Shabaana Abdul Khader

Tuberculosis (TB), caused by the bacterium Mycobacterium tuberculosis ( Mtb ), infects approximately one-fourth of the world’s population. Inbred mouse models of TB do not reflect the pathological states and heterogeneity seen in human TB disease. Thus, we recently established a model of TB in diversity outbred (DO) mice, which displayed heterogeneity in inflammatory and protective responses following aerosol Mtb infection. In the current study, we show that DO mice vaccinated with M. bovis Bacille Calmette Guerin (BCG) are significantly protected upon Mtb HN878 infection, and protection is associated with the induction of transcriptional pathways involved in transforming growth factor B (TGF-β) and Toll-like receptor (TLR)-10 signaling. Targeting lung innate pathways in BCG-vaccinated DO mice using adjuvants also further improved protection upon Mtb infection by inducing genes associated with cellular responses to external stimuli, B-cell responses, as well as IL-17-producing CD4 ⁺ T-cell responses. Depletion of CD4 ⁺ T cells resulted in loss of vaccine-induced protection in DO BCG-vaccinated and adjuvant-treated Mtb -infected mice. Together, our new results show that innate targeting of the lung by activating TLR pathways could induce protective pathways in T cells that significantly improve upon the protection induced by BCG vaccination. Additionally, the DO mouse model of vaccination and Mtb infection can provide novel insights into immune pathways that are important for improving vaccine-induced protection against TB. IMPORTANCE: Bacille Calmette Guerin (BCG) vaccination in genetically diverse outbred (DO) mice provides significant protection against Mycobacterium tuberculosis ( Mtb) challenge. This protection induced pathways associated with transforming growth factor B (TGF-β) receptor complex, genes associated with lung repair, and Toll-like receptor (TLR)-10 pathway. The enhanced protection observed in BCG-vaccinated mice correlated with improved formation of B-cell follicles and IL-17-producing CD4 ⁺ T-cell responses. CD4 ⁺ T-cell responses mediated the enhanced protection in the lungs of DO mice vaccinated with BCG + adjuvant, as depletion of CD4 ⁺ T-cell responses reversed the enhanced protection. The DO mouse model of tuberculosis vaccination is a highly relevant model to probe mechanisms of vaccine-induced protection and provide novel insights into lung pathways that mediate protection. The study also found that genes associated with lung repair, including TGF-β receptor complex pathways, were induced in BCG-vaccinated Mtb -infected DO mouse lungs. The study suggests that the activation of lung innate pathways in BCG vaccination through the use of mucosal Amph CpG delivery, CD40L activation, and IL-10 neutralization could significantly enhance protection upon Mtb challenge.

Tuberculosis (TB), caused by the bacterium Mycobacterium tuberculosis ( Mtb ), infects approximately one-fourth of the world’s population. We reported an increased accumulation of mast cells (MCs) in the lungs of macaques with active pulmonary TB (PTB), compared with those with latent TB infection (LTBI). MCs respond in vitro to Mtb exposure via degranulation and by inducing proinflammatory cytokines. In the current study, we demonstrate an increased production of chymase by MCs in granulomas of humans and macaques with PTB. Single-cell (sc) RNA sequencing analysis revealed distinct MC transcriptional programs between LTBI and PTB, with PTB-associated MCs enriched in interferon gamma, oxidative phosphorylation, and MYC signaling. In a mouse model, MC deficiency led to improved control of Mtb infection that coincided with reduced accumulation of lung myeloid cells and diminished lung inflammation at chronic stages of infection. Airway transfer of MCs into wild-type Mtb -infected mice showed increased neutrophils, decreased recruited macrophages, and elevated Mtb dissemination to the spleen. Together, these findings highlight MCs as active drivers of TB pathogenesis and potential targets for host-directed therapies for TB.

