Multi-omics analysis reveals that linoleic acid metabolism is associated with variations of trained immunity induced by distinct BCG strains.

Trained immunity is one of the mechanisms by which BCG vaccination confers persistent nonspecific protection against diverse diseases. Genomic differences between the different BCG vaccine strains that are in global use could result in variable protection against tuberculosis and therapeutic effects on bladder cancer. In this study, we found that four representative BCG strains (BCG-Russia, BCG-Sweden, BCG-China, and BCG-Pasteur) covering all four genetic clusters differed in their ability to induce trained immunity and nonspecific protection. The trained immunity induced by BCG was associated with the Akt-mTOR-HIF1α axis, glycolysis, and NOD-like receptor signaling pathway. Multi-omics analysis (epigenomics, transcriptomics, and metabolomics) showed that linoleic acid metabolism was correlated with the trained immunity-inducing capacity of different BCG strains. Linoleic acid participated in the induction of trained immunity and could act as adjuvants to enhance BCG-induced trained immunity, revealing a trained immunity-inducing signaling pathway that could be used in the adjuvant development.

Natural products in anti-tuberculosis host-directed therapy.

Given that the disease progression of tuberculosis (TB) is primarily related to the host's immune status, it has been gradually realized that chemotherapy that targets the bacteria may never, on its own, wholly eradicate Mycobacterium tuberculosis, the causative agent of TB. The concept of host-directed therapy (HDT) with immune adjuvants has emerged. HDT could potentially interfere with infection and colonization by the pathogens, enhance the protective immune responses of hosts, suppress the overwhelming inflammatory responses, and help to attain a state of homeostasis that favors treatment efficacy. However, the HDT drugs currently being assessed in combination with anti-TB chemotherapy still face the dilemmas arising from side effects and high costs. Natural products are well suited to compensate for these shortcomings by having gentle modulatory effects on the host immune responses with less immunopathological damage at a lower cost. In this review, we first summarize the profiles of anti-TB immunology and the characteristics of HDT. Then, we focus on the rationale and challenges of developing and implementing natural products-based HDT. A succinct report of the medications currently being evaluated in clinical trials and preclinical studies is provided. This review aims to promote target-based screening and accelerate novel TB drug discovery.

High accuracy of recombinant fusion protein early secretory antigenic target protein 6-culture filtrate protein 10 skin test for the detection of tuberculosis infection: a phase III, multi-centered, double-blind, hospital-based, randomized controlled trial.

OBJECTIVES: To verify the diagnostic utility of recombinant fusion protein ESAT6-CPF10 (EC), a novel skin test reagent to detect Mycobacterium tuberculosis infection. METHODS: A multi-centered, double-blind, randomized controlled trial was conducted from December 17, 2015, to March 2, 2018. Participants involved in this study included those with active tuberculosis (TB), suspected pulmonary TB, or non-TB pulmonary disease. Each participant received three tests simultaneously, TB-specific enzyme-linked immunospot assay (T-SPOT.TB), tuberculin skin test (TST), and EC skin test (ECST), and adverse events were reported. RESULTS: Diagnostic accuracy was analyzed using data from 1085 protocol-compliant participants. The sensitivities of the ECST, TST, and T-SPOT.TB were 91.2% (95% CI, 89.0-93.2%), 91.4% (95% CI, 89.1-93.3%), and 92.1% (95% CI, 89.9-93.9%), respectively. The specificities of the ECST (69.7%, 95% CI, 64.5-74.5%) and T-SPOT.TB (76.1%, 95% CI, 71.2-80.5%) were significantly higher than the TST (54.4%, 95% CI, 48.9-59.7%). The agreements between ECST and TST (kappa = 0.632) and between ECST and T-SPOT.TB (kappa = 0.780) were substantial. No severe adverse event was reported. CONCLUSION: The diagnostic performance of the ECST was close to the T-SPOT.TB assay in the detection of TB infection and indicated good potential for clinical application in common scenarios.

An Updated Review on MSMD Research Globally and A Literature Review on the Molecular Findings, Clinical Manifestations, and Treatment Approaches in China.

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.

Research Advances for Virus-vectored Tuberculosis Vaccines and Latest Findings on Tuberculosis Vaccine Development.

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.

Trained immunity: A Yin-Yang balance.

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.

A two-dose optimum for recombinant S1 protein-based COVID-19 vaccination.

BACKGROUND: Recombinant protein subunit vaccination is considered to be a safe, fast and reliable technique when combating emerging and re-emerging diseases such as coronavirus disease 2019 (COVID-19). Typically, such subunit vaccines require the addition of adjuvants to attain adequate immunogenicity. AS01, which contains adjuvants MPL and saponin QS21, is a liposome-based vaccine adjuvant system that is one of the leading candidates. However, the adjuvant effect of AS01 in COVID-19 vaccines is not well described yet. METHODS: In this study, we utilized a mixture of AS01 as the adjuvant for an S1 protein-based COVID-19 vaccine. RESULTS: The adjuvanted vaccine induced robust immunoglobulin G (IgG) binding antibody and virus-neutralizing antibody responses. Importantly, two doses induced similar levels of IgG binding antibody and neutralizing antibody responses compared with three doses and the antibody responses weakened only slightly over time up to six weeks after immunization. CONCLUSION: These results suggested that two doses may be enough for a clinical vaccine strategy design using MPL & QS21 adjuvanted recombinant protein, especially in consideration of the limited production capacity of COVID-19 vaccine in a public health emergency.

Mycobacterium tuberculosis Rv3628 isan effective adjuvant via activationof dendritic cells for cancer immunotherapy.

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.

