Preclinical immunogenicity and efficacy of optimized O25b O-antigen glycoconjugates to prevent MDR ST131 E. coli infections

Multivalent O-antigen polysaccharide glycoconjugate vaccines are under development to prevent invasive infections caused by pathogenic Enterobacteriaceae. Sequence type 131 Escherichia coli of serotype O25b has emerged as the predominant lineage causing invasive multi-drug resistant extraintestinal pathogenic E. coli infections. We observed the prevalence of E. coli O25b ST131 among a contemporary collection of isolates from US bloodstream infections (2013-2016, n=455) and global urinary tract infections (2014-2017,n=103) to be 24 percent and 23 percent, respectively. To maximize immunogenicity of the serotype O25b O31 antigen, we investigated glycoconjugate properties including CRM197 carrier protein cross linking (single-end vs cross-linked lattice) and conjugation chemistry (RAC/DMSO vs (2-((2-oxoethyl)thio)ethyl)carbamate (eTEC) linker). Using opsonophagocytic assays (OPA) to measure serum functional antibody responses to vaccination, we observed that higher molecular mass O25b long chain lattice conjugates showed improved immunogenicity in mice compared with long or short chain O-antigens conjugated via single-end attachment. The lattice conjugates protected mice from lethal challenge with acapsular O25b ST131 strains as well as against hypervirulent O25b isolates expressing K5 or K100 capsular polysaccharides. A single 1 microgram dose of long chain O25b lattice conjugate constructed with both chemistries also elicited robust serum IgG and OPA responses in cynomolgus macaques. Our findings show that key properties of the O-antigen carrier protein conjugate such as saccharide epitope density and degree of intermolecular cross-linking can significantly enhance functional immunogenicity.