Immune recognition of Klebsiella pneumoniae O2v1 and O2v2 O-antigen subtypes

PROJECT SUMMARY This research project will enhance our understanding of the immune responses elicited by the O-antigen of Klebsiella pneumoniae (Kp), an important target in putative vaccine development. The long-term goal of this proposal is to further our knowledge of the innate and adaptive immune responses elicited by two subtypes of the common serogroup O2 of Kp, O2v1 and O2v2. We seek to understand how the addition of the single branched galactose of O2v2 changes its bacterial fitness and host immune susceptibility relative to O2v1. Further, we will determine cross-reactivity between these two subtypes and whether infection or vaccination with one subtype confers cross-protection against the other, information critical to the design of vaccines that can broadly target this increasingly antibiotic-resistant organism. Kp infections, including pneumonia, urinary tract infection, and bloodstream infection, are sharply on the rise among hospitalized patients; CDC has declared that infections with Kp and other carbapenem-resistant Enterobacteriales demand a threat level of urgent. This project builds on the PI's background in bacteriology, murine models of infection, and pathogenesis studies of Gram-negative bacteria, by focusing specifically on the Kp virulence factor O-antigen and the host immunogenic response. We have previously demonstrated that use of bioconjugate vaccines targeting Kp are beneficial in preventing disease and seek to increase our understanding and breadth of coverage of future vaccines. Kp historically has eleven known serogroups of O-antigen. Recently, additional O-antigen subtypes within these serogroups have been identified. Yet, differences in pathogenic fitness, immunogenicity, functional antibody response, and cross-protection between related subtypes are not well understood. We have constructed a mutant of a classical Kp strain expressing O-antigen subtype O2v2 to generate an otherwise isogenic strain expressing O2v1. We will test if these bacteria exhibit similar phenotypic expression of virulence factors by measuring capsule, hypermucoviscosity, fimbriae and biofilm. A well-established murine model of pneumonia will be leveraged to determine pulmonary fitness and delineate host innate immune response to each pathogen. Neutrophil and complement-mediated bacterial killing assays will be employed to further explore bacterial resistance to innate immune attack, which the literature indicates may be enhanced in O2v2 strains. Next, we will perform a range of experiments testing if O2v1 and O2v2 are cross-protective. Using classically immunological techniques including ELISA and flow cytometry, we will determine the IgG subclasses and effector cytokines elicited by respiratory tract infection with these pathogens. Lastly, using novel O2v1 or O2v2 bioconjugate vaccines followed by challenge with O2v1 or O2v2 strains, we will assess cross-protection against weight loss and mortality from pneumonia. We will further characterize antibody functionality using serum bactericidal and opsonophagocytic killing assays. These studies will significantly advance our understanding of immune response to Kp O-antigen and aid in vaccine design to combat this pathogen.