Author: Ke Hui Fan Lin
Affiliation: Department of Tuberculosis, Shanghai Pulmonary Hospital, Tongji University, Shanghai Clinical Research Center for Infectious Diseases (Tuberculosis), Shanghai Key Laboratory of Tuberculosis (Pulmonary).
Cite this article: Ke Hui, Fan Lin. Annual progress in tuberculosis immunotherapy 2023 [J] . Chinese Journal of Tuberculosis and Respiration, 2024, 47(4) : 371-375. DOI: 10.3760/cma.j.cn112147-20231031-00283.
summary
As a chronic infectious disease, tuberculosis is closely related to immune regulation and immune effects. As one of the important means of comprehensive treatment of tuberculosis, immunotherapy can improve the efficacy of tuberculosis and control the spread of tuberculosis. From October 2022 to September 2023, a number of immunotherapy studies on tuberculosis were conducted at home and abroad, providing new possibilities for the treatment of multidrug-resistant and XDR-TB. The research of host-targeted therapy and therapeutic vaccines is a new direction for the exploration of adjuvant treatment for tuberculosis.
Tuberculosis caused by Mycobacterium tuberculosis (MTB) infection remains the leading cause of death from infectious diseases. According to the World Health Organization's Global TB Report 2023, the global estimated number of TB cases in 2022 was 10.6 million, 1.3 million deaths, and 410,000 cases of multidrug/rifampicin-resistant TB (MDR/RR-TB) [1]. Tuberculosis is a chronic infectious disease, the onset and progression of the disease are closely related to the immune status of the host, immunotherapy is one of the important methods for the treatment of tuberculosis, tuberculosis can regulate the immune effect of the body through immunotherapy, so as to accelerate the transformation of sputum bacteria into negative, improve the success rate of treatment, and shorten the course of treatment. This article intends to review the progress of tuberculosis immunotherapy research from October 2022 to September 2023 from three levels: cell experiments, animal experiments, and clinical studies.
1. Research related to cell experiments
Host-oriented therapy is one of the main strategies for immunotherapy for tuberculosis in recent years, which shifts the direction of antimicrobial treatment to target cells and proteins of the host, and regulates the immune function of the host through small molecules, including the reuse of old drugs, nutritional preparations, monoclonal antibodies, immunomodulators, etc., to achieve the purpose of immunotherapy by regulating the immune function of the host [2]. MTB has a variety of antigens that interact with host cells through various pattern recognition receptors, among which Toll-like receptors (TLR) 2 and 4 are the two main pattern recognition receptors, and different MTB ligands activate TLR2/4 receptors to alter the host immune response by activating downstream signaling cascade antagonism or agonism[3]. Understanding the signaling networks and cross-conversations between immune networks is important for the development of host-directed immunization strategies against MTB. It is urgent and meaningful to identify host-targeted targets and molecular markers of immune response for tuberculosis immunotherapy. Previous studies have demonstrated that autophagy plays an important role in both the infectious process and post-infection pathogenesis of tuberculosis, and is related to the prognosis of tuberculosis treatment. Cytokine therapy and cytokine-mediated autophagy have been used as primary host-directed therapies to inhibit the growth of MTBs in host cells. Mishra et al. [4] investigated the antituberculous activity of soybean lectin (SBL) in THP-1 (dTHP-1) cells induced by cytokine-mediated autophagy. After SBL treatment, the expression of interleukin (IL)-6 in MTB-infected dTHP-1 cells was significantly increased through the P2X-ligand-gated ion channel 7 (P2RX7)-mediated pathway. SBL interacts with P2RX7 to regulate the PI3K/Akt/CREB network and promote the secretion and release of IL-6 from dTHP-1 cells, which in turn activates the JAK2/STAT3/Mcl-1 pathway, regulates autophagy by interacting with IL-6Rα, and ultimately controls the growth of MTB in macrophages. In addition, Xiao et al. [5] found that sulforaphane (SFN) reduces intracellular bacterial load by enhancing autophagy in THP-1 cells, and showed that autophagy-related genes are associated with immune infiltration of tuberculosis, and SFN can regulate three key genes related to autophagy, especially the