An In Silico-Designed Chimeric Antigen Confers Broad Protection Against Major Diarrheal Pathogens (Shigella, ETEC, and EHEC) in Preclinical Models.

Diarrheagenic E. coli (DEC) and Shigella species are leading causes of under-5 mortality globally, prompting the development of a broad-spectrum vaccine. Here, we rationally designed a novel chimeric antigen ("SEISL") incorporating five virulence determinants: StxB and EspA (EHEC), LTB and a detoxified STa (ETEC), and the N-terminal domain of IpaD (Shigella). Bioinformatically optimized for maximal antigenicity, SEISL was expressed in E. coli BL21(DE3), purified from inclusion bodies under denaturing conditions, and confirmed by western blot. Immunization with SEISL + Freund's adjuvants in pathogen-specific models elicited robust protection in all models.In rabbits (ETEC challenge), serum IgG significantly increased and ileal loop assays with virulent ETEC H10408 showed a significant reduction in fluid accumulation (p<0.05). The Guinea pigs exhibited 71.4% protection against Shigella flexneri-induced keratoconjunctivitis in Sereny tests, and finally the mice (EHEC challenge) demonstrated elevated serum IgG and significant reduction in fecal shedding of EHEC O157:H7 post-challenge (p<0.05). These results demonstrate that the SEISL antigen elicits broad protection immune responses against three critical diarrheal pathogens in preclinical models, providing proof-of-concept support for its continued development as a multipathogen vaccine candidate.