Understanding Papillomavirus Virion Proteins and Vaccines

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Papillomaviruses (PVs) infect the epithelia of animals and man, where they generally induce benign proliferation at the site of infection. However, there is a strong association between malignant progression of human genital, anal and oropharyngeal lesions and certain human papillomavirus (HPV) types, most frequently HPV 16. Our research is primarily concerned with development of vaccines and other agent to prevent and treat HPV infections and the neoplasia they induce, the elucidation of the HPV life cycle, and using the insights obtained in these studies to develop treatments against other cancers and chronic diseases. We have developed a simple and efficient strategy for generating papillomavirus-like particles (VLPs) and high titers of infectious papillomavirus particles that transduce encapsidated marker plasmids, i.e. pseudovirions. We have exploited these technologies in our basic virologic and translational research efforts. We have used our pseudovirus technology to develop the first cervicovaginal challenge model for HPVs and used this assay to define the molecular mechanism used by HPV to infect its target tissue and to determine how the antibodies induced L1- and L2-based prophylactic vaccines prevent infection. Key to the process is an obligatory binding to the heparan sulfate proteoglycans (HSPGs) on the basement membrane of a disrupted epithelium. In a just published study we further shown that under certain conditions mimicking a wound healing environment, the capsid can also utilize specifically modified forms of chondroitan sulfate proteoglycans as initial attachment factors. Our development of a method to induce efficient HPV pseudovirus infection of the female genital tract after transient disruption with the over-the-counter spermicide nonoxonol-9 has proven to be the key to our development of an effective, and we believe practical, intravaginal vaccination strategy. We have found that intravaginal pseudovirus vaccination of N-9 treated mice induces strong systemic and mucosal T and B cell responses to target antigens transduced by the pseudovirions. Most of the induced T are long lived intraepithelial tissue resident effector memory CD8 T cells (Trms). Critically, CD8 IEL's were not induced after systemic vaccination with peptides or viral vectors. Intravaginal pseudovirus vaccination is a promising approach for focusing immune responses to the female genital tract and so should increase the effectiveness of vaccines directed against HSV infections and against HPV induced neoplasia. In collaboration with Dr. Jeff Cohen, NIAID, vectors expressing HSV antigens are were tested in mouse and guinea pig HSV-2 challenge models. Intravaginal vaccination with these vectors reduced local pathology induced by intravaginal HSV inoculation, but standard parenteral vaccination did not. In a CRADA collaborated with Crucell/Janssen, we have determined that Ad26 and Ad35 vectors induce similar locals T cell responses as intravaginal delivery of HPV pseudovirions and are superior at inducing systemic T cell responses. Crucell has extensive expertise in GMP production of their adenovirus vectors. The company was moving our findings toward a clinical trial but these plans were waylaid by the COVID-19 epidemic, in part because their vaccine is based on the same adenovirus vectors. To more generally evaluate the potential of HPV pseudoviruses as gene transfer vehicles, we conducted a broad in vivio binding infection tropism survey. We demonstrated that intact murine epithelium at all sites, whether simple, columnar, or squamous, was highly resistant to both virion binding and infection, whereas disrupted epithelium was susceptible. In contrast, virtually all human-derived carcinoma and melanoma cell lines in the NCI-60 panel were highly susceptible to VLP/psuedovirus binding/infection in vitro. We have now published binding studies on over 120 cancer lines. The remarkable specificity of HPV VLP/pseudovirus binding and infection is mediated by specific HSPG modifications on the tumor cell surfaces that mimic those normally found on the basement membrane. The results suggest that HPV VLPs/pseudovirions may be useful in tumor diagnostic or tumor-directed cytotoxic gene or drug conjugate applications. In proof of concept studies, we documented highly specific binding and infection, and dramatic imaging, of human ovarian tumor nodules implanted in nude mouse peritoneum after intraperitoneal injection of RFP-expressing pseudovirus. A CRADA with Aura Biosciences was initiated to facilitate further development and clinical testing of this approach to tumor therapy. Based on earlier antibody-based studies by CCR's Peter Choyke, we are investigating tumor therapies based on HPV capsids coupled to an infrared dye, IR700. Tumor treatment studies of the intravenous delivery of the dye-coupled VLPs followed by local tumor illumination in several mouse models and particularly in a rabbit zenograft model of human uveal melanoma have produced exceptionally encouraging results. We published on the profound change in the tumor microenvironment induced by the this treatment. These studies have led to an FDA approved phase 1 and 2 trials for treatment of ocular melanoma. Based up the positive safety and efficacy results obtained, a company-sponsored phase 3 study should commence in Q4 2023. In preclinical murine studies, for which we a obtained an NCI Director's Innovation Award, we have testing the hypothesis that preexisting immune responses to CMV can be effectively direct to tumors to both kill tumor cells and initiate/amplify protect responses to tumor neoantigens, initially by expressing viral antigens specifically in the tumors via pseudovirus transduction. While significant tumor regression was observed, we found that intratumoral injection of immunodominant minimal CD8 and/or CD4 restricted CMV peptides induced more potent tumor killing and antigen spreading, such that, under an optimized protocol, it induced complete long term regression and protection from tumor rechallenge in almost all treated mice. In an extension of these studies we have determined that vaccine-specific T cells can also be recruited to treat cancer. Together with CCR's Comparative oncology unit, we secured CCR funding to conduct a proof-of concept cancer therapy trial in companion dogs which will commence in Q4 2023. A long-standing collaboration with DCEG colleagues has resulted in many publications related to the NCI-sponsored prophylactic HPV vaccine clinical trial in Costa Rica (CVT). In the last year, we published papers on long term protection against non-targeted high risk types, on long term antibody avidity maturation after one vs 3 doses, and quantified the extent of "unmasking" of CIN3 arising from non-targeted vaccine types. Based on our published post hoc analyses from CVT indicating that even a single dose of Cervarix induces long term protection (now 11 years), we have worked with our DCEG colleagues to design, secure NCI and Gates Foundation funding, and initiate an efficacy trial of one versus two doses of Cervarix and Gardasil-9 in Costa Rican girls. In addition, an immunogenicity non-inferiority trial comparing one dose of Cervarix to three doses of Gardasil has largely been completed and we now conducting the ELISA and in vitro neutralization assays to assess the antibody responses. We have also just initiated a trial to evaluate the the antibody response to a single dose in "older" women, i.e. over 26 years of age.