coinfection that allows the testing of underlying mechanisms.

Advancing Adjuvants for Mycobacterium tuberculosis Therapeutics.

Tuberculosis (TB) remains one of the leading causes of death worldwide due to a single infectious disease agent. BCG, the only licensed vaccine against TB, offers limited protection against pulmonary disease in children and adults. TB vaccine research has recently been reinvigorated by new data suggesting alternative administration of BCG induces protection and a subunit/adjuvant vaccine that provides close to 50% protection. These results demonstrate the need for generating adjuvants in order to develop the next generation of TB vaccines. However, development of TB-targeted adjuvants is lacking. To help meet this need, NIAID convened a workshop in 2020 titled "Advancing Vaccine Adjuvants for Mycobacterium tuberculosis Therapeutics". In this review, we present the four areas identified in the workshop as necessary for advancing TB adjuvants: 1) correlates of protective immunity, 2) targeting specific immune cells, 3) immune evasion mechanisms, and 4) animal models. We will discuss each of these four areas in detail and summarize what is known and what we can advance on in order to help develop more efficacious TB vaccines.

Characterizing Early T Cell Responses in Nonhuman Primate Model of Tuberculosis.

Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), remains a leading infectious disease killer worldwide with 1.4 million TB deaths in 2019. While the majority of infected population maintain an active control of the bacteria, a subset develops active disease leading to mortality. Effective T cell responses are critical to TB immunity with CD4+ and CD8+ T cells being key players of defense. These early cellular responses to TB infection have not yet been studied in-depth in either humans or preclinical animal models. Characterizing early T cell responses in a physiologically relevant preclinical model can provide valuable understanding of the factors that control disease development. We studied Mtb-specific T cell responses in the lung compartment of rhesus macaques infected with either a low- or a high-dose of Mtb CDC1551 via aerosol. Relative to baseline, significantly higher Mtb-specific CD4+IFN-γ+ and TNF-α+ T cell responses were observed in the BAL of low dose infected macaques as early as week 1 post TB infection. The IFN-γ and TNF-a response was delayed to week 3 post infection in Mtb-specific CD4+ and CD8+T cells in the high dose group. The manifestation of earlier T cell responses in the group exposed to the lower Mtb dose suggested a critical role of these cytokines in the antimycobacterial immune cascade, and specifically in the granuloma formation to contain the bacteria. However, a similar increase was not reflected in the CD4+ and CD8+IL-17+ T cells at week 1 post infection in the low dose group. This could be attributed to either a suppression of the IL-17 response or a lack of induction at this early stage of infection. On the contrary, there was a significantly higher IL-17+ response in Mtb-specific CD4+ and CD8+T cells at week 3 in the high dose group. The results clearly demonstrate an early differentiation in the immunity following low dose and high dose infection, largely represented by differences in the IFN-γ and TNF-α response by Mtb-specific T cells in the BAL. This early response to antigen expression by the bacteria could be critical for both bacterial growth control and bacterial containment.

Schistosoma mansoni Infection Is Associated With a Higher Probability of Tuberculosis Disease in HIV-Infected Adults in Kenya.

BACKGROUND: Helminth infections can modulate immunity to Mycobacterium tuberculosis (Mtb). However, the effect of helminths, including Schistosoma mansoni (SM), on Mtb infection outcomes is less clear. Furthermore, HIV is a known risk factor for tuberculosis (TB) disease and has been implicated in SM pathogenesis. Therefore, it is important to evaluate whether HIV modifies the association between SM and Mtb infection. SETTING: HIV-infected and HIV-uninfected adults were enrolled in Kisumu County, Kenya, between 2014 and 2017 and categorized into 3 groups based on Mtb infection status: Mtb-uninfected healthy controls, latent TB infection (LTBI), and active TB disease. Participants were subsequently evaluated for infection with SM. METHODS: We used targeted minimum loss estimation and super learning to estimate a covariate-adjusted association between SM and Mtb infection outcomes, defined as the probability of being Mtb-uninfected healthy controls, LTBI, or TB. HIV status was evaluated as an effect modifier of this association. RESULTS: SM was not associated with differences in baseline demographic or clinical features of participants in this study, nor with additional parasitic infections. Covariate-adjusted analyses indicated that infection with SM was associated with a 4% higher estimated proportion of active TB cases in HIV-uninfected individuals and a 14% higher estimated proportion of active TB cases in HIV-infected individuals. There were no differences in estimated proportions of LTBI cases. CONCLUSIONS: We provide evidence that SM infection is associated with a higher probability of active TB disease, particularly in HIV-infected individuals.

