Long noncoding RNAs (lncRNAs) have emerged as critical regulators of gene expression, yet their role in shaping human responses to vaccination remains largely uncharacterized. Here, we analyzed RNA-sequencing data from three independent human cohorts vaccinated with the rVSVΔG-ZEBOV-GP Ebola vaccine to profile lncRNA expression dynamics. Using differential expression analysis and correlation meta-analysis across cohorts, we identified an expression signature with several lncRNAs, including LEF1-AS1 and DOCK8-AS1, that exhibit conserved transcriptional activation following vaccination. Correlation of lncRNA expression with gene targets and IgG titers revealed putative roles for lncRNAs in regulating and/or participate in both innate immune responses and adaptive antibody production. Functional enrichment of lncRNA co-expressed protein-coding genes highlighted involvement in T-cell differentiation, interferon signaling, and leukocyte activation. Integrating global run-on sequencing data and comparative transcriptomic analysis across other vaccine studies suggests that LEF1-AS1 modulation is distinctively associated with Ebola vaccination. Our findings demonstrate that lncRNAs are potential integral components of the human vaccine response and provide a foundation for future mechanistic studies targeting noncoding RNA regulation of immunity.

The human immune system employs both innate and adaptive mechanisms to control pathogens, with antibodies playing a pivotal role in immune memory and defense, in particular against viral infections. In tuberculosis, antibody titers have long been used to assess immune responses, but their presence alone fails to predict protective efficacy. Recent studies highlight that antibody functionality is critical for effective immune activity. Despite widespread detection of Mycobacterium tuberculosis (Mtb)-reactive antibodies in individuals with active disease, Mtb infection, and even in healthy controls, their potential to control Mtb growth is variable and only detected in a proportion of individuals. This perspective emphasizes the need for robust functional assessment of antibodies to better understand their role in mycobacterial control and inform vaccine development. Notably, antibodies binding to purified protein derivative of Mtb, a mixture of degraded antigens from Mtb cultures, are widespread but not universally functional, underscoring the importance of Fc characteristics and epitope specificity. Initial high-throughput screening using phagocytosis and direct mycobacterial binding assays is an active indicator of antibody function. By refining and combining existing assays, as recommended in this perspective, we can better characterize antibody contributions, particularly their immunomodulatory potential, toward improved control of Mtb. Albeit antibodies may not be essential in natural protection, functional antibodies induced by vaccination may be of added value and contribute to host protection.

[This corrects the article DOI: 10.3389/fimmu.2025.1695514.].

Tuberculosis (TB) is a curable infectious disease that requires prolonged treatment with multiple antibiotics. To better understand how the immune system contributes to the clearance of Mycobacterium tuberculosis (Mtb), in vitro assays are essential for monitoring functional immune changes during infection, therapy, and following vaccination. In this study, we investigated whether mycobacterial growth control in thirty patients with TB disease changes over the course of treatment. Comprehensive immune profiling of peripheral blood mononuclear cells (PBMCs), using a 30-color spectral flow cytometry panel, identified dynamic shifts in immune cell subsets related to functional activity. Notably, in addition to memory and effector T cells, a subset of naive B cells changed during treatment. Most sera contained antibodies binding to purified protein derivative (PPD) and Mtb-specific antigens ESAT-6/CFP-10, and enhanced phagocytic activity. A functional mycobacterial growth inhibition assay (MGIA) revealed growth control, which appeared to be heterogeneous but generally sustained throughout longitudinal follow-up. We conclude that T and B cell responses change in response to antibiotic treatment of TB disease, but that mycobacterial growth control capacity is a property of the individual, which is not influenced by disease activity or antibiotic treatment.

The recombinant vesicular stomatitis virus-vectored Zaire ebolavirus (rVSVΔG-ZEBOV-GP) vaccine has been recently approved for children. Here, the transcriptomic response to this vaccine was characterised for the first time in children using a targeted gene panel. In a randomised controlled trial in Lambaréné, Gabon, children (n = 114, age 1–12 years old) were immunised with the rVSVΔG-ZEBOV-GP or varicella-zoster vaccine as control. Gene perturbation peaked at D1, and returned to baseline levels between D2 and D14. Genes related to type I/II interferon-signalling, pattern recognition receptors, myeloid cells and cell activation modules were upregulated, while downregulated genes were related to T cell and cytotoxicity modules. Perturbation of type-I IFN genes positively correlated with children’s age. Correlates of D28 ZEBOV-GP antibody titres were identified at D7 post-vaccination. Concluding, these findings provide insights into the vaccine-induced immune regulation at play in children.

