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Abstract The absence of validated correlates of protection (CoPs) hampers the rational design and clinical development of new tuberculosis vaccines. In this review, we provide an overview of the potential CoPs in tuberculosis vaccine research. Major hindrances and potential opportunities are then discussed. Based on recent progress, it is reasonable to anticipate that success in the ongoing efforts to identify CoPs would be a game-changer in tuberculosis vaccine development.

One-quarter of the world’s population is infected with Mycobacterium tuberculosis (Mtb). After initial exposure, more immune-competent persons develop asymptomatic latent tuberculosis infection (LTBI) but not active diseases, creates an extensive reservoir at risk of developing active tuberculosis. Previously, we constructed a novel recombinant Sendai virus (SeV)-vectored vaccine encoding two dominant antigens of Mtb, which elicited immune protection against acute Mtb infection. In this study, nine Mtb latency-associated antigens were screened as potential supplementary vaccine candidate antigens, and three antigens (Rv2029c, Rv2028c, and Rv3126c) were selected based on their immune-therapeutic effect in mice, and their elevated immune responses in LTBI human populations. Then, a recombinant SeV-vectored vaccine, termed SeV986A, that expresses three latency-associated antigens and Ag85A was constructed. In murine models, the doses, titers, and inoculation sites of SeV986A were optimized, and its immunogenicity in BCG-primed and BCG-naive mice were determined. Enhanced immune protection against the Mtb challenge was shown in both acute-infection and latent-infection murine models. The expression levels of several T-cell exhaustion markers were significantly lower in the SeV986A-vaccinated group, suggesting that the expression of latency-associated antigens inhibited the T-cell exhaustion process in LTBI infection. Hence, the multistage quarter-antigenic SeV986A vaccine holds considerable promise as a novel post-exposure prophylaxis vaccine against tuberculosis.

Abstract Background Mycobacteria bloodstream infections are common in immunocompromised people and usually have disastrous consequences. As the primary phagocytes in the bloodstream, monocytes and neutrophils play critical roles in the fight against bloodstream mycobacteria infections. In contrast to macrophages, the responses of monocytes infected with the mycobacteria have been less investigated. Results In this study, we first established a protocol for infection of non-adherent monocyte-like THP-1 cells (i.e. without the differentiation induced by phorbol 12-myristate 13-acetate (PMA) by bacillus Calmette-Guérin (BCG). Via the protocol, we were then capable of exploring the global transcriptomic profiles of non-adherent THP-1 cells infected with BCG, and found that NF-κB, MAPK and PI3K-Akt signaling pathways were enhanced, as well as some inflammatory chemokine/cytokine genes (e.g. CCL4, CXCL10, TNF and IL-1β) were up-regulated. Surprisingly, the Akt-HIF-mTOR signaling pathway was also activated, which induces trained immunity. In this in vitro infection model, increased cytokine responses to lipopolysaccharides (LPS) restimulation, higher cell viability, and decreased Candida albicans loads were observed. Conclusions We have first characterized the transcriptomic profiles of BCG-infected non-adherent THP-1 cells, and first developed a trained immunity in vitro model of the cells.

Summary: Bacillus Calmette-Guérin (BCG), the only licensed tuberculosis vaccine, provides non-specific protection against non-tuberculosis diseases that is mediated by trained immunity, a functional reprogramming mediated by innate immune memory. Here, we present a protocol for analyzing BCG-induced trained immunity in murine bone marrow-derived macrophages (BMDMs). We describe steps for preparing BCG single bacterial suspensions, isolating BMDM cells, and the training process. This protocol can assist researchers to conveniently utilize BMDM cells to study trained immunity.For complete details on the use and execution of this protocol, please refer to Xu et al.1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

IntroductionThe unique dormancy of Mycobacterium tuberculosis plays a significant role in the major clinical treatment challenge of tuberculosis, such as its long treatment cycle, antibiotic resistance, immune escape, and high latent infection rate.MethodsTo determine the function of MtrA, the only essential response regulator, one strategy was developed to establish its regulatory network according to high-quality genome-wide binding sites.Results and discussionThe complex modulation mechanisms were implied by the strong bias distribution of MtrA binding sites in the noncoding regions, and 32.7% of the binding sites were located inside the target genes. The functions of 288 potential MtrA target genes predicted according to 294 confirmed binding sites were highly diverse, and DNA replication and damage repair, lipid metabolism, cell wall component biosynthesis, cell wall assembly, and cell division were the predominant pathways. Among the 53 pathways shared between dormancy/resuscitation and persistence, which accounted for 81.5% and 93.0% of the total number of pathways, respectively, MtrA regulatory genes were identified not only in 73.6% of their mutual pathways, but also in 75.4% of the pathways related to dormancy/resuscitation and persistence respectively. These results suggested the pivotal roles of MtrA in regulating dormancy/resuscitation and the apparent relationship between dormancy/resuscitation and persistence. Furthermore, the finding that 32.6% of the MtrA regulons were essential in vivo and/or in vitro for M. tuberculosis provided new insight into its indispensability. The findings mentioned above indicated that MtrA is a novel promising therapeutic target for tuberculosis treatment since the crucial function of MtrA may be a point of weakness for M. tuberculosis.

