Umesh D. Gupta

MicroRNA let-7 Modulates the Immune Response to Mycobacterium tuberculosis Infection via Control of A20, an Inhibitor of the NF-κB Pathway

The outcome of the interaction between Mycobacterium tuberculosis (Mtb) and a macrophage depends on the interplay between host defense and bacterial immune subversion mechanisms. MicroRNAs critically regulate several host defense mechanisms, but their role in the Mtb-macrophage interplay remains unclear. MicroRNA profiling of Mtb-infected macrophages revealed the downregulation of miR-let-7f in a manner dependent on the Mtb secreted effector ESAT-6. We establish that let-7f targets A20, a feedback inhibitor of the NF-κB pathway. Expression of let-7f decreases and A20 increases with progression of Mtb infection in mice. Mtb survival is attenuated in A20-deficient macrophages, and the production of TNF, IL-1β, and nitrite, which are mediators of immunity to Mtb, is correspondingly increased. Further, let-7f overexpression diminishes Mtb survival and augments the production of cytokines including TNF and IL-1β. These results uncover a role for let-7f and its target A20 in regulating immune responses to Mtb and controlling bacterial burden.

Immunogenicity and Protective Efficacy of “Mycobacterium w” against Mycobacterium tuberculosis in Mice Immunized with Live versus Heat-Killed M. w by the Aerosol or Parenteral Route

ABSTRACT As the disease caused by Mycobacterium tuberculosis continues to be a burden, there is a concerted effort to find new vaccines to combat this problem. One of the important vaccine strategies is whole bacterial vaccines. This approach relies on multiple antigens and built-in adjuvanticity. Other mycobacterial strains which share cross-reactive antigens with M. tuberculosis have been considered as alternatives to M. bovis for vaccine use. One such strain, “Mycobacterium w”, had been evaluated for its immunomodulatory properties in leprosy. A vaccine against leprosy based on killed M. w is approved for human use, where it has resulted in clinical improvement, accelerated bacterial clearance, and increased immune responses to Mycobacterium leprae antigens. M. w shares antigens not only with M. leprae but also with M. tuberculosis, and initial studies have shown that vaccination with killed M. w induces protection against tuberculosis in Mycobacterium bovis BCG responder, as well as BCG nonresponder, strains of mice. Hence, we further studied the protective potential of M. w and the underlying immune responses in the mouse model of tuberculosis. We analyzed the protective efficacy of M. w immunization in both live and killed forms through the parenteral route and by aerosol immunization, compared with that of BCG. Our findings provide evidence that M. w has potential protective efficacy against M. tuberculosis. M. w activates macrophage activity, as well as lymphocytes. M. w immunization by both the parenteral route and aerosol adminstration gives higher protection than BCG given by the parenteral route in the mouse model of tuberculosis.

Inhaled Microparticles Containing Clofazimine Are Efficacious in Treatment of Experimental Tuberculosis in Mice

ABSTRACT Inhalable clofazimine-containing dry powder microparticles (CFM-DPI) and native clofazimine (CFM) were evaluated for activity against Mycobacterium tuberculosis in human monocyte-derived macrophage cultures and in mice infected with a low-dose aerosol. Both formulations resulted in 99% killing at 2.5 μg/ml in vitro. In mice, 480 μg and 720 μg CFM-DPI inhaled twice per week over 4 weeks reduced numbers of CFU in the lung by as much as log10 2.6; 500 μg oral CFM achieved a log10 0.7 reduction.

Development and characterization of spray-dried porous nanoaggregates for pulmonary delivery of anti-tubercular drugs

Abstract Tuberculosis, MTB or tubercle bacillus (TB) is a lethal, infectious disease mainly caused by various strains of mycobacteria, usually Mycobacterium tuberculosis. In this study, guar gum-based porous nanoaggregates were formulated by precipitation technique with two frontline antitubercular drugs, i.e. isoniazid and rifampicin. The formulations were optimized on the basis of various evaluation parameters such as morphology, density, entrapment efficiency and in vitro drug release. The optimized formulations were administered by inhalable route to Wistar rats for the evaluation of drugs in different organs (lungs, liver and kidneys). High drug encapsulation efficiency was achieved in guar gum porous nanoaggregates, ranging from 50% to 60%. A single pulmonary dose resulted in therapeutic drug concentrations of 30%–50% in the lungs and in other organs (less than 5%) for 24 h. From this study, we can conclude that delivering drugs through pulmonary route is advantageous for local action in lungs. Furthermore, the formulation showed sustained drug release pattern, which could be beneficial for reducing the drug dose or frequency of dosing, thus helpful in improving patient compliance.

