A pan-HPV vaccine based on bacteriophage PP7 VLPs displaying broadly cross-neutralizing epitopes from the HPV minor capsid protein, L2

Abstract

Background: Current human papillomavirus (HPV) vaccines that are based on virus-like particles (VLPs) of the major capsid protein L1 largely elicit HPV type-specific antibody responses. In contrast, immunization with the HPV minor capsid protein L2 elicits antibodies that are broadly cross-neutralizing, suggesting that a vaccine targeting L2 could provide more comprehensive protection against infection by diverse HPV types. However, L2-based immunogens typically elicit much lower neutralizing antibody titers than L1 VLPs. We previously showed that a conserved broadly neutralizing epitope near the N-terminus of L2 is highly immunogenic when displayed on the surface of VLPs derived from the bacteriophage PP7. Here, we report the development of a panel of PP7 VLP-based vaccines targeting L2 that protect mice from infection with carcinogenic and non-carcinogenic HPV types that infect the genital tract and skin.

Methodology/principal findings: L2 peptides from eight different HPV types were displayed on the surface of PP7 bacteriophage VLPs. These recombinant L2 VLPs, both individually and in combination, elicited high-titer anti-L2 IgG serum antibodies. Immunized mice were protected from high dose infection with HPV pseudovirus (PsV) encapsidating a luciferase reporter. Mice immunized with 16L2 PP7 VLPs or 18L2 PP7 VLPs were nearly completely protected from both PsV16 and PsV18 challenge. Mice immunized with the mixture of eight L2 VLPs were strongly protected from genital challenge with PsVs representing eight diverse HPV types and cutaneous challenge with HPV5 PsV.

Conclusion/significance: VLP-display of a cross-neutralizing HPV L2 epitope is an effective approach for inducing high-titer protective neutralizing antibodies and is capable of offering protection from a spectrum of HPVs associated with cervical cancer as well as genital and cutaneous warts.

Figure 1. Amino acid sequence alignment of L2 (residue 17–31, or equivalent) from selected carcinogenic, genital, and/or cutaneous HPV types.

The consensus sequence was derived from an alignment of 15 carcinogenic HPVs, 2 genital HPVs, 4 cutaneous HPVs plus 2 animal papillomaviruses using ClustalW2. Recombinant L2 PP7 VLPs displaying these peptide sequences were constructed. Amino acids are shown in single-letter code, dots indicate that the amino acid is identical to consensus sequence, and amino acid differences from the consensus are shown for each HPV type. A plus (+) symbol indicates that there is no consensus amino acid at this position.

Figure 2. Transmission electron micrographs of selected PP7 VLPs.

Shown are A) wild-type PP7 VLPs, B) 16L2 PP7 VLPs, and C) 18L2 PP7 VLPs. VLPs were visualized at a magnification of 40,000×.

Figure 3. Reactivity of the RG-1 monoclonal antibody with recombinant L2 PP7 VLPs.

500 ng of wild-type PP7 VLPs or L2 PP7 VLPs were used to coat ELISA plates. Binding of a 1∶5,000 dilution of RG-1 was detected using a horseradish peroxidase-conjugated goat anti-mouse IgG secondary antibody followed by development with 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS). Reactivity was determined by measuring the mean optical density (OD) values at 405 nm. Error bars indicate standard error of the mean (SEM) of duplicate wells.

Figure 4. Serum IgG antibody responses in mice immunized with individual L2 PP7 VLPs.

Mice were immunized i.m. twice with 5 µg of PP7 or L2 PP7 VLPs with IFA and their sera collected 2 weeks after the last immunization. Serum anti-L2 IgG titer was determined by end-point dilution ELISA against streptavidin-conjugated HPV L2 peptides (amino acid 14–40) from HPV1 (squares), HPV5 (inverted triangles), HPV6 (upright triangles), HPV16 (white-filled circles), and HPV18 (diamonds). Black-filled circles denote reactivity of PP7 sera with the different five L2 peptides. Each datum point shows antibody titer from each mouse and lines represent the geometric mean titers for each group.

Figure 5. Cross-reactivity of sera elicited upon immunization with individual L2 PP7 VLPs with diverse L2 peptides.