Abstract Fluorescent‐magneto nanoemitters have gained considerable attention for their applications in mechanical controlling‐assisted optical signaling. However, the incompatibility between magnetic and fluorescent components often leads to functional limitations in traditional magneto@fluorescence nanostructure. Herein, we introduce a new compact‐discrete spatial arrangement on a “fluorescence@magneto” core–shell nanostructure consisting of a close‐packed aggregation‐induced emission luminogen (AIEgen) core and a discrete magnetic shell. This structural design effectively eliminates the optical and magnetic interferences between the dual components by facilitating AIEgens loading in core region and reducing the magnetic feeding amount through effective exposure of the magnetic units. Thereby, the resulting magneto‐AIEgen nanoparticle (MANP) demonstrates “win‐win” performances: (i) high fluorescent intensity contributed by AIEgens stacking‐enhanced photoluminescence and reduced photons loss from the meager magnetic shell; (ii) marked magnetic activity due to magneto extraposition‐minimized magnetic shielding. Accordingly, the dual functions‐retained MANP provides a proof of concept for construction of an immunochromatographic sensing platform, where it enables bright fluorescent labeling after magnetically enriching and separating procalcitonin and lipoarabinomannan in clinical human serum and urine, respectively, for the clinical diagnosis of bacterial infections‐caused inflammation and tuberculosis. This study not only inspires the rational design of magnetic‐fluorescent nanoemitter but also highlights promising potential in magneto‐assisted point‐of‐care test and biomedicine applications.

ObjectivesLipoarabinomannan (LAM), an abundant cell wall glycolipid of mycobacteria including Mycobacterium tuberculosis (Mtb), is a promising TB diagnostic marker. The current commercially available urine LAM assays are not sufficiently sensitive, and more novel detection strategies are urgently needed to fill the current diagnostic gap.MethodsA proteinase K-pretreated Concanavalin A (ConA)-based ELISA assay was developed. Diagnostic performance was assessed by several bacterial strains and clinical urine samples.ResultsThe limit of detection (LoD) of the assay against ManLAM was 6 ng/ml. The assay reacted strongly to Mtb H37Rv and M. bovis BCG, intermediately to M. smegmatis mc2155, and weakly to four non-mycobacteria pathogens. This method could distinguish TB patients from healthy controls (HCs) and close contacts (CCs) in 71 urine samples treated with proteinase K, which increases urine LAM antibody reactiveness. In TB+HIV+ and TB+HIV− patients, the sensitivity was 43.8 and 37.5%, respectively, while the specificity was 100.0%. The areas under ROC curves (AUCs) were 0.74 and 0.82, respectively.ConclusionThis study implies that ConA can be paired with antibodies to detect LAM. Proteinase K treatment could effectively enhance the sensitivity by restoring the reactiveness of antibodies to LAM.

Tuberculosis (TB), also known as the “White Plague”, is caused by Mycobacterium tuberculosis (Mtb). Before the COVID-19 epidemic, TB had the highest mortality rate of any single infectious disease. Vaccination is considered one of the most effective strategies for controlling TB. Despite the limitations of the Bacille Calmette-Guérin (BCG) vaccine in terms of protection against TB among adults, it is currently the only licensed TB vaccine. Recently, with the evolution of bioinformatics and structural biology techniques to screen and optimize protective antigens of Mtb, the tremendous potential of protein subunit vaccines is being exploited. Multistage subunit vaccines obtained by fusing immunodominant antigens from different stages of TB infection are being used both to prevent and to treat TB. Additionally, the development of novel adjuvants is compensating for weaknesses of immunogenicity, which is conducive to the flourishing of subunit vaccines. With advances in the development of animal models, preclinical vaccine protection assessments are becoming increasingly accurate. This review summarizes progress in the research of protein subunit TB vaccines during the past decades to facilitate the further optimization of protein subunit vaccines that may eradicate TB.

