Mtb In Lungs Research Articles

CD8+ lymphocytes are critical for early control of tuberculosis in macaques.

The functional role of CD8+ lymphocytes in tuberculosis remains poorly understood. We depleted innate and/or adaptive CD8+ lymphocytes in macaques and showed that loss of all CD8α+ cells (using anti-CD8α antibody) significantly impaired early control of Mycobacterium tuberculosis (Mtb) infection, leading to increased granulomas, lung inflammation, and bacterial burden. Analysis of barcoded Mtb from infected macaques demonstrated that depletion of all CD8+ lymphocytes allowed increased establishment of Mtb in lungs and dissemination within lungs and to lymph nodes, while depletion of only adaptive CD8+ T cells (with anti-CD8β antibody) worsened bacterial control in lymph nodes. Flow cytometry and single-cell RNA sequencing revealed polyfunctional cytotoxic CD8+ lymphocytes in control granulomas, while CD8-depleted animals were unexpectedly enriched in CD4 and γδ T cells adopting incomplete cytotoxic signatures. Ligand-receptor analyses identified IL-15 signaling in granulomas as a driver of cytotoxic T cells. These data support that CD8+ lymphocytes are required for early protection against Mtb and suggest polyfunctional cytotoxic responses as a vaccine target.

Replication-Deficient Lymphocytic Choriomeningitis Virus-Vectored Vaccine Candidate for the Induction of T Cell Immunity against Mycobacterium tuberculosis.

Mycobacterium tuberculosis (Mtb) represents a major burden to global health, and refined vaccines are needed. Replication-deficient lymphocytic choriomeningitis virus (rLCMV)-based vaccine vectors against cytomegalovirus have proven safe for human use and elicited robust T cell responses in a large proportion of vaccine recipients. Here, we developed an rLCMV vaccine expressing the Mtb antigens TB10.4 and Ag85B. In mice, rLCMV elicited high frequencies of polyfunctional Mtb-specific CD8 and CD4 T cell responses. CD8 but not CD4 T cells were efficiently boosted upon vector re-vaccination. High-frequency responses were also observed in neonatally vaccinated mice, and co-administration of rLCMV with Expanded Program of Immunization (EPI) vaccines did not result in substantial reciprocal interference. Importantly, rLCMV immunization significantly reduced the lung Mtb burden upon aerosol challenge, resulting in improved lung ventilation. Protection was associated with increased CD8 T cell recruitment but reduced CD4 T cell infiltration upon Mtb challenge. When combining rLCMV with BCG vaccination in a heterologous prime-boost regimen, responses to the rLCMV-encoded Mtb antigens were further augmented, but protection was not significantly different from rLCMV or BCG vaccination alone. This work suggests that rLCMV may show utility for neonatal and/or adult vaccination efforts against pulmonary tuberculosis.

Recombinant Ag85B vaccine by taking advantage of characteristics of human parainfluenza type 2 virus vector showed Mycobacteria-specific immune responses by intranasal immunization

Viral vectors are promising vaccine candidates for eliciting suitable Ag-specific immune response. Since Mycobacterium tuberculosis (Mtb) normally enters hosts via the mucosal surface of the lung, the best defense against Mtb is mucosal vaccines that are capable of inducing both systemic and mucosal immunity. Although Mycobacterium bovis bacille Calmette-Guérin is the only licensed tuberculosis (TB) vaccine, its efficacy against adult pulmonary forms of TB is variable. In this study, we assessed the effectiveness of a novel mucosal TB vaccine using recombinant human parainfluenza type 2 virus (rhPIV2) as a vaccine vector in BALB/c mice. Replication-incompetent rhPIV2 (M gene-eliminated) expressing Ag85B (rhPIV2–Ag85B) was constructed by reverse genetics technology. Intranasal administration of rhPIV2–Ag85B induced Mtb-specific immune responses, and the vaccinated mice showed a substantial reduction in the number of CFU of Mtb in lungs and spleens. Unlike other viral vaccine vectors, the immune responses against Ag85B induced by rhPIV2–Ag85B immunization had an advantage over that against the viral vector. In addition, it was revealed that rhPIV2–Ag85B in itself has an adjuvant activity through the retinoic acid-inducible gene I receptor. These findings provide further evidence for the possibility of rhPIV2–Ag85B as a novel TB vaccine.