Tailored co-localization analysis of intracellular microbes and punctum-distributed phagosome-lysosome pathway proteins using ImageJ plugin EzColocalization.

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.

Stool-based Xpert MTB/RIF Ultra assay as a tool for detecting pulmonary tuberculosis in children with abnormal chest imaging: A prospective cohort study.

OBJECTIVES: To evaluate the diagnostic efficacy of stool-based Xpert MTB/RIF Ultra assay versus other assays for the detection of paediatric pulmonary tuberculosis (PTB). METHODS: A prospective head-to-head comparative study was conducted from Dec 2017 to May 2019 in Shanghai Public Health Clinical Centre. Samples were collected from children (< 15 years) with abnormal chest imaging (X-ray or CT scan) results for the following tests: Ultra on stool sample (Ultra-Stool), Ultra on respiratory tract sample (Ultra-RTS), Xpert MTB/RIF assay (Xpert) on RTS (Xpert-RTS), acid-fast bacilli smear on RTS (AFB-RTS), and Mycobacterium tuberculosis (Mtb) culture on RTS (Culture-RTS). The results were compared with a composite reference standard. RESULTS: A total of 126 cases with paired results were analysed. Against a composite reference standard, Ultra-RTS demonstrated the highest sensitivity (52%) and specificity (100%). Ultra-Stool showed 84.1% concordance with Ultra-RTS, demonstrating 45.5% sensitivity and 94.7% specificity (kappa = 0.65, 95% CI= 0.51-0.79). The sensitivity of Ultra-Stool was similar to Mtb culture (45.5%, p = 1.000) and higher than AFB-RTS (27.3%, p < 0.05). Assay positivity was associated with age and infiltration range in chest imaging. CONCLUSIONS: When RTS is difficult to obtain, stool sample-based Ultra is a comparable alternative.

Decreased Expression of CD69 on T Cells in Tuberculosis Infection Resisters.

BACKGROUND: CD69 is a biomarker of T-cell activation status, but its activation status in human Mycobacterium tuberculosis (Mtb) infection remains elusive. METHODS: A 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. RESULTS: The 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. CONCLUSION: Our 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.

Probe Signal Values in mRNA Arrays Imply an Excessive Involvement of Neutrophil FCGR1 in Tuberculosis.

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.

Heterologous prime-boost vaccination against tuberculosis with recombinant Sendai virus and DNA vaccines.

In an earlier study, a novel Sendai virus-vectored anti-tuberculosis vaccine encoding Ag85A and Ag85B (SeV85AB) was constructed and shown to elicit antigen-specific T cell responses and protection against Mycobacterium tuberculosis (Mtb) infection in a murine model. In this study, we evaluate whether the immune responses induced by this novel vaccine might be elevated by a recombinant DNA vaccine expressing the same antigen in a heterologous prime-boost vaccination strategy. The results showed that both SeV85AB prime-DNA boost (SeV85AB-DNA) and DNA prime-SeV85AB boost (DNA-SeV85AB) vaccination strategies significantly enhanced the antigen-specific T cell responses induced by the separate vaccines. The SeV85AB-DNA immunization regimen induced higher levels of recall T cell responses after Mtb infection and conferred better immune protection compared with DNA-SeV85AB or a single immunization. Collectively, our study lends strong evidence that a DNA vaccine boost might be included in a novel SeV85AB immunization strategy designed to enhance the immune protection against Mtb. KEY MESSAGES: A heterologous prime-boost regimen with a novel recombinant SeV85AB and a DNA vaccine increase the T cell responses above those from a single vaccine. The heterologous prime-boost regimen provided protection against Mtb infection. The DNA vaccine might be included in a novel SeV85AB immunization strategy designed to enhance the immune protection against Mtb.

Differential T cell responses against DosR-associated antigen Rv2028c in BCG-vaccinated populations with tuberculosis infection.

The IFN-γ release assays (IGRAs) based on region of difference 1 (RD1) antigens have improved diagnosis of Mycobacterium tuberculosis (Mtb) infection. However, IGRAs with these antigens could not distinguish latent tuberculosis infection (LTBI) from active tuberculosis (ATB). DosR regulon genes are thought to be important for Mtb dormancy, and their products have higher immunogenicity in LTBI than ATB individuals, suggesting protective immunity mediated by DosR regulon-encoded antigens and potential utility of them for differential diagnostics of Mtb-infected populations or development of therapeutic vaccines against tuberculosis (TB). Among them, Rv2028c is a dormancy-related antigen that has demonstrated potential use in TB control, but its immunological characteristics in the BCG-vaccinated Chinese population are unknown. In this study, a total of 148 individuals, including 98 patients with ATB, 20 cases with LTBI and 30 healthy controls, were tested for Rv2028c-specific T cell responses by using an IFN-γ ELISA assay. The results showed that the T-cell responses in LTBI individuals were almost always higher than those in ATB patients, regardless of the site of infection or the results of bacteriological examination in the patients. This allowed for good differentiation between these two groups of Mtb-infected individuals even in the BCG-vaccinated high TB-incidence setting that pertains in China. In addition, the diagnostic efficacy for ATB was enhanced by combining the results from Rv2028c and RD1 antigen-based IFN-γ ELISA assays. In conclusion, Rv2028c-specific T-cell responses might contribute to natural protection against dormant Mtb infection, and the determination of these responses can aid discrimination between healthy LTBI individuals and ATB patients in the Mtb-infected populations.

The Profile of T Cell Responses in Bacille Calmette-Guérin-Primed Mice Boosted by a Novel Sendai Virus Vectored Anti-Tuberculosis Vaccine.

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.