In order to deepen the understanding of the role and regulation mechanisms of prokaryotic global transcription regulators in complex processes, including virulence, the associations between the affinity and binding sequences of Mycobacterium tuberculosis MtrA have been explored extensively. Analysis of MtrA 294 diversified 26 bp binding sequences revealed that the sequence similarity of fragments was not simply associated with affinity. The unique variation patterns of GC content and periodical and sequential fluctuation of affinity contribution curves were observed along the sequence in this study. Furthermore, docking analysis demonstrated that the structure of the dimer MtrA-DNA (high affinity) was generally consistent with other OmpR family members, while Arg 219 and Gly 220 of the wing domain interacted with the minor groove. The results of the binding box replacement experiment proved that box 2 was essential for binding, which implied the differential roles of the two boxes in the binding process. Furthermore, the results of the substitution of the nucleotide at the 20th and/or 21st positions indicated that the affinity was negatively associated with the value of minor groove width precisely at the 21st position. The dimerization of the unphosphorylated MtrA facilitated by a low-affinity DNA fragment was observed for the first time. However, the proportion of the dimer was associated with the affinity of substrate DNA, which further suggested that the affinity was actually one characteristic of the stability of dimers. Based on the finding of 17 inter-molecule hydrogen bonds identified in the interface of the MtrA dimer, including 8 symmetric complementary ones in the conserved α4-β5-α5 face, we propose that hydrogen bonds should be considered just as important as salt bridges and the hydrophobic patch in the dimerization. Our comprehensive study on a large number of binding fragments with quantitative affinity values provided new insight into the molecular mechanism of dimerization, binding specificity and affinity determination of MtrA and clues for solving the puzzle of how global transcription factors regulate a large quantity of target genes.

ObjectiveTo compare the diagnostic performance of laboratory assays on the ultrasound-guided core needle biopsy samples for diagnosis of extra-pulmonary tuberculosis (EPTB) in HIV-positive and HIV-negative patients.MethodsA total of 217 patients suspected to have EPTB underwent lesion biopsy from 2017 to 2020. Results of laboratory tests on the biopsy and non-biopsy samples were collected with clinical data for retrospective analysis of test utility. The calculated diagnostic accuracy of the tests was stratified according to the specimen types and HIV status.ResultsThe cohort contained 118 patients with a final positive diagnosis of extrapulmonary tuberculosis (EPTB group, 54.4%) and 99 finally diagnosed as without TB (non-EPTB group, 45.6%). The risk factor for EPTB was HIV co-infection (OR 2.22, 95% CI 1.17-4.28, p = 0.014). In biopsy samples, GeneXpert (Xpert) showed higher sensitivity (96.6% [91.6-98.7], p < 0.0001) than culture (56.1% [47.0-64.9]). Regardless of HIV status, Xpert had the highest sensitivity (>95%) and specificity (nearly 100%) of any methods. In non-biopsy samples, only T-SPOT.TB (T-SPOT) showed higher sensitivity than culture (90.9% [62.3-99.5] vs 35.3% [17.3-58.7], p = 0.0037). Furthermore, the sensitivities of Xpert were lower in non-biopsy samples (60.0% [23.1-92.9], p = 0.022) than in biopsy samples (100% [86.7-100]). Even in smear-negative biopsy samples, Xpert still had higher sensitivity than culture and retained high specificity (100% [95.7-100]).ConclusionSuperior performance of Xpert in diagnosing EPTB was observed regardless of HIV status and specimen types. Nevertheless, the biopsy samples still substantially facilitated the accurate diagnosis of extrapulmonary tuberculosis.

Tumor antigens (Ags) are weakly immunogenic and elicit inadequate immune responses, thus induction of antigen-specific immune activation via the maturation of dendritic cells (DCs) is a strategy used for cancer immunotherapy. In this study, we examined the effect of Rv3628 from Mycobacterium tuberculosis (Mtb) on activation of DCs and anti-tumor immunity in vivo. Intravenous injection of mice with Rv3628 promoted DC activation of spleen and lymph nodes. More importantly, Rv3628 also induced activation of DCs and enhanced Ag presentation in tumor-bearing mice. In mice bearing ovalbumin (OVA)-expressing tumors, combination treatment with Rv3628 and OVA peptide promoted OVA-specific T cell activation and accumulation of interferon (IFN)-γ and tumor necrosis factor (TNF)-α-producing OT-I and OT-II cells in tumor-draining lymph nodes. Moreover, three different tumor Ags in three different tumor models showed enhanced anti-tumor activity with Rv3628 as adjuvant, including inhibition of growth of OVA-expressing B16 melanoma, CT26 carcinoma, and B16 melanoma tumors, and a synergistic effect with anti-programmed cell death protein 1 (PD-1) antibody treatment. Additionally, potential application against human tumors was indicated by similar activation of human peripheral blood DCs by Rv3628. Taken together, these data demonstrate that Rv3628 could be an effective adjuvant in tumor immunotherapy via enhanced capacity of DC activation and Ag presentation.

