Klebsiella pneumoniae (Kp) is a Gram-negative bacterium, and a leading cause of neonatal sepsis in low- and middle-income countries, often associated with anti-microbial resistance. Two types of polysaccharides are expressed on the Kp cell surface and have been proposed as key antigens for vaccine design: capsular polysaccharides (known as K-antigens, K-Ags) and O-antigens (O-Ags). Historically, Kp has been classified using capsule serotyping and although 186 distinct genotypes have been predicted so far based on sequence analysis, many structures are still unknown. In contrast, only 11 distinct OAg serotypes have been described. The characterization of emerging strains requires the development of a high-throughput purification method to obtain sufficient K- and O-Ag material to characterize the large collection of serotypes and gain insight on structural features and potential cross-reactivity that could allow vaccine simplification. Here, this was achieved by adapting our established method for the simple purification of O-Ags, using mild acetic acid hydrolysis performed directly on bacterial cells, followed by filtration and precipitation steps. The method was successfully applied to purify the surface carbohydrates from different Kp strains, thereby demonstrating the robustness and general applicability of the purification method developed. Further, antigen characterization showed that the purification method had no impact on the structural integrity of the polysaccharides and preserved labile substituents such as O-acetyl and pyruvyl groups. This method can be further optimized for scaling up and manufacturing to support the development of high-valency saccharide-based vaccines against Kp.

Klebsiella pneumoniae is a leading cause of nosocomial infections, neonatal sepsis, and childhood mortality worldwide. A drastic rise in antibiotic-resistant isolates poses an urgent threat to humanity, and the World Health Organization (WHO) has classified this as a critical-priority antimicrobial-resistant (AMR) pathogen. Recent advancements in developing vaccines against Klebsiella pneumoniae have highlighted the lack of standardized assays to evaluate immunogenicity, complicating comparison among different vaccines under development and the establishment of a serological threshold of risk reduction (SToRR). Here, we describe the development of a ten-plex multiplex assay to measure IgG against capsular polysaccharides (K2, K25, K102, K149), O antigens (O1v1, O1v2, O2v1, O2v2 and O5), and a conserved protein (MrkA). A standard curve was established by pooling human sera from naturally exposed subjects and then calibrated in terms of Relative Luminex Units/mL. The assay was fully characterized in terms of specificity, precision, linearity, and repeatability. This immunoassay demonstrates performance suitable for future clinical trials, as well as to perform sero-epidemiological studies to gain insights into naturally occurring immunity, potentially contributing to the establishment of a serological threshold of risk reduction against Klebsiella pneumoniae.