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Efforts towards the development of TB vaccine

Protective efficacy of Cellular and Secretary antigens of Mycobaterium habana against M. tuberculosis infection in mouse

Cell wall, cell membrane cytosol, Peripheral(PM) and integral membrane(IM) proteins of M. habana TMC 5135 (M simiae serovar-1) bacilli were evaluated for their protective efficacy against M. tuberculosis infection in mice. Results revealed that protective antigens are distributed in both peripheral and integral membrane compartments though such effect is dominant in the former. Polysaccharide staining showed that LAM, LM and PIMs have a preference for the detergent phase.

Protective efficacy Protein antigens of M. habana secreted into the culture medium during the mid-exponential growth phase (secretory proteins, SPs) also showed protection against M.tb infection in mice.

Mice immunized with cellular antigens or SPs of M. habana showed lesser bacterial (M. tuberculosis) load (assessed as CFUs) in their lung and spleen and survived longer than unimmunized controls. Findings suggest that both cellular and secretary antigens of M. habana are protective against tuberculosis infection.

Efforts towards the Sero-diagnosis of Extra-pulmonary TB

Role of integral membrane antigens of Mycobacterium habana

Performance of the antigens belong to the integral membrane (IMAs) of M. habana was evaluated in detecting antimycobacterial antibodies in serum and body fluids (CSF, ascetic fluid, gastric aspirate, pleural fluid and Urine) of patients of extra-pulmonary tuberculosis by ELISA. The IMAs were recovered from the detergent phase during Triton X-114 treatment of the plasma membrane of M. habana bacilli. Anti-M. habana IMA antibody detection increased the positivity rate from that obtained by culture (26%) and smear microscopy (10%), to 86%. M. tuberculosis antigens were also found in about 80% anti-M. habana IMA antibody-positive cases. Interestingly, all 11 culture-positive cases were also found positive for anti-M. habana IMA antibodies. M. habana IMAs may be promising non-tubercular candidate antigens in ELISA-based serodiagnosis of extrapulmonary tuberculosis with substantial sensitivity, specificity and safety.

Role of Mycobacterial antigen specific circulating immune complexes (CICs)

Mycobacterial antigens were detected in the CICs precipitated from the serum (by polyethylene glycol, PEG 6000) and in the respective body fluids (CSF, ascetic fluid, gastric aspirate, pleural fluid and Urine) of patients suffering from different forms of extra-pulmonary TB, by ELISA. Antigen detection enhanced the positivity to 76% from the 26% by culture positivity. Findings of the study suggest that detection of Mycobacterial antigens in CICs may be potentially useful tool for the rapid diagnosis of extra-pulmonary forms of TB where results of conventional methods of diagnosis are disappointing or difficult to carry out.

Anti-TB Drug development

Evaluation of anti-TB activity in vitro, ex vivo and in vivo systems

In vitro Agar proportion assay determines the potential of the test sample to kill (or inhibit the multiplication) of M. tuberculosis H37Rv/ H37Ra

Ex vivo macrophage (mouse bone marrow derived) model mimics growth environment of natural infection. It demonstrates ability of the candidate molecule to penetrate host cell membrane and phagocytic vacuole, bacilli residing within the vacuole and reach the desired drug targets. In addition, it also serves as a model for hypoxia induced latent TB infection. The effect of selected samples (non-toxic, with MIC ≤ 6.25 μg/ml) on the survival and multiplication of M. tuberculosis H37Rv/ H37Ra within macrophages is evaluated by light microscopy and counting of colony-forming units (CFU).

In vivo model, mouse infected with a high number (CFUs) of virulent M. tuberculosis by the i.v. route, harbors a bacillary population that is similar in number and in metabolic state to that present in the lung cavity of human TB. Efficacy of a sample is monitored by survival/mortality rate, evaluation of body weight, extent of gross lesions, and the enumeration of the CFUs in organs (spleen or lungs or both).

Cytotoxicity evaluation of in vitro `HITs’

Cytotoxicity of the in vitro active samples (MIC ≤ 3.12 for synthetic molecules and ≤ 50.0 µg/ml for the extracts of natural products ) is tested in the mammalian cell lines (VERO C1008)followed by in primary host cells (mouse macrophages) by MTT assay. A sample is considered as potentially toxic if its IC50 (concentration causing 50% loss in cell viability) was ≤ 10 times its MIC for M. tuberculosis H37Rv/ H37Ra.

Determination of Minimum Bactericidal Concentration (MBC)

This is done to determining the capacity of test compounds to kill the bacilli, in addition to inhibiting their multiplication. In the assays killing by the sample is recorded with respect to the seed culture (inoculum).

Mechanism-of-action studies with the in vivo active compounds:

Experiments are performed to elucidate the mechanism of action of in vivo actives in terms of their ability to inhibit the biosynthesis of fatty acids, proteins or nucleic acids by mycobacteria. The experiment involves inhibition in the uptake of radioactive precursor(s) of the respective macromolecules.

Model development

Development of model for screening of molecules against Multidrug resistant M. tuberculosis

New and better drugs are needed for tuberculosis (TB), particularly for the multi-drug resistant (MDR) disease. However, the highly infectious nature of MDR Mycobacterium tuberculosis restricts its use for large scale screening of probable drug candidates. We evaluated the potential of a screen based on a ‘fast grower’non-pathogenic mycobacterium (smegmatis) to shortlist compounds which could be active against MDR M. tuberculosis.

RDevelopment of an in vitro model to select new molecules active against latent TB.

Currently available in vitro dormancy models (use single stress,mostly hypoxia or multiple stress factors) fail to generate a truly dormant M tb population that can be correlated to in vivo dormancy. Further, the in vitro models cannot accurately simulate the complex host-pathogen interactions involved in bacillary containment during human LTBI.

We made an attempt to developed an in vitro model in which M. tuberculosis bacilli isolated from the mouse macrophages were used. Bacilli adopted a true latent metabolic state (shown by the higher accumulation/production of Triglycerides, 16kDa protein and relative resistance to INH and RFM and susceptibility to Mz) under the influence of multiple stresses in a natural host environment comprising low pH, oxygen limitation, nitric oxide (NO), nutrient starvation, and several other anti-microbial effectors.