Targeting mycobacterial PPE65 protein to develop a multi-epitope vaccine candidate against tuberculosis

Tuberculosis is a deadly infectious disease, and the current BCG vaccine being inconsistent in protecting against it, underscores the urgent need to develop better therapeutics. PPE65 is a surface-localized and highly immunogenic protein of Mycobacterium tuberculosis, and has been proposed as a potential vaccine target because of its ability to induce both humoral and cellular immunity. Hence, the present study outlines the systematic design and detailed computational and in vitro assessment of a novel multi-epitope vaccine against M. tuberculosis utilizing PPE65 protein as a core antigen. The designed vaccine construct (MtbPV), which integrates chosen immunodominant epitopes alongside TLR4 agonist RS-09 as an adjuvant, was found to be non-allergenic, non-toxic and highly antigenic. The 3D structure of MtbPV was constructed, and subsequent in silico docking and simulation analyses confirmed binding and steady interactions with human toll-like receptors. Computational disulfide engineering further enhanced structural stability, while immune simulations indicated strong activation of immune responses. Codon-adapted MtbPV sequence was cloned into pET-28a(+) expression vector, the protein was expressed in a bacterial system, and then purified using affinity chromatography. In vitro assays employing THP-1 macrophages revealed MtbPV to elicit proinflammatory cytokines and activation of NF-κB signaling pathway, where macrophage activation and maturation surface markers were also observed to be upregulated. Blocking of TLR2/TLR4 hampered the cytokine production that indicate involvement of these host receptors in MtbPV-mediated immune response. Altogether, a polyepitopic vaccine construct, based on a mycobacterial virulence protein, was successfully designed, highlighting its potential as a promising vaccine candidate to combat tuberculosis.