Development and Protective Evaluation of an O18-CRM197 Glycoconjugate Candidate Preparation Against APEC O18...

Avian pathogenic Escherichia coli (APEC) is an important cause of colibacillosis in poultry, and increasing antimicrobial resistance has highlighted the need for alternative preventive strategies. This study aimed to develop an O18-cross-reacting material 197 (O18-CRM197) glycoconjugate vaccine candidate using an Escherichia coli-based protein glycan coupling technology (PGCT) platform and to evaluate its immunogenicity and preliminary protective performance in specific-pathogen-free (SPF) chickens. A CLM24-derived production host, Y03, was generated by sequential deletion of lpxM, zwf, and gldA using a CRISPR/Cas9- and λ-Red-mediated genome editing system. An O18 O-polysaccharide expression plasmid and a co-expression plasmid encoding PglB and a modified CRM197 carrier protein were introduced into Y03 for in vivo glycoconjugate production. Western blotting showed high-molecular-weight signals recognized by both anti-CRM197 antibody and anti-O18 serum, supporting production of an O18-CRM197 glycoconjugate candidate. Immunization with the adjuvanted purified candidate induced O18-specific serum immunoglobulin G (IgG) responses in SPF chickens. After homologous APEC O18 challenge at 1 × 10⁹ colony-forming units (CFU)/bird, immunized chickens showed reduced gross lesion scores and bacterial burdens in blood and organs, with the 50 μg dose showing more consistent protective performance than the 30 μg dose. These findings provide preliminary evidence that the adjuvanted purified O18-CRM197 candidate preparation has immunogenicity and protective potential against homologous APEC O18 challenge.