Human papillomaviruses (HPVs) replicate only in the terminally differentiating epithelium of the skin and mucosa. While infection of basal keratinocytes is considered a requirement for permissive infection, it remains unclear whether virions can specifically target basal cells for adsorption and uptake following epithelial wounding. We present evidence that HPV binds specifically to laminin 5 (LN5), a component of the extracellular matrix (ECM) secreted by migrating and basal keratinocytes. HPV type 11 capsids colocalized with LN5 in the ECM secreted by vaginal keratinocytes. Binding of both virions and virus-like particles to purified LN5 and to the LN5-rich ECM secreted by cultured keratinocytes was effectively blocked by pretreatment with anti-LN5 antibodies. HPV capsid binding to human cervical mucosa sections included the basement membrane which contains LN5. Cultured keratinocytes expressing ␣6 integrin, a transmembrane protein known to bind LN5, were readily infected by virions preadsorbed to LN5-containing substrates, whereas mutant keratinocytes lacking ␣6 integrin were relatively resistant to infection via this route. These findings suggest a model of natural HPV infection in which proliferating keratinocytes expressing ␣6 integrin at the site of epithelial wounding might be targeted by virions adsorbed transiently to LN5 secreted by migrating keratinocytes.Human papillomavirus (HPV) particles have been shown to adsorb to the plasma membranes of cultured cells via membrane-associated heparan sulfate proteoglycans (HSPGs) (18,20,33) or ␣6 integrin (CD49f) (15,26). Multiple HSPGs including CD44, syndecans and glypicans are expressed on the membranes of keratinocytes throughout the epidermis and mucosa (22,29). ␣6 integrin expression is generally restricted to basal keratinocytes where this transmembrane protein pairs with ␤4 integrin and contributes to the nucleation of hemidesmosomes connecting the keratin cytoskeleton to the basement membrane (BM) (reviewed in reference 28). Results from experiments utilizing several in vitro infection models suggest that the importance of a particular receptor in HPV adsorption/infection may differ between cell lines and viral genotypes (12,30,33).In addition to binding directly to membrane-associated glycoproteins, we recently found that HPV capsids are also capable of binding a component of the extracellular matrix (ECM) secreted by keratinocytes, but not by nonkeratinocyte cell lines (12). Here we show evidence that this secreted HPV adsorption receptor is laminin 5 (LN5), an epithelial laminin secreted by migrating keratinocytes as they invade wounded epithelium (reviewed in reference 27). In the context of the ECM secreted by cultured keratinocytes, HPV virions can use LN5 as an extracellular "transreceptor" by transiently binding LN5 and subsequently transferring to entry receptors on adjacent cells. In another viral system, human immunodeficiency virus (HIV) is hypothesized to transiently bind DC-SIGN (CD209) on immature dendritic cells within the epithelium an...

Human papillomavirus (HPV) hybrid virus-like particles (VLPs) were prepared using complementary regions of the major capsid L1 proteins of HPV-11 and -16. These hybrid L1 proteins were tested for assembly into VLPs, for presentation and mapping of conformational neutralizing epitopes, and as immunogens in rabbits and mice. Two small noncontiguous hypervariable regions of HPV-16 L1, when replaced into the HPV-11 L1 backbone, produced an assembly-positive hybrid L1 which was recognized by the type-specific, conformationally dependent HPV-16 neutralizing monoclonal antibody (N-MAb) H16.V5. Several new N-MAbs that were generated following immunization of mice with wild-type HPV-16 L1 VLPs also recognized this reconstructed VLP, demonstrating that these two hypervariable regions collectively constituted an immunodominant epitope. When a set of hybrid VLPs was tested as immunogens in rabbits, antibodies to both HPV-11 and -16 wild-type L1 VLPs were obtained. One of the hybrid VLPs containing hypervariable FG and HI loops of HPV-16 L1 replaced into an HPV-11 L1 background provoked neutralizing activity against both HPV-11 and HPV-16. In addition, conformationally dependent and type-specific MAbs to both HPV-11 and HPV-16 L1 VLP were obtained from mice immunized with hybrid L1 VLPs. These data indicated that hybrid L1 proteins can be constructed that retain VLP-assembly properties, retain type-specific conformational neutralizing epitopes, can map noncontiguous regions of L1 which constitute type-specific conformational neutralizing epitopes recognized by N-MAbs, and trigger polyclonal antibodies which can neutralize antigenically unrelated HPV types.

