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John T. Schiller, Ph.D.
Papillomaviruses: Basic Biology and Vaccine Development
Papillomaviruses (PVs) infect the squamous epithelia of a wide variety of animals and man. Infections are species-specific and generally induce localized benign proliferation. However, the lesions induced by certain PVs can undergo malignant progression. There is a strong association between malignant progression of human genital lesions and certain human (H) PV types, most frequently HPV16. The major recent goals of the laboratory have been to elucidate the mechanisms of papillomavirus virion assembly and infection and to develop safe and effective vaccines, and other interventions, to prevent or treat genital HPV-induced disease.
Difficulties in studying PV virions, due to the lack of efficient in vitro propagation methods, were partially overcome by our demonstration that noninfectious virus-like particles (VLPs) and infectious pseudovirions can be produced by over expression of the virion proteins in eukaryotic cells. Procedures for efficent generation of papillomavirus pseudovirions with titers of over 10e10 per ml were recently developed in the lab. These technical developments have served as the basis for the projects outlined below.
Questions into the basic cell biology and biochemistry of virion assembly and infection are being examined. We determined that the L1 major capsid protein alone has the capacity to self-assemble into structures that closely resemble authentic virions but the L2 minor is also required for generating infectious capsids. We have found that L1 determinants are involved in successive cell surface interactions with heparin sulfate proteoglycans and a second, as yet unidentified, receptor. We determined that L2 must be cleaved by furin, a cellular protease at an early phase of infection, but L2 appears to primarily function later by enabling escape from endocytic vesicles and by leading the viral genome to specific subnuclear structures designated ND10 bodies (or PODs) and thereby facilitating viral gene expression. We are currently conducting a detailed analysis of the infection process and the mechanisms by which neutralizing antibodies to L1 and L2 vaccines (outlined below) prevent infection.
HPV L1 VLP-based vaccines were developed to prevent cervical and other genital HPV-associated cancers. We and our colleagues demonstrated type-specific protection from high-dose experimental infection after VLP vaccination in rabbit and cattle models. To facilitate the evaluation of immune responses in human vaccine trials, we have developed high throughput in vitro infectivity and neutralization assays based on the HPV pseudovirions. The possibility that VLPs of one type could cross protect against infection by other types was evaluated using these assays. The results strongly predicted the type-restricted protection of VLP-based vaccines that has recently been observed in humans trials. Our phase I and II trials of an HPV-16 L1 VLP vaccine demonstrated the induction of high titers of neutralizing antibodies, even without adjuvant, and low reactogenicity. Polyvalent VLP vaccines manufactured by Merck and GlaxoSmithKline were subsequently shown to provide almost 100% protection against cervical neoplasia associated with the vaccine HPV types. The vaccines were recently approved for routine vaccination of adolescent and young adult females and males in many countries.
In preclinical vaccine studies, we determined that L2 can induce broadly HPV type cross-neutralizing antibodies when it is removed from its normal context in an HPV capsid structure. Although neutralizing titers to L2 are lower than titers to VLPs, these finds raise the possibility of the development of a monovalent broadly cross-protective HPV vaccine. With academic and industry collaborators we are examining several approaches to increase the neutralizing titers generated by L2 immunogens.
We developed the first cervicovaginal challenge models for a papillomavirus based on our pseudovirus technology. In basic studies, we found that PVs cannot infect or even bind intact epithelial surfaces of the murine genital tract. Infection required trauma that results in exposure and capsid binding to the underlying basement membrane. Interestingly, nonoxonol-9, the active ingredient in most spermicides, is known to disrupt genital tract epithelium, and it greatly potentiated infection in our model. In a screen to identify HPV infection inhibitors, we identified carrageenan, a widely utilized inexpensive gelling polysaccharide extracted from red algae, as an extremely potent and broad spectrum inhibitor of in vitro infection by genital HPV types. Carrageenan also prevented HPV infection of the female genital tract in our challenge model, even in the presence of nonoxonol-9. Since carrageenan is already used as the principal gelling agent in some over-the-counter sexual lubricants, it is an attractive candidate for an HPV topical microbicide. Clinical trials with NCI collaborators are now underway.
Based on the understanding of in vivo infection obtained is studies using the cervicovaginal challenge model, we have recently developed an in vitro neutralization assay that is a much more sensitive measure of L2-specific neutralizing antibodies. This assay will permit a better assessment of L2 vaccines in animal studies and early phase clinical trials.
We are exploring the use of PV pseudovirus as gene transfer vehicles. The tropism of the pseudovirions is remarkably limited to traumatized epithilium. However, almost all epithelial tumor-derived cell lines are readily infected. This suggests that wounded keratinocytes and tumors share a fundamental property essential for PV infection. Practically, it suggests that PV pseudoviruses might be useful in tumor diagnosis or cytotoxic therapy since the vectors do not infect or even bind normal tissue. With NCI and industry partners we are evalutating PsV therapies involving tumor targeting/killing via cytotoxic genes, radioactive alpha emitters and intrared dyes.
With other NIH colleagues, we are evaluating the pseudovirions as vehicles for genetic immunization of the female genital tract. Studies in mice and monkeys indicate that cervicovaginal instillation of pseudovirus in the presence of nonoxonal-9 induces potent systemic and mucosal immune responses and is particularly effective at inducing intraepithelial CD8+ T cells.
Finally, we have initiated virological and immunological analyses of the recently identified MusPV, the first domestic mouse papillomavirus.
This page was last updated on 11/7/2013.