autophagy-related gene FOXO1, which will provide new targets for clinical tuberculosis diagnosis and immunotherapy strategies. Since MTB mainly persists in macrophages after infecting the host, targeting macrophages with anti-TB drugs can help improve the anti-TB effect. Phosphatidylserine (PS) is a biofilm phospholipid that is commonly found in the inner part of cell membranes. During apoptosis, PS can flip from the inside of the cell membrane to the surface of the cell membrane, showing a specific "eat-me" signal that is recognized by specific receptors on macrophages. Tian et al. [6] used this macrophage targeting property of PS to construct PCN-CpG@PS nanocomposites to form a targeted drug delivery system. In this study. CpG oligodeoxynucleotides (CpG ODNs) are synthetic oligodeoxyribonucleotide sequences that induce a Th1-type immune response to kill MTB by stimulating TLRs in mammalian immune cells. However, due to the negative charge and easily degradable nature of CpG ODNs, nanomaterials are needed to transport them into cells. PCN-CpG@PS is designed to target macrophages at the lesion site and kill intracellular MTB, and studies have shown that the targeted nanocomposites have certain anti-tuberculosis effects and good safety in vitro. Kumari et al. [7] explored the role of withaferin A (WA) as an immunotherapy drug for tuberculosis in another study, suggesting that WA can inhibit intracellular surviving MTB by enhancing the host's immune response, and can also guide host macrophages to switch to defensive M1 polarization, enhancing the host's Th1 and Th17 immune responses to MTB infection. These results suggest that WA can be used as an effective adjuvant immunomodulator to enhance the function of host protective memory cells by promoting STAT signaling, thereby improving the host's defense against MTB.
2. Research related to animal experiments
1. Targeting the glutamine metabolic antagonist JHU083: MTB has evolved a series of different determinants to disrupt host immunity and alter host metabolic patterns. However, the metabolic mechanisms by which pathogens interfere with hosts are still poorly understood. Parveen et al. [8] demonstrated that a new glutamine metabolism antagonist, JHU083, could inhibit the proliferation of MTB in vitro and in vivo, which showed that the weight gain, survival rate of mice treated with JHU083 increased, the bacterial load in the lungs decreased by 2.5 log at 35 days after infection, and the pathological damage in the lungs was reduced. Compared with the uninfected and rifampicin-treated control group, JHU083 treatment initiates T cell recruitment earlier, increases pro-inflammatory myeloid cell infiltration, and decreases the aggregation of suppressor myeloid cells. Metabolomic analysis of lung tissues of JHU083-treated MTB-infected mice showed decreased glutamine levels, citrulline accumulation, increased nitric oxide (NO) synthase activity, and decreased levels of quinoline acid (produced by the immunosuppressive metabolite kynurenine). JHU083-treated macrophages produced more NO and enhanced the antimicrobial activity of macrophages. When tested in a mouse model of immunocompromised MTB infection, a reduced therapeutic effect of JHU083 was found, suggesting that the host-oriented immunomodulatory effects induced by the drug predominate in the efficacy. Therefore, JHU083, as a glutamine metabolism antagonist, has dual effects of immunomodulatory and antibacterial effects on MTB, and has potent host-oriented anti-tuberculosis activity. 2. Protease-based therapy: Cathepsin in host cells can regulate MTB and reduce the proteolytic activity of macrophages, thereby improving the survival rate of pathogens, which is a promising therapeutic target. Mandal et al. [9] showed that the enzyme protein inhibitor cystatin F was significantly increased in host cells during MTB infection. The use of siRNA silencing cystatin F can improve the proteolytic activity of cathepsin S, L, and B, and significantly affect the killing capacity of pathogens in macrophages, suggesting that cystatin F can be used as a potential immunotherapy for tuberculosis, especially multidrug-resistant and XDR-TB. In addition, in MTB, caseinolytic protease P (ClpP) proteolysis includes ClpP1, ClpP2, and AAA1 chaperone proteins, among which ClpP and its AAA1 chaperone can maintain protein homeostasis in MTB cells, which is essential for bacterial survival and is one of the important biological targets [10], with the potential to develop new drugs for the treatment of multidrug-resistant tuberculosis. Over the past 10 years, a number of modulators targeting MTB ClpP1P2 have been identified and characterized, and some of them have shown potent antituberculous activity [10]. 