Galina S. Shepelkova

Background. Ongoing research is investigating the interaction between mycobacteria and macrophages. Upon infection with mycobacteria, macrophages release cytokines. These include IFNy, TNF, IL-1, IL-6, and IL-12p40, among others. These substances play a critical role in the host response to tuberculosis and other mycobacterial infections. Aim. To investigate and compare the cytokine patterns produced by human macrophages in response to interaction with antigens from both pathogenic and conditionally pathogenic mycobacterial strains in vitro. Methods. Blood monocytes were cultured for five days in the presence of 10 % active human serum. The resulting macrophages were then cultured with mycobacterial antigens from Mycobacterium tuberculosis, Mycobacterium avium, and Mycobacterium kansasii for 24 hours. Changes in macrophage gene expression profiles and the production of pro-inflammatory and anti-inflammatory cytokines were then assessed. A control group of “naive” macrophages cultured without any mycobacterial antigen was also used. Results . Stimulation of human macrophages with antigens from different mycobacterial species resulted in the formation of distinct cell phenotypes: protective (M. tuberculosis), characterized by the expression of IL6, IFNG, and TNF; pro-inflammatory (M. kansasii), with active expression of IL6, IL1B, and TNF; and pro-inflammatory (M. avium), expressing IL6 and IL12. The cytokine production findings were fully consistent with the gene expression data. Conclusion . These findings on macrophage-mycobacterial antigen interactions provide insight into the mechanisms by which both tuberculous and nontuberculous mycobacterial infections establish chronic macrophage infection.

Background. Among the factors contributing to unsuccessful treatment outcomes in patients with drug-resistant pulmonary tuberculosis-particularly multidrug-resistant tuberculosis (MDR-TB)-considerable importance is attributed to interindividual variability in pharmacological response, which is largely determined by patients’ genetic characteristics. Recent advances in MDR-TB chemotherapy are closely associated with the introduction of bedaquiline. Bedaquiline is primarily metabolized by the cytochrome P450 enzyme CYP3A4; however, the impact of CYP3A4 gene polymorphisms on bedaquiline pharmacokinetics and treatment efficacy in patients with drug-resistant TB remains insufficiently studied. Personalized therapy based on a patient’s genetic profile represents a key strategy for optimizing dosing regimens, improving treatment efficacy, and reducing the risk of developing further drug resistance. Aims — to evaluate the influence of CYP3A4 polymorphic alleles (*1B/rs2740574 and *1G/rs2242480) on bedaquiline pharmacokinetic parameters and chemotherapy efficacy in patients with drug-resistant pulmonary tuberculosis, including those with MDR-TB. Methods. A prospective, observational cohort study was conducted involving 143 patients with pulmonary tuberculosis and confirmed drug-resistant Mycobacterium tuberculosis (including MDR, pre-XDR, and XDR-TB) treated at the Central TB Research Institute (CTRIT), Russia, between 2022 and 2024 (66 women and 77 men). Three observation groups were formed based on genotype: Group 1 — wild-type CYP3A4*1 (n = 99); Group 2 — *1B (rs2740574) carriers (n = 10); Group 3 — *1G (rs2242480) carriers (n = 34). Genotyping was performed using polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP). Bedaquiline was administered as part of individualized chemotherapy regimens at a dose of 400 mg once daily for 2 weeks, followed by 200 mg three times weekly for up to 6 months. Bedaquiline pharmacokinetic parameters were assessed in 30 patients using high-performance liquid chromatography. Treatment efficacy was evaluated based on sputum culture conversion by month 6. Statistical analysis employed nonparametric Kruskal–Wallis and Mann–Whitney tests for group comparisons. Results. Patients carrying the CYP3A4 *1G (rs2242480) allele showed significantly higher bedaquiline exposure compared to wild-type individuals: AUC₀–₂₄ (49.06 vs. 41.99 µg·h/mL; p 0.05), Cmax (3.13 vs. 2.21 µg/mL; p 0.05), and AUC₀–₂₄/MIC ratio (196.24 vs. 167.94; p 0.05), suggesting reduced metabolic clearance. In contrast, the *1B variant was associated with lower AUC₀–₂₄. Although culture conversion rates at 6 months did not differ significantly (p = 0.87), a trend toward higher efficacy was observed in *1G carriers (95.8%) versus wild-type patients (84.5%). Conclusion. The CYP3A4 *1G (rs2242480) polymorphism is associated with decreased bedaquiline metabolism, leading to increased systemic drug exposure and a potential improvement in treatment response. These findings highlight the role of CYP3A4 genetics in bedaquiline pharmacokinetics and support the integration of CYP3A4 genotyping into personalized MDR-TB treatment strategies. The *1G allele may serve as a promising pharmacogenetic biomarker for optimizing bedaquiline dosing in drug-resistant tuberculosis.