Promiscuous Peptide of 16 kDa Antigen Linked to Pam2Cys Protects Against Mycobacterium tuberculosis by Evoking Enduring Memory T-Cell Response

One of the main reasons considered for BCG failure in tuberculosis-endemic areas is impediment by environmental mycobacteria in its processing and generation of memory T-cell response. To overcome this problem, we developed a unique lipopeptide (L91) by linking the promiscuous peptide (sequence 91-110) of 16 kDa antigen of Mycobacterium tuberculosis to Pam2Cys. L91 does not require extensive antigen processing and generates enduring Th1 memory response. This is evidenced by the fact that L91 significantly improved the activation, proliferation, and generation of protective T cells. Furthermore, L91 surmounts the barrier of major histocompatibility complex polymorphism and induces better protection than BCG. This peptide has self-adjuvanting properties and activates dendritic cells. Importantly, L91 activates T cells isolated from purified protein derivative-positive healthy volunteers that responded weakly to free peptide (F91). In essence, L91 can be a potent future vaccine candidate against tuberculosis.

A booster vaccine expressing a latency-associated antigen augments BCG induced immunity and confers enhanced protection against tuberculosis.

Background In spite of a consistent protection against tuberculosis (TB) in children, Mycobacterium bovis Bacille Calmette-Guerin (BCG) fails to provide adequate protection against the disease in adults as well as against reactivation of latent infections or exogenous reinfections. It has been speculated that failure to generate adequate memory T cell response, elicitation of inadequate immune response against latency-associated antigens and inability to impart long-term immunity against M. tuberculosis infections are some of the key factors responsible for the limited efficiency of BCG in controlling TB. Methods/Principal Findings In this study, we evaluated the ability of a DNA vaccine expressing α-crystallin- a key latency antigen of M. tuberculosis to boost the BCG induced immunity. ‘BCG prime – DNA boost’ regimen (B/D) confers robust protection in guinea pigs along with a reduced pathology in comparison to BCG vaccination (1.37 log10 and 1.96 log10 fewer bacilli in lungs and spleen, respectively; p<0.01). In addition, B/D regimen also confers enhanced protection in mice. Further, we show that B/D immunization in mice results in a heightened frequency of PPD and antigen specific multi-functional CD4 T cells (3+) simultaneously producing interferon (IFN)γ, tumor necrosis factor (TNF)α and interleukin (IL)2. Conclusions/Significance These results clearly indicate the superiority of α-crystallin based B/D regimen over BCG. Our study, also demonstrates that protection against TB is predictable by an increased frequency of 3+ Th1 cells with superior effector functions. We anticipate that this study would significantly contribute towards the development of superior booster vaccines for BCG vaccinated individuals. In addition, this regimen can also be expected to reduce the risk of developing active TB due to reactivation of latent infection.

Coadministration of Interleukins 7 and 15 with Bacille Calmette-Guérin Mounts Enduring T Cell Memory Response against Mycobacterium tuberculosis

BACKGROUND The bacille Calmette-Guérin (BCG) vaccine renders protection against tuberculosis in childhood but not in adulthood. This may be due to its failure to induce long-lasting memory T cells. T cell memory is dependent on crucial cytokine signals during the priming phases. Therefore, coadministering the BCG vaccine with cytokines may improve its efficacy. METHODS A combination of the cytokines interleukin 7 (IL-7) and interleukin 15 (IL-15) or a combination of the cytokines interleukin 1 (IL-1), interleukin 6 (IL-6), and tumor necrosis factor alpha (TNF-alpha), which are known to influence memory T cell generation, were administered along with BCG to mice. The animals were rested for a period of 240 d before they were challenged with Mycobacterium tuberculosis. Five weeks later, they were killed to study the T cell memory response. RESULTS Administration of IL-7 and IL-15, but not IL-1, IL-6, and TNF-alpha, with BCG resulted in an improved CD4 and CD8 T cell memory response. Mice injected with BCG supplemented with IL-7 and IL-15 showed enhanced T cell proliferation, T helper 1-type cytokine production, and an increased pool of multifunctional M. tuberculosis-specific memory T cells. Furthermore, there was a statistically significant reduction in the mycobacterial burden in the lungs. CONCLUSION Our results indicate that supplementation of the BCG vaccine with IL-7 and IL-15 would substantially improve its efficacy by enhancing the T cell memory response.

