PROJECT SUMMARY Klebsiella pneumoniae is an encapsulated human pathogen capable of causing a myriad of human infections. Recently, K. pneumoniae has also emerged as one the most common causes of secondary bacterial pneumonia in COVID-19 patients. Over the last 40 years, K. pneumoniae has evolved into two distinct pathotypes, known as classical K. pneumoniae (cKp) and hypervirulent K. pneumoniae (hvKp). cKp commonly acts as an opportunistic pathogen causing disease in hospitalized or immunocompromised individuals. In fact, cKp is annually responsible for 5% of all healthcare-associated infections and is the leading cause of nosocomial pneumonia in the US. Furthermore, cKp isolates are often carbapenem-resistant (CR), limiting treatment options. In the US, K. pneumoniae multilocus sequence type 258 (ST258) strains account for ~70% of all carbapenem- resistant K. pneumoniae infections. Conversely, hvKp usually cause community-acquired infections in healthy hosts that frequently manifest as community-acquired pneumonia. Like ST258 infections, hvKp infections have high mortality rates approaching 40-60%. Currently, there are no licensed vaccines available to prevent K. pneumoniae infections and none in clinical trials. Nevertheless, preliminary data demonstrate both cKp and hvKp infections can be prevented by vaccines that target their capsular polysaccharide (CPS). Conjugate vaccines consist of a CPS covalently attached to an immunogenic carrier protein. While the clinical benefits of conjugate vaccines are well documented, the development of new conjugate vaccines targeting K. pneumoniae is lagging, likely due to the high technological barriers to entry and high costs associated with conjugate vaccine production. In addition, most conjugate vaccines are multivalent, further increasing manufacturing complexities. In order to simplify conjugate vaccine production, we have developed an in vivo conjugation platform termed bioconjugation. Bioconjugation allows for the simultaneous production of the CPS, the carrier protein and their subsequent covalent linkage all within E. coli. Key to our bioconjugation platform is our patented conjugating enzyme, PglS, which attaches virtually any polysaccharide to a unique amino acid sequence fused to the carrier protein. Furthermore, bioconjugation is modular, allowing for rapid production of multiple, different CPS-protein conjugates. Using our bioconjugation platform, we are developing a multivalent CPS-based bioconjugate vaccine to prevent the majority of K. pneumoniae infections. In this Phase I STTR program, four serotypes were initially selected (K1, K2, KL106, KL107) as these serotypes are associated with >80% of all hvKp (K1 and K2) isolates worldwide and >70% of ST258 (KL106 and KL107) isolates in the US. In Aim 1, we will produce a tetravalent (K1, K2, KL106, KL107) bioconjugate vaccine on a modified carrier protein glycosylated at an internal site, which is expected to improve conjugate characteristics su...