BackgroundStudy on effect of fertilization methods on maternal and perinatal outcomes with respect to TB during pregnancy was scarce. This study aimed to analyze maternal and perinatal outcomes in active TB cases after in vitro fertilization (IVF) treatment vs. normal pregnancy.MethodsClinical data of 80 pregnant women with active TB hospitalized at Shanghai Public Health Clinical Center between June 1st, 2014 and November 30th, 2020 were extracted and retrospectively analyzed. History of receiving IVF was recorded at admission and its association with maternal and perinatal outcomes were assessed using multivariable logistic regression models with adjustment for potential confounders.ResultsOf the 80 pregnant women with active TB, 28 (35.0%) received IVF treatment and 52 (65.0%) did not receive IVF treatment. After adjusting for potential confounders, receiving IVF was associated with worse maternal and perinatal outcomes, including maternal criticality (21.4 vs. 2.0%, adjusted OR = 28.3, P = 0.015), miliary TB (89.3 vs. 13.5%, adjusted OR = 75.4, P < 0.001), TB meningitis (32.1 vs. 7.7%, adjusted OR = 6.2, P = 0.010), and perinatal mortality (64.3 vs. 28.8%, adjusted OR = 9.8, P = 0.001).ConclusionThe additional risk of TB to women receiving IVF treatment is a public health challenge specific to countries with a high tuberculosis burden. Increased awareness of latent tuberculosis infection in women receiving IVF treatment is needed.

The development of heterologous prime-boost regimens utilizing Bacille Calmette–Guerin (BCG) as the priming vaccine is a promising approach to improve the efficacy of vaccination against tuberculosis (TB). In this study, we examined the ability of a DNA vaccine that expressed a fusion of antigens Rv2299c and Ag85A to boost BCG immunity and protection against Mycobacterium tuberculosis (Mtb) in Balb/c mice. The fusion DNA vaccine was moderately immunogenic and afforded some protection when used on its own. After a priming BCG vaccination, the DNA boost significantly amplified Th1-type cell-mediated immunity compared to that resulting from either BCG or DNA immunization. In the DNA-boosted mice, Ag-specific CD4+ and CD8+ T cells that were mono-positive for IFN-γ alone were the most prominently expanded in infected lungs. The protective efficacy afforded by BCG against challenge infection was greatly improved by the DNA boost; bacterial loads were significantly reduced in both spleen and lung and histological damage in the lung was less. The use of a DNA vaccine containing the fusion antigens Rv2299c and Ag85A to boost BCG may be a good choice for the rational design of an efficient vaccination strategy against TB.

Mendelian susceptibility to mycobacterial disease (MSMD) arises from a group of rare inherited errors of immunity that result in selective susceptibility of otherwise healthy people to clinical disease caused by low virulence strains of mycobacteria, such as Mycobacterium bovis Bacille Calmette-Guérin (BCG) and environmental mycobacteria. Patients have normal resistance to other pathogens and no overt abnormalities in routine immunological and hematological evaluations for primary immunodeficiencies. At least 19 genes and 34 clinical phenotypes have been identified in MSMD. However, there have been no systematic reports on the clinical characteristics and genetic backgrounds of MSMD in China. In this review, on the one hand, we summarize an update findings on molecular defects and immunological mechanisms in the field of MSMD research globally. On the other hand, we undertook a systematic review of PubMed (MEDLINE), the Cochrane Central Register of Controlled Trials (CENTRAL), Web of Science, EMBASE, CNKI, and Wanfang to identify articles published before Jan 23, 2022, to summarize the clinical characteristics, diagnosis, treatment, and prognosis of MSMD in China. All the English and Chinese publications were searched without any restriction on article types.

