Identification and characterisation of of vaccine antigens for Klebsiella pneumoniae

Klebsiella pneumoniae is a Gram–negative bacterium, which causes septicaemia, respiratory tract infections, urinary tract infections and soft tissue infections. K. pneumoniae is prevalent among nosocomial infections, accounting for up to 8% of all nosocomial infections in the Europe. It is a major opportunistic pathogen and is also a typical antimicrobial resistant pathogen, resistant to ampicillin, amoxicillin, second– and third–generation cephalosporins, carbapenems, and colistins. This pathogen is associated with high mortality rates and is more prevalent in Latin America and Asia–Pacific regions than in North America and Europe. It was the third most commonly reported bacterial species in 2021, accounting for 11.9 % of all reported cases in the EU/EEA. Therefore, there is an urgent need to develop a vaccine against K. pneumoniae infection. Vaccines are considered as a promising approach against AMR pathogens, as they pose no substantial selection pressure on the environment and do not contribute to antimicrobial resistance. Despite a long history of investigating of K. pneumoniae vaccines, there is no efficacious vaccine approved to date. Bacterial proteins involved in host cell attachment have been identified as promising vaccine candidates. The McClean Lab has developed a cell blot approach to identify bacterial proteins involved in host cell attachment as potential vaccine antigens against Burkholderia cenocepacia, Burkholderia multivorans, and Verocytotoxigenic E. coli. In this study, the attachment of K. pneumoniae CIP52.145 wild–type strain and CIP52.145 Δcps strain to two lung epithelial cell lines, A549 and 16HBE14o– were examined. The CIP52.145 wild–type strain showed 7.3–fold greater binding to 16HBE14o– cells than the mutant strain (p = 0.0215), but a lower attachment ability to A549 cells (4.7–fold, p = 0.0081). In total, 31 bacterial proteins involved in host cell attachment from K. pneumoniae were identified, 24 of which were novel and common to both K. pneumoniae strains studied. Five antigens were selected for further analysis: LysM/BON family protein (LysM/BON), DNA protection during starvation protein (Dps), OMPP1/FadL/TodX family outer membrane transporter (OMPP1), TolC family protein (TolC) and UTP––glucose–1–phosphate uridylyltransferase (GalU). LysM/BON, Dps, OMPP1 and GalU were confirmed to play a role in K. pneumoniae attachment to lung cells in vitro, as BL21 cells expressing recombinant proteins demonstrated increased levels of attachment to 16HBE14o– cells by 14.3–fold, 7.22–fold, 6.48–fold, and 13.3–fold respectively compared to BL21 cells transformed with empty plasmid. LysM/BON, Dps and OMPP1 were individually shown to be protective in mouse immunisation and challenge studies. In each case, lower levels of K. pneumoniae burden were observed in peritoneal cavities and spleens of antigen immunised mice, with the expression of high antigen–specific antibody responses in mice compared to the control group. T–cell responses induced by the immunisation of rLysM/BON or rOMPP1 demonstrated that the stimulation of IL–17 and IL–22 expression was most important against K. pneumoniae, especially in NK cells. Although GalU also stimulated high antibody responses, mice immunised with rGalU demonstrated higher K. pneumoniae colonisation level in the lungs. Anti–GalU antibodies produced by mice increased host cell attachment, suggesting antibody dependent enhancement (ADE) of infection in mice. This immunisation and