The treatment of Mycobacterium avium (Mav) infection, responsible for over 80% of nontuberculous mycobacterial pulmonary disease, remains challenging due to rising antibiotic resistance and unsatisfactory success rates. Hence, there is a need for a deeper understanding of host-pathogen interactions to inform the development of alternative therapeutic approaches, like host-directed therapy (HDT), aimed at improving host antimycobacterial defenses. However, compared to Mycobacterium tuberculosis (Mtb) infections, knowledge of host-pathogen interactions for Mav infection is still limited. To address this knowledge gap, we performed a genome-wide host transcriptomic analysis of Mav-infected primary human macrophages-the primary host cell-alongside Mtb-infected macrophages to leverage insights from Mtb research. Our findings show substantial overlap in the gene expression patterns between Mav-infected and Mtb-infected macrophages, including induction of cytokine responses and modulation of various G-protein coupled receptors (GPCRs) involved in (lipid-mediated) macrophage immune functions. Notable differences were observed in the expression of immediate early genes (IEGs), phospholipases, and genes of the GTPase of immunity-associated protein (GIMAP) family. This study laid a foundation for identifying both shared and Mav-specific host response pathways, providing direction for future investigations into host-pathogen interactions during Mav infection and the identification of novel targets for HDT.

INTRODUCTION:Despite the high global prevalence of Mycobacterium tuberculosis (Mtb) infection in humans, most infected individuals achieve a stable immunological equilibrium, without showing clinical signs and symptoms of tuberculosis (TB). Although the role of antibodies in TB is assumed to be relatively small compared with cell-mediated immunity, their role in TB has been documented in a few recent studies. METHODS:In this cross-sectional study, we quantitated antibody responses to Mtb antigens, lipoarabinomannan (LAM), and heparin-binding hemagglutinin adhesin (HBHA) by determining antigen-specific immunoglobulin A(IgA) and G(IgG) secretion levels using enzyme-linked immunosorbent assay in serum and saliva of pulmonary TB patients (PTB), their household contacts, and community controls (determined by QuantiFERON TB Gold assay QFT-test result). RESULTS:The HBHA-specific IgA levels were significantly higher in both saliva and serum in household contacts groups compared with PTB patients (P = 0.013, P = 0.023). Exposed contacts, who were QFT-negative, had higher serum HBHA-specific IgA responses compared with PTB patients (P = 0.04). QFT-negative household contacts and QFT-positive community controls showed higher HBHA and lipoarabinomannan-specific IgG responses (P = 0.006, P = 0.002, P = 0.0009, P = 0.006, respectively) than PTB patients. Generally, lipoarabinomannan and HBHA-specific IgA levels were significantly higher in saliva compared with serum (P < 0.0001) in all study groups. CONCLUSION:Overall, the observed higher levels of IgA and IgG in controls, and exposed but QFT-negative contacts suggest a correlation with, and perhaps a role for these antibodies in preventing the development of active TB. The findings highlighted the potential involvement of saliva IgA in the immune response to Mtb, underscoring the relevance of mucosal immunity in TB infection.

Lung epithelial cells present the first line of defense against pathogens like Mycobacterium tuberculosis and other related species. Studying their responses is instrumental to understand early infection stages of tuberculosis. We present a protocol to study the infection potential of mycobacteria in differentiated primary human bronchial epithelial cell cultures. We describe mycobacterial and epithelial cell culture techniques. We then detail how to perform infections in epithelial cells and determine intracellular bacterial load using flow cytometry and colony-forming unit assays. For complete details on the use and execution of this protocol, please refer to Barclay et al.1,2.

