Phase 1 Open-Label Dose Escalation Trial for the Development of a Human Bacillus Calmette-Guérin Challenge Model for Assessment of Tuberculosis Immunity In Vivo.

BACKGROUND: A controlled human infection model for assessing tuberculosis (TB) immunity can accelerate new vaccine development. METHODS: In this phase 1 dose escalation trial, 92 healthy adults received a single intradermal injection of 2 × 106 to 16 × 106 colony-forming units of Bacillus Calmette-Guérin (BCG). The primary endpoints were safety and BCG shedding as measured by quantitative polymerase chain reaction, colony-forming unit plating, and MGIT BACTEC culture. RESULTS: Doses up to 8 × 106 were safe, and there was evidence for increased BCG shedding with dose escalation. The MGIT time-to-positivity assay was the most consistent and precise measure of shedding. Power analyses indicated that 10% differences in MGIT time to positivity (area under the curve) could be detected in small cohorts (n = 30). Potential biomarkers of mycobacterial immunity were identified that correlated with shedding. Transcriptomic analysis uncovered dose- and time-dependent effects of BCG challenge and identified a putative transcriptional TB protective signature. Furthermore, we identified immunologic and transcriptomal differences that could represent an immune component underlying the observed higher rate of TB disease incidence in males. CONCLUSIONS: The safety, reactogenicity, and immunogenicity profiles indicate that this BCG human challenge model is feasible for assessing in vivo TB immunity and could facilitate the vaccine development process. CLINICAL TRIALS REGISTRATION: NCT01868464 (ClinicalTrials.gov).

Reversed isoniazids: Design, synthesis and evaluation against Mycobacterium tuberculosis.

Novel reversed isoniazid (RINH) agents were synthesized by covalently linking isoniazid with various efflux pump inhibitor (EPI) cores and their structural motifs. These RINH agents were then evaluated for anti-mycobacterial activity against sensitive, isoniazid mono-resistant and MDR clinical isolates of M. tuberculosis and a selected number of compounds were also tested ex vivo for intracellular activity as well as in the ethidium bromide (EB) assay for efflux pump inhibition efficacy. The potency of some compounds against various strains of M. tuberculosis (4a-c, 7 and 8; H37Rv-MIC99 ≤1.25 µM, R5401-MIC99 ≤2.5 µM, X_61-MIC99 ≤5 µM) demonstrated the potential of the reversed anti-TB agent strategy towards the development of novel anti-mycobacterial agents to address the rapidly growing issue of resistance. Further, macrophage activity with >90% inhibition by 1a-c and 3b (MIC90 ≤13.42 µM) and inhibition of EB efflux demonstrated by these compounds are encouraging.

Mycobacterium-Specific γ9δ2 T Cells Mediate Both Pathogen-Inhibitory and CD40 Ligand-Dependent Antigen Presentation Effects Important for Tuberculosis Immunity.

Numerous pathogens, including Mycobacterium tuberculosis, can activate human γ9δ2 T cells to proliferate and express effector mechanisms. γ9δ2 T cells can directly inhibit the growth of intracellular mycobacteria and may also act as antigen-presenting cells (APC). Despite evidence for γδ T cells having the capacity to function as APC, the mechanisms involved and importance of these effects on overall tuberculosis (TB) immunity are unknown. We prepared M. tuberculosis-specific γ9δ2 T cell lines to study their direct protective effects and APC functions for M. tuberculosis-specific αß T cells. The direct inhibitory effects on intracellular mycobacteria were measured, and the enhancing effects on proliferative and effector responses of αß T cells assessed. Furthermore, the importance of cell-to-cell contact and soluble products for γ9δ2 T cell effector responses and APC functions were investigated. We demonstrate, in addition to direct inhibitory effects on intracellular mycobacteria, the following: (i) γ9δ2 T cells enhance the expansion of M. tuberculosis-specific αß T cells and increase the ability of αß T cells to inhibit intracellular mycobacteria; (ii) although soluble mediators are critical for the direct inhibitory effects of γ9δ2 T cells, their APC functions do not require soluble mediators; (iii) the APC functions of γ9δ2 T cells involve cell-to-cell contact that is dependent on CD40-CD40 ligand (CD40L) interactions; and (iv) fully activated CD4(+) αß T cells and γ9δ2 T cells provide similar immune enhancing/APC functions for M. tuberculosis-specific T cells. These effector and helper effects of γ9δ2 T cells further indicate that these T cells should be considered important new targets for new TB vaccines.

New Verapamil Analogs Inhibit Intracellular Mycobacteria without Affecting the Functions of Mycobacterium-Specific T Cells.

