Exploration of hypothetical proteins and reverse vaccinology approach for novel multi-epitope vaccine design against multidrug-resistant clinical isolate Pseudomonas aeruginosa JJPA01.

Antimicrobial resistance endangers global health by rapidly disseminating Multidrug-resistant (MDR) pathogens that undermine antibiotic therapies. P.aeruginosa, a high-priority ESKAPE pathogen, exemplifies the crisis with complex resistance mechanisms that demand alternative strategies beyond conventional antibiotics. Despite its clinical significance, no licensed vaccine currently prevents P. aeruginosa infections. Targeting the MDR clinical strains, rather than the reference laboratory strain, is crucial to ensure real-world efficacy against the resistant strains. This study explored the hypothetical proteins from an MDR clinical isolate, P. aeruginosa JJPA01, to identify novel antigenic targets for Multi-Epitope Vaccine (MEV) design, which is overlooked in the conventional vaccine design pipeline. Functional annotation of 430 hypothetical proteins led to the identification of 26 High degree of confidence (HDC) proteins. Among them, three outer membrane proteins exhibiting antigenic and non-allergenic properties were prioritized. Reverse vaccinology-based screening of these proteins shortlisted 8 B-cell, 19 cytotoxic T-cell, and 8 helper T-cell epitopes. The final MEV macromolecule was engineered to enhance HLA coverage using selected epitopes linked with human β-defensin and PADRE sequence. Among the four models constructed, V4 showed the most favourable binding towards TLR2, TLR4, MHC I, and MHC II with 99.73 % global population coverage. Molecular Dynamics confirmed complex stability, and in-silico cloning with pET-28a(+) vector supported its expression potential. Immune simulation demonstrated potent B and T cell vaccine responses with effective memory generation. This MEV lays the groundwork for future in vivo validation against P. aeruginosa infections.