Efficacy of Mycobacterium indicus pranii Immunotherapy as an Adjunct to Chemotherapy for Tuberculosis and Underlying Immune Responses in the Lung

Background The 9-month-long chemotherapy of tuberculosis often results in poor compliance and emergence of drug-resistant strains. So, improved therapeutic strategy is urgently needed. Immunotherapy could be beneficial for the effective management of the disease. Previously we showed the protective efficacy of Mycobacterium indicus pranii (MIP) when given as prophylactic vaccine in animal models of tuberculosis. Methods We sought to investigate whether MIP can be used as an adjunct to the chemotherapy in guinea pig models of tuberculosis. Efficacy of MIP was evaluated when given subcutaneously or by aerosol. Results MIP-therapy as an adjunct to the chemotherapy was found to be effective in accelerating bacterial killing and improving organ pathology. MIP-immunotherapy resulted in higher numbers of activated antigen-presenting cells and lymphocytes in the infected lungs and also modulated the granulomatous response. Early increase in protective Th1 immune response was observed in the immunotherapy group. Following subsequent doses of MIP, decrease in the inflammatory response and increase in the immunosuppressive response was observed, which resulted in the improvement of lung pathology. Conclusion MIP immunotherapy is a valuable adjunct to chemotherapy for tuberculosis. Aerosol route of immunotherapy can play a crucial role for inducing immediate local immune response in the lung.

Inhalable microparticles of nitric oxide donors induce phagosome maturation and kill Mycobacterium tuberculosis.

Nitric oxide (NO) kills Mycobacterium tuberculosis (Mtb) in vitro, but gaseous NO is difficult to administer to patients. We evaluated the consequences of intracellular delivery of NO using inhalable microparticles (MP) containing NO donors. MP containing 10% w/w of NO donors alone, or in addition to 25% each of isoniazid (INH) and rifabutin (RFB) in a polylactide-co-glycolide (PLGA) matrix were prepared by spray drying. THP-1-derived macrophages infected with Mtb H37Rv were exposed to MP or soluble NO donors. Phagosome-lysosome fusion (PLF) and bacterial killing were monitored. Colony forming units (cfu) in lungs and spleen of mice infected with a low-dose aerosol and administered inhalations of MP were enumerated. Bacterial DNA in these tissues was estimated by real-time PCR. In vitro studies indicated a bacteriostatic effect of NO donors despite significant enhancement of PLF. Daily inhalation of MP containing 10% diethylenetriamine nitric oxide adduct (DETA/NO) alone reduced log10 cfu in the lungs from 6.1 to 4.4 at the highest dose in four weeks, but did not significantly affect cfu in the spleen. Inhalations of MP containing DETA/NO in combination with INH and RFB significantly (P < 10(-5), ANOVA) reduced cfu in lungs and spleens by 4 log. Gross morphology and histology of the lungs and spleen indicated that inhaled particles were well-tolerated. Inhalable MP containing NO donors need further investigation as an adjunct to standard anti-tuberculosis chemotherapy.

Protective efficacy of Mycobacterium indicus pranii against tuberculosis and underlying local lung immune responses in guinea pig model.

Tuberculosis kills two million people each year. As the current vaccine BCG fails to prevent adult cases of TB, an improved vaccine and/or vaccination strategy is urgently needed to combat TB. Previously we reported the higher protective efficacy of Mycobacterium indicus pranii (MIP), formerly known as Mycobacterium w (M.w) as compared to BCG in murine model of TB. In this study we further evaluated the protective efficacy of MIP in guinea pig model of TB. Modulation of post infection immune response was analyzed in the lungs of MIP immunized and control groups. We found reduced bacterial loads, improved pathology and organized granulomatous response at different post infection time points in the MIP-immunized group as compared to the BCG-immunized group. Combined results suggest that MIP-immunization results in heightened protective Th1 response as compared to BCG group, early after infection with M.tb and a balanced Th1 versus immunosuppressive response at late chronic stage of infection. The study demonstrates the higher antigen presenting cells function both inside the granuloma as well as in the single cell suspension of the lung in the MIP-immunized group. We further demonstrate that live MIP is safe to use in vivo as we observed quick clearance of MIP from the body and no untoward reaction was found. Aerosol route of immunization provided higher protection. Further this study provides evidence that MIP-immunization gives significantly better long term protection as compared to BCG against TB.