Most HIV-negative individuals exposed to Mycobacterium tuberculosis (Mtb) control infection as latent TB infection (LTBI), but HIV co-infection greatly increases progression to tuberculosis (TB), the leading cause of death in people living with HIV (PLHIV). Although combination antiretroviral therapy (cART) reduces LTBI reactivation, immune control of Mtb is not fully restored, as shown by persistent TB incidence in PLHIV on cART. In macaques, skewed pulmonary effector memory CD4⁺ T-cell (T_EM) responses and new TB lesions persist despite cART. We hypothesize that concurrent anti-TB therapy with cART would improve bacterial control and immune restoration compared to cART alone. Using rhesus macaques (RM) with LTBI and Simian Immunodeficiency Virus (SIV) co-infection, we tested three months of weekly isoniazid and rifapentine (3HP) plus daily cART. Concurrent cART+3HP improves clinical and microbiological outcomes but fails to fully restore lung CD4⁺ T-cell immunity. Treated RMs retain caseous granulomas with high FDG uptake and incomplete CD4⁺ T-cell reconstitution, marked by persistent activation, exhaustion, and inflammation. CD4⁺ T_EM cells remain depleted. Concurrent therapy induces Type I IFN signatures and enhances Mtb-specific T_H1/T_H17—but reduces TNFα—responses. These findings reveal persistent pulmonary immune defects underlying TB risk in HIV co-infection and identify potential targets for host-directed adjunctive therapies.

Tuberculosis (TB), caused by the bacterium Mycobacterium tuberculosis (Mtb), infects approximately one-fourth of the world’s population. We reported an increased accumulation of mast cells (MCs) in the lungs of macaques with active pulmonary TB (PTB), compared with those with latent TB infection (LTBI). MCs respond in vitro to Mtb exposure via degranulation and by inducing proinflammatory cytokines. In the current study, we demonstrate an increased production of chymase by MCs in granulomas of humans and macaques with PTB. Single-cell (sc) RNA sequencing analysis revealed distinct MC transcriptional programs between LTBI and PTB, with PTB associated MCs enriched in interferon gamma, oxidative phosphorylation, and MYC signaling. In a mouse model, MC deficiency led to improved control of Mtb infection that coincided with reduced accumulation of lung myeloid cells and diminished lung inflammation at chronic stages of infection. Airway transfer of MCs into wild-type Mtb infected mice showed increased neutrophils, decreased recruited macrophages, and elevated Mtb dissemination to the spleen. Together, these findings highlight MCs as active drivers of TB pathogenesis and potential targets for host-directed therapies for TB.

The COVID-19 pandemic, triggered by the SARS-CoV-2 virus, has led to global consequences since its emergence in late 2019, impacting health, economies, and societies worldwide. Understanding the early responses following infection will provide crucial insights into the immune mechanisms governing disease progression and protection. We employed the single-cell RNA-sequencing approach using bronchoalveolar lavage cells of rhesus macaques at 0–3 days post-infection (dpi) in a non-human primate model of SARS-CoV-2. Our results show the increased abundance of two subsets of macrophages that express IFN-responsive gene signatures, inflammatory dendritic cells (DCs), plasmacytoid DCs, and conventional DCs at 3 dpi, coinciding with peak viremia. Moreover, our investigation underscores the enrichment of IFN-α/β, IFN-γ, and IL-1 signaling pathways within myeloid populations at 2–3 dpi in comparison to baseline. These findings substantially deepen our comprehension, elucidating the transcriptional landscape at the single-cell level during the early phases of COVID-19 within a highly relevant model. IMPORTANCE: Understanding the immune response in the early stages of COVID-19 is crucial for developing better treatments and vaccines. In this study, we used advanced single-cell RNA sequencing to examine the immune cells in the lungs of rhesus macaques, a model for human SARS-CoV-2 infection, within the first few days after infection. We identify key immune cell types, such as macrophages and dendritic cells, that are activated early on and produce important signals to fight the virus. These findings help clarify how the immune system responds to SARS-CoV-2 at the single-cell level, offering valuable insights that could guide future therapeutic approaches.

