*2.2. Modular Antigen Display*

CLP-display of large and complex protein antigens is not easily obtained by genetic fusion. Thus, an alternative strategy has been to attach protein antigens to the surface of preassembled CLPs. Importantly, when using such modular approaches, the resultant epitope display is directly dependent on the applied conjugation method, as well as the specific surface geometry of the employed CLP backbone. In addition, these technologies allow for separate recombinant production of antigens in various expression systems, ensuring high quality and correct protein processing before CLP conjugation [39–42]. The different conjugation systems used for modular CLP vaccine development is described in detail elsewhere [43]. Here, the main techniques are listed in Table 1 to provide a comparative overview of their relative practicality and ability to facilitate a high-quality epitope display. This evaluation is done in recognition that HPV CLPs represent a highly immunogenic viral epitope display, and on that basis, modular platforms should likewise facilitate ordered, high-density and unidirectional presentation of antigens in their native conformation. This cannot, to a similar degree, be achieved by all available antigen conjugation methods.


**Table 1.** Properties of different conjugation strategies used for capsid-like particles (CLP) antigen display.

+ indicates property can be achieved, − indicates property not readily achieved, +/− indicates property can only partly be achieved or has not been experimentally validated.

### 2.2.1. Chemical Conjugation and Click Chemistry

One of the most widely used techniques for modular CLP antigen display has been through chemical conjugation, which is compatible with most capsid backbones. A common approach is through cross-linking of lysine residues on the CLP surface to cysteine residues present or incorporated into the antigen [45,46,52]. This vaccine design has been shown to induce high titers of antigen-specific antibodies, and several vaccines have shown promising results in preclinical studies, as well as in clinical testing (e.g., Nicotine-Qβ [53]). However, the CLP surface often contains multiple lysine residues, resulting in uneven antigen distribution, and little control over antigen orientation. In addition, introduction of a reactive cysteine may cause antigen misfolding [54,55]. In some cases, chemical conjugation can lead to destabilization of the CLP, and cannot always facilitate high-density antigen display, due to suboptimal coupling efficiency [46,56]. Some of the limitations associated with standard chemical conjugation have been resolved by the development of click chemistry [35,48,57]. This method uses the incorporation of unnatural amino acids, allowing for increased control over site-specificity. This technique requires minimal change to the VLP and antigen, while providing a highly specific and fast coupling reaction. However, the scalability of this technology remains a concern [33,58].

#### 2.2.2. Affinity-Based Conjugation

Affinity-based conjugation systems, such as streptavidin/biotin [42,49,59], and to a lesser extent His-tag/Ni-NTA [50], have been used to facilitate unidirectional display. This is due to the single attachment site on each antigen and promotes an even display of heterologous epitopes. In addition, these methods have the advantage of being able to display larger and complex protein antigens, enabling the induction of broad, polyclonal humoral responses without compromising CLP formation and stability. For further development of the streptavidin/biotin conjugation system, engineered monomeric streptavidin (mSA) was used for antigen display on HPV CLPs containing a biotin acceptor site (AviTagTM) [60]. A potential disadvantage of this strategy, is that the coupling is based on a non-covalent interaction, and thus there is a risk of antigen disengagement. This can affect the coupling efficiency and diminish antigen density. Additionally, it has been hypothesized that dissociation due to an altered chemical environment in vivo, may reduce the biological efficacy of the vaccine.

#### 2.2.3. Split-Protein (Tag/Catcher) Conjugation

Split-protein (Tag/Catcher) conjugation systems have been developed [61–70] and used for covalent anchoring of vaccine antigens onto CLPs [39,51]. The split-protein technology is based on the separation of a bacterial pili protein, into a reactive peptide (Tag) and corresponding protein binding partner (Catcher). Upon mixing in solution, the Tag and Catcher rapidly react to form a spontaneous isopeptide bond [61]. In the following section, we discuss key features of the Tag/Catcher-AP205 technology and emphasize the versatility of this platform, as well as its ability to effectively mediate highly immunogenic antigen display.

The Tag/Catcher-AP205 platform was developed by genetic incorporation of the SpyTag [39] and SpyCatcher [39,51] into the capsid protein of the *Acinetobacter phage* AP205, yielding particles of 180 subunits with a diameter of approximately 36 nm and 43 nm, respectively. Since its development, the Tag/Catcher-AP205 platform has been utilized to display structurally and functionally diverse vaccine antigens, ranging in size from small peptides (e.g., toxins of 19 amino acids [71]) to large (>300 kDa) trimeric proteins [72]. These studies have repeatedly demonstrated a remarkable ability to achieve complete and even decoration of the CLP surface, with coupling efficiencies reaching 100% for smaller vaccine antigens, which are not limited by steric hindrance. Importantly, the resultant CLP-display induces antibody titers of high quality [55], affinity [41] and avidity [39]. The platform is additionally capable of effectively overcome B-cell tolerance and induce strong antibody responses against a variety of self-antigens, including IL-5, CTLA-4, PD-L1 and Her2 [39,41].

Several features of the AP205 CLP make it attractive as a vaccine backbone, including its structure, intrinsic immunogenicity and manufacturability [73,74]. While the overall capsid structure within the RNA bacteriophage family is similar, the surface exposed regions available for genetic fusion differ substantially [74,75]. The AP205 capsid is remarkable in that both the N- and C- termini are surface exposed and evenly distributed on the assembled CLP [74]. Moreover, AP205 CLPs tolerate genetic fusion at both the N- and C-terminus of the subunit protein, while maintaining stable CLP assembly [39,44]. For future large-scale manufacturing and clinical development, the Tag/Catcher-AP205 platform can be cost-effectively produced at very high yield in *E. coli* [39,74]. In fact, although the scalability of the platform has previously been questioned [76,77], our results show that fermentation can enable the production and purification of correctly assembled CLPs in the scale of grams per liter bacterial cell culture (manuscript in preparation).
