Comparison of Immunogenicity of Cood-Cotd, CfaB-st-CfaE, and Cssa-Cssb-ltb Chimeric Proteins and Their Chitosan Nanoparticles Against ETEC in an Animal Model.

Enterotoxigenic Escherichia coli (ETEC) remains a major cause of diarrheal disease, and colonization factors together with enterotoxins are key targets for vaccine development. In this study, three ETEC-derived chimeric proteins, Cssa-Cssb-LTB, CfaB-ST-CfaE-LTB, and CooD-CotD, were produced and evaluated in free and chitosan nanoparticle-formulated forms. The recombinant proteins were expressed in E. coli BL21 (DE3) using the pET-28a vector, purified by Ni-NTA affinity chromatography, and confirmed by western blotting. Chitosan nanoparticles containing each recombinant protein were prepared and used for immunization of BALB/c mice. Antigen-specific antibody responses were assessed by ELISA. Cssa-Cssb-LTB was purified under soluble/native conditions, whereas CfaB-ST-CfaE-LTB and CooD-CotD were purified under denaturing conditions. The mean sizes of nanoparticles containing Cssa-Cssb-LTB, CfaB-ST-CfaE-LTB, and CooD-CotD were 121.5, 24.93, and 63.22 nm, respectively. Immunological evaluation showed that the cocktail formulation induced the strongest immune response, followed by the oral-injection regimen. The recombinant chimeric proteins induced marked humoral immune responses, and formulation in chitosan nanoparticles enhanced their immunogenic potential. These findings support the potential of chimeric antigen-loaded chitosan nanoparticles as a promising strategy for ETEC vaccine development.