Since the Mycobacterium tuberculosis genome was decoded in 1998, our understanding of this dangerous pathogen – which has become the leading cause of bacterial infection-related deaths in modern human history – has expanded significantly. This review examines genetic, physiological, and metabolic factors believed to play a key role in the adaptation strategies of M. tuberculosis to survival within the human host. These strategies underpin critical aspects of the bacterium life cycle, including infection, persistence, dissemination, and transmission to the new hosts. We also discuss how M. tuberculosis adapts to the primary factors of the host’s innate and adaptive immunity, including their roles in disease progression. Granulocyte migration, phagosome damage and repair, autophagy, and cell death are pivotal processes that determine outcome of the host–mycobacterium interactions. To date, experimental evidence has accumulated indicating that small bacterial RNAs play a crucial role in regulating numerous physiological processes and key stages of the pathogen life cycles. Our review explores the hypothesis that M. tuberculosis small RNAs may not only adapt the bacterial transcriptome to changing conditions but also interact with the transcriptome of the infected host, interfering with antibacterial defense processes. Given the growing recognition of the significance of asymptomatic infection and transmission of M. tuberculosis, we argue for stronger integration between the laboratory and clinical research in this field.

The success of mRNA-based vaccine formulations against viral infections motivated many researchers to develop mRNA vaccines against bacterial infections. The development of new anti-tuberculosis vaccine is an urgent task since the only approved BCG vaccine is not effective enough in terms of infection prevention, despite the fact that it reduces the risk of severe disease. The study aimed to compare two anti-tuberculosis mRNA vaccines based on the classic linear mRNA (mRNA-MTBmEp-5-1) and circular RNA (circRNA-MTB-mEp-5-1) by immunogenicity and the capability of protecting I/St mice against M. tuberculosis infection. The efficacy of mRNA vaccines in the formulations with lipid nanoparticles was compared with the BCG efficacy. The findings suggest that immunization with the mRNA vaccine based on the linear mRNA resulted in the cell-based and humoral immune response (OD IgG = 0.36 ± 0.12) that was less pronounced than after BCG vaccination (OD IgG = 0.54 ± 0.14). At the same time, immunization with the mRNA vaccine and BCG ensured comparable reduction of bacterial load in the lung and spleen of experimental mice (CFU in lung tissue for BCG: 4.00 × 105 ± 2.13 × 105, p = 0.0068; mRNA: 4.72 × 105 ± 3.44 × 105, p = 0.0059; LNP: 4.91 × 106 ± 3.89 × 106, ns; PBS: 4.01 × 106 ± 1.69 × 106) and increased survival of mice after getting infected with M. tuberculosis. Immunization with the vaccine based on the circular RNA resulted in developing humoral mmunity only (OD IgG = 0.52 ± 0.13) and did not ensure protection after getting infected with M. tuberculosis (CFU in the lung for circRNA: 2.12 × 106 ± 5.30 × 105, p = 0.85). Thus, in our studies, anti-tuberculosis vaccines based on circular RNAs are inferior in effectiveness to formulations based on linear RNAs.

The pivotal component in the protection against TB is the tissue macrophages (Mф). These cells have been demonstrated to play a crucial role in the elimination of pathogens and mycobacterial killing. Elucidation of the molecular and phenotypic events that determine the outcome of infection in Mф is fundamental to understanding the key features of these cells that are so important in fighting infection. Mф activation is driven by cytokines and other inflammatory mediators secreted by T lymphocytes. The interaction between Mycobacterium tuberculosis (Mtb) and host Мф has been the subject of extensive in vitro research. This dynamic interplay represents a pivotal step in the progression of mycobacterial infection because pulmonary macrophages constitute the primary line of defense against the pathogen, thereby serving as the initial immune cells to which Mtb must adapt to establish a replicative foothold within the host. Our studies have demonstrated that highly differentiated Th1 effectors with the CD27^low phenotype exhibit superior efficacy in activating both peritoneal (Mф: T cell ratio ranging from 125:1 to 625:1) and pulmonary macrophages (Mф: T cell ratio = 5:1) compared to cells with the CD27^high phenotype. Furthermore, our findings indicate that this activation mechanism is not contingent upon the production of reactive nitrogen species. To effectively activate the bacteriostatic function of macrophages, CD27^high T lymphocytes must differentiate into effectors with the CD27^low phenotype.

