High resolution immunoinformatic profiling of Zonula occludens toxin reveals a conserved multiepitope vaccine...

Acinetobacter baumannii is a multidrug-resistant (MDR) nosocomial pathogen associated with exceptionally high mortality, underscoring the urgent need for immune-based strategies that operate independently of antibiotic susceptibility. The Zonula occludens toxin (Zot), a barrier-disrupting virulence factor embedded within mobile genetic elements, has recently been proposed as a conserved component of clinically relevant A. baumannii lineages, however, its immunological architecture remains poorly defined. Here, we applied an integrated immunoinformatic and evolutionary framework to evaluate Zot as a target for adaptive immune recognition and immunotherapeutic intervention. Comparative analysis of 62 full-length Zot homologs revealed structured sequence diversity superimposed on near-complete alignment continuity, consistent with structural constraint maintaining protein integrity. Structural modeling demonstrated that deep sequence divergence between historical and contemporary isolates does not disrupt the overall Zot fold, indicating conservation of antigenic architecture. High-resolution epitope mapping identified multiple potential B-cell-accessible regions and dense clusters of conserved MHC Class I and Class II binding motifs, particularly within a compact C-terminal domain exhibiting strong sequence and structural constraint. Notably, predicted T-cell epitopes remained resilient across divergent strains, consistent with conserved MHC anchor residue architecture. Together, these findings suggest that Zot may represent a structurally constrained immunological target with features consistent with both humoral and cell-mediated immune engagement across genetically diverse A. baumannii backgrounds. This immune-centric characterization provides a mechanistic foundation for immunotherapeutic targeting of Zot and supports its prioritization for further experimental evaluation in vaccine development pipelines targeting MDR A. baumannii.