To radically diminish tuberculosis (TB) incidence and mortality by 2035, as set out by the WHO End TB Strategy, there is a desperate need for improved TB therapies and a more effective vaccine against the deadly pathogen Mycobacterium tuberculosis . Aerosol vaccination with the MtbΔsigH mutant protects 2 species of nonhuman primates against lethal TB challenge by invoking vastly superior T and B cell responses in the lungs through superior antigen presentation and interferon conditioning. Since the Geneva Consensus on essential steps toward the development of live mycobacterial vaccines recommends that live TB vaccines incorporate at least 2 independent gene knockouts, we have now generated several rationally designed, double-knockout (DKO) and triple-knockout (TKO) mutants in Mtb , each containing the ΔsigH deletion. Here, we report preclinical studies in the rhesus macaque model of aerosol infection and SIV/HIV coinfection, aimed at assessing the safety of these MtbΔsigH -based DKOs and TKOs. We found that most of these mutant strains were attenuated in both immunocompetent and SIV-coinfected macaques, and combinatorial infection with these generated strong cellular immune responses in the lung, akin to MtbΔsigH . Aerosol infection with these KO strains elicited inducible bronchus-associated lymphoid tissue, which is a correlate of protection from TB.

Infections with nontuberculous mycobacterium (NTM) are on the rise. Here, we investigated an uncommon NTM infection, by M . haemophilum ( Mh , n = 4), from a shared geographic location in the United States. All patients had underlying immunosuppressive conditions or treatments. We identified that all these individuals had a nonsynonymous mutation in GATA2 gene, which was absent in healthy controls (HCs, n = 4) from the same geographic area (Missouri, USA). Whole blood from these individuals had attenuated cytokine responses to Mh stimulation for IL-1β, IL-6, IL-8, MIP-1α and MIP-1β, but not to phytohemagglutinin (PHA) or another NTM, M . abscessus . Impaired whole blood transcriptional responses in individuals with GATA2 mutation included heightened Ras-homolog (Rho) guanosine triphosphate hydrolases (GTPase) and lowered TGF-β responses, among others. Our results highlight that, comparatively, M . abscessus and Mh elicit differential immune responses in humans. We identify a 23-gene signature that distinguished host response to Mh and M . abscessus and show that in vitro GATA2 siRNA knockdown indeed attenuated cytokine responses to Mh . Thus, we provide evidence that links GATA2 mutation and immune dysfunction in individuals with compromised immunity to Mh infection in humans and outline host factors associated with the immune response of this clinically relevant NTM.

The need for novel vaccination strategies to control tuberculosis (TB) is underscored by the limited and variable efficacy of the currently licensed vaccine, Bacille Calmette-Guerin (BCG). SigH is critical for Mycobacterium tuberculosis (Mtb) to mitigate oxidative stress, and in its absence Mtb is unable to scavenge host oxidative/nitrosative bursts. The MtbΔsigH (ΔsigH) isogenic mutant induces signatures of the innate immunity in macrophages and protects rhesus macaques from a lethal Mtb challenge. To understand the immune mechanisms of protection via mucosal vaccination with ΔsigH, we employed the resistant cynomolgus macaque model; and our results show that ΔsigH vaccination significantly protects against lethal Mtb challenge in this species. ΔsigH-vaccinated macaques are devoid of granulomas and instead generate inducible bronchus associated lymphoid structures, and robust antigen-specific CD4⁺ and CD8⁺ T cell responses, driven by a hyper-immune, trained immunity-like phenotype in host macrophages with enhanced antigen presentation. Correlates of protection in ΔsigH-vaccinated macaques include gene signatures of T cell activation, IFNG production, including IFN-responsive, activated T cells, concomitant with IFNG production, and suppression of IDO⁺ Type I IFN-responsive macrophage recruitment. Thus, ΔsigH is a promising lead candidate for further development as an antitubercular vaccine.