(A) ELISA plates were coated with the streptavidin-conjugated L2 peptides described in Figure 4 and reacted with a 1∶160 dilution of serum from mice immunized with wild-type PP7 VLPs or L2 PP7 VLPs followed by secondary antibody. Shown are the averages of the optical density (OD₄₀₅) values of sera from three individual mice in each group. Error bars represent SEM. (B) A summary of these results. “++++” indicates a mean OD₄₀₅>1.2, “+++” indicates a mean OD₄₀₅ between 0.8 and 1.2, “++” indicates a mean OD₄₀₅ between 0.4 and 0.8, “+” indicates a mean OD₄₀₅ between 0.2 and 0.4, and “−” indicates a mean OD₄₀₅ below 0.2.

Figure 6. Mice immunized with 16L2 PP7 VLPs or 18L2 PP7 VLPs are protected from vaginal challenge with HPV18 and HPV16 PsV.

Groups of 5 BALB/c mice were immunized i.m. twice with 5 µg of PP7 VLPs, 16L2 PP7 VLPs or 18L2 PP7 VLPs with IFA. Three weeks after the second immunization, mice were vaginally challenged with 1.3×10⁵ (PsV18) or 3.0×10⁶ (PsV16) IU of PsV. Forty-eight hours later, luciferin was instilled vaginally and images were taken 3 minutes post-luciferin instillation. Images showing the magnitude of vaginal infection with PsV18 or PsV16 are shown in panels A and B, respectively. The colors reflect the intensity of luciferase expression. Colors are scaled for each image and shown to the right of the image. Quantitative data (shown in each panel) was extracted by drawing equally sized regions of interests surrounding the site of PsV instillation and determining average radiance (p/s/cm²/sr) by using Living Image 3.2 software. Background radiance (determined by gating on another region of the mouse) was subtracted from this value. Black-filled circles denote mice immunized with control wild-type PP7 VLPs and white-filled circles denote mice immunized with either 16L2 PP7 VLPs or 18L2 PP7 VLPs. Lines reflect the geometric mean radiance for each group.

Figure 7. Immunization with a mixture of L2-PP7 VLPs induces broad anti-L2 IgG responses.

Shown is the reactivity of sera from mice immunized with mixed L2 PP7 VLPs to L2 peptides. Mice were immunized i.m. three times with 10 µg of wild-type PP7 VLPs or 10 µg of mixed L2 PP7 VLPs. Sera were collected 2 weeks after the last immunization and serum anti-L2 IgG titer was determined by end-point dilution ELISA against streptavidin-conjugated HPV L2 peptides (amino acid 14–40) from HPV1, HPV5, HPV6, HPV16, and HPV18. Black-filled circles denote mice immunized with PP7 VLPs and diamonds denote mice immunized with mixed L2 PP7 VLPs.

Figure 8. Mice immunized with mixed L2 PP7 VLPs are protected from vaginal challenge with diverse PsVs.

Groups of 5 Balb/c mice were immunized i.m. three times with 10 µg of PP7 VLPs or mixed L2 PP7 VLPs. Three weeks after the last immunization, mice were vaginally challenged with 1.3×10⁵– 6.5×10⁶ IU of PsV5, PsV6, PsV16, PsV18, PsV31, PsV45, PsV52, or PsV58. Forty-eight hours later, luciferin was instilled vaginally and images were taken 3 minutes post-luciferin instillation. Average radiance (p/s/cm²/sr) values for each animal were determined as described in Figure 6. Black-filled circles denote mice immunized with wild-type PP7 VLPs and white-filled circles denote mice immunized with mixed L2 PP7 VLPs.

Figure 9. Mice immunized with mixed L2 PP7 VLPs are protected from cutaneous challenge with PsV5.

Balb/c mice were immunized i.m. as described in Figure 8. Three weeks after the last immunization, mice were subcutaneously challenged in the belly with 6.0×10⁵ IU of PsV5. Three days post-challenge, mice were anesthetized and 0.7 mg luciferin was injected subcutaneously. Images of mice immunized with control PP7 VLPs and mixed L2 PP7 VLPs are shown in panels A and B, respectively. Average radiance values for the two groups are shown in panel C. Black-filled circles denote mice immunized with PP7 VLPs and white-filled circles denote mice immunized with mixed L2 PP7 VLPs.