*4.3. Ferritin-Based Nanoparticle*

Ferritin is an intracellular globular protein that stores iron and releases it in a controlled fashion [99]. The protein is expressed in many tissues, mainly as a cytosolic protein, but it can be also found at low concentration in the serum where it functions as an iron carrier [100]. Ferritin is a four-helix bundle protein consisting of two couples of anti-parallel α-helices connected by a long loop and a short C-terminal α-helix (Figure 3A). The monomer can dimerize and further self-assemble to form a nanocage of 24 protein subunits, which has an internal and external diameter of 8 and 12 nm, respectively [101]. Ferritin nanoparticle (ferritin-NP) presents the advantage of being resistant to thermal and chemical stress [102]. Moreover, antigen display is achieved through genetic fusion at the N-terminus of each ferritin protomer. Ferritin-NP self-arranges as an octahedron composed of eight trimeric units (Figure 3B), each with a 3-fold symmetry axis that allows the correct presentation of trimeric antigens at the nanoparticle surface (Figure 3C).

**Figure 3.** Structure of ferritin nanoparticle. (**A**) The antigen of interest is fused to the N-terminus of the ferritin polypeptide (shown in red). (**B**) Ferritin assembles to form symmetry units with threefold axes. (**C**) Fully assembled nanoparticle with surface-exposed N-termini colored in red. The figure was prepared using PDB: 3bve using PyMOL software (The PyMOL Molecular Graphics System, Version 4.5 Schrödinger, LLC).

A recent development and one of the major advantages of this carrier is the possibility to generate multivalent displays of different antigenic proteins. For example, ferritin-NP was first used to present HIV-1 and influenza antigens on the same particle [103]. Furthermore, Kanekiyo and coworkers at the Vaccine Research Center in the United States. further developed and exploited the ferritin-NP system using a ferritin hybrid protein [89] to generate a universal vaccine against influenza. The team co-displayed the receptor binding domain (RBD) of the last 90 years H1N1 influenza viruses [104] and showed that immunization with this mosaic RBD-NP elicited a broad antibody response covering all the known H1N1 strains. Multiple displays on ferritin-NP have opened up new possibilities for novel vaccines against different pathogens or against different antigens of a given pathogen.

In the case of HCMV, we can speculate that a mosaic ferritin-NP displaying both pentamer and gB ECDs should generate a potent humoral immune reaction. The antibodies generated targeting the gHgL and gB subunits will block infection of connective tissues and stroma (containing fibroblasts). On the other hand, antibodies directed against the pUL subunits will block infection of epithelial tissues.