Mycobacterium avium (Mav) complex is the leading cause of pulmonary diseases associated with non-tuberculous mycobacterial (NTM) infections worldwide. The inherent and increasing acquired antibiotic resistance of Mav hampers the treatment of Mav infections and emphasizes the urgent need for alternative treatment strategies. A promising approach is host-directed therapy (HDT), which aims to boost the host's immune defenses to combat infections. In this study, we show that phenothiazines, particularly trifluoperazine (TFP) and chlorproethazine (CPE), restricted Mav survival in primary human macrophages. Notably, TFP and CPE did not directly inhibit mycobacterial growth at used concentrations, confirming these drugs function through host-dependent mechanisms. TFP and CPE induced a mild, albeit not statistically significant, increase in autophagic flux along with the nuclear intensity of transcription factor EB (TFEB), the master transcriptional regulator of autophagy. Inhibition of autophagic flux with bafilomycin, however, did not impair the improved host infection control by TFP and CPE, suggesting that the host (auto)phagolysosomal pathway is not causally involved in the mechanism of action of TFP and CPE. Additionally, TFP and CPE increased the production of both cellular and mitochondrial reactive oxygen species (ROS). Scavenging mitochondrial ROS did not impact, whereas inhibition of NADPH oxidase (NOX)-mediated ROS production partially impaired the HDT activity of TFP and CPE, indicating that oxidative burst may play a limited role in the improved host control of Mav infection by these drugs. Overall, our study demonstrates that phenothiazines are promising HDT candidates that enhance the antimicrobial response of macrophages against Mav, through mechanism(s) that were partially elucidated.

Cutaneous leishmaniasis (CL) is the most prevalent type of leishmaniasis disease and causes skin lesions, mainly ulcers, on exposed parts of the body. The Americas, Mediterranean basin, Middle East, and Central Asia account for approximately 95% of all CL cases. Leishmania (L.) major and L. tropica are the most significant species causing CL. A better understanding of the molecular mechanisms of CL caused by Leishmania parasite species in patients' skin lesions may help inform intervention approaches. Using dual-color reverse transcriptase multiplex ligation-dependent probe amplification (dcRT-MLPA), we evaluated the expression of 144 host immune-related genes in lesions from CL patients infected with two Leishmania species, L. major and L. tropica, in Morocco and Iran, respectively. Distinct gene expression patterns were identified in the lesions of patients infected with L. major and L. tropica. The results revealed that L. tropica-infected patients had rather more significant gene expression than L. major-infected patients relative to healthy volunteers. However, CD14 and IFI6 (interferon alpha inducible protein 6), were two common genes expressed in the lesions of patients infected with L. major and L. tropica. Our analysis revealed that gene expression changes related to the IFN signaling pathway were significant in both lesion groups. This research advances our understanding of the host immune response to zoonotic and anthroponotic leishmaniasis and shows immune transcript signatures in the skin lesions of CL patients infected with L. major and L. tropica. These findings can inform further investigation into the processes underpinning immunity and immunopathology of CL caused by L. major and L. tropica.

The virtually monomorphic antigen presentation molecule HLA-E can present self- and non-self peptides to the NKG2A/CD94 co-receptor inhibitory complex expressed on natural killer (NK) cells and to T cell receptors (TCRs) expressed on T cells. HLA-E presents self-peptides to NKG2A/CD94 to regulate tissue homeostasis, whereas HLA-E restricted T cells mediate regulatory and cytotoxic responses toward pathogen-infected cells. In this study, we directly compared HLA-E/peptide recognition and signaling between NKG2A/CD94 and 2 HLA-E restricted TCRs that can recognize self-peptides or identical peptide mimics from the viral UL40 protein of cytomegalovirus using position substituted peptide variants. We show that position 7 is critical for interaction with NKG2A/CD94, whereas position 8 is important for interaction with the TCRs. The Arginine at position 5 of these peptides is an essential residue for recognition by both receptors. Thus, NKG2A/CD94 and TCRs have different requirements for recognition of peptides presented in HLA-E.

Tuberculosis (TB) displays several characteristics commonly linked to biofilm-associated infections, including recurrence of infection and resistance to antibiotics. Studying biofilm formation by mycobacteria on relevant mucosal surfaces advances our understanding of its role in TB pathogenesis and drug tolerance. Here, we present a protocol to promote biofilm formation by mycobacteria on differentiated primary pseudostratified human bronchial epithelial cell cultures. We then detail procedures to visualize and quantify biofilm matrix and biomass using electron and confocal microscopy and crystal violet staining. For complete details on the use and execution of this protocol, please refer to Barclay et al.1.

