An integrated immunoinformatics and experimental pipeline identifies an LCL-scaffolded OmpA loop 3 construct as a...

Acinetobacter baumannii has emerged as a critical nosocomial pathogen due to its extensive antimicrobial resistance and limited treatment options, highlighting the urgent need for effective vaccines. In this study, we developed and evaluated a rationally engineered epitope-based vaccine targeting extracellular loop 3 of outer membrane protein A (OmpA). Using an integrated immunoinformatics and experimental pipeline, the selected OmpA loop was grafted onto a loopless C-lobe (LCL) scaffold derived from transferrin-binding protein B to preserve native-like conformation and enhance epitope presentation. Four hybrid constructs were designed and comprehensively evaluated through computational analyses, including antigenicity prediction, structural modeling, molecular dynamics simulation, Toll-like receptor docking, and MHC-II binding assessment. Among them, OmpAL3-P2 exhibited the most favorable structural stability, surface accessibility, and epitope preservation profile. Experimental validation demonstrated successful recombinant expression and purification of the selected construct. Immunization of C57BL/6 mice with OmpAL3 induced strong antigen-specific humoral and cellular immune responses characterized by elevated IgG, predominant IgG2c production, enhanced splenocyte proliferation, and increased IFN-γ secretion, indicating a Th1-skewed response. In murine sepsis challenge models, OmpAL3 conferred significant protection, reduced bacterial burdens in major organs, and preserved tissue integrity. Passive immunization experiments further confirmed the protective contribution of vaccine-induced antibodies. Collectively, these findings demonstrate that LCL-scaffolded OmpA loop 3 is a promising subunit vaccine candidate against multidrug-resistant A. baumannii and supports the utility of scaffold-based structural vaccinology for rational vaccine development.