Conjugated microporous polymers (CMP) as porous functional materials have received considerable attention due to their unique structures and fascinating properties for the adsorption and degradation of dyes. Herein, a triazine-conjugated microporous polymer material with rich N-donors at the skeleton itself was successfully synthesized via the Sonogashira–Hagihara coupling by a one–pot reaction. These two polymers had Brunauer–Emmett–Teller (BET) surface areas of 322 and 435 m2g−1 for triazine-conjugated microporous polymers (T-CMP) and T-CMP-Me, respectively. Due to the porous effects and the rich N-donor at the framework, it displayed a higher removal efficiency and adsorption performance compared to cationic-type dyes and selectivity properties for (methylene blue) MB+ from a mixture solution of cationic-type dyes. Furthermore, the T-CMP-Me could quickly and drastically separate MB+ and (methyl orange) MO− from the mixed solution within a short time. Their intriguing absorption behaviors are supported by 13C NMR, UV−vis absorption spectroscopy, scanning electron microscopy, and X-ray powder diffraction studies. This work will not only improve the development of porous material varieties, but also demonstrate the adsorption or selectivity of porous materials for dyes from wastewater.

Abstract Immunofluorescence is indispensable to monitor redistribution of proteins involved in phagosome–lysosome association pathway-relevant (P–LApr) proteins. The software digitizing the signals of these proteins in an unbiased and automated manner is generally costly and not widely available. The open-source ImageJ plugin EzColocalization, which is for co-localization analysis of reporters in cells, was not straightforward and sufficient for such analysis. We describe here the input of custom Java code in a novel tailored protocol using EzColocalization to digitize the signals of punctum-distributed P–LApr proteins co-localized with phagosomes and to calculate percentages of phagosomes engaged. We showed that SYBR Gold nucleic acid dye could visualize intracellular mycobacteria that did not express a fluorescent protein. This protocol was validated by showing that IFN-γ enhanced the co-localization of a punctum-distributed P–LApr protein (LC3) with Mycobacterium bovis BCG in the monocyte/macrophage-like RAW264.7 cells and that there was greater co-localization of LC3 with BCG than with M. tuberculosis H37Rv in bone marrow-derived macrophages (BMDMs). Although BCG and a derived strain (rBCG-PA) showed a similarly high degree co-localization with LC3 in BMDMs, in RAW264.7 cells BCG showed much less co-localization with LC3 than rBCG-PA indicating the need for caution in interpreting biological significance from studies in cell lines.

Tuberculosis (TB), caused by respiratory infection with Mycobacterium tuberculosis, remains a major global health threat. The only licensed TB vaccine, the one-hundred-year-old Bacille Calmette-Guérin has variable efficacy and often provides poor protection against adult pulmonary TB, the transmissible form of the disease. Thus, the lack of an optimal TB vaccine is one of the key barriers to TB control. Recently, the development of highly efficacious COVID-19 vaccines within one year accelerated the vaccine development process in human use, with the notable example of mRNA vaccines and adenovirus-vectored vaccines, and increased the public acceptance of the concept of the controlled human challenge model. In the TB vaccine field, recent progress also facilitated the deployment of an effective TB vaccine. In this review, we provide an update on the current virus-vectored TB vaccine pipeline and summarize the latest findings that might facilitate TB vaccine development. In detail, on the one hand, we provide a systematic literature review of the virus-vectored TB vaccines are in clinical trials, and other promising candidate vaccines at an earlier stage of development are being evaluated in preclinical animal models. These research sharply increase the likelihood of finding a more effective TB vaccine in the near future. On the other hand, we provide an update on the latest tools and concept that facilitating TB vaccine research development. We propose that a pre-requisite for successful development may be a better understanding of both the lung-resident memory T cell-mediated mucosal immunity and the trained immunity of phagocytic cells. Such knowledge could reveal novel targets and result in the innovative vaccine designs that may be needed for a quantum leap forward in vaccine efficacy. We also summarized the research on controlled human infection and ultra-low-dose aerosol infection murine models, which may provide more realistic assessments of vaccine utility at earlier stages. In addition, we believe that the success in the ongoing efforts to identify correlates of protection would be a game-changer for streamlining the triage of multiple next-generation TB vaccine candidates. Thus, with more advanced knowledge of TB vaccine research, we remain hopeful that a more effective TB vaccine will eventually be developed in the near future.

