Abstract
Diarrheal diseases caused by Shigella and enterotoxigenic Escherichia coli (ETEC) are significant health burdens, especially in resource-limited regions with high child mortality. In response to the lack of licensed vaccines and rising antibiotic resistance for these pathogens, this study developed a recombinant Shigella flexneri strain with the novel incorporation of the eltb gene for the heat-labile enterotoxin B (LTB) subunit of ETEC directly into Shigella's genome, enhancing stability and consistent production. This approach combines the immunogenic potential of LTB with the antigen delivery properties of S. flexneri outer membrane vesicles (OMVs), aiming to provide cross-protection against both bacterial pathogens in a stable, non-replicating vaccine platform. We confirmed successful expression through GM1-capture ELISA, achieving levels comparable to ETEC. Additionally, proteomic analysis verified that the isolated vesicles from the recombinant strains contain the LTB protein and the main outer membrane proteins and virulence factors from Shigella, including OmpA, OmpC, IcsA, SepA, and Ipa proteins, and increased expression of Slp and OmpX. Thus, our newly designed S. flexneri OMVs, engineered to carry ETEC's LTB toxin, represent a promising strategy to be considered as a subunit vaccine candidate against S. flexneri and ETEC.