Epstein-Barr virus is a ubiquitous human herpesvirus associated with epithelial and lymphoid tumors. EBV is transmitted between human hosts in saliva and must cross the oral mucosal epithelium before infecting B lymphocytes, where it establishes a life-long infection. The latter process is well understood because it can be studied in vitro, but our knowledge of infection of epithelial cells has been limited by the inability to infect epithelial cells readily in vitro or to generate cell lines from EBV-infected epithelial tumors. Because epithelium exists as a stratified tissue in vivo, organotypic cultures may serve as a better model of EBV in epithelium than monolayer cultures. Here, we demonstrate that EBV is able to infect organotypic cultures of epithelial cells to establish a predominantly productive infection in the suprabasal layers of stratified epithelium, similar to that seen with Kaposi's-associated herpesvirus. These cells did express latency-associated proteins in addition to productive-cycle proteins, but a population of cells that exclusively expressed latency-associated viral proteins could not be detected; however, an inability to infect the basal layer would be unlike other herpesviruses examined in organotypic cultures. Furthermore, infection did not induce cellular proliferation, as it does in B cells, but instead resulted in cytopathic effects more commonly associated with productive viral replication. These data suggest that infection of epithelial cells is an integral part of viral spread, which typically does not result in the immortalization or enhanced growth of infected epithelial cells but rather in efficient production of virus.Epstein-Barr virus | epithelial | organotypic culture | productive replication A lthough the association between Epstein-Barr virus and epithelial malignancies has been known for more than three decades, the EBV life cycle within the epithelial milieu is still only poorly understood. In contrast, our broad understanding of the biology of EBV within the B-cell compartment has been facilitated by the ability of EBV to infect and immortalize primary B cells in vitro and by the ability of some EBV-positive B-cell tumors to give rise to cell lines that maintain restricted programs of latency gene expression similar to those seen in vivo. Although in primary EBV infection the entire complement of EBV latencyassociated nuclear proteins (EBNAs 1, 2, 3A, 3B, 3C, and LP) and membrane proteins (LMPs 1, 2A, and 2B) promote cellular proliferation and survival (Latency III), EBV gene expression must be progressively silenced (Latency II; EBNA1 and LMPs 1 and 2) so that the most restricted program, Latency 0 (in which EBV gene expression is believed to be completely silenced

Clinical trials evaluating the efficacy of nonoxynol-9 (N-9) as a topical microbicide concluded that N-9 offers no in vivo protection against human immunodeficiency virus type 1 (HIV-1) infection, despite demonstrated in vitro inactivation of HIV-1 by N-9. These trials emphasize the need for better model systems to determine candidate microbicide effectiveness and safety in a preclinical setting. To that end, time-dependent in vitro cytotoxicity, as well as in vivo toxicity and inflammation, associated with N-9 exposure were characterized with the goal of validating a mouse model of microbicide toxicity. In vitro studies using submerged cell cultures indicated that human cervical epithelial cells were inherently more sensitive to N-9-mediated damage than human vaginal epithelial cells. These results correlated with in vivo findings obtained by using Swiss Webster mice in which intravaginal inoculation of 1% N-9 or Conceptrol gel (containing 4% N-9) resulted in selective and acute disruption of the cervical columnar epithelial cells 2 h postapplication accompanied by intense inflammatory infiltrates within the lamina propria. Although damage to the cervical epithelium was apparent out to 8 h postapplication, these tissues resembled control tissue by 24 h postapplication. In contrast, minimal damage and infiltration were associated with both short-and long-term exposure of the vaginal mucosa to either N-9 or Conceptrol. These analyses were extended to examine the relative toxicity of polyethylene hexamethylene biguanide (PEHMB), a polybiguanide compound under evaluation as a candidate topical microbicide. In similar studies, in vivo exposure to 1% PEHMB caused minimal damage and inflammation of the genital mucosa, a finding consistent with the demonstration that PEHMB was >350-fold less cytotoxic than N-9 in vitro. Collectively, these studies highlight the murine model of toxicity as a valuable tool for the preclinical assessment of toxicity and inflammation associated with exposure to candidate topical microbicides.

