Monoclonal Antibodies Directed Against Surface Antigens of Klebsiella Pneumoniae as Potential Immunoprophylactic Treatment Against Bacterial Infection

Abstract

Antimicrobial resistance represents a significant threat to global health, contributing to millions of deaths annually. The rapid emergence of multidrug-resistant (MDR) pathogens, such as Klebsiella pneumoniae, has exacerbated the challenge, particularly in the absence of new antibiotics. K. pneumoniae is responsible for a wide range of infections, including pneumonia, bloodstream infections, and urinary tract infections, often leading to increased morbidity and mortality due to its resistance to multiple antibiotics. This critical global situation requires urgent need for alternative therapeutics, for example, immunotherapies based on highly effective monoclonal antibodies (mAbs). We hypothesize that mAbs targeting critical K. pneumoniae surface proteins, such as outer membrane proteins and siderophores, can serve as an effective prophylactic or therapeutic strategy against MDR infections. In this study, we developed murine mAbs against two key proteins of K. pneumoniae, outer membrane protein (OmpW) and the siderophore receptor (FepA), both of which are associated with bacterial virulence and survival. We expressed both proteins in E. coli and used the recombinant proteins as adjuvanted immunogens for immunization and induction of activated B cells in BALB/c mice. We generated mAbs using standard hybridoma fusion techniques with SP2/0 myeloma cells. Supernatants from positive hybrids, as determined microscopically, were screened for antibody specificity using enzyme-linked immunosorbent assay (ELISA). Hybridomas that produced high titers of antibodies were cloned by limiting dilution to achieve monoclonal antibodies. Culture supernatants from the clonal population were screened again, and the developed K. pneumoniae mAbs were characterized for their class and isotype as well as their ability to bind to the immunogen and K. pneumoniae. mAbs of each specificity were also evaluated for their ability to engage in opsonophagocytosis (OP) using murine phagocytic macrophage cell lines, which express Fc receptors crucial for antibody-mediated clearance. The OP assay, based on flow cytometry, was optimized using fixed and pHrodo Red-labeled K. pneumoniae to quantify the phagocytic uptake by macrophages. Results demonstrated that mAbs targeting OmpW-2, but not OmpW-1 significantly increased OP, as determined by the mean fluorescence intensity, compared to the control mAbs. We also evaluated the OmpW and FepA mAbs for their efficacy to prevent K. pneumoniae infection using the Galleria mellonella worm model. Measuring the survival of G. mellonella larvae for either 24 or 48 hours in the presence of mAbs, we observed that while some mAbs were quite effective (e.g., anti-OmpW-1), mAbs targeting the other specificities, OmpW-2 and FepA, were ineffective. Overall, these data suggest that targeting surface proteins of K. pneumoniae with mAbs could enhance bacterial clearance in vitro and in vivo, although further studies are needed to optimize mAbs dosage and improve synergistic effects for potential clinical applications.