Pulmonary Mycobacterium tuberculosis control associates with CXCR3- and CCR6-expressing antigen-specific Th1 and Th17 cell recruitment.

Mycobacterium tuberculosis-specific (M. tuberculosis-specific) T cell responses associated with immune control during asymptomatic latent tuberculosis infection (LTBI) remain poorly understood. Using a nonhuman primate aerosol model, we studied the kinetics, phenotypes, and functions of M. tuberculosis antigen-specific T cells in peripheral and lung compartments of M. tuberculosis-infected asymptomatic rhesus macaques by longitudinally sampling blood and bronchoalveolar lavage, for up to 24 weeks postinfection. We found substantially higher frequencies of M. tuberculosis-specific effector and memory CD4+ and CD8+ T cells producing IFN-γ in the airways compared with peripheral blood, and these frequencies were maintained throughout the study period. Moreover, M. tuberculosis-specific IL-17+ and IL-17+IFN-γ+ double-positive T cells were present in the airways but were largely absent in the periphery, suggesting that balanced mucosal Th1/Th17 responses are associated with LTBI. The majority of M. tuberculosis-specific CD4+ T cells that homed to the airways expressed the chemokine receptor CXCR3 and coexpressed CCR6. Notably, CXCR3+CD4+ cells were found in granulomatous and nongranulomatous regions of the lung and inversely correlated with M. tuberculosis burden. Our findings provide insights into antigen-specific T cell responses associated with asymptomatic M. tuberculosis infection that are relevant for developing better strategies to control TB.

Antiretroviral therapy does not reduce tuberculosis reactivation in a tuberculosis-HIV coinfection model.

While the advent of combination antiretroviral therapy (ART) has significantly improved survival, tuberculosis (TB) remains the leading cause of death in the HIV-infected population. We used Mycobacterium tuberculosis/simian immunodeficiency virus-coinfected (M. tuberculosis/SIV-coinfected) macaques to model M. tuberculosis/HIV coinfection and study the impact of ART on TB reactivation due to HIV infection. Although ART significantly reduced viral loads and increased CD4+ T cell counts in blood and bronchoalveolar lavage (BAL) samples, it did not reduce the relative risk of SIV-induced TB reactivation in ART-treated macaques in the early phase of treatment. CD4+ T cells were poorly restored specifically in the lung interstitium, despite their significant restoration in the alveolar compartment of the lung as well as in the periphery. IDO1 induction in myeloid cells in the inducible bronchus-associated lymphoid tissue (iBALT) likely contributed to dysregulated T cell homing and impaired lung immunity. Thus, although ART was indispensable for controlling viral replication, restoring CD4+ T cells, and preventing opportunistic infection, it appeared inadequate in reversing the clinical signs of TB reactivation during the relatively short duration of ART administered in this study. This finding warrants the modeling of concurrent treatment of TB and HIV to potentially reduce the risk of reactivation of TB due to HIV to inform treatment strategies in patients with M. tuberculosis/HIV coinfection.

Tryptophan catabolism reflects disease activity in human tuberculosis.

There is limited understanding of the role of host metabolism in the pathophysiology of human tuberculosis (TB). Using high-resolution metabolomics with an unbiased approach to metabolic pathway analysis, we discovered that the tryptophan pathway is highly regulated throughout the spectrum of TB infection and disease. This regulation is characterized by increased catabolism of tryptophan to kynurenine, which was evident not only in active TB disease but also in latent TB infection (LTBI). Further, we found that tryptophan catabolism is reversed with effective treatment of both active TB disease and LTBI in a manner commensurate with bacterial clearance. Persons with active TB and LTBI also exhibited increased expression of indoleamine 2,3-dioxygenase-1 (IDO-1), suggesting IDO-1 mediates observed increases in tryptophan catabolism. Together, these data indicate IDO-1-mediated tryptophan catabolism is highly preserved in the human response to Mycobacterium tuberculosis and could be a target for biomarker development as well as host-directed therapies.