Abstract Traditionally, immune memory is regarded as an exclusive hallmark of adaptive immunity. However, a growing body of evidence suggesting that innate immune cells show adaptive characteristics has challenged this dogma. In the past decade, trained immunity, a de facto innate immune memory, has been defined as a long‐term functional reprogramming of cells of the innate immune system: the reprogramming is evoked by endogenous or exogenous insults, the cells return to a nonactivated state and subsequently show altered inflammatory responses against a second challenge. Trained immunity became regarded as a mechanism selected in evolution to protect against infection; however, a maladaptive effect might result in hyperinflammation. This dual effect is consistent with the Yin‐Yang theory in traditional Chinese philosophy, in which Yang represents active, positive, and aggressive factors, whereas Yin represents passive, negative, and inhibitory factors. In this review, we give a brief overview of history and latest progress about trained immunity, including experimental models, inductors, molecular mechanisms, clinical application and so on. Moreover, this is the first time to put forward the theory of Yin‐Yang balance to understand trained immunity. We envision that more efforts will be focused on developing novel immunotherapies targeting trained immunity in the coming years.

The neuron derived synaptic adhesion molecular neuroligin-3 (NLGN3) plays an important role in glioma growth. While the role of autocrine NLGN3 in glioma has not been well-studied. The expression of NLGN3 in glioma was detected using immunohistochemistry. We further explored its function and regulatory mechanism in U251 and U87 cells with high expression of NLGN3. Knockdown of endogenous NLGN3 significantly reduced the proliferation, migration, and invasion of glioma cells and down-regulated the activity of the PI3K-AKT, ERK1/2, and LYN signaling pathways. In comparison, overexpression of NLGN3 yielded opposite results. Our results further demonstrate that LYN functions as a feedback mechanism to promote NLGN3 cleavage. This feedback regulation was achieved by upregulating the ADAM10 sheddase responsible for NLGN3 cleavage. Inhibition of ADAM10 suppressed the proliferation, migration, and invasion of glioma cells; oppositely, the expression of ADAM10 was correlated with a higher likelihood of lower grade glioma (LGG) in the brain. Our study demonstrates that glioma-derived NLGN3 promotes glioma progression by upregulating activity of LYN and ADAM10, which in turn promote NLGN3 cleavage to form a positive feedback loop. This pathway may open a potential therapeutic window for the treatment of human glioma.

While Baccillus Calmette-Guerin (BCG) is used worldwide, tuberculosis (TB) is still a global concern due to the poor efficacy of BCG. Novel vaccine candidates are therefore urgently required. In this study, two attenuated recombinant Listeria strains, LMΔ-msv and LIΔ-msv were constructed by deletion of actA and plcB and expression of a fusion protein consisting of T cell epitopes from four Mycobacterium tuberculosis (Mtb) antigens (Rv2460c, Rv2660c, Rv3875, and Rv3804c). The safety and immunogenicity of the two recombinant strains were evaluated in C57BL/6J mice. After intravenous immunization individually, both recombinant strains entered liver and spleen but eventually were eliminated from these organs after several days. Simultaneously, they induced antigen-specific cell-mediated immunity, indicating that the recombinant Listeria strains were immunogenic and safe in vivo. LMΔ-msv immunization induced stronger cellular immune responses than LIΔ-msv immunization, and when boosted with LIΔ-msv, antigen-specific IFN-γ CD8+ T cell responses were notably magnified. Furthermore, we evaluated the protection conferred by the vaccine candidates against mycobacterial infection via challenging the mice with 1 × 107 CFU of BCG. Especially, we tested the feasibility of application of them as heterologous BCG supplement vaccine by immunization of mice with BCG firstly, and boosted with LMΔ-msv and LIΔ-msv sequentially before challenging. Combination immune strategy (LMΔ-msv prime-LIΔ-msv boost) conferred comparable protection efficacy as BCG alone. More importantly, BCG-vaccinated mice acquired stronger resistance to Mycobacterial challenge when boosted with LMΔ-msv and LIΔ-msv sequentially. Our results inferred that heterologous immunization with Listeria-based recombinant strains boosted BCG-primed protection against pulmonary mycobacterial infection.

BackgroundCD69 is a biomarker of T-cell activation status, but its activation status in human Mycobacterium tuberculosis (Mtb) infection remains elusive.MethodsA set of cohorts of patients with different tuberculosis (TB) infection status including active TB patients (ATB), latent tuberculous infection patients (LTBI) and close contacts (CCs) of ATB was designed, and the expression profiles of CD69 and several T-cell markers were determined on Mtb antigen-stimulated T cells by flow cytometry.ResultsThe frequencies of CD4+ and CD8+ T cells were both comparable among Mtb-infected individuals including ATB and LTBI, which guaranteed the consistency of the background level. A t-Distributed Stochastic Neighbor Embedding (tSNE) analysis on a panel of six phenotypic markers showed a unique color map axis gated on T cells in the CCs group compared with ATB and LTBI populations. By further gating on cells positive for each individual marker and then overlaying those events on top of the tSNE plots, their distribution suggested that some markers were expressed differently in the CCs group. Further analysis showed that the expression levels of CD69 on both CD4+ and CD8+ T cells were significantly lower in the CCs group, especially in interferon-γ-responding T cells.ConclusionOur findings suggest that the T-cell activation status of CD69 is associated with Mtb infection and may have the potential to distinguish LTBI from those populations who have been exposed continuously to Mtb but have not become infected.

