*4.2. Hepatitis B Core Antigen Nanoparticle*

The core protein of the hepatitis B virus (HBc) self-assembles into highly immunogenic virus-like structures with icosahedral symmetry. The hepatitis B core nanoparticle (HBc) is assembled from dimers of 183 or 185 amino acids and occurs in two size classes with a T = 3 or T = 4 symmetry. Depending on the symmetry, HBc is formed by 90 or 120 dimeric subunits with a respective diameter of 24 or 31 nm [93]. The antigen of interest needs to be inserted between alpha helices 3 and 4 of the major immune-dominant region (MIR) domain (amino acids 78–82) (Figure 2A) of the HBc protein. This insertion allows the antigen to be exposed on spike structures at the surface of the assembled nanoparticle. HBc protomers first dimerize and then multimerize (Figure 2B,C). Several developments led to the success of this platform, such as splitting of the HBc protein between the α3 and α4 helices or fusing two monomers together using a flexible glycine serine linker that allowed the introduction of larger protein domains [94,95]. However, one limitation of this technology is the need for both the Nand C-termini of the inserted antigen to fit with the geometry of the core acceptor site, a requirement that is not always successfully met by natively folded proteins.

**Figure 2.** Hepatitis B core antigen and relative nanoparticle. (**A**) Polypeptide fold and structure of HBcAg monomer. The antigen of interest needs to be inserted in the MIR loop connecting alpha helices 3 and 4. (**B**) Two HBcAg monomers spontaneously interact to form a dimer, which acts as an intermediate in the nanoparticle formation. (**C**) Assembled nanoparticle containing either 90 or 120 dimers. The figure was prepared with PDB: 6htx using Pymol software (The PyMOL Molecular Graphics System, Version 4.5 Schrödinger, LLC).

In summary, the HBc is a powerful platform to generate nanoparticles and it is particularly useful in the case of small antigens. Indeed, it is unlikely that complex envelope glycoproteins such as gB or the gHgL complexes could be correctly displayed on HBc particles. Nevertheless, HBc could be very efficient to generate strong T cell responses [96]. For example, the viral-encoded G protein-coupled receptor US28 was recently shown to aid in the establishment and the maintenance of viral latency. US28 modulates host–cell proteins to suppress viral processes associated with lytic replication and thereby represent an important target promoting latent infection [97]. HBc particles displaying US28 peptides recognized by T cells could provide an important advancement to boost the existing immune responses necessary to control reactivation of latently infected individuals [98].
