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Antimicrobial Agents and Chemotherapy, April 2008, p. 1374-1381, Vol. 52, No. 4
0066-4804/08/$08.00+0     doi:10.1128/AAC.01467-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Sulfated K5 Escherichia coli Polysaccharide Derivatives as Wide-Range Inhibitors of Genital Types of Human Papillomavirus{triangledown}

David Lembo,1* Manuela Donalisio,1 Marco Rusnati,2 Antonella Bugatti,2 Maura Cornaglia,1 Paola Cappello,3 Mirella Giovarelli,3 Pasqua Oreste,4 and Santo Landolfo5

Department of Clinical and Biological Sciences, University of Turin, San Luigi Gonzaga Hospital, 10043 Orbassano, Turin, Italy,1 Department of Biomedical Sciences and Biotechnology, University of Brescia, 25123 Brescia, Italy,2 Department of Medicine and Experimental Oncology, University of Turin, and Center for Experimental Research and Medical Studies, San Giovanni Battista Hospital, 10126 Turin, Italy,3 Glycores 2000 Srl, 20155 Milan, Italy,4 Department of Public Health and Microbiology, University of Turin, 10126 Turin, Italy5

Received 13 November 2007/ Returned for modification 22 December 2007/ Accepted 28 January 2008


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ABSTRACT
 
Genital human papillomaviruses (HPV) represent the most common sexually transmitted agents and are classified into low or high risk by their propensity to cause genital warts or cervical cancer, respectively. Topical microbicides against HPV may be a useful adjunct to the newly licensed HPV vaccine. A main objective in the development of novel microbicides is to block HPV entry into epithelial cells through cell surface heparan sulfate proteoglycans. In this study, selective chemical modification of the Escherichia coli K5 capsular polysaccharide was integrated with innovative biochemical and biological assays to prepare a collection of sulfated K5 derivatives with a backbone structure resembling the heparin/heparan biosynthetic precursor and to test them for their anti-HPV activity. Surface plasmon resonance assays revealed that O-sulfated K5 with a high degree of sulfation [K5-OS(H)] and N,O-sulfated K5 with a high [K5-N,OS(H)] or low [K5-N,OS(L)] sulfation degree, but not unmodified K5, N-sulfated K5, and O-sulfated K5 with low levels of sulfation, prevented the interaction between HPV-16 pseudovirions and immobilized heparin. In cell-based assays, K5-OS(H), K5-N,OS(H), and K5-N,OS(L) inhibited HPV-16, HPV-18, and HPV-6 pseudovirion infection. Their 50% inhibitory concentration was between 0.1 and 0.9 µg/ml, without evidence of cytotoxicity. These findings provide insights into the design of novel, safe, and broad-spectrum microbicides against genital HPV infections.


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INTRODUCTION
 
Human papillomaviruses (HPVs) are members of the Papillomaviridae family of DNA viruses. More than 100 HPV types have been identified so far, over 30 of which infect the genital area (19). Genital HPV infections are estimated to be the most common sexually transmitted infection. Although the majority of infections cause no symptoms and are self-limiting, genital HPVs have become a major public health concern because persistent infection with certain types can cause cervical cancer, which kills about 250,000 women worldwide each year (2). Genital HPVs are classified according to their association with cervical cancer. Infections with low-risk types (primarily types 6 and 11) can cause benign or low-grade cervical cell changes and genital warts but are not associated with cervical cancer. Infection with high-risk types (primarily types 16, 18, 31, and 45) can cause low-grade and high-grade cervical cell abnormalities that are precursors to cancer (23). Types 44, 53, 70, and 74 rarely cause genital warts or cervical cancer but are relatively common worldwide. These types are an important public health concern because they are not covered by the currently available vaccine and yet can lead to positive Papanicolaou (Pap) test results.

Cervical cancer is relatively uncommon in countries where widespread cervical Pap testing detects precancerous lesions before they can develop into cancer. In many developing countries where screening activities are limited, however, cervical cancer is the most common cancer in women (2). HPVs have also been implicated in a substantial fraction of other anogenital cancers at other sites including the vulva, vagina, penis, and anus (30), as well as some head and neck cancers (17).

Current treatments are ablative and directed to abnormal cells associated with HPV rather than the virus itself; no direct antiviral treatment is available. The prevention of genital HPV infection is essential for reducing the prevalence of genital warts and abnormal Pap tests, as well as cervical cancer. Since male condoms have been reported to provide only partial protection against HPV transmission, they cannot be recommended as a primary prevention strategy (18, 24).

Recently, a highly effective vaccine was approved to prevent infections by four HPV types that together cause about 70% of cervical cancers (HPV-16 and HPV-18) and 90% of genital warts (HPV-6 and HPV-11) worldwide (13, 14). However, women may remain exposed to the risk of becoming infected with some types of high-risk HPVs that can cause cervical cancer but are not targeted by the current vaccine. Moreover, the vaccine is relatively expensive, and it may not be initially available to all women, especially those in developing countries. In this scenario, a topical microbicide, a compound that could block the full spectrum of genital HPV infections at the portal of entry, would be a useful complement to vaccination programs.

Papillomaviruses replicate exclusively in stratified squamous epithelial tissues such as the skin or the genital mucosa (19). Because the viral life cycle is closely linked to cellular differentiation in these tissues, papillomaviruses cannot be cultured using conventional monolayer cell lines. This has hampered targeted screens to identify molecules that might inhibit the infectious entry of papillomaviruses. Recently, John Schiller and coworkers developed systems to efficiently produce high-titer HPV-based gene delivery vectors, known as papillomavirus pseudovirions (PsV) in cultured cell lines (6, 7, 8). HPV PsV, which are capable of efficiently delivering reporter plasmids to a wide variety of cell lines, have rapidly become a useful tool for studying the initial infectious entry phase of the HPV life cycle.

A main objective in the development of novel microbicides against HPV infections is to block the interaction between the virion and the cell surface heparan sulfate proteoglycans (HSPG) that mediate the attachment of HPV to the target cell and its infectivity (15, 20, 31). Associated with