There is a growing interest in repurposing mycobacterial efflux pump inhibitors, such as verapamil, for tuberculosis (TB) treatment. To aid in the design of better analogs, we studied the effects of verapamil on macrophages and Mycobacterium tuberculosis-specific T cells. Macrophage activation was evaluated by measuring levels of nitric oxide, tumor necrosis factor alpha (TNF-α), interleukin-1 beta (IL-1ß), and gamma interferon (IFN-γ). Since verapamil is a known autophagy inducer, the roles of autophagy induction in the antimycobacterial activities of verapamil and norverapamil were studied using bone marrow-derived macrophages from ATG5(flox/flox) (control) and ATG5(flox/flox) Lyz-Cre mice. Our results showed that despite the well-recognized effects of verapamil on calcium channels and autophagy, its action on intracellular M. tuberculosis does not involve macrophage activation or autophagy induction. Next, the effects of verapamil and norverapamil on M. tuberculosis-specific T cells were assessed using flow cytometry following the stimulation of peripheral blood mononuclear cells from TB-skin-test-positive donors with M. tuberculosis whole-cell lysate for 7 days in the presence or absence of drugs. We found that verapamil and norverapamil inhibit the expansion of M. tuberculosis-specific T cells. Additionally, three new verapamil analogs were found to inhibit intracellular Mycobacterium bovis BCG, and one of the three analogs (KSV21) inhibited intracellular M. tuberculosis replication at concentrations that did not inhibit M. tuberculosis-specific T cell expansion. KSV21 also inhibited mycobacterial efflux pumps to the same degree as verapamil. More interestingly, the new analog enhances the inhibitory activities of isoniazid and rifampin on intracellular M. tuberculosis. In conclusion, KSV21 is a promising verapamil analog on which to base structure-activity relationship studies aimed at identifying more effective analogs.

Flagellin adjuvanted F1/V subunit plague vaccine induces T cell and functional antibody responses with unique gene signatures.

Yersinia pestis, the cause of plague, could be weaponized. Unfortunately, development of new vaccines is limited by lack of correlates of protection. We used pre- and post-vaccination sera and peripheral blood mononuclear cells from a flagellin adjuvanted F1/V vaccine trial to evaluate for protective markers. Here, we report for the first time in humans that inverse caspase-3 levels, which are measures of protective antibody, significantly increased by 29% and 75% on days 14 and 28 post-second vaccination, respectively. In addition, there were significant increases in T-cell responses on day 28 post-second vaccination. The strongest positive and negative correlations between protective antibody levels and gene expression signatures were identified for IFNG and ENSG00000225107 genes, respectively. Flagellin/F1/V subunit vaccine induced macrophage-protective antibody and significant CD4+ T-cell responses. Several genes associated with these responses were identified that could serve as potential correlates of protection.

BCG Vaccination Induces M. avium and M. abscessus Cross-Protective Immunity.

Pulmonary non-tuberculous mycobacterial (NTM) infections particularly caused by Mycobacterium avium complex (MAC) and Mycobacterium abscessus (MAB) are becoming major health problems in the U.S. New therapies or vaccines which will help prevent the disease, shorten treatment duration and/or increase treatment success rates are urgently needed. This study was conducted with the objective of testing the hypothesis that Bacillus Calmette Guerin (BCG), a vaccine used for prevention of serious forms of tuberculosis (TB) in children and adolescents in tuberculosis hyperendemic countries, induces cross-protective T cell immunity against Mycobacterium avium (MAV) and MAB. Human TB and NTM cross-protective T cells were quantified using flow cytometric assays. The ability of BCG expanded T cells to inhibit the intracellular growth of MAV and MAB was assessed in co-cultures with infected autologous macrophages. In both BCG-vaccinated and M. tuberculosis (Mtb)-infected mice, NTM cross-reactive immunity was measured using IFN-γ ELISPOT assays. Our results demonstrate the following key findings: (i) peripheral blood mononuclear cells from TB skin test-positive individuals contain MAV and MAB cross-reactive T cells, (ii) both BCG vaccination and Mtb infection of mice induce MAV and MAB cross-reactive splenic cells, (iii) BCG-expanded T cells inhibit intracellular MAV and MAB, (iv) CD4, CD8, and γδ T cells play important roles in inhibition of intracellular MAV and MAB and (v) BCG vaccination of healthy volunteers induces TB and NTM cross-reactive T cells. In conclusion, BCG-vaccination induces NTM cross-reactive immunity, and has the potential for use as a vaccine or immunotherapy to prevent and/or treat pulmonary NTM disease.

Design, Synthesis, and Evaluation of Novel Hybrid Efflux Pump Inhibitors for Use against Mycobacterium tuberculosis.

Efflux pumps are considered a major potential contributor to the development of various forms of resistance in Mycobacterium tuberculosis leading to the emergence of multidrug-resistant tuberculosis (TB). Verapamil (VER) and tricyclic chemosensitizers such as the phenothiazines are known to possess efflux pump inhibition properties and have demonstrated significant efficacy in various TB disease models. Novel hybrid molecules based on fusion of the VER substructure with various tricyclic, as well as nontricyclic, chemosensitizer cores or their structural motifs are described. These hybrid compounds were evaluated in vitro and ex vivo individually for their intrinsic activity and in combination for their potentiating potential with the frontline anti-TB drugs, rifampin and isoniazid. In addition, efflux pump inhibition was assessed in an ethidium bromide assay. This study led to the identification of novel compounds, termed hybrid efflux pump inhibitors, with intrinsic antimycobacterial activities (MIC90 ≤ 3.17 µg/mL) and intracellular activity in macrophages at a low concentration (≤6.25 µg/mL).