Optimizing vaccine design for prevention of neonatal sepsis

Over the last years, protein nanoparticles (NPs) have raised increasing interest as innovative technology to develop next generation vaccines. Protein NPs are molecules with the intrinsic ability to self-assemble into highly stable and symmetric structures and they have been largely investigated as a platform to display vaccine antigens in multicopy ordered patterns with the overall aim to enhance their immunogenicity. Klebsiella pneumoniae (Kp) is one of the major causes of neonatal sepsis in low- and middle-income countries (LMICs). Sepsis occurs in the first 72 hours after birth and the increase of multi-drug resistant strains makes more urgent the development of a vaccine with a particular attention to maternal vaccination. With the aim to allow a strong response after a single immunization, in this work type 3 fimbriae major subunit MrkA belonging to Kp was selected as model antigen to be genetically fused to a panel of self-assembling protein NPs, differing for size and number of subunits composing the final structure. MrkA is a well conserved protein expressed in approximately 60% of Kp strains, including the hypervirulent ones. MrkA has been proposed as vaccine antigen and demonstrated able to stimulate a protective immune response in vivo. However, its 3D-molecular structure is not yet known. An in-depth structural investigation of MrkA by X-ray crystallography and Nuclear Magnetic Resonance (NMR) was performed to inform about the optimal antigen design and to determine its structure. MrkA was successfully displayed on NPs and tested in in vivo studies. In particular, by using larger scaffolds combined with different multicopy display of the MrkA antigen, an efficient anti-MrkA immune response was obtained for all the NPs investigated already after the first injection compared to the recombinant MrkA antigen, both in mice and in rabbits. In conclusion, this work demonstrates and supports the ability of self-assembling NP to increase the immune response of a bacterial protein antigen like MrkA and contribute to better characterize this antigen from a structural point of view.