Untranslated Region Sequences and the Efficacy of mRNA Vaccines against Tuberculosis.

mRNA vaccines have been shown to be effective in combating the COVID-19 pandemic. The amount of research on the use of mRNAs as preventive and therapeutic modalities has undergone explosive growth in the last few years. Nonetheless, the issue of the stability of mRNA molecules and their translation efficiency remains incompletely resolved. These characteristics of mRNA directly affect the expression level of a desired protein. Regulatory elements of RNA-5' and 3' untranslated regions (UTRs)-are responsible for translation efficiency. An optimal combination of the regulatory sequences allows mRNA to significantly increase the target protein's expression. We assessed the translation efficiency of mRNA encoding of firefly luciferase with various 5' and 3'UTRs in vitro on cell lines DC2.4 and THP1. We found that mRNAs containing 5'UTR sequences from eukaryotic genes HBB, HSPA1A, Rabb, or H4C2, or from the adenoviral leader sequence TPL, resulted in higher levels of luciferase bioluminescence 4 h after transfection of DC2.4 cells as compared with 5'UTR sequences used in vaccines mRNA-1273 and BNT162b2 from Moderna and BioNTech. mRNA containing TPL as the 5'UTR also showed higher efficiency (as compared with the 5'UTR from Moderna) at generating a T-cell response in mice immunized with mRNA vaccines encoding a multiepitope antigen. By contrast, no effects of various 5'UTRs and 3'UTRs were detectable in THP1 cells, suggesting that the observed effects are cell type specific. Further analyses enabled us to identify potential cell type-specific RNA-binding proteins that differ in landing sites within mRNAs with various 5'UTRs and 3'UTRs. Taken together, our data indicate high translation efficiency of TPL as a 5'UTR, according to experiments on DC2.4 cells and C57BL/6 mice.

Lung Inflammation Signature in Post-COVID-19 TB Patients.

Tuberculosis (TB) remains a leading cause of infectious disease mortality worldwide, despite the COVID-19 pandemic. The mechanisms by which SARS-CoV-2 affects tuberculosis progression have not yet been established. Here, we compared the level of inflammation in the wall of the tuberculoma and in the parenchymal lung tissue of 30 patients diagnosed with tuberculoma without a history of COVID-19 and 30 patients diagnosed with tuberculoma 3 months after COVID-19. We also characterized TB activity in these patients using a panel of TB-associated miRNAs. Histopathological changes were examined in the resection material, and the expression level of cytokine/chemokine genes was determined by qRT-PCR. In patients with a history of COVID-19, the histological data obtained suggested activation of tuberculosis. In the same group of patients, as opposed to those without a history of COVID-19, equally high levels of pro-inflammatory cytokines/chemokines were expressed both in the tuberculoma wall and in the periphery of the resected specimen. A full set of miRNAs (miR-191, miR-193a, miR-222, miR-223, miR-155, miR-26a, and miR-150) were downregulated in the sera of patients with TB and active COVID-19 co-infection compared to controls. Our observations indicate signs of tuberculosis activation resulting from COVID-19 infection.

Therapeutic Effect of Recombinant Mutated Interleukin 11 in the Mouse Model of Tuberculosis.

Earlier we demonstrated that blocking of interleukin 11 (IL-11) by systemic administration of anti-IL-11 antibodies attenuates severity of Mycobacterium tuberculosis infection in mice. The substitution W147A in the IL-11 molecule creates the form of cytokine capable to disrupt gp130/IL11R signaling complex formation, thus serving as a high-affinity specific antagonist of IL-11-mediated signaling. We hypothesized that this mutant form of IL-11 may serve as an effective tool for inhibition of native IL-11 activity in vivo. We established the recombinant W147A mutant form of IL-11 in an optimized Escherichia coli expression system and administered it as the aerosol in the lungs of M. tuberculosis-susceptible I/St mice infected with M. tuberculosis Our results show that this therapeutic approach markedly inhibits tuberculous inflammation in lungs, increases the survival time of infected animals, and decreases expression of key inflammatory factors at the RNA and protein levels. These findings are a step toward clinical evaluation of the anti-IL-11 therapy for tuberculosis.

MicroRNAs as Biomarkers of Active Pulmonary TB Course.

