An Immunoinformatics-Based Study of Mycobacterium tuberculosis Region of Difference-2 Uncharacterized Protein (Rv1987) as a Potential Subunit Vaccine Candidate for Preliminary Ex Vivo Analysis.

Mycobacterium tuberculosis (Mtb) is the pathogen that causes tuberculosis and develops resistance to many of the existing drugs. The sole licensed TB vaccine, BCG, is unable to provide a comprehensive defense. So, it is crucial to maintain the immunological response to eliminate tuberculosis. Our previous in silico study reported five uncharacterized proteins as potential vaccine antigens. In this article, we considered the uncharacterized Mtb H37Rv regions of difference (RD-2) Rv1987 protein as a promising vaccine candidate. The vaccine quality of the protein was analyzed using reverse vaccinology and immunoinformatics-based quality-checking parameters followed by an ex vivo preliminary investigation. In silico analysis of Rv1987 protein predicted it as surface localized, secretory, single helix, antigenic, non-allergenic, and non-homologous to the host protein. Immunoinformatics analysis of Rv1987 by CD4 + and CD8 + T-cells via MHC-I and MHC-II binding affinity and presence of B-cell epitope predicted its immunogenicity. The docked complex analysis of the 3D model structure of the protein with immune cell receptor TLR-4 revealed the protein's capability for potential interaction. Furthermore, the target protein-encoded gene Rv1987 was cloned, over-expressed, purified, and analyzed by mass spectrometry (MS) to report the target peptides. The qRT-PCR gene expression analysis shows that it is capable of activating macrophages and significantly increasing the production of a number of key cytokines (TNF-α, IL-1ß, and IL-10). Our in-silico analysis and ex vivo preliminary investigations revealed the immunogenic potential of the target protein. These findings suggest that the Rv1987 be undertaken as a potent subunit vaccine antigen and that further animal model immuno-modulation studies would boost the novel TB vaccine discovery and/or BCG vaccine supplement pipeline.

In silico and in vitro study of Mycobacterium tuberculosis H37Rv uncharacterized protein (RipD): an insight on tuberculosis therapeutics.

Tuberculosis caused by Mycobacterium tuberculosis (Mtb) is responsible for the highest global health problem, with the deaths of millions of people. With prevalence of multiple drug resistance (MDR) strains and extended therapeutic times, it is important to discover small molecule inhibitors against novel hypothetical proteins of the pathogen. In this study, a virtual screening protocol was carried out against MtbH37Rv hypothetical protein RipD (Rv1566c) for the identification of potential small molecule inhibitors. The 3D model of the protein structure binding site was used for virtual screening (VS) of inhibitors from the Pathogen Box, followed by its validation through a molecular docking study. The stability of the protein-ligand complex was assessed using a 150 ns molecular dynamics simulation. MM-PBSA and MM-GBSA are the two approaches that were used to perform the trajectory analysis and determine the binding free energies, respectively. The ligand binding was observed to be stable across the entire time frame with an approximate binding free energy of -22.9916 kcal/mol. The drug-likeness of the inhibitors along with a potential anti-tuberculosis compound was validated by ADMET prediction software. Furthermore, a CFU inhibition assay was used to validate the best hit compound's in vitro inhibitory efficacy against a non-pathogenic Mycobacterium smegmatis MC2155 under low nutrient culture conditions. The study reported that the compound proposed in our study (Pathogen Box ID: MMV687700) will be useful for the identification of potential inhibitors against Mtb in future.

Glycoconjugates, hypothetical proteins, and post-translational modification: Importance in host-pathogen interaction and antitubercular intervention development.

With the emergence of multidrug-resistant bacteria, insufficiency of the established chemotherapy, and the existing vaccine BCG, tuberculosis (TB) subsists as the chief cause of death in different parts of the world. Thus, identification of novel target proteins is urgently required to develop more effective TB interventions. However, the novel vaccine and drug target knowledge based on the essentiality of the pathogen cell envelope components such as glycoconjugates, glycans, and the peptidoglycan layer of the lipid-rich capsule are limited. Furthermore, most of the genes encoding proteins are characterized as hypothetical and functionally unknown. Correspondingly, some researchers have shown that the lipid and sugar components of the envelope glycoconjugates are largely in charge of TB pathogenesis and encounter many drugs and vaccines. Therefore, in this review we provide an insight into a comprehensive study concerning the importance of cell envelope glycoconjugates and hypothetical proteins, the impact of post-translational modification, and the bioinformatics-based implications for better antitubercular intervention development.

Computational discovery and ex-vivo validation study of novel antigenic vaccine candidates against tuberculosis.

Tuberculosis (TB) is a complex infectious bacterial disease, which has evolved with highly successful mechanisms to interfere with host defenses and existing classes of antibiotics to resist eradication. The single obtainable TB vaccine, Bacille Calmette-Guerin (BCG) has failed to provide regular defense for respiratory TB in adults. In this study, a bioinformatics and immunoinformatics approach was applied on Mycobacterium tuberculosis (Mtb) H37Rv proteomes to discover the potential subunit vaccine candidates that elicit both tuberculosis-specific T-cells and B-cell immune response. A total of 4049 proteins of MtbH37RvMtbH37Rv were retrieved and subjected to in silico sequence-based analysis. Finally, five (P9WL69 (Rv2599), P9WIG1 (Rv0747), P9WLQ1 (Rv1987), O53608 (Rv0063), O06624 (Rv1566c)) novel putative proteins were selected. Among the five putative antigenic vaccine candidates, P9WL69 protein was selected for the ex-vivo validation study. The P9WL69 protein encoding gene was amplified and cloned on pET21b vector. The success of the recombinant clone (pET21b-RV2599) was confirmed by colony PCR, insert release test and sequencing. Furthermore, the identified epitopes of the P9WL69 protein were considered for in silico docking and molecular dynamics simulation study using Toll-like Receptors (TLRs) (TLR-2, TLR-4, TLR-9), Mannose receptor, and Myeloid differentiation 88 (MYD88) to understand their binding affinity towards the development of immunogenic vaccines against tuberculosis.