Optimizing a Subunit Vaccine of <i>Mycobacterium tuberculosis</i> Using In-Silico and In-Vitro Approaches

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 3 , showed a detectable titer, and copy numbers (10 9 /mL-10 11 /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.