Tuberculosis (TB) remains a major cause of global mortality. Understanding the underlying immune response to the pathogen, Mycobacterium tuberculosis (Mtb), is essential for the development of vaccines. Evidence is accumulating supporting a contribution of innate immune cells and antibodies in Mtb control. Here, we focus on the functional capacity of antibodies from individuals with TB disease, both before and after TB treatment, individuals with TB infection, and healthy uninfected individuals, using an adapted in vitro mycobacterial growth inhibition assay, measuring Bacillus Calmette-Guérin (BCG) growth inhibition. Sera displayed heterogeneous impact on mycobacterial growth control. This was correlated with enhanced phagocytic capacity, which was abrogated by blocking Fc receptors (FcR) and depletion of antibodies. This phenotype negatively associated with mono- and digalactosylated Fc-glycans of IgG. Together, we demonstrate disease state independent direct effects of sera to mycobacterial growth control and antibody-FcR interactions modulating phagocytic capacity.

Background:Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), is among the leading causes of death from an infectious agent among children worldwide. Children represent a particularly vulnerable population due to the greater challenges in diagnosis and the higher risk of progression to severe forms of the disease. However, whether different pediatric outcomes relate to distinct immunologic responses remains incompletely understood. Emerging data suggest that Mtb-specific humoral immune responses represent a correlate of protection against Mtb both following natural infection and vaccination. Methods:To determine if immune profiles can distinguish children across the spectrum from Mtb infection to TB disease, as well as children with TB from non-TB lower respiratory tract infection, we mapped the humoral immune response across a panel of 4 dozen Mtb antigens across children presenting with symptoms of active TB (ATB), children with evidence of latent TB infection (LTBI) and children exhibiting non-TB lower respiratory tract infection (non-TB LRTI). Using a custom Luminex assay, Mtb-specific antibody subclass/isotype, Fc receptor (FcR) binding profiles, and functions were profiled across the pediatric groups. Findings:A robust humoral immune response was observed in children with active TB compared to non-TB LRTI, marked by a strong IgA response, that exhibited high FcαR binding. Conversely, children with LTBI uniquely elicited Mtb-specific antibodies with enhanced opsinophagocytic FcγR2A binding, as well as a higher capacity to activate NK cells and neutrophils. Interpretation:There are significant differences in humoral immune profiles across the landscape of pediatric TB, potentially contributing to differential mycobacterial control, and highlighting biomarkers that could guide both diagnostic and therapeutic approaches. Funding:US National Institutes of Health.

Human leukocyte antigen E (HLA-E) communicates cellular health to natural killer (NK) cells through presentation of peptides derived from the leader sequence of classical major histocompatibility complex class I (MHC-I), inhibiting NK cell activation and lysis of healthy cells. Besides this canonical role, HLA-E can also present peptides from pathogens such as Mycobacterium tuberculosis (Mtb) to T cells and can inhibit phagocytosis by engaging with LILRB1/2. To identify additional HLA-E binding surface molecules, we utilized a CRISPR/Cas9 activation screen with HLA-E tetramers, which identified Stabilin (STAB)1 and STAB2 as novel interactors. This interaction depended on the nature of the peptide/HLA-E complex, whereby high affinity peptides did not permit the interaction while low affinity peptides did. Functionally, expression of STAB1 or STAB2 on THP-1 monocytes increased phagocytic uptake of HLA-E coated microbeads. These results provide the first evidence of an interaction between Stabilin receptors and specific HLA-E conformations.

INTRODUCTION:Repeated exposures to Mycobacterium tuberculosis (Mtb) and related species may influence host responses, which in turn may affect vaccine efficacy and could even render the host less or more susceptible to progression to active tuberculosis (TB) disease. METHODS:Using well-differentiated primary human bronchial epithelial cells (PBEC), we investigated the effect of a prior exposure of the epithelium to Mtb and Mycobacterium bovis vaccine strain Bacille Calmette-Guerin (BCG) on the intracellular infection efficiency of Mtb and Mycobacterium avium (Mav) during a second exposure and measured cytokine and antimicrobial peptide secretion. RESULTS:PBEC that were first exposed to BCG were significantly more resistant to subsequent infection with Mtb. A similar trend was observed in PBEC that were previously exposed to Mtb, although to a lesser magnitude compared to BCG pre-exposure. Furthermore, while the first exposure to mycobacteria induced inflammatory cytokine secretion by PBEC, cytokine secretion was dampened upon a secondary exposure to Mtb, most strongly in previously BCG-exposed cells. Secretion of the antimicrobial peptide hBD-2 was not affected by sequential exposures. CONCLUSION:Repeated exposure of differentiated airway epithelial cells to mycobacteria reduced intracellular infection and inflammation.