Background/Objectives. Tuberculosis is a deadly bacterial disease and the second most common cause of death from monoinfectious diseases worldwide. Comprehensive measures taken by health authorities in various countries in recent decades have saved tens of millions of lives, but the number of new cases of this infection has been steadily increasing in the last few years and already exceeds 10 million new cases annually. The development of new vaccines against tuberculosis is a priority area in the prevention of new cases of the disease. mRNA vaccines have already shown high efficacy against COVID-19 and other viral infections and can currently be considered a promising field of antituberculosis vaccination. In our previous study, we assessed the immunogenicity and protective activity of several types of antituberculosis mRNA vaccines with different 5′ untranslated regions, but the efficacy of these vaccines was either comparable with or lower than that of BCG. Methods. Here, we conducted a comprehensive experiment to investigate the effects of cotranscriptional capping conditions and of cap structure on the magnitude of the mRNAs’ translation in HEK293T and DC2.4 cells. The most effective cap version was used to create an antituberculosis mRNA vaccine called mEpitope-ESAT6. Results and Conclusions. We compared immunogenicity and protective activity between mEpitope-ESAT6 and BCG and found that the vaccine with the new cap type is more immunogenic than BCG. Nonetheless, the increased immunogenicity did not enhance vaccine-induced protection. Thus, the incorporation of different cap analogs into mRNA allows to modulate the efficacy of mRNA vaccines.

Targeted treatment of tuberculosis-associated lung damage requires an understanding of the precise mechanisms of immunopathology. A major obstacle to the longitudinal study of tuberculosis (TB) immunopathogenesis in humans is the lack of serial lung biopsies during disease progression and treatment, which could be used to characterize local immune pathways involved in tissue damage. Understanding of the immunobiology of lung tissue damage in tuberculosis has largely been based on animal models. Our study looked for signs of inflammation in TB patients’ lung biopsies. Results were compared between a site of infection and relatively healthy tissue outside the site. The most significant differences in the expression of microRNAs (miRs) and cytokine/chemokines were observed between the non-decayed tuberculoma and the surrounding parenchyma. In addition, these parameters showed almost no differences between the cavitary wall and surrounding tissue. This is an indication that the inflammatory process is more prevalent in fibrotic cavitary tuberculosis (FCT). In FCT subjects, no difference was observed between the cavity wall and the parenchyma in the production of key inflammatory factors such as IL-6, IL-11, IL-17, and IFNγ. This is an indication that the limits of the inflammatory response are broader in FCT. The expression levels of miR-191, miR-193a, miR-222, miR-223, miR-18, miR-155, miR-376c, miR-26a, miR-150, and miR-124 were not significantly different between the cavernous wall and lung tissue in patients with FCT, further confirming the spread of inflammatory and destructive processes beyond the focus of infection.

MRNA vaccines turned out to be highly effective in combating the COVID-19 pandemic and other viral infections. Despite extensive study of mRNA vaccines in the last five years, the issue of safety of their use is still relevant. The study aimed to assess immunogenicity of two anti-tuberculosis mRNA vaccine doses in female and male rats 2 and 4 weeks after vaccination. Hematological and biochemical parameters of blood were determined within the same timeframe. The dose-dependent nature of mRNA vaccine immunogenicity was confirmed in both females and males. Vaccination led to moderate lymphopenia and neutrophilia in male rats, as well as to apparent dose-dependent and sex-related changes in blood biochemistry parameters at various time points. The findings suggest moderate toxicity of the anti-tuberculosis mRNA vaccine and the importance of assessing its toxic effects at various time points in animals of both sexes.

