*2.1. Genetic Fusion*

The first widely used approach for CLP-based antigen display was by genetic fusion of a heterologous antigen sequence to the capsid subunit protein [27–29]. When successful, each subunit of the CLP will display the inserted antigenic sequence in a pattern matching that of the underlying CLP. Although this approach can deliver foreign epitopes in a viral-like configuration, it is generally limited to small peptide antigens that do not inhibit particle assembly [30–32]. However, in some cases, even small peptide antigens prevent CLP assembly. Thus, the success of genetic fusion is difficult to predict, and structural characteristics of the CLP as well as biochemical properties of the peptide must be taken into account [33,34]. Studies on HBcAg CLPs show that highly charged and hydrophobic peptides tend to prevent CLP assembly [30,35]. Due to the small size of the incorporated antigen, CLP vaccines made by genetic fusion generally induce narrow epitope-specific antibody responses [36]. The challenges associated with genetic fusion have to some extent been relieved by creating mosaic CLPs, consisting of both native and genetically modified CLP subunits. However, this is at the cost of reduced antigen density [37,38].
