Protective immunity elicited by distinct polysaccharide antigens of classical and hypervirulent Klebsiella

PROJECT SUMMARY This project will enhance our understanding of the humoral immune response to Klebsiella pneumoniae (Kp) and its polysaccharide antigens with the long-term goal of guiding and optimizing broad vaccine development. Kp infections, including pneumonia, urinary tract infection, and bacteremia, are sharply on the rise among hospitalized patients; CDC has declared infections with Kp and other carbapenem-resistant Enterobacteriales (CRE) demand a threat level of urgent. Beyond classical Kp typically seen in the US, emerging hypervirulent Kp strains, capable of causing liver abscess, bacteremia, and meningitis in healthy hosts, are spreading globally. This work builds on the PI's background in bacteriology, Gram-negative bacterial pathogenesis, and modeling of host adaptive immune responses, to investigate antibodies targeting Kp's polysaccharide capsule (K-type) and O-antigen. We have found that, while mice are able to produce antibodies targeting Kp capsule and O- antigen, capsule may directly interfere with O-antibody binding and killing of Kp. With our collaborators at Omniose, we have developed and are testing novel bioconjugate vaccines targeting the most prevalent K- and O-types. Bioconjugation is an alternative manufacturing process that uses recombinant E. coli strains to concurrently produce the capsule or O-antigen and an engineered carrier protein, and to enzymatically link the two. We have produced multiple K- and O-bioconjugates that have demonstrated promising efficacy in mice. As both O- and K- vaccines are under development, we will use our novel bioconjugate vaccines in murine protection experiments to determine the relative effectiveness of O-antigen or K-antigen bioconjugates against classical and hypervirulent Kp isolates. Further, we will challenge O-immunized mice with strains of closely related O-antigen structural subtypes that have not been included to date in vaccine formulations being developed commercially. Potential masking of O-antigen by capsule will be determined through mouse serum IgG ELISAs. Serum bactericidal assays (SBAs) and opsonophagocytic killing assays (OPKAs) will be developed and correlated with murine protection. Further, with bacterial mutants, complemented strains, and capsule inhibitors, we will determine the specificity of Kp O-antibody inhibition by capsule, utilizing multiple techniques including biolayer interferometry, immunofluorescence microscopy, and transmission immunoelectron microscopy. Finally, we will perform a first-ever longitudinal study of human patients with Kp infection, analyzing their sera for antibodies specific to Kp polysaccharides and their functional activity against the inciting Kp strain. At the conclusion of these studies, the relative efficacy of both K- and O-type bioconjugate vaccines will be determined, cross-protection among O-antigen subtypes will be resolved, correlates of protective immunity will be established, and mechanisms of O-antigen masking will be defined. Our results will illuminate human antibody responses to Kp infection and guide vaccine development to target this worrisome pathogen.