Differential Expression of Activation Markers by Mycobacterium tuberculosis-specific CD4+ T Cell Distinguishes Extrapulmonary From Pulmonary Tuberculosis and Latent Infection.

BACKGROUND: Diagnosis of active tuberculosis (ATB) currently relies on detection of Mycobacterium tuberculosis (Mtb). Identifying patients with extrapulmonary TB (EPTB) remains challenging because microbiological confirmation is often not possible. Highly accurate blood-based tests could improve diagnosis of both EPTB and pulmonary TB (PTB) and timely initiation of anti-TB therapy. METHODS: A case-control study was performed using discriminant analyses to validate an approach using Mtb-specific CD4+T-cell activation markers in blood to discriminate PTB and EPTB from latent TB infection (LTBI) as well as EPTB from PTB in 270 Brazilian individuals. We further tested the effect of human immunodeficiency virus (HIV) coinfection on diagnostic performance. Frequencies of interferon-γ +CD4+T cells expressing CD38, HLADR, and/or Ki67 were assessed by flow cytometry. RESULTS: EPTB and PTB were associated with higher frequencies of CD4+T cells expressing CD38, HLADR, or Ki67 compared with LTBI (all P values < .001). Moreover, frequencies of HLADR+ (P = .03) or Ki67+ (P < .001) cells accurately distinguished EPTB from PTB. HIV infection did not affect the capacity of these markers to distinguish ATB from LTBI or EPTB from PTB. CONCLUSIONS: Cell activation markers in Mtb-specific CD4+T cells distinguished ATB from LTBI and EPTB from PTB, regardless of HIV infection status. These parameters provide an attractive approach for developing blood-based diagnostic tests for both active and latent TB.

Isoniazid and Rifapentine Treatment Eradicates Persistent Mycobacterium tuberculosis in Macaques.

Rationale: Direct evidence for persistence of Mycobacterium tuberculosis (Mtb) during asymptomatic latent tuberculosis infection (LTBI) in humans is currently lacking. Moreover, although a 12-week regimen of once-weekly isoniazid and rifapentine (3HP) is currently recommended by the CDC as treatment for LTBI, experimental evidence for 3HP-mediated clearance of persistent Mtb infection in human lungs has not been established.Objectives: Using a nonhuman primate (NHP) model of TB, we sought to assess 3HP treatment-mediated clearance of Mtb infection in latently infected macaques.Methods: Sixteen NHPs were infected via inhalation with ∼10 cfu of Mtb CDC1551, after which asymptomatic animals were either treated with 3HP or left untreated. Pharmacokinetics of the 3HP regimen were measured. Following treatment, animals were coinfected with simian immunodeficiency virus to assess reactivation of LTBI and development of active TB disease.Measurements and Main Results: Fourteen NHPs remained free of clinical signs or microbiological evidence of active TB following infection with Mtb and were subsequently either treated with 3HP (n = 7) or left untreated (n = 7). Untreated NHPs were asymptomatic for 7 months but harbored persistent Mtb infection, as shown by reactivation of latent infection following simian immunodeficiency virus coinfection. However, none of the treated animals developed TB reactivation disease, and they remained without clinical or microbiological evidence of persistent bacilli, suggesting treatment-mediated clearance of bacteria.Conclusions:Mtb can persist in asymptomatic macaques for at least 7 months. Furthermore, 3HP treatment effectively cleared bacteria and prevented reactivation of TB in latently infected macaques.

Mechanisms of reactivation of latent tuberculosis infection due to SIV coinfection.

HIV is a major driver of tuberculosis (TB) reactivation. Depletion of CD4+ T cells is assumed to be the basis behind TB reactivation in individuals with latent tuberculosis infection (LTBI) coinfected with HIV. Nonhuman primates (NHPs) coinfected with a mutant simian immunodeficiency virus (SIVΔGY) that does not cause depletion of tissue CD4+ T cells during infection failed to reactivate TB. To investigate the contribution of CD4+ T cell depletion relative to other mechanisms of SIV-induced reactivation of LTBI, we used CD4R1 antibody to deplete CD4+ T cells in animals with LTBI without lentiviral infection. The mere depletion of CD4+ T cells during LTBI was insufficient in generating reactivation of LTBI. Instead, direct cytopathic effects of SIV resulting in chronic immune activation, along with the altered effector T cell phenotypes and dysregulated T cell homeostasis, were likely mediators of reactivation of LTBI. These results revealed important implications for TB control in HIV-coinfected individuals.