The perturbed genes from transcriptomes are often presented in terms of relative expressions against control samples. However, the probe signal values (PSVs) of genes, implying protein abundances, are often ignored. Here, we explored the PSVs in tuberculosis (TB)-relevant signature genes. The signatures from Mycobacterium tuberculosis-infected THP-1 cells were defined as induced (TMtb-i, with a derived TMtb-iNet) and repressed (TMtb-r). The signature from human blood was defined as a pulmonary TB (PTB)-specific signature (PTBsig). The analysis showed that before infection, TMtb-i and TMtb-iNet had lower PSVs and TMtb-r genes had average PSVs. In the blood of healthy donors, PTBsig (divided into up-regulated PTBsigUp and down-regulated PTBsigDn) displayed average PSVs. This was partly due to masking by the cellular heterogeneity of blood; diverse PSVs were seen in constituent cell populations (CD4/8+ T, monocytes and neutrophils). Specifically, the PSVs of PTBsigUp in the neutrophils of healthy donors were higher (implying higher protein abundances), and much higher in the neutrophils of PTB (implying excessive protein abundances). Based on the PSV patterns of PTBsigUp in four cell populations, we identified three representative highly homologous genes (FCGR1A, FCGR1B, and the pseudogene FCGR1CP, which were often poorly distinguished), of which the summed PSVs were the highest in the neutrophils of PTB patients and healthy donors. The three genes were all up-regulated and responsive to chemotherapy in the blood of PTB, as validated in an RNA-seq-based analysis. This PSV-based study confirms the excessive involvement of neutrophil FCGR1 in PTB.

A simple mRNA vaccine was shown to protect mice against tuberculosis more than 15 years ago. Like COVID-19, tuberculosis is a respiratory infection killing over a million people per year. It too presents a global emergency. Can the stunning success of RNA vaccination against COVID-19 be replicated for TB?

Mycobacterial lipoproteins are considered to be involved in both virulence and immunoregulatory processes during Mycobacterium tuberculosis (M.tb) infection. In our previous investigations on the immunoreactivity of more than 30 M.tb proteins in active TB patients, we identified mycobacterial lipoprotein Z (LppZ) as one of the most immune dominant antigens. How LppZ triggers immune responses is still unclear. In this study, we analyzed LppZ-mediated innate and adaptive immunity using a murine air pouch model and an M.tb infection model, respectively. We found that LppZ could not only recruit inflammatory cells but also induce the production of proinflammatory cytokines inside the pouches. LppZ could also induce strong Th1 responses following immunization and confer protection against challenge with M.tb virulent strain H37Rv at a similar level to BCG vaccination but with less pathological damage in the lungs. Furthermore, we revealed the presence of LppZ-specific functional CD4+ T cells in the lungs of the challenged mice that were capable of secreting double or triple cytokines, including IFN-γ, IL-2, and TNF-α. Our study thus demonstrates that LppZ is of strong immunogenicity during M.tb infection in both humans and mice and has the ability to trigger effective innate and cellular immunity. Considering the limitations of candidate antigens in the pipeline of TB vaccine development, LppZ-mediated immune protection against M.tb challenge in the mouse model implies its potential application in vaccine development.

The kinds of vaccine-induced T cell responses that are beneficial for protection against Mycobacterium tuberculosis (Mtb) infection are not adequately defined. We had shown that a novel Sendai virus vectored vaccine, SeV85AB, was able to enhance immune protection induced by bacille Calmette–Guérin (BCG) in a prime-boost model. However, the profile of T cell responses boosted by SeV85AB was not determined. Herein, we show that the antigen-specific CD4+ and CD8+ T cell responses were both enhanced by the SeV85AB boost after BCG. Different profiles of antigen-specific po T cell subsets were induced in the local (lung) and systemic (spleen) sites. In the spleen, the CD4+ T cell responses that were enhanced by the SeV85AB boost were predominately IL-2 responses, whereas in the lung the greater increases were in IFN-γ- and TNF-α-producing CD4+ T cells; in CD8+ T cells, although IFN-γ was enhanced in both the spleen and lung, only IL-2+TNF-α+CD8+ T subset was boosted in the latter. After a challenge Mtb infection, there were significantly higher levels of recall IL-2 responses in T cells. In contrast, IFN-γ-producing cells were barely boosted by SeV85AB. After Mtb challenge a central memory phenotype of responding CD4+ T cells was a prominent feature in SeV85AB-boosted mice. Thus, our data strongly suggest that the enhanced immune protection induced by SeV85AB boosting was associated with establishment of an increased capacity to recall antigen-specific IL-2-mediated T cell responses and confirms this Sendai virus vector system as a promising candidate to be used in a heterologous prime-boost immunization regimen against TB.