Background:Influenza viruses with pandemic potential and possible burden of post-viral sequelae are a global concern. To prepare for future pandemics and the development of improved vaccines, it is vital to identify the immunological changes underlying influenza disease severity. Methods:We combined unsupervised high-dimensional single-cell mass cytometry with gene expression analyses, plasma CXCL13 measurements, and antigen-specific immune cell assays to characterize the immune profiles of hospitalized patients with severe and moderate seasonal influenza disease during active infection and at 6-month follow-up. We used age-matched healthy donors as controls. Results:Severe disease was associated with a distinct immune profile, including lower frequencies of ICOS+ mucosal-associated invariant T (MAIT) cells, and CXCR5+ memory B and CD4+CXCR5+CD95+ICOS+ and CD8+CXCR3+CD95+PD-1+TIGIT+ memory T cells, as well as lower CD4 gene expression. Higher frequencies of CD16+CD161+ NK cells, CD169+ monocytes, CD123+/- dendritic cells, and CD38high plasma cells and high CXCL13 plasma levels were also associated with severe disease. Alterations in immune cell subpopulations persisted at convalescence for the severely ill patients only. Conclusions:Our results indicated a reduction in regulatory MAIT cells and memory T and B cells and an increase in the inhibitory subpopulations of monocytes and NK cells in severe influenza that persisted at convalescence. These immune cell alterations were associated with higher age and the presence of several underlying conditions that may contribute to frailty. This study illustrates the power and sensitivity of high-dimensional single-cell analyses in identifying potential cellular biomarkers for disease severity after influenza infection.

Tuberculosis (TB) remains a significant global health challenge, latently affecting around a quarter of the global population. The sole licensed TB vaccine, Mycobacterium bovis Bacillus Calmette-Guérin (BCG), shows variable efficacy, particularly among adolescents and adults, underscoring the pressing need for more effective vaccination strategies. The administration route is crucial for vaccine efficacy, and administration via the skin, being rich in immune cells, may offer advantages over conventional subcutaneous routes, which lack direct access to abundant antigen-presenting cells.This study compared the immunogenic effects of intradermal versus subcutaneous administration of a candidate TB vaccine delivering a Ag85B-ESAT6-Rv2034 (AER) multiphase fusion recombinant protein, in lipid-poly(D,L-lactic-co-glycolic acid) (lipid-PLGA) nanoparticles in mice. In-depth evaluation of immune responses in splenocytes was performed using 27-marker spectral flow cytometry. Both routes elicited significant T-cell responses. However, intradermal administration uniquely increased polyfunctional CD4+ and CD8+ T-cells producing IL-2, IFNγ, and TNFα, associated with protection against TB. Additionally, it significantly increased CD69+ B-cell counts and induced higher AER-specific antibody titers, particularly IgG2a. These results underscore the superior immunogenic potential of intradermal vaccine administration by effectively inducing immune cells associated with TB protection, highlighting its significance in the development of new vaccine strategies.

Tuberculosis (TB) is a major global health problem, and the development of effective and safe vaccines is urgently needed. CD8+ T-cells play an important role alongside CD4+ T-cells in the protective immune response against TB. pH-sensitive liposomes are hypothesized to boost CD8+ T-cell responses by promoting class I presentation through a mechanism involving pH-dependent endosomal escape and the cytosolic transfer of antigens. The aim of the study was to explore the potential of pH-sensitive liposomes as a novel delivery system for a multi-stage protein subunit vaccine against TB in primary human cells. The liposomes were formulated with the fusion antigen Ag85b-ESAT6-Rv2034 (AER), which was previously shown to be effective in reducing bacterial load in the lungs HLA-DR3 transgenic mice and guinea pigs. The liposomes were assessed in vitro for cellular uptake, cell viability, upregulation of cell surface activation markers, induction of cytokine production using human monocyte-derived dendritic cells (MDDCs), and activation of human antigen-specific T-cells. Liposome DOPC:DOPE:DOBAQ:EPC (3:5:2:4 M ratio) was effectively taken up, induced several cell surface activation markers, and production of CCl3, CCL4, and TNFα in MDDCs. It also induced upregulation of CD154 and IFNγ in T-cell clones in an antigen-specific manner. Thus, cationic pH-sensitive liposome-based TB vaccines have been demonstrated to be capable of inducing robust protective Mtb-specific immune responses, positioning them as promising candidates for effectiveTBvaccination.