Immunology of Mycobacterium tuberculosis Infections.

Tuberculosis (TB) is a serious global public health challenge that results in significant morbidity and mortality worldwide. TB is caused by infection with the bacilli Mycobacterium tuberculosis (M. tuberculosis), which has evolved a wide variety of strategies in order to thrive within its host. Understanding the complex interactions between M. tuberculosis and host immunity can inform the rational design of better TB vaccines and therapeutics. This chapter covers innate and adaptive immunity against M. tuberculosis infection, including insights on bacterial immune evasion and subversion garnered from animal models of infection and human studies. In addition, this chapter discusses the immunology of the TB granuloma, TB diagnostics, and TB comorbidities. Finally, this chapter provides a broad overview of the current TB vaccine pipeline.

Mucosal-activated invariant T cells do not exhibit significant lung recruitment and proliferation profiles in macaques in response to infection with Mycobacterium tuberculosis CDC1551.

TB is a catastrophic infectious disease, affecting roughly one third of the world's population. Mucosal-associated invariant T (MAIT) cells are innate-like T cells that recognize vitamin B metabolites produced by bacteria, possess effector memory phenotype, and express tissue-homing markers driving migration to sites of infection. Previous research in both Mtb and HIV infections has shown that MAIT cells are depleted in the human periphery, possibly migrating to the tissue sites of infection. We investigated this hypothesis using rhesus macaques (RMs) with active TB, latent TB (LTBI), and SIV-coinfection to explore the effects of different disease states on the MAIT cell populations in vivo. Early in infection, we observed that MAIT cells increased in the blood and bronchoalveolar lavage fluid (BAL) of all infected RMs, irrespective of clinical outcome. However, the frequency of MAIT cells rapidly normalized such that they had returned to baseline levels prior to endpoint. Furthermore, following infection, the chemokines expressed on MAIT cells reflected a strong shift towards a Th1 phenotype from a shared Th1/Th17 phenotype. In conclusion, MAIT cells with enhanced Th1 functions migrating to the site of Mtb-infection. The anti-mycobacterial effector functions of MAIT cells, particularly during the early stages of Mtb infection, had been of interest in promoting protective long-term TB immunity. Our research shows, however, that they have relatively short-acting responses in the host.

High Frequencies of Caspase-3 Expressing Mycobacterium tuberculosis-Specific CD4+ T Cells Are Associated With Active Tuberculosis.

Antigen-specific CD4+ T cell responses to Mycobacterium tuberculosis (Mtb) infection are important for host defense against tuberculosis (TB). However, Mtb-specific IFN-γ-producing T cells do not distinguish active tuberculosis (ATB) patients from individuals with asymptomatic latent Mtb infection (LTBI). We reasoned that the immune phenotype of Mtb-specific IFN-γ+CD4+ T cells could provide an indirect gauge of Mtb antigen load within individuals. We sought to identify immune markers in Mtb-specific IFN-γ+CD4+ T cells and hypothesized that expression of caspase-3 Mtb-specific CD4+ T cells would be associated with ATB. Using polychromatic flow cytometry, we evaluated the expression of caspase-3 in Mtb-specific CD4+ T cells from LTBI and ATB as well as from ATB patients undergoing anti-TB treatment. We found significantly higher frequencies of Mtb-specific caspase-3+IFN-γ+CD4+ T cells in ATB compared to LTBI. Caspase-3+IFN-γ+CD4+ T cells were also more activated compared to their caspase-3-negative counterparts. Furthermore, the frequencies of caspase-3+IFN-γ+CD4+ T cells decreased in response to anti-TB treatment. Our studies suggest that the frequencies of caspase-3-expressing antigen-specific CD4+ T cells may reflect mycobacterial burden in vivo and may be useful for distinguishing Mtb infection status along with other host biomarkers.