*4.1. The Display of L2 Peptides on Bacteriophage VLP Platforms*

Different HPV L2 peptides (shown in Figure 3) have been displayed, by genetic insertion or by chemical conjugation, on VLPs derived from viruses (MS2, PP7, Qβ, AP205) that infect bacteria. Genetic insertion of L2 allows for DNA sequences that code for L2 peptides to be inserted by polymerase chain reaction on the single-chain dimer (two fused copies) of the coat proteins of bacteriophages MS2, PP7 or to be inserted on the coat protein monomer of bacteriophage AP205 in expression vectors (Table 1). Expression of the chimeric coat proteins in a suitable bacterial host allows 90 copies of the coat protein dimers (for MS2 and PP7) and 180 copies of coat protein monomers (AP205) to spontaneously assemble into VLPs, thus displaying 90 copies and 180 copies of L2 on the VLPs, respectively [5,11,57–60]. Chemical conjugation, on the other hand, allows chemically synthesized L2 peptides to be displayed mostly on Qβ VLPs as follows. Qβ VLPs are first expressed and purified from bacterial cells. Synthesized L2 peptides, carrying a terminal cysteine residue, are then cross-linked to lysine residues on the VLPs using a bi-functional cross-linker, succinimidyl 6-((beta-maleimidopropionamido)hexanoate) [61]. This allows for at least 360 copies of L2 to be displayed on Qβ VLPs.


Candidate bacteriophage VLP-based L2 HPV vaccines.

**Table 1.**

240

Ref: Reference.

#### *Viruses* **2020**, *12*, 18

As mentioned above, peptides representing different L2 epitopes have been displayed on bacteriophage VLPs. These peptides, which range in size from 12 amino acids (aa) to 110 aa, can be grouped into three categories based on the source of peptides:


Of all the above HPV L2 peptides that have been displayed on bacteriophage VLPs, only bacteriophage VLPs displaying peptides representing aa 17–31 from HPV5, 6, 16, 18 [5,58,59], bacteriophage VLPs displaying consensus aa 65–85 or 69–86 [57,61], and bacteriophage VLPs displaying peptides from the L2 concatemers (16L2/31L2 and 16L2/18L2/31L2/35L2/52L2) [57,60,62] elicited broadly neutralizing/protective antibodies against diverse HPV types (Table 1). For instance, immunization with a mixture of eight PP7 VLPs each displaying L2 peptide (aa 17–31) from eight different HPV types offered broader and better protection against HPV pseudoviruses 5/6/16/18/31/45/52/58 (Table 1) [58]. Each L2 peptide was inserted on the AB-loop of PP7 coat protein. Similarly, immunization with a mixture of two MS2–L2 VLPs (mixed MS2-L2 VLPs; one VLP displaying a concatemer peptide, aa 17–31 from 16L2 and aa 20–31 from 31L2 and another VLP displaying a consensus peptide from aa 69–86) offered robust broad protection against genital and oral infections with HPV pseudoviruses 11/16/18/31/33/35/39/45/52/53/56/58 (Table 1) [57,62]; the L2 peptides were inserted on the N-terminus of MS2 coat protein. Mixed MS2-L2 VLPs have the potential to protect against all eleven HPV types (tested so far) associated with ~95% of cervical cancer and against ~99% of HPV-associated head and neck cancers. Additionally, they can protect against one HPV type (HPV11, tested so far) associated with 36% of genital warts and ~32% of recurrent respiratory papillomatosis [57,62]. More HPV types need to be tested to assess the spectrum of protection. Thus, mixed MS2-L2 is an excellent next-generation candidate vaccine against HPV.