Tuberculosis, a disease caused by the bacterium Mycobacterium tuberculosis, is a major public health concern. Innate and adaptive immunity provide robust defense against pathogens. However, M. tuberculosis, which co-evolved with humans, has acquired many mechanisms to evade the immune response and ensure its intracellular existence and long-term survival within the host. Moreover, emerging evidence suggests that this secretive bacterium can alter expression of regulatory noncoding RNAs (including microRNAs), leading to dysregulation of biological processes underlying tuberculosis pathogenesis. For example, miR-222-3p has been shown to regulate the functional reprogramming of macrophages and is involved in the regulation of host innate immunity. Previously, we demonstrated the important role of miR-222-3p as a biological marker of tuberculosis activity. To confirm their biological targets and understand their role in the pathogenesis of tuberculosis, many research groups are working to establish functional relationships between miRNA expression under different conditions and their actual biological action using molecular biology and bioinformatics methods. In the present study, we demonstrated the effect of miR-222-3p overexpression on several functions of human macrophages of monocytic origin activated with M. tuberculosis antigens in in vitro culture. Specifically, we found that miR-222-3p overexpression significantly decreased IL-6 and IFNγ expression and increased IL-1β and cxcl10 expression in cultures of uninfected macrophages. Infected macrophages overexpressing miR-222-3p were characterized by increased NF-κB and IL-6 expression, as were infected macrophages without transfection. Another important finding was that miR-222-3p overexpression caused a small but significant increase in reactive nitrogen species production by infected macrophages, but did not affect their bacteriostatic activity against M. tuberculosis. Elucidating the functions of different microRNAs in regulating different pathogenic pathways in TB may lead to discovering new therapeutic targets. The detailed study of microRNAs that regulate immune-associated pathways will be useful for the design of miRNA mimetic molecules, either as inhibitors or as activators. Immune effects induced by miRNA drugs are currently a major challenge for miRNA therapeutics.

S uchetom togo, chto adaptivnyj immunnyj otvet vazhen dlya kontrolya i ustraneniya virusnyh infekcij, vyzyvayushchih zabolevaniya u lyudej, krajne vazhna ocenka adaptivnogo otveta na SARS-CoV-2. Nejtralizuyushchie antitela i T-limfocity CD4+/CD8+ sposobstvuyut kontrolyu SARS-CoV-2. Tuberkulez do sih por ostaetsya glavnoj prichinoj smerti sredi bakterial'nyh infekcij v mire. Na dannyj moment lechenie tuberkuleza oslozhneno koinfekciej COVID-19. Cel'yu raboty bylo issledovat' obrazovanie nejtralizuyushchih antitel protiv SARS-CoV-2 i specifichnyh dlya SARS-CoV-2 T-kletok CD4+ i CD8+ u pacientov s TB legkih. Urovni nejtralizuyushchih antitel k SARS-CoV-2 i kolichestvo specifichnyh k SARS-CoV-2 T-kletok ocenivali v dvuh vremennyh tochkah (cherez 3 i cherez 6 mesyacev posle perenesennogo COVID-19) u bol'nyh s diagnozom tuberkulez legkih (69 chelovek: 33 zhenshchiny i 36 muzhchin ot 18 do 70 let). V kontrol'nuyu gruppu voshli pacienty, perenesshie COVID-19 bez tuberkuleza (35 chelovek: 25 zhenshchin i 10 muzhchin ot 18 do 70 let). V rezul'tate issledovaniya byli zaregistrirovany odinakovye urovni nejtralizuyushchih antitel k SARS-CoV-2 v obeih gruppah cherez 3 mesyaca posle perenesennogo COVID-19. Urovni antitel snizilis' v dvuh gruppah cherez 6 mesyacev posle COVID-19 po sravneniyu s 3 mesyacami. Uroven' antitel byl dostoverno nizhe v gruppe bol'nyh TB (r = 0,01). Kolichestvo SARS-CoV-2-specifichnyh T-kletok bylo nizhe u bol'nyh TB cherez 6 mesyacev posle perenesennogo COVID-19 (r < 0,001) po sravneniyu s kontrol'noj gruppoj. Takim obrazom, koinfekciya TB snizhaet specificheskij immunnyj otvet protiv SARS-CoV-2 cherez 6 mesyacev posle perenesennogo COVID-19.