A rationally designed PE/PPE-based multi-epitope vaccine candidate engaging TLR4 to induce balanced humoral and cellular immunity against Mycobacterium tuberculosis

TB is a serious health challenge globally, with more than 10 million new cases every year and rising MDR and XDR variants diminishing the efficacy of existing chemotherapeutics. Although BCG vaccination confers protection in children, its limited ability to prevent adult pulmonary TB reveals the urgent need for next-generation vaccines proficient in inducing robust and durable immunity. PE and PPE protein families of M. tb , constituting nearly 10% of the genome, play pivotal roles in antigenic variation, immune modulation, autophagy inhibition, and host-pathogen interactions, making them compelling vaccine targets. In this study, we rationally constructed a MEV incorporating immunodominant B-cell, CTL, and HTL epitopes from seven infection-induced PE/PPE proteins, PE23, PE34, PPE22, PPE23, PPE29, PPE59, and PPE61, combined with an RpfE-derived TLR4 agonist adjuvant. Comprehensive immunoinformatics tools were employed for epitope prediction, antigenicity screening, allergenicity and toxicity filtration, epitope assembly, and ab initio structural modeling. The final 483-residue MEV demonstrated high antigenicity, structural stability, favorable physicochemical properties, strong TLR4 binding affinity, and robust stability during 200-ns molecular dynamics simulation. In silico immune profiling predicted strong Th1-skewed responses, characterized by elevated IFN-γ, IL-2, and IgG subclasses, alongside expansion of memory B- and T-cell subsets. Codon optimization produced a CAI of 1.0 and optimal GC content, encouraging efficient expression in Escherichia coli (E. coli). To ensure the in silico immunogenicity predictions were biologically relevant, we expressed and purified the MEV (~ 46 kDa) from E . coli BL21(DE3) and found that it induced significantly higher TNF-α, IL-6, IL-12p70 and IL-10 in RAW264.7 macrophages versus untreated and heat-inactivated controls, confirming its integrity and predicted TLR4-linked, Th1-biased immunostimulatory activity. Together, these findings present a structurally coherent, immunologically potent, and non-allergenic MEV targeting seven PE/PPE antigens, offering a promising next-generation vaccine candidate for preclinical evaluation against M. tb .