Previous transcriptomic analysis revealed a 393-transcript signature (PTBsig), which is dominated by interferon inducible genes, in whole blood of pulmonary tuberculosis (PTB) patients. Comparisons with a limited set of interferon-driven genes among separated monocytes, CD4+ T cells, CD8+ T cells, and neutrophils indicated that the signature is due to changes in neutrophils, the overwhelmingly predominant cell type. By extending the analysis to the entire 393 transcripts of PTBsig and by switching the cell proportions between separated monocytes, CD4+ T cells, CD8+ T cells, and neutrophils, we create putative PTBsig for whole blood (pPTBsig) in which CD4+ or CD8+ T cells or monocytes predominated or in which the cell proportions were unchanged. These putative signatures are then compared to the actual reported PTBsig. We show that, because of their predominance in peripheral blood and their larger transcriptional responses, neutrophils were indeed almost exclusively responsible for PTBsig. We caution that the functional significance of changes in other cell types might escape notice in transcriptome analysis that is based upon whole blood.

One approach for improving BCG efficacy is to utilize BCG as vehicle to develop recombinant BCG (rBCG) strains overexpressing Mycobacterium tuberculosis (M. tb) antigens. Also expression level of a candidate antigen should impact the final T cell responses conferred by rBCG. In this study, based on our previously constructed differential expression system, we developed two rBCG strains overexpressing M. tb chimeric antigen Ag856A2 (coding a recombinant ag85a with 2 copies of esat-6 inserted at Acc I site of ag85a) at differential levels under the control of the subtly modified furA promoters. These two rBCG strains were used to vaccinate C57BL/6 mice and exploit dose of incorporated antigen in rBCG to optimize immune response and protective efficiency against M. tb challenge in mouse model. The results showed that rBCG strains overexpressing Ag856A2 at differential levels induced different antigen-specific IFN-γ production and comparable number of M. tb-specific CD4 T cells expressing IL-2. M. tb challenge experiment showed that rBCG strains afforded enhanced but comparable immune protection characterized by reduced bacillary load, lung pathology, and inflammation. These results suggested that the dose of antigens incorporated in rBCG can impact T cell immune responses but imposed no significantly differential protective efficacies.

Generation of reactive oxygen species and reactive nitrogen species in phagocytes is an important innate immune response mechanism to eliminate microbial pathogens. It is known that deoxynucleotides (dNTPs), the precursor nucleotides to DNA synthesis, are one group of the significant targets for these oxidants and incorporation of oxidized dNTPs into genomic DNA may cause mutations and even cell death. Here we show that the mycobacterial dNTP pyrophosphohydrolase MazG safeguards the bacilli genome by degrading 5-OH-dCTP, thereby, preventing it from incorporation into DNA. Deletion of the (d)NTP pyrophosphohydrolase-encoding mazG in mycobacteria leads to a mutator phenotype both under oxidative stress and in the stationary phase of growth, resulting in increased CG to TA mutations. Biochemical analyses demonstrate that mycobacterial MazG can efficiently hydrolyze 5-OH-dCTP, an oxidized nucleotide that induces CG to TA mutation upon incorporation by polymerase. Moreover, chemical genetic analyses show that direct incorporation of 5-OH-dCTP into mazG-null mutant strain of Mycobacterium smegmatis (Msm) leads to a dose-dependent mutagenesis phenotype, indicating that 5-OH-dCTP is a natural substrate of mycobacterial MazG. Furthermore, deletion of mazG in Mycobacterium tuberculosis (Mtb) leads to reduced survival in activated macrophages and in the spleen of infected mice. This study not only characterizes the mycobacterial MazG as a novel pyrimidine-specific housecleaning enzyme that prevents CG to TA mutation by degrading 5-OH-dCTP but also reveals a genome-safeguarding mechanism for survival of Mtb in vivo.