Although the precise host-defence mechanisms are not completely understood, T-cell-mediated immune responses are believed to play a pivotal role in controlling parasite infection. In this study, the potential HLA*A2 restricted peptides were predicted and the ability of peptides to bind HLA-A*02 was confirmed by a MHC stabilization assay. Two of the peptides tested stabilized HLA-A*02: (a) LLATTVSGL (P1) and (b) LMTNGPLEV (P3). The potential of the peptides to generate protective immune response was evaluated in patients with treated visceral leishmaniasis as well as in healthy control subjects. Our data suggest that CD8 T-cell proliferation against the selected peptide was significantly higher compared to unstimulated culture conditions. The stimulation of peripheral blood mononuclear cells with epitopes individually or as a cocktail upregulated IFN-γ production, which indicates its pivotal role in protective immune response. The IFN-γ production was mainly in a CD8 T-cells-dependent manner, which suggested that these epitopes had an immunoprophylactic potential in a MHC class I-dependent manner. Moreover, no role of the CD3 T cell was observed in the IL-10 production against the selected peptides, and no role was found in disease pathogenesis. Further studies on the role of these synthetic peptides may contribute significantly to developing a polytope vaccine idea towards leishmaniasis.

Glucose-1-Phosphate Thymidylyltransferase (RmlA) is one of the enzymes in rhamnose biosynthesis pathway, where rhamnose acts as linker of peptidoglycan and arabinogalacton in the cell wall, therefore RmlA is a potential enzyme for the survival of Mycobacterium tuberculosis (Mtb). To go into the depth of the structure for exploring binding regions, homology model of RmlA was built in Prime, Schrodinger v9.2. The model with lowest Discrete Optimized Potential Energy (DOPE) score of -35524.17 kcal/mol and RMSD of 0.1 Å with the template (1H5R_B) was subjected to Molecular Dynamics Simulation (MDS) for 5 ns to achieve its stable folding state. The tertiary structure of the proposed model is composed of α/β/α sandwich type protein with quasi-Rossmann type folding pattern. The substrate, deoxy Thymidine tri phosphate (dTTP) comprises of triphosphate (R1) and methyl (R2) side chains where, R1 is highly essential for the survival of Mtb. Therefore, nineteen side chain analogues of dTTP were designed by substituting R1 and R2 chain of dTTP using Combi Glide, Schrodinger v9.2 and docked with the target RmlA protein. Out of which two analogues such as, 6-[(2R,3S,5R)-5-[5-(2- aminoethyl)-2,4-dioxo-1,2,3,4-tetrahydropyrimidin-1-yl]-3-hydroxyoxolan-2 yl] hexanoic acid (COMP- 11) and 4-(2-{1-[(1S,3S,4S)-3-(5-carboxypentyl)-4-hydroxy-2-methylidenecyclopentyl]-2,4-dioxo- 1,2,3,4-tetrahydropyrimidin-5-yl}ethyl)morpholin-4-ium (COMP-12) showed the highest GLIDE score (-12.55 Kcal/mol and -11.58 Kcal/mol respectively) than that of substrate (-9.725 Kcal/mol). During simulations, hydrogen bonding profile between the two top hits and protein ranges up to 5 strong polar contacts which were much stronger than that of substrate. Similarly, the computational binding free energy of both the analogues was found to be less than -70 Kcal/mol which is much lower than that of substrate (-52.84 Kcal/mol). All these results suggest that these two compounds have more stable interaction than that of substrate inside the solvent condition and can be used as competitive inhibitors.

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