Noninvasive and practical techniques to longitudinally track viral infection are sought after in clinical practice. We report a proof-of-principle study to monitor the viral DNA copy number using a newly established mouse papillomavirus (MmuPV1) mucosal infection model. We hypothesized that viral presence could be identified and quantified by collecting lavage samples from cervicovaginal, anal and oral sites. Nude mice infected at these sites with infectious MmuPV1 were tracked for up to 23 weeks starting at 6 weeks post-infection. Viral DNA copy number was determined by SYBR Green Q-PCR analysis. In addition, we tracked viral DNA load through three complete oestrous cycles to pinpoint whether there was a correlation between the DNA load and the four stages of the oestrous cycle. Our results showed that high viral DNA copy number was reproducibly detected from both anal and cervicovaginal lavage samples. The infection and disease progression were further confirmed by histology, cytology, in situ hybridization, immunohistochemistry and transmission electron microscopy. Interestingly, the viral copy number fluctuated over the oestrous cycle, with the highest level at the oestrus stage, implying that multiple sampling might be necessary to provide a reliable diagnosis. Virus DNA was detected in oral lavage samples at a later time after infection. Lower viral DNA load was found in oral samples when compared with those in anal and vaginal tracts. To our knowledge, our study is the first in vivo study to sequentially monitor papillomavirus infection from mucosal anal, oral and vaginal tracts in a preclinical model.

Baculovirus-expressed human papillomavirus type 11 (HPV-11) major capsid protein (L1) virus-like particles (VLPs) were produced in insect cells and purified on CsC1 density gradients. The VLPs retained conformational neutralizing epitopes that were detected by a series of HPV-11-neutralizing monoclonal antibodies. Electron microscopy determined that the HPV-11 L1 VLPs were variable in size with a surface topography similar to that of infectious HPV-I 1. The VLPs were very antigenic, and induced high titres of neutralizing antibodies in rabbits and mice when used as an immunogen without commercial preparations of adjuvant. These VLP reagents may be effective vaccines for protection against HPV infections.

Human papillomaviruses (HPVs) have previously been shown to adsorb to cultured cells via membrane-associated heparan sulfate (HS) and alpha6 integrin. We demonstrate that cultured keratinocytes uniquely secrete a component into the basal extracellular matrix (ECM) which can function to adsorb HPV particles which can then be internalized by adherent cells. This uncharacterized basal ECM adsorption receptor was secreted by normal human epidermal keratinocytes (NHEK) and by each of the four keratinocyte-derived cell lines we examined, but not by non-keratinocyte cell lines. Multiple HPV types bound preferentially to this keratinocyte-specific receptor over the membrane-associated receptor, and binding to the basal ECM adsorption receptor was refractory to inhibition by heparin. Like the membrane-associated receptor, this basal ECM component was functional as an adsorption receptor in our in vitro infection model using HPV-11. Unlike particle adsorption, however, successful infection with HPV-11 virions remained sensitive to the pretreatment of virions with heparin. The secreted basal ECM receptor did not colocalize with antibodies against HS, perlecan, or alpha6 integrin, but colocalized with antibody against laminin-5, a marker of keratinocyte ECM and an abundant component of the basement membrane in mucosa and skin. These findings suggest a model for natural infections in which HPV virions, nonspecifically adsorbed to HS on suprabasal keratinocytes throughout an epithelial wound, might be transferred to mitotically active migrating keratinocytes via an intermediate association with the ECM secreted by these cells as they reestablish the basement membrane.

Papillomavirus disease poses a special challenge to people with compromised immune systems. Appropriate models to study infections in these individuals are lacking. We report here the development of a model that will help to address these deficiencies. The MmuPV1 genome was synthesized and used successfully to produce virus from DNA infections in immunocompromised mice. In these early studies, we have demonstrated both primary and secondary infections, expanded tissue tropism, and extensive dysplasia. Papillomaviruses are double-stranded DNA tumor viruses of about 8 kb. These viruses are ubiquitous in nature, and 241 types have been identified to date in both humans and many other animal species (http://pave.niaid.nih.gov). A subset of human papillomaviruses is linked to cancers, most notably cervical cancer, but also cancers of the head and neck, skin, and other anogenital sites. Papillomaviruses require fully differentiating cells for the completion of the viral life cycle and thus are not easily studied in cell culture systems. For a review of these viruses, see reference 1. The cottontail rabbit papillomavirus (CRPV) in vivo model has proven to be very useful in our hands for the study of cutaneous papillomavirus disease (2-8). An in vivo system to study both cutaneous and mucosal disease, papillomavirus pathology, and