3. Vγ2Vδ2 T cell immunotherapy: Previous studies have suggested that phosphoantigen-specific Vγ2Vδ2 T cells can play a protective role in tuberculosis immunity. Enhancement of protective Vγ2Vδ2 effector T cells improves treatment outcomes in patients with MDR-TB. Shen et al. [11] used the clinically approved drugs zoledronate sodium (ZOL) and interleukin-2 (IL-2) to induce Vγ2Vδ2 effector T cells with antituberculous effects as adjuvant immunotherapy for macaques infected with MDR-TB strains. The study suggested that the use of ZOL/IL-2 as an adjuvant treatment to chemotherapy in macaques infected with MDR-TB strains could significantly expand Vγ2Vδ2 T cells, enhance and maintain the secretion of protective cytokines by the Vγ2Vδ2 effector T cell subset, and the effect could be maintained until the 21st week after treatment. ZOL/IL-2 can significantly increase and maintain the number of CD4+Th1 and CD8+Th1-like effector cells in the blood circulation while increasing the number of Vγ2Vδ2 T cells, and γδ T cells or αβ effector T cells can migrate to the airways at the 3rd week after MDR-TB infection, and the effect can be maintained until 19 or 21 weeks. The results showed that compared with the control group, the adjuvant use of ZOL/IL-2 significantly reduced the bacterial load in the host lungs and alleviated the pathological damage in the host body of MDR-TB. It can be seen that zoledronate sodium combined with IL-2 can increase the number of Vγ2vδ2 T cells and αβ effector T cells with anti-tuberculosis effect, thereby improving the therapeutic effect of MDR-TB. 4. Mesenchymal stem cell immunotherapy: Mesenchymal stem cells (MSCs) have a variety of functions, including regeneration, promoting wound healing, and regulating immune signaling. These pluripotent stem cells also play a crucial role in regulating various aspects of the immune system, and MTB can infect mesenchymal stem cells and evade their elimination by the immune system, which has been shown to be used as an adjuvant therapy for tuberculosis. Chenari et al. [12] established a model of pneumonia in BALB/c mice infected with Bacillus Calmette-Guérin (BCG) to investigate the therapeutic effect of nasal administration of MSC supernatant. Mesenchymal stem cells were isolated from mouse adipose tissue, and after the third passage, cell culture fluid was collected. The results showed that the ratio of TNF-α/IL-10 in alveolar lavage fluid of mice infected with BCG group decreased significantly after being given mesenchymal stem cell supplementation, suggesting that MSC treatment could reduce lung tissue damage and induce effective immune protective response. 5. Therapeutic vaccines: Traditional BCG vaccination is an intradermal injection, and providing BCG through another route may enhance the depth and breadth of protection. Kurtz et al. [13] used diversity diplomacy (DO) mice to examine the protective effect induced by BCG when administered by intravenous injection. The study found that intravenously inoculated DO mice had a greater BCG distribution throughout their organs compared to subcutaneously vaccinated mice, but there was no significant reduction in MTB burden in the lungs and spleen, and there was no significant alteration in lung inflammation. Nonetheless, DO mice injected with intravenous BCG had a higher survival rate than mice inoculated by the conventional route, and it has been detected in different small animal models that intravenous route delivery of BCG can enhance the protective effect. MTB Ag85A and Ag85B proteins are secreted proteins and antigens recognized by host innate immune cells in the early stage, and have good immunogenicity. Previous studies have developed ag85a/b chimeric DNA vaccines that can induce significant Th1 and CTL cell immune responses by intramuscular injection (IM) and electroporation (EP) inoculation in MTB mouse models, but the mechanism of action between the two vaccine immunization methods is still unclear. Wang et al. [14] suggested that most of the upregulated differential genes in MTB mice were related to nutrient digestion and absorption or neuroendocrine, while most of the down-regulated differential genes were related to cell structure and functional proteins, especially those of alveolar epithelial cells. In the Ag85a/b