A Novel Vaccine for Bovine Diarrhea Complex Utilizing Recombinant enterotoxigenic Escherichia coli and Salmonella Expressing Surface-Displayed Chimeric Antigens from Enterohemorrhagic Escherichia coli O157:H7

Enterohemorrhagic Escherichia coli (EHEC) O157:H7, primarily found in cattle, is a zoonotic pathogen associated with Hemolytic Uremic Syndrome (HUS) in humans that is typically transmitted through contaminated food. Among its many virulence factors, the Type Three Secretion System (T3SS) is responsible for gut colonization. Neonatal calf diarrhea (NCD), in contrast, is primarily caused by pathogens such as enterotoxigenic Escherichia coli (ETEC), Salmonella spp, Bovine Coronavirus (BCoV) and Bovine Rotavirus type A (BRoVA). In this study, we engineered and expressed a chimeric protein combining EspB and Int280γ—two key components of the T3SS—in the membrane of Salmonella dublin and ETEC. We confirmed successful membrane anchorage, stability and preservation of the chimera and assessed its immunogenicity in murine and guinea pig models. Immune response evaluations showed that combining recombinant bacteria did not enhance immunogenicity, indicating either bacterium could be effective in a single formulation. Chimeric expression achieved equivalent immunogenicity to 10 µg of recombinant chimera protein, with similar antibody titers across doses, indicating that a single vaccination may suffice. IgG1 and IgG2a levels, along with Th1, Th2, and Th17 markers, suggest a mixed immune response, providing broad humoral and cellular protection. Additionally, the immune response to BCoV, BRoVA, ETEC and Salmonella antigens remained high and showed no interference with the chimera-specific responses, which could enhance the overall efficacy of an NCD vaccine. The results underscore the robust immunogenicity of the chimera, supporting its potential as a commercially viable and effective vaccine candidate against EHEC O157:H7. This strategy could enhance the valency of NCD vaccines by offering broader protection against calf diarrhea and contribute to public health by reducing the risk of HUS transmission from cattle to humans.