Summary
Klebsiella pneumoniae (Kp) is a significant cause of hospital-acquired infections, leading to severe diseases such as pneumonia, sepsis, and urinary tract infections. This pathogen causes large-scale outbreaks in healthcare settings, with rapid transmission and severe clinical outcomes. The prevalence of the K64 capsular type, associated with the hyper-virulent sequence type ST147, has risen dramatically from 2% of reported genomes in 2012 to over 15% in recent years, underscoring the urgency for targeted vaccine development.
This project proposes to synthesize and compare multiple glycoconjugate vaccines targeting the K64 capsular polysaccharide (CPS). By conjugating K64 CPS with selected homologous carrier proteins, we aim to induce broader protective immunity, potentially offering cross-protection against different Kp serotypes. Mice will be immunized with the different glycoconjugates, and the induced immunogenicity evaluated by analyzing sera for antigen-specific antibody responses (IgG and IgM) and functional assays such as serum bactericidal activity and opsonophagocytic killing capacity against various Kp strains.
The expected outcomes include identifying the most effective carrier protein for the K64-based glycoconjugate, developing a vaccine that provides cross-protection against multiple serotypes, and demonstrating strong immunogenicity in animal models.
This project will also pave the way for future research, such as applying the developed methodology to other capsular types and pathogens, optimizing vaccine formulations, and advancing promising candidates to clinical trials in collaboration with LMIC partners and industry. The successful development of this vaccine could significantly reduce Kp infections in both hospital and community settings, particularly benefiting LMICs where the burden of such infections is high.
Project Outcomes
Klebsiella pneumoniae is a leading Gram-negative cause of healthcare-associated infections (HAIs), implicated in pneumonia, bloodstream, and urinary tract infections worldwide. Surveillance and genomic studies consistently rank K. pneumoniae among the most prevalent nosocomial pathogens, with growing antimicrobial resistance burdens. Among capsular (K) types, K64 has repeatedly featured in high-risk, multidrug-resistant lineages such as ST147, driving hospital outbreaks and underscoring the rationale for capsular-antigen vaccination.
Because polysaccharides are T-independent antigens, they elicit suboptimal, poorly boosting responses in naïve hosts; covalent conjugation to protein converts the response to T-cell-dependent immunity with improved class switching, affinity maturation, and memory—principles validated by licensed conjugate vaccines. Building on this, we generated K64 glycoconjugates comprising purified K64 capsular polysaccharide (CPS) linked to carrier proteins. Alongside benchmark carriers (CRM197, ovalbumin), we evaluated homologous carriers derived from K. pneumoniae (i.e. OmpA, OmpA-displaying nanoparticle (OmpA-NP) and a DUF-family surface protein) to test whether adding conserved bacterial protein epitopes could broaden strain coverage beyond K64 alone. OmpA has independent support as a Klebsiella vaccine antigen, and nanoparticle display is a recognized strategy to enhance B-cell activation and breadth.
We extracted and purified K64 CPS, then optimized controlled sonication to balance chain length and conjugation efficiency. Conjugation employed 1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP) random chemistry, and products were characterized by orthogonal analytics: SDS-PAGE (high-molecular-weight, polydisperse smears diagnostic of CPS–protein coupling), HPLC-SEC, and sugar/protein colorimetry, with SEC and HIC for polishing.
In mice (prime/boost on days 0/15, alum-adjuvanted, 5 µg/mL sugar dose), all K64 conjugates elicited robust anti-K64 CPS IgG, with immune sera bound whole K64 K. pneumoniae and K64-poly-L-lysine in ELISA, indicating recognition of capsule also in its native context. Immune sera also bound to heterologous K1 and K2 strains by ELISA, with breadth most evident for DUF, and with some extent OmpA-NP, with other conjugates giving lower responses. Preliminary serum bactericidal assays (SBA) demonstrated complement-dependent killing, strongest for OVA glycoconjugate. Notably, DUF also showed a reproducible complement-dependent effect, consistent with its high ELISA titers, although further assays are needed to confirm the extent of functional protection. Opsonophagocytic assays (OPA) have been established with controls and will be applied to immune sera in follow-up studies.
Together, these findings provide proof-of-concept for a K64 conjugate vaccine and support the idea that homologous Klebsiella proteins, particularly DUF, may augment capsule immunogenicity and broaden strain coverage. The immediate next steps are to complete SBA/OPA assays and move toward efficacy testing in challenge models.