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 lesions and certain human papillomavirus (HPV) types, most frequently HPV 16. Our research is primarily concerned with development of vaccines and other infection inhibition strategies against HPV and the elucidation of the PV life cycle. We have developed a simple and efficient strategy for generating high titers of infectious papillomavirus particles that transduce encapsidated marker plasmids, referred to hereafter as pseudovirions. This methodology represents a technical breakthrough in papillomavirus research. We have exploited this technology in our basic virologic and translational research efforts. Pseudovirus production technology is being used to explore the basic features of papillomavirus infection and mechanisms of antibody-mediated neutralization. We have determined that a cleavage of the terminus of L2 by the cellular proprotein convertase furin is required for papillomavirus infection. This cleavage presumably exposes a binding site for a conserved cellular receptor, because in other studies we have determined that broadly cross-type neutralizing L2 antibodies bind the peptide immediately downstream of the furin cleavage site. This cleavage site is not exposed on muture virions in solution. Rather it becomes exposed only after engagement of the cell surface and/or extracellular matrix and a resulting conformation change in the capsid. We have determined that, surprisingly, the major class of neutralizing antibodies induced by the licensed L1 VLP vaccines does not prevent cell surface binding. Rather these antibodies prevent the change in virion conformation required for furin cleavage of L2, resulting in retention of the virions on the cell surface. In contrast, the antibodies induced by our broadly cross-neutralizing L2 vaccines induce the disassociation of the virions from the cell surface, presumably by preventing engagement of a secondary cell surface receptor. We have used our pseudovirus technology to develop the first cervicovaginal challenge model for HPVs. We have found that the infection of the female mouse genital tract, even of monolayer endocervical cells, requires exposure the basement membrane to the virus. The capsids bind avidly to the basement membrane but not to the apical surfaces of intact columnar or stratified squamous epithelia. Using a quantitative assay based on whole tissue fluorecence imaging after infection with red fluorescent protein (RFP) expressing pseudovirions, we have further determined that nonoxynol-9 (N-9) and Conceptrol, an over the counter spermacide that containing N-9, dramatically potentiate in vivo papillomavirus infection, presumably due to it's ability to permeablize the epithelial layers and thereby expose the basement membrane to the virus. Interesting, no infection was detected if nonoxynol-9 was formulated in carrageenan rather than its normal gelling agent. Carrageenan is an algal polysaccharide widely used in processed food and cosmetics and is the main gelling agent in some over-the-counter lubricants. We had previously shown that carragenen, and products that contain it, are extremely potent inhibitors of HPV infection in vitro. The results suggest that women using N-9 spermacides may be at increased risk of acquiring genital HPV infection and that this risk might be eliminated by reformulation of N-9 in a carrageenan based gel. We are currently working with the NCI extramural division DCP to initiate a clinical trial of carrageenan as a microbicide to prevent genital HPV infection in young women. Our studies in the mouse cervicovaginal challenge model suggested that other interventions that compromise the integrity of the genital epithelium might also potentiate HPV infection. Acquisition of ecto- and endocervical cells for cytology (Pap) screening disrupts the epithelium by design. Therefore we sought to determine whether the cytology specimen (Pap smear) collection procedure renders the cervix more susceptible to HPV infection in a rhesus macaque model. In unpublished studies, we found that the Pap smear collection procedure greatly potentiated infection by RFP expressing HPV16 pseudovirus. However, use of a carrageenan gel rather than Surgilube as the lubricant used for the internal digital exam after specimen collection largely abrogated the infection enhancing effect. These findings suggest that cytology screening in women might lead to a transient enhancement of susceptibility to HPV infection and that use of a carrageenan-based gel during the examine might mitigate this enhancement. Our development of a method to induce efficient HPV pseudovirus infection of the female genital tract after transient disruption with N-9 has proven to be the key to our recent development of an effective intravaginal vaccination strategy. In patent pending studies, 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. Systemic responses rival those induced by intramuscular injection of optimized Ad5 vectors. Intravaginal vaccination could serve to focus immune responses to the female genital tract and so increase the effectiveness of vaccines directed against HSV and HIV infections and for immunotheraphy of HPV induced neoplasia. To more generally evaluate the potential of HPV pseudoviruses as gene transfer vehicles, we have conducted a broad infection tropism survey. In patent pending studies, we demonstrated that intact murine epithelium at all sites test, whether simple, columnar, or squamous, was highly resistant to both virion binding and infection, whereas disrupted epithelium was susceptible. In contrast, virtually all human epithelial tumor lines in the NC1-60 panel were highly susceptible to infection in vitro. The remarkable specificity of HPV pseudovirus binding and infection suggests that they may be useful in tumor diagnostic or cytotoxic gene therapy 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 instillation of RFP-expressing pseudovirus.
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