Tehran University of Medical Sciences Iranian Journal of Public Health 2251-6085 54 9 2025 10 04 1938 1953 Zaiqin Ling Department of Tuberculosis, Shandong Public Health Clinical Center, Shandong University, Jinan, 250013, China Muhammad Naeem 1. College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, China 2. Department of Basic Sciences (Pharmacology), University of Veterinary and Animal Sciences, Lahore, Pakistan (Narowal Campus- 51600) 2024 12 03 2025 02 18 Background: The present study addresses the development of a novel subunit vaccine (SV) to combat tuberculosis (TB). Methods: The research used immunoinformatics to develop a subunit vaccine with 7 MHC-I, 3 MHC-II, and 7 B-cell epitopes joined by AAV, GPGPG, and KK linkers. It involved Mtb protein Rv0577 and PADRE sequence as an adjuvant. TLR2 binding affinity (Kd, nM) was determined through PRODIGY. In-silico evaluations determined allergenicity, antigenicity, and physicochemical properties. The vaccine was presented in an AAVDj/8 system, intracellular expression was verified, and the copy number was identified using qPCR and qRT-PCR. Results: The web tools confirmed the stability, non-allergenicity, and high immunogenicity of the vaccine (0.5673 < 0.4). PRODIGY tool depicted good SV-TLR2 binding (ΔG = -8.8 kcal/mol, Kd = 330 nM) with 59 intermolecular contacts, indicating possible TLR2 activation. Indirect immunofluorescence showed the expression of intracellular proteins. Viral titers, determined by 10-fold serial dilution up to 10³, showed a detectable titer, and copy numbers (10⁹/mL–10¹¹/mL) proved productive viral replication and significant vaccine effectiveness. Conclusion: This comprehensive methodology, from epitope selection to in-vitro testing, establishes a robust foundation for further exploring and advancing this SV.  https://ijph.tums.ac.ir/index.php/ijph/article/view/37410 https://ijph.tums.ac.ir/index.php/ijph/article/download/37410/8650