Enhancement of the long term efficacy of BCG vaccine against experimental tuberculosis in mice through rapamycin induced modulation of CD4 and CD8 T cell functions (166.17)

Abstract BCG vaccine protects children but does not induce long-term immunity against tuberculosis. Rapamycin has been found to directly act on MTOR of T cells and extend the longevity of CD8 T cell response to viral infection in mice. We hypothesized that rapamycin co-administration during BCG vaccination may alter the quality of CD8 function to enable long-term immunity. Methods: C57Bl/6 mice were given one dose of BCG vaccine (1 million per mouse; s.c.) or BCG with doses of rapamycin (75 microgram/mg given daily for 30 days) followed by aerosol infection with virulent Mycobacterium tuberculosis (Mtb). At 4 and 8 weeks, antigen specific CD8 T cells, memory specific markers and tetramers specific for Mtb antigens-ESAT6 and TB10.4 were analysed using flow cytometry. Log10 decline of Mtb counts in lungs was determined to correlate T cell phenotype with protection after vaccination. Results: Rapamycin coadministration with BCG enhanced the expansion of antigen-specific CD8 T cells in mice that correlated with a decline in the counts of Mtb in lungs and spleens of mice. Rapamycin increased both CD8 and CD4 T cells in the lungs and spleens and enhanced central memory (Tcm) CD8 T cells in lymphoid organs. Tcm levels, in turn, correlated with an increase in expression of Eomes transcription factor by CD8 T cells. Since Eomes is a determinant of Tcm lineage, we suggest that the long-term efficacy of BCG vaccine can be enhanced through modulation of MTOR dependent pathways of T cells.

Characterization of M. Tuberculosis-derived IL-12-inducing material by alveolar macrophages

We have investigated the substance derived from Mycobacterium tuberculosis (Mtb) that induces interleukin (IL)-12 production by alveolar macrophages (AMs) in vitro. The cytosol fraction of live Mtb H37Rv induced IL-12 production by AMs in a dose-dependent manner. The addition of interferon-gamma (IFN-γ) augmented IL-12 production. IL-12-inducing activity by AMs (termed as surely active keeping rescue antigen, SAKRA) was purified by gel filtration and ion exchange column chromatography, and the molecular weight of SAKRA was estimated by gel filtration to be more than 700kDa. SDS-polyacrylamide gel electrophoresis (PAGE) and Western blotting of SAKRA using rabbit anti-SAKRA antibody suggested that SAKRA is composed with several low molecular weight proteins. Amino acids sequence analysis of several bands after SDS-PAGE suggested that SAKRA is a part of ribosomes. RT-PCR showed that SAKRA induced not only expression of IL-12 p40 mRNA, but expression of tumor necrosis factor (TNF)-α and inducible nitric oxide synthase (iNOS) mRNA at least 6h after stimulation, suggesting that SAKRA activates the bactericidal activity of macrophages. To investigate the potential use of SAKRA as a vaccine against tuberculosis, SAKRA was administered to BALB/c mouse that had been immunized with BCG for 18 months, and mouse were infected with Mtb H37Rv via a respiratory route. Replication of Mtb in lungs and spleens was examined 6 weeks after infection. Administration of SAKRA to BCG-vaccinated mice significantly reduced the numbers of Mtb in lungs and spleens as compared with BCG-vaccinated control mice. Taken together, these results suggest that SAKRA is one of the Mtb-derived immunomodulatory substances which induce IL-12 production during infection and also increases mycobactericidal activities of macrophages, and that SAKRA may be a promising new vaccine candidate against tuberculosis.