Regulated delayed attenuation improves vaccine efficacy in preventing infection from avian pathogenic Escherichia coli O78 and Salmonella typhimurium

Avian pathogenic Escherichia coli (APEC) O-78 and Salmonella typhimurium (S. Typhimurium) are two leading bacterial pathogens that cause significant economic loss in the poultry industry. O-antigen is an important immunogen of these two bacteria to induce host protective immune responses during infection. To develop a bivalent vaccine against APEC O-78 and S. Typhimurium, the attenuated Salmonella ST01 (Delta asd Delta rfbp Delta crp) was genetically constructed to deliver APEC O-78 O-antigen polysaccharide (OPS), which stably expresses OPS with asd(+) balanced-lethal system in vitro and in vivo. After oral immunization, the recombinant attenuated Salmonella vaccine (RASV) strain ST01 (pSS26-O-78) provided insufficient protection against the APEC O-78 challenge. Therefore, the regulated delayed attenuation strain ST02 (Delta asd Delta rfbp Delta pcrp::TTaraC P-BAD crp) was further constructed by regulating cyclic AMP receptor protein (crp) with araC PBAD cassette to better present the heterologous O-antigen to the host immune system. The innovative recombinant strain ST02 (pSS26-O-78) stimulated robust antibody responses against APEC O-78 and S. Typhimurium OPS, with serum titers over 1:800 for both IgG and IgA, thereby providing the complement-mediated bactericidal activity and stronger protection against APEC O-78 and S. Typhimurium infection. Collectively, this study demonstrates a biologically-conjugated polysaccharide vaccine candidate that can enhance homologous protection against APEC O-78 and S. Typhimurium.