A proof-of-concept study for the design of a VLP-based combinatorial HPV and placental malaria vaccine

Abstract In Africa, cervical cancer and placental malaria (PM) are a major public health concern. There is currently no available PM vaccine and the marketed Human Papillomavirus (HPV) vaccines are prohibitively expensive. The idea of a combinatorial HPV and PM vaccine is attractive because the target population for vaccination against both diseases, adolescent girls, would be overlapping in Sub-Saharan Africa. Here we demonstrate proof-of-concept for a combinatorial vaccine utilizing the AP205 capsid-based virus-like particle (VLP) designed to simultaneously display two clinically relevant antigens (the HPV RG1 epitope and the VAR2CSA PM antigen). Three distinct combinatorial VLPs were produced displaying one, two or five concatenated RG1 epitopes without obstructing the VLP’s capacity to form. Co-display of VAR2CSA was achieved through a split-protein Tag/Catcher interaction without hampering the vaccine stability. Vaccination with the combinatorial vaccine(s) was able to reduce HPV infection in vivo and induce anti-VAR2CSA IgG antibodies, which inhibited binding between native VAR2CSA expressed on infected red blood cells and chondroitin sulfate A in an in vitro binding-inhibition assay. These results show that the Tag/Catcher AP205 VLP system can be exploited to make a combinatorial vaccine capable of eliciting antibodies with dual specificity

A Pan-HPV Vaccine Based on Bacteriophage PP7 VLPs Displaying Broadly Cross-Neutralizing Epitopes from the HPV Minor Capsid Protein, L2

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.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.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

VLPs displaying a single L2 epitope induce broadly cross-neutralizing antibodies against human papillomavirus.

Virus-like Particles (VLPs) display can be used to increase the immunogenicity of heterologous antigens. Here, we report the use of a bacteriophage MS2-based VLP display platform to develop a monovalent vaccine targeting a broadly neutralizing epitope in the minor capsid protein human papillomavirus (HPV) that provides broad protection from diverse HPV types in a mouse pseudovirus infection model.Peptides spanning a previously described cross-neutralizing epitope from HPV type 16 were genetically inserted at the N-terminus of MS2 bacteriophage coat protein. Three of the four recombinant L2-coat proteins assembled into VLPs. L2-VLPs elicited high-titer anti-L2 antibodies in mice, similar to recombinant VLPs that we had previously made in which the L2 peptide was displayed on a surface-exposed loop on VLPs of a related bacteriophage, PP7. Somewhat surprisingly, L2-MS2 VLPs elicited antibodies that were much more broadly cross-reactive with L2 peptides from diverse HPV isolates than L2-PP7 VLPs. Similarly, mice immunized with L2-MS2 VLPs were protected from genital and cutaneous infection by highly diverse HPV pseudovirus types.We show that peptides can be displayed in a highly immunogenic fashion at the N-terminus of MS2 coat protein VLPs. A VLP-based vaccine targeting HPV L2 elicits broadly cross-reactive and cross-protective antibodies to heterologous HPV types. L2-VLPs could serve as the basis of a broadly protective second generation HPV vaccine

Characterization of a spray-dried candidate HPV L2-VLP vaccine stored for multiple years at room temperature

HPV infections are associated with human cancers. Although three prophylactic vaccines have been approved to protect against HPV infections, the vaccines require cold-chain storage and may not be suitable for third world countries with less developed refrigeration facilities. We previously developed a bacteriophage L2 virus-like particle (VLP)-based candidate vaccine, which elicited broadly protective antibodies against diverse HPV types. Spray-drying of MS2-16L2 VLPs into a dry powder enhanced the stability of these VLPs. Building on these studies, we assessed the long-term stability and immunogenicity of the spray-dried VLPs. Mice immunized with a single dose of spray-dried MS2-16L2 VLPs, which had been stored for 14 months at room temperature (RT), were partially protected from challenge with a high dose of HPV16, one year after immunization. However, immunization with two doses of MS2-16L2 VLPs stored at RT for 34 months elicited high titer anti-HPV antibodies. More importantly, this group of mice showed significant protection from HPV16, 4 months after immunization. These results suggest that spray-dried MS2-16L2 VLPs retain their effectiveness after long-term storage at RT, and may be suitable in third world countries with less developed refrigeration facilities. Keywords: HPV vaccine, Bacteriophage L2-VLPs, Longevity, Formulation, Spray-drying, Thermostabilit

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

<p>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.</p

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

<p>Balb/c mice were immunized i.m. as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0023310#pone-0023310-g008" target="_blank">Figure 8</a>. 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.</p

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

<p>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.</p

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

<p>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.</p

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

<p>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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0023310#pone-0023310-g006" target="_blank">Figure 6</a>. Black-filled circles denote mice immunized with wild-type PP7 VLPs and white-filled circles denote mice immunized with mixed L2 PP7 VLPs.</p