DNA IM group and EP group, most of the abnormal up- or down-regulated differential genes were restored. The results showed that MTB infection caused accelerated catabolism and slowed down anabolism in mice, and the effective dose of ag85a/b DNA vaccine could significantly up-regulate immune-related pathways and restore metabolic disorders and damage caused by MTB. The new multi-component vaccine combines a variety of immune dominant antigens to form a vaccine with a broad spectrum of antigens, and it is the development trend of tuberculosis vaccine to induce protective immune response. Wang et al. [15] constructed three antigen panels using the epitope-rich protein subunits of T cells: EPC002, ECA006, and EPCP009. Purified proteins EPC002f, ECA006f, EPCP009f and recombinant purified protein mixtures EPC002m (a mixture of CFP-10, ESAT-6 and nPPE18), ECA006m (a mixture of CFP-10, ESAT-6 and Ag85B) and EPCP009m (a mixture of CFP-10, ESAT-6, nPPE18 and nPstS1) were used as antigens and formulated with gelatin adjuvant. The immunogenicity and efficacy of BALB/c mice were analyzed by immunoassay. In vitro MTB growth inhibition experiments showed that EPCP009m had the strongest inhibitory effect on MTB growth, which was significantly better than that of the other four vaccine candidates. EPCP009m, which contains 4 immunodominant antigens, has good immunogenicity and ability to inhibit MTB growth in vitro, and may be a promising vaccine candidate for tuberculosis control. In addition, Yu et al. [16] selected ESAT-6, CFP-10, two full-length antigens, and the T cell epitope peptide antigen nPstS1 of PstS1 to form a multicomponent protein antigen, named ECP001, which includes two types, one is the mixed protein antigen ECP001m, and the other is the fusion expression protein antigen ECP001f, as candidates for protein subunit vaccines. A novel subunit vaccine was constructed by mixing or fusing three proteins in combination with aluminum hydroxide adjuvant, and the immunogenicity and protective performance of the vaccine were evaluated in mice. The results showed that ECP001 stimulated mice to produce high titers of IgG, IgG1 and IgG2a antibodies. At the same time, mouse splenocytes secrete high levels of IFN-γ and a variety of specific cytokines. In addition, ECP001 inhibits MTB proliferation in vitro with comparable ability to BCG. The study suggests that ECP001 is a novel and effective multi-component subunit vaccine candidate, which has the potential to be used as a BCG initial immunization - ECP001 booster immunization or a vaccine for the treatment of MTB infection. 6. Metformin: Metformin controls its growth by increasing host cell viability and a direct and independent pro-inflammatory response to MTB. MTB growth rates were 14.2 times higher in the metformin-naïve control group than in the metformin group [17]. The effect of metformin combined with isoniazid in controlling the growth of MTB was slightly better than that of isoniazid alone, and metformin was better than isoniazid in regulating cytokine and chemokine responses within 72 h [17]. In addition, metformin can play an auxiliary anti-tuberculosis role by regulating multiple signaling pathways to reduce inflammation and eliminate bacteria. Huang et al. [18] established an experimental model of THP-1 macrophages infected with a bovine MTB attenuated strain (M. bovis), and metformin could up-regulate the expression of peroxisome proliferator-activated receptor γ γ (PPARγ) in MTB-infected macrophages, and further inhibit p38 mitogen-activated protein kinase protein kinase, p38 MAP) to inhibit the inflammatory response of M bovis infected macrophages. PPARγ and p38MAPK, as regulators of inflammatory response, are expected to be candidate targets for tuberculosis prevention and treatment. Metformin enhances the immune response and promotes bacterial clearance, making it a candidate for adjuvant antituberculosis therapy. 7. Allicin: Allicin is the main bioactive component in garlic, which has anti-inflammatory, antioxidant, antibacterial and immunomodulatory effects. Studies have reported that allicin can inhibit inflammatory response, reduce oxidative stress level, improve lung pathological damage, and then exert a protective effect on MTB-infected rats. Zhao Chunyan and Gao Jian [19] investigated the effect of allicin on the phosphatidylinositol-3-kinase (PI3K)/protein kinase B (AKT) pathway on a mouse model of MTB infection. The results showed that the body weight of the mice in the infected group decreased, the number of MTB increased, and the pathological damage of lung tissue was serious. The corresponding trends of mice in the low-dose allicin group, high-dose allicin group and isoniazid group were opposite to those mentioned above (all P<0.05). 