The spread of drug-resistant forms of TB dictates the need for surgical treatment in the complex of anti-tuberculosis measures in Russia. Most often, surgical intervention is performed in the case of pulmonary tuberculoma or fibrotic cavitary tuberculosis (FCT). This study is devoted to the search for biomarkers that characterize the course of disease in surgical TB patients. It is assumed that such biomarkers will help the surgeon decide on the timing of the planned operation. A number of serum microRNAs, potential regulators of inflammation and fibrosis in TB, selected on the basis of PCR-Array analysis, were considered as biomarkers. Quantitative real time polymerase chain reaction and receiver operating curves (ROC) were used to verify Array data and to estimate the ability of microRNAs (miRNAs) to discriminate between healthy controls, tuberculoma patients, and FCT patients. The study showed that miR-155, miR-191 and miR-223 were differentially expressed in serum of tuberculoma with "decay" and tuberculoma without "decay" patients. Another combination (miR-26a, miR-191, miR-222 and miR-320) forms a set to differentiate between tuberculoma with "decay" and FCT. Patients with tuberculoma without "decay" diagnosis differ from those with FCT in serum expression of miR-26a, miR-155, miR-191, miR-222 and miR-223. Further investigations are required to evaluate these sets on a larger population so as to set cut-off values that could be applied in laboratory diagnosis.

Analysis of the lung transcriptome in Mycobacterium tuberculosis-infected mice reveals major differences in immune response pathways between TB-susceptible and resistant hosts.

Using whole genome microarrays, we compared changes in gene expression patterns in the lungs of TB-resistant A/Sn and TB-susceptible I/St mice at day 14 following infection with Mycobacterium tuberculosis H37Rv. Analyses of differentially expressed genes for representation of gene ontology terms and activation of regulatory pathways revealed interstrain differences in antigen presentation, NK, T and B cell activation pathways. In general, resistant A/Sn mice exhibited a more complex pattern and stronger activation of host defense pathways compared to the TB-susceptible I/St mouse strain. In addition, in I/St mice elevated activation of genes involved in neutrophil response was observed and confirmed by quantitative RT-PCR and histopathology. Furthermore, a specific post infection upregulation of cysteine protease inhibitors was found in susceptible I/St mice.

Interleukin-11 drives early lung inflammation during Mycobacterium tuberculosis infection in genetically susceptible mice.

IL-11 is multifunctional cytokine whose physiological role in the lungs during pulmonary tuberculosis (TB) is poorly understood. Here, using in vivo administration of specific antibodies against IL-11, we demonstrate for the first time that blocking IL-11 diminishes histopathology and neutrophilic infiltration of the lung tissue in TB-infected genetically susceptible mice. Antibody treatment decreased the pulmonary levels of IL-11 and other key inflammatory cytokines not belonging to the Th1 axis, and down-regulated IL-11 mRNA expression. This suggests the existence of a positive feedback loop at the transcriptional level, which is further supported by up-regulation of IL-11 mRNA expression in the presence of rIL-11 in in vitro cultures of lung cells. These findings imply a pathogenic role for IL-11 during the early phase of Mycobacterium tuberculosis-triggered disease in a genetically susceptible host.

CD27low CD4 T lymphocytes that accumulate in the mouse lungs during mycobacterial infection differentiate from CD27high precursors in situ, produce IFN-gamma, and protect the host against tuberculosis infection.

The generation of effector, IFN-gamma producing T lymphocytes and their accumulation at sites of infection are critical for host protection against various infectious diseases. The activation and differentiation of naive T lymphocytes into effector memory cells starts in lymphoid tissues, but it is not clear whether the Ag-experienced cells that leave lymph nodes (LN) are mature or if they undergo further changes in the periphery. We have previously shown that CD44(high)CD62L(low) effector CD4 T lymphocytes generated during the course of mycobacterial infection can be segregated into two subsets on the basis of CD27 receptor expression. Only the CD27(low) subset exhibited a high capacity for IFN-gamma secretion, indicating that low CD27 expression is characteristic of fully differentiated effector CD4 T lymphocytes. We demonstrate now that CD27(low) IFN-gamma-producing CD4 T lymphocytes accumulate in the lungs but are rare in LNs. Several factors contribute to their preferential accumulation. First, CD27(low) CD4 T lymphocytes present in the LN are highly susceptible to apoptosis. Second, circulating CD27(low) CD4 T cells do not enter the LN but efficiently migrate to the lungs. Third, CD27(high) effector CD4 T cells that enter the lungs down-regulate CD27 expression in situ. In genetically heterogeneous mice that exhibit varying susceptibility to tuberculosis, the accumulation of mature CD27(low) CD4 T cells in the lungs correlates with the degree of protection against infection. Thus, we propose that terminal maturation of effector CD4 T lymphocytes in the periphery provides the host with efficient local defense and avoids potentially harmful actions of inflammatory cytokines in lymphoid organs.