Immunoinformatics-Based Design of a Dual-Adjuvanted Multi-Epitope Vaccine Targeting PE_PGRS3 and Immune-Evasion Antigens of Drug- resistant Mycobacterium tuberculosis

The global rise of drug-resistant Mycobacterium tuberculosis (Mtb) strains has reduced the effectiveness of current therapeutic strategies, underscoring the urgent need for improved vaccination approaches. Moreover, the limited and variable protective efficacy of the BCG vaccine, particularly against adult pulmonary tuberculosis, highlights critical gaps in current Tuberculosis (TB) prevention. Together, these challenges emphasize the importance of developing next-generation vaccines capable of eliciting robust and protective immunity. The selected antigens play key roles in immune evasion and host–pathogen interactions that induces Mtb persistence. Targeting these proteins provides a rational strategy to overcome limitations of traditional vaccine antigens and enhance protective immunity. A novel multi-epitope vaccine (MEV) incorporating dual peptide adjuvant domains (LL-37 and β-defensin-3) was designed using an integrated immunoinformatics and molecular modeling framework. The designed vaccine exhibited a molecular weight of 57.5 kDa and favorable physicochemical properties, including structural stability (instability index: 36.38), moderate thermostability (aliphatic index: 54.74), and high hydrophilicity (GRAVY: −0.845). Secondary structure analysis revealed 28% α-helices, 16.5% β-strands, and 55.5% random coils, indicating conformational flexibility conducive to molecular interactions. Three-dimensional structure modeling, refinement, and validation confirmed appropriate geometry and residue distribution. Protein–protein docking demonstrated strong interactions with toll-like receptor (TLR) 4 and TLR8, suggesting the vaccine’s ability to engage innate immune recognition pathways. Molecular dynamics simulations demonstrated stable binding, structural adaptability, and compactness, supporting the vaccine’s dynamic stability under physiological conditions. Overall, these results support the designed MEV as a stable candidate for further experimental evaluation and emphasize the relevance of rational MEV design against Mycobacterium tuberculosis.