Harnessing Human Papillomavirus’ Natural Tropism to Target Tumors

Human papillomaviruses (HPV) are small non-enveloped DNA tumor viruses established as the primary etiological agent for the development of cervical cancer. Decades of research have elucidated HPV’s primary attachment factor to be heparan sulfate proteoglycans (HSPG). Importantly, wounding and exposure of the epithelial basement membrane was found to be pivotal for efficient attachment and infection of HPV in vivo. Sulfation patterns on HSPG’s become modified at the site of wounds as they serve an important role promoting tissue healing, cell proliferation and neovascularization and it is these modifications recognized by HPV. Analogous HSPG modification patterns can be found on tumor cells as they too require the aforementioned processes to grow and metastasize. Although targeting tumor associated HSPG is not a novel concept, the use of HPV to target and treat tumors has only been realized in recent years. The work herein describes how decades of basic HPV research has culminated in the rational design of an HPV-based virus-like infrared light activated dye conjugate for the treatment of choroidal melanoma

Role of Heparan Sulfate in Attachment to and Infection of the Murine Female Genital Tract by Human Papillomavirus▿

The host factors required for in vivo infection have not been investigated for any papillomavirus. Using a recently developed murine cervicovaginal challenge model, we evaluated the importance of heparan sulfate proteoglycans (HSPGs) in human papillomavirus (HPV) infection of the murine female genital tract. We examined HPV type 16 (HPV16) as well as HPV31 and HPV5, for which some evidence suggests that they may differ from HPV16 in their utilization of HSPGs as their primary attachment factor in vitro. Luciferase-expressing pseudovirus of all three types infected the mouse genital tract, although HPV5, which normally infects nongenital epidermis, was less efficient. Heparinase III treatment of the genital tract significantly inhibited infection of all three types by greater than 90% and clearly inhibited virion attachment to the basement membrane and cell surfaces, establishing that HSPGs are the primary attachment factors for these three viruses in vivo. However, the pseudoviruses differed in their responses to treatment with various forms of heparin, a soluble analog of heparan sulfate. HPV16 and HPV31 infections were effectively inhibited by a highly sulfated form of heparin, but HPV5 was not, although it bound the compound. In contrast, a N-desulfated and N-acylated variant preferentially inhibited HPV5. Inhibition of infection paralleled the relative ability of the variants to inhibit basement membrane and cell surface binding. We speculate that cutaneous HPVs, such as HPV5, and genital mucosal HPVs, such as HPV16 and -31, may have evolved to recognize different forms of HSPGs to enable them to preferentially infect keratinocytes at different anatomical sites

Vaccination with Human Papillomavirus Pseudovirus-Encapsidated Plasmids Targeted to Skin Using Microneedles

<div><p>Human papilloma virus-like particles (HPV VLP) serve as the basis of the current licensed vaccines for HPV. We have previously shown that encapsidation of DNA expressing the model antigen M/M2 from respiratory syncytial virus (RSV) in HPV pseudovirions (PsV) is immunogenic when delivered intravaginally. Because the HPV capsids confer tropism for basal epithelium, they represent attractive carriers for vaccination targeted to the skin using microneedles. In this study we asked: 1) whether HPV16 VLP administered by microneedles could induce protective immune responses to HPV16 and 2) whether HPV16 PsV-encapsidated plasmids delivered by microneedles could elicit immune responses to both HPV and the antigen delivered by the transgene. Mice immunized with HPV16 VLP coated microneedles generated robust neutralizing antibody responses and were protected from HPV16 challenge. Microneedle arrays coated with HPV16-M/M2 or HPV16-F protein (genes of RSV) were then tested and dose-dependent HPV and F-specific antibody responses were detected post-immunization, and M/M2-specific T-cell responses were detected post RSV challenge, respectively. HPV16 PsV-F immunized mice were fully protected from challenge with HPV16 PsV and had reduced RSV viral load in lung and nose upon intranasal RSV challenge. In summary, HPV16 PsV-encapsidated DNA delivered by microneedles induced neutralizing antibody responses against HPV and primed for antibody and T-cell responses to RSV antigens encoded by the encapsidated plasmids. Although the immunogenicity of the DNA component was just above the dose response threshold, the HPV-specific immunity was robust. Taken together, these data suggest microneedle delivery of lyophilized HPV PsV could provide a practical, thermostable combined vaccine approach that could be developed for clinical evaluation.</p></div

HPV16 mmunogenicity is retained by inclusion of L2 and DNA using the HPV PsV.

<p>Animals received one of three variations of the HPV16 particle: L1 only, L1/L2 empty particles or L1/L2 encapsidating the RSV F protein expression plasmid (HPV16-F PsV). Approximately 25 ng of HPV L1 equivalent was coated onto each microneedle array (MN Array), and mice received either 10, 3 or 1 microneedle arrays. The control arm received 10 microneedles coated with coating buffer only. Animals were immunized three times, each three weeks apart. Three weeks after the last immunization, serum was drawn and mice were cervicovaginally challenged with 10⁸ IU of HPV16-Luc. (A) nAb were detected in all groups and a dose response was observed. Data reported as EC50, the reciprocal dilution at which 50% of the pseudovirus was neutralized compared to positive control wells. (B) Protection from challenge was observed in all groups as measured by <i>in vivo</i> bioluminescence after challenge with HPV16-Luc. The “No PsV” group was not challenged in order to serve as a negative control group for luminescence. Data reported as average radiance; n = 5. *p = 0.03, **p<0.01.</p

HPV16-M/M2 PsV delivered by microneedles primes for an RSV specific CD4⁺ and CD8⁺ T-cell response.

<p>Animals received a single administration of microneedles (10, 3 or 1 arrays) coated with HPV16-M/M2 PsV and the control group received 3 microneedle arrays coated with HPV16-Luc PsV. One month after immunization, mice were challenged with 10⁷ pfu of RSV and five days after challenge, RSV M/M2 tetramer-specific (A) CD8⁺ and (B) CD4⁺ T-cells were measured in the lungs. (C) A second group of animals were boosted IM with 10⁷ pfu of rAd5-M/M2 and CD8⁺ M2 specific T-cells were measured in the blood 14 days later. Data are reported as percentage of tetramer positive cells as determined by flow cytometry, n = 3–5/group. *p = 0.04.</p