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JOURNAL OF VIROLOGY, Aug. 2007, p. 8784–8792 0022-538X/07/$08.00 0 doi:10.1128/JVI.00552-07

Vol. 81, No. 16

Neutralization of Human Papillomavirus with Monoclonal Antibodies Reveals Different Mechanisms of Inhibition
Patricia M. Day,* Cynthia D. Thompson, Christopher B. Buck, Yuk-Ying S. Pang, Douglas R. Lowy, and John T. Schiller
Laboratory of Cellular Oncology, Center for CancerResearch, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892
Received 15 March 2007/Accepted 24 May 2007

The mechanisms of human papillomavirus (HPV) neutralization by antibodies are incompletely understood. We have used HPV16 pseudovirus infection of HaCaT cells to analyze how several neutralizing monoclonal antibodies (MAbs) generated against HPV16 L1 interfere withthe process of keratinocyte infection. HPV16 capsids normally bind to both the cell surface and extracellular matrix (ECM) of HaCaT cells. Surprisingly, two strongly neutralizing MAbs, V5 and E70, did not prevent attachment of capsids to the cell surface. However, they did block association with the ECM and prevented internalization of cell surface-bound capsids. In contrast, MAb U4 preventedbinding to the cell surface but not to the ECM. The epitope recognized by U4 was inaccessible when virions were bound to the cell surface but became accessible after endocytosis, presumably coinciding with receptor detachment. Treatment of capsids with heparin, which is known to interfere with binding to cell surface heparan sulfate proteoglycans (HSPGs), also resulted in HPV16 localization to theECM. These results suggest that the U4 epitope on the intercapsomeric C-terminal arm is likely to encompass the critical HSPG interaction residues for HPV16, while the V5 and E70 epitopes at the apex of the capsomer overlap the ECM-binding sites. We conclude that neutralizing antibodies can inhibit HPV infection by multiple distinct mechanisms, and understanding these mechanisms can add insight tothe HPV entry processes. Human papillomavirus (HPV) infections are extremely common, with estimates suggesting that approximately 75% of women will become infected with one or more of the sexually transmitted HPV types at some point after initiating sexual activity (1). Infection with a subset of sexually transmitted HPVs, especially HPV16, is considered a necessary factor in the development ofvirtually all cases of cervical cancer (3). A neutralizing antibody response to L1, the major structural viral protein, is known to effectively prevent papillomavirus (PV) infection, as demonstrated by studies in animal models and the successes of the recently developed HPV vaccine (33). However, the mechanisms by which these neutralizing antibodies act to prevent infection are unclear. L1 canself-assemble to form empty capsids known as viruslike particles that resemble authentic capsids morphologically and immunologically (28) and are the basis for current HPV vaccines. Although L1 is overall a highly conserved PV protein, anti-L1 neutralizing antibodies are type restricted, because the conserved residues are largely confined to the portions of L1 that are poorly exposed on the surface of thecapsid, whereas the antibody responses are typically generated against epitopes found on the external loops, where the L1 sequences are highly divergent (11, 38). These external loops, which form the apex of the L1 capsomer, are relatively unstructured and, based on analogy to the polyomavirus major structural protein, VP1, have been proposed as candidate regions for receptor interaction (10). In* Corresponding author. Mailing address: Laboratory of Cellular Oncology, Room 4112, Building 37, National Institutes of Health, Bethesda, MD 20892. Phone: (301) 594-6945. Fax: (301) 480-5322. E-mail: Published ahead of print on 6 June 2007. 8784

fact, despite minimal sequence homology among their major capsid proteins, the PV capsid structure is very similar to that of...
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