Uncovering novel OMPs of Vibrio cholerae: Biophysical insights and epitope prediction of AcfA, Opa, VxrD, LamB, and VpsM for immunotherapy and vaccine design.

AcfA, Opa, VxrD, LamB, and VpsM are outer membrane proteins (OMPs) in Vibrio cholerae that contribute significantly to its virulence and show strong promise as components of subunit vaccines. Owing to their inherent β-sheet-rich structures, these proteins tend to aggregate into inclusion bodies during recombinant expression. To achieve proper folding and restore their native-like configurations, multiple detergents were assessed for their ability to facilitate in vitro refolding. We systematically investigated the performance of both non-ionic and zwitterionic detergents, identifying the highly effective candidates for promoting correct structural recovery. Structural confirmation was achieved through circular dichroism spectroscopy and intrinsic tryptophan fluorescence, while additional validation using heat-induced mobility shifts and proteolytic stability assays confirmed that the refolded proteins adopted compact and stable conformations. Furthermore, immunoinformatic screening revealed immunologically relevant B-cell and T-cell epitopes localized in the extracellular loops of AcfA, Opa, VxrD, LamB, and VpsM regions that are surface-accessible and immunodominant. These findings underscore the therapeutic potential of these OMPs, highlighting their immunogenic properties, predicted safety, and ability to trigger comprehensive immune responses aimed at controlling cholera.