740 Y-P (PI3K activator) attenuated the inhibitory effect of high-dose allicin on inflammatory response and the improvement of immune function in mice. Therefore, allicin can protect the immune function of MTB-infected mice by inhibiting the PI3K/Akt pathway. 8. Berberine: Berberine is a quaternary ammonium alkaloid extracted from the traditional Chinese medicine Coptis chinensis, which has anti-inflammatory, antioxidant, anti-atherosclerosis, antibacterial, antitumor and neuroprotective effects. Continuous stimulation of TB-specific antigens induces T cell depletion, and berberine can inhibit T cell glycolysis, regulate T cell activation, and prevent T cell depletion [20]. In addition, berberine can enhance the host's innate defense mechanism against MTB, stimulate the differentiation of Th1/Th17-specific effector memory (TEM), central memory (TCM) and tissue-resident memory (TRM) responses, and enhance the host's resistance to drug-sensitive and drug-resistant Mycobacterium tuberculosis infection. Proteome-wide analysis of PBMCs from healthy individuals with a tuberculin test positive found that berberine-regulated NOTCH3/PTEN/AKT/FOXO1 pathway is the primary mechanism for elevated TEM and TRM responses in human CD4+ T cells [20]. In addition, berberine induces glycolysis leading to enhanced Th1/Th17 responses in human and mouse T cells. Berberine's regulation of T cell memory significantly enhanced BCG-induced anti-tuberculosis immunity and reduced the rate of tuberculosis recurrence and reinfection. These results suggest that adjustment of immune memory is a feasible method to enhance host resistance to tuberculosis, and berberine can be used as a potential adjunct to tuberculosis immunotherapy and immunoprophylaxis. 9. Some antibiotics: In addition to bactericidal effects, some antibiotics can interfere with the host's immune system. Biapenem (BPM) is a carbapenem antibiotic that significantly increases the activation status of innate immune macrophages by enhancing p38 signaling, which further activates CD4+ and CD8+ T cells, adaptive immune cells in the lungs and spleen of MTB-infected mice. BPM treatment significantly enhances the polarization of T lymphocytes to inflammatory subsets such as Th1 and Th17, promoting the production of central memory T cell subsets that can survive for a long time. In mouse models, BPM therapy promoted the generation of a subset of central memory T lymphocytes and significantly reduced the reactivation and reinfection of tuberculosis bacteria, suggesting that BPM could be used as an effective immunomodulator in the adjuvant treatment of tuberculosis [21]. In addition, there is growing evidence that combination therapy with antibiotics and immunomodulators has a better therapeutic effect. Clofazimine (CFZ) promotes the production of central memory T (TCM) cells by blocking Kv1.3+ potassium ion channels; Rapamycin (Rapa) promotes MTB clearance by inducing autophagy. The combination of CFZ and Rapa has effectively reduced multidrug-resistant and extensively drug-resistant MTB clinical isolates in mouse models by inducing potent T memory cells and central memory T cells [22]. As components of the innate immune response, antimicrobial peptides are thought to have potent antimicrobial activity against MTB. Palacios et al. [23] evaluated WBCATH, an antimicrobial peptide from Indian buffalo, to determine its antituberculous activity, cytotoxicity, and effect of the antimicrobial peptide on bacterial burden and cytokine secretion in alveolar macrophages in MTB mice. The results showed that WBCATH had bactericidal activity against both drug-sensitive and multidrug-resistant MTB (MTB structural damage could be observed under electron microscopy), and improved the ability of mouse macrophages to kill MTB and induce the production of protective cytokines. WBCATH can also reduce the bacterial load in drug-susceptible and multidrug-resistant MTB mice. When first-line antibiotics are used in combination with WBCATH, a synergistic effect can be observed. These suggests that this buffalo-derived antimicrobial peptide can enhance the protective immune response and shorten the duration of antibiotic treatment, which may be an immunotherapy method for current anti-tuberculosis drugs.
3. Research related to clinical trials
1. Natural killer cell group 2C (NKG2C): Cytotoxic T lymphocytes (T-CTLs) exert immunoprotection against MTB infection by strongly inhibiting the growth of intracellular MTB. Shen et al. [24] found that NKG2C was highly expressed in T-CTL and was a potent activator of cytotoxic activity in CD3+ T cells. NKG2C+CD3+ T cells can effectively inhibit the growth of intracellular MTB. In tuberculosis patients, the proportion of NKG2C+ cells in CD3+ and CD8+ T cells was significantly reduced, in contrast, NKG2A, an inhibitor of T cytotoxic activity, was highly expressed in T-CTL of CD3+ and CD8+ T cells in TB patients. Studies suggest that the decrease in CTL activity in patients with tuberculosis is attributed to the low expression of cytotoxic granular molecules and the high expression of inhibitory NKG2A receptors, thereby inhibiting the agonist receptor NKG2C. This study suggests that NKG2 receptors are potential targets for immunotherapy for tuberculosis, especially multidrug-resistant tuberculosis. 2. Cytokine immunotherapy: Cytokines are small molecule proteins that coordinate innate and adaptive immune responses by affecting functions such as cell development, transport, and immune effects. An Huiru et al. [25] systematically evaluated the clinical efficacy and safety of γ-interferon (IFN-γ) combined with anti-tuberculosis drugs in the treatment of pulmonary tuberculosis, and the analysis results showed that compared with the control group, the sputum conversion rate, lesion absorption rate and cavitation closure rate of patients in the IFN-γ treatment group at the end of 2~3 months and 6~9 months were significantly higher than those in the control group (all P<0.05). In addition, compared with the control group, the percentage of CD4+ T cells in peripheral blood in the observation group increased more significantly and the percentage of CD8+ T cells decreased more significantly after treatment (all P<0.05). There was no significant difference in the incidence of adverse reactions between the two groups (all P>0.05). The results of meta-analysis suggest that IFN-γ combined with anti-tuberculosis chemotherapy in the treatment of pulmonary tuberculosis can promote the absorption of lesions and the conversion rate of sputum bacteria to negative in patients. 3. Some antibiotics: Delamanib (DLM) is an inhibitor of mycobacterial cell wall fungal acid synthesis and can also exert immunotherapeutic effects by regulating macrophages. In one study, 23 patients with MDR-TB were included, of which 13 were treated with an optimized background regimen (OBR) + DLM regimen (OBR+DLM) and 10 were treated with OBR+placebo, and the CXCL10 levels in the OBR+DLM group were significantly lower than those in the control group after treatment; In cellular models, DLM inhibits the JAK/STAT pathway and inhibits the migration of PBMCs, resulting in significant inhibition of CXCL10 expression [26]. These results suggest that DLM inhibits the expression of CXCL10 by regulating the JAK2/STAT1 signaling pathway, and is associated with the reduction of inflammatory response in patients with multidrug-resistant tuberculosis. The study suggests that DLM can be used as a potential drug for immunotherapy in patients with elevated CXCL10 response. In addition, the combination of CFZ and Rapa can reduce the expression of MTB latencies-related genes in human macrophages [22], which is expected to treat patients with multidrug-resistant and XDR-TB.
Fourth, summary and outlook
At present, the treatment of tuberculosis advocates a comprehensive treatment mode with chemotherapy as the main treatment and immunotherapy as adjuvant therapy, especially for the immunotherapy demand for multidrug-resistant and extensively drug-resistant tuberculosis. From October 2022 to September 2023, the immunotherapy of tuberculosis has made some progress in using host autophagy-related genes as therapeutic targets, host macrophages, toxic T lymphocytes and proteases as therapeutic targets, and glutamine metabolism as research targets. Cellular immunotherapy represented by Vγ2Vδ2 T cells and mesenchymal stem cells has achieved certain clinical efficacy. Therapeutic vaccines and some antibiotics and anti-tuberculosis drugs have certain immunotherapeutic effects. In the current research progress, the exploration of new targets of host-targeted therapy and the research of new therapeutic vaccines are the hotspots and focuses of immunotherapy, and the ultimate goal of many immunotherapy studies is to improve the protective effect of host immunity and the improvement of cell bactericidal ability by regulating the innate immune effect and adaptive immune effect of host cells. At present, the mechanism of immunotherapy is still not fully elucidated, and the timing and duration of immunotherapy are not fully understood, and more high-quality clinical studies are needed to evaluate and explore immunotherapy. Immunotherapy for tuberculosis is of great significance for improving the treatment outcomes of tuberculosis patients, cutting the transmission chain, and controlling infectious diseases.
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