Leveraging MrkA Display on Self-Assembling Protein Nanoparticles for Enhanced Immune Activation

: Self-assembling protein nanoparticles (NPs) offer a versatile platform to enhance antigen immunogenicity through a multivalent display. Klebsiella pneumoniae (Kp) is a critical multidrug-resistant pathogen for which no licensed vaccine exists, posing a significant global health threat. This study aimed to develop and evaluate protein nanoparticle-based vaccine candidates presenting MrkA, a highly conserved type 3 fimbrial subunit and promising Kp vaccine target, to elicit robust protective immune responses. We genetically fused MrkA to four distinct self-assembling protein nanoparticle scaffolds: Helicobacter pylori ferritin, Thermotoga maritima encapsulin, computationally designed mI3, and Mycobacterium tuberculosis dodecin. The resulting MrkA-NPs were expressed and purified, and their structural integrity was confirmed by electron microscopy, dynamic light scattering, and nuclear magnetic resonance spectroscopy. These constructs were then evaluated for their immunogenicity in animal models. All MrkA-displaying NPs significantly enhanced MrkA-specific IgG responses, eliciting significantly higher antibody titers compared to recombinant MrkA. Importantly, passive transfer of immune sera from rabbits immunized with MrkA-dodecin NP conferred significant protection against a lethal Kp challenge, resulting in 90% survival compared to the control group. Our findings confirmed the ability of NPs to significantly enhance the immune responses against a bacterial protein such as MrkA, confirming their potential as transformative tools in vaccine development. This study also offers promising results for the development of a vaccine against Kp, addressing antimicrobial resistance and improving global health.