**2. HPV Vaccines**

Approximately 42 HPV types can be transmitted sexually via anogenital to anogenital sex or anogenital to oral sex [16]. Out of these HPVs, ~19 types called high-risk types (oncogenic types; types 16, 18, 26, 31, 33, 35, 39, 45, 51–53, 56, 58, 59, 66, 68, 70, 73, and 82) are associated with

cancers [17,18]. The remaining types, known as low-risk HPV types (types 6, 11, 40–44, 54, 61, 72, 81, etc.), are associated with genital warts and recurrent respiratory papillomatosis. VLP-based HPV vaccines have recently been shown to prevent cases of cervical intraepithelial neoplasias, with protection levels lasting for at least 10 years [19,20]. Moreover, recent studies show that a single dose of the HPV VLP-based vaccine can lead to long-lasting protection from HPV infection [21]. These vaccines are based on VLPs derived from over-expression of the capsid proteins in yeast (Gardasil vaccines) or in insect cells (Cervarix vaccine). HPV capsid is composed of two capsid proteins, the major capsid protein (L1) and the minor capsid protein (L2) (Figure 2). The L1 protein forms pentamers, and 72 copies of the pentamer assemble to form a capsid [22]. The L2 protein is suggested to be present as canyons at the vertices of pentamers and it is only transiently exposed following binding of the virion to heparan sulfate proteoglycans (HSPG) on the basement membrane [23,24]. The exact number of L2 protein on a virion is debatable. Studies suggest that about 12–72 copies of L2 proteins are present on a virion [25,26]. It is worth mentioning that L1 can assemble into the capsid without L2. L2 enhances encapsidation of a double-stranded circular DNA genome into the capsid, thus forming a virion [27]. The L2 protein also has other functions in the life cycle of the virus. Binding of a virion to HSPG promotes conformational change that exposes L2 on the capsid, and thus L2 enhances binding of the virion to epithelial cells [23,28]. It also promotes egress of the virion from the endosome [29] and trafficking of the viral genome towards the nucleus for replication [30,31].

**Figure 2.** A schematic of HPV (human papillomavirus virion). A double-stranded DNA genome (red) is surrounded by the capsid, which is composed of two proteins: the major capsid protein (L1, shown in light green) and the minor capsid protein (L2, shown in brown color). The L1 protein forms pentamers, and L2 protein is inserted on vertices of the pentamers. Seventy-two copies of the pentamers and about 12–72 copies of L2 protein assemble to form a virion.

Although current L1-based HPV vaccines are highly immunogenic, they protect mostly against the HPV types included in the vaccines [32–36]; the L1 protein is not conserved among HPV types. For example, the most recent HPV vaccine (Gardasil-9, an upgrade of Gardasil-4) offers protection against seven HPV types (HPV16, 18, 31, 45, 33, 52, 58) associated with ~90% of cervical cancer and two HPV types (HPV6 and 11) associated with ~90% genital warts [17]. Thus, complete protection from HPV-associated cancers/warts may require the addition, to current HPV vaccines, of VLPs from HPV types that are not currently included in the vaccines. With this in consideration and given the fact that L1-based vaccines offer little cross-protection against other HPV types, the L2 protein has been explored within the last two decades to develop next-generation HPV vaccines. As shown in Figure 3, the L2 protein is conserved among different HPV types. This thus suggests that vaccines targeting L2 are going to be broadly protective against different HPV types. In fact, results from preclinical studies targeting L2 protein look very promising [37–41].

**Figure 3.** Sequence alignment of L2 (aa 1–135) from different HPV types. Sequence alignment was done using sequences from both high-risk (HPVs 16, 18, 26, 31, 33, 34, 35, 39, 45, 51–53, 56, 58, 59, 66, 68, 73, and 82) and low-risk HPV types (HPVs 6, 11, 43, and 44). Alignment was done using Jalview software. Residues that are highly conserved among different HPV types are highlighted, below the alignment, in gold bars. A consensus sequence of amino acid 17–36 from different HPV types is shown below the bars. Amino acid residues (in the consensus sequence) that differ from HPV16 L2 are highlighted in yellow background. The numbers above sequence alignment represent peptides (amino acid residues) that have been displayed on different VLP platforms (see Tables 1 and 2 for details).

## **3. L2 Protein**

Immunization with the N-terminus of L2 protein (out of the context of the L1 protein) elicits antibodies that protect and cross-protect against heterologous HPV types. For example, unlike antibodies elicited against L1 VLPs, antibodies elicited against an L2 peptide representing amino acids 1–88 from HPV type 18 or even from L2 of bovine papillomavirus (BPV) cross-protect, albeit at low titers, against diverse mucosal (HPV6, 11, 16, 18, 31) and cutaneous HPV types (HPV5) [37–41]. Given these data, efforts to develop next-generation HPV vaccines have focused on eliciting protective antibodies against L2 protein, especially against the first 130 amino acids of L2 peptide. Unfortunately, the L2 protein cannot form VLPs like L1 protein. Thus, studies to develop an HPV L2 vaccine have relied on using protein/peptide antigens. As mentioned earlier, L2 peptide antibody titers are very low. This could be explained by the fact that peptide antigens are very unstable in serum and are rapidly degraded following immunization [42,43]. As such, different strategies have been explored in preclinical studies to enhance protective antibody responses against L2 protein. For example, immunizations with an L2 peptide conjugated to thioredoxin [44,45], immunizations with concatemers of L2 protein fused to a self-adjuvanting protein (flagellin) [46,47] as well as immunizations with a concatemer of L2 proteins derived from different HPV types [48,49] have been used. More recently, an L2 polypeptide and a heptamerizing coiled-coil polypeptide OVX313 fused to a nanoparticle derived from a thermostable thioredoxin has been used [50–52]. These approaches have enhanced the immunogenicity of L2 peptides, especially immunization with the nanoparticle thioredoxin-L2-OVX313 candidate vaccine. The nanoparticle candidate vaccine offers protection against more than 14 HPV types [50]. Although the aforementioned strategies enhanced immune responses, the responses were observed mostly when large doses (up to 25 μg) of antigens, coupled with large amount of adjuvant with multiple immunizations regimens were used. With this in consideration, we and others have focused on immunizing with L2 peptides displayed multivalently on the surface of VLPs.

## **4. Multivalent Display of HPV L2 on VLPs**

Not only can VLPs be used to develop vaccines against the virus from which the structural proteins are derived from, they can also be used as platforms to display heterologous antigens from other viruses [53], bacteria [54], pathophysiological diseases like cholesterol [55], and even tumor-associated antigens [56]. The goal of a chimeric VLP is to induce antibodies against a heterologous antigen displayed on the platform, but not against the platform. This technology has been exploited to develop VLP-based L2 vaccines against HPV, targeting some of the L2 peptides or epitopes shown in Figure 3. VLPs from viruses that infect bacteria (bacteriophages) or from viruses that infect eukaryotic cells have both been explored as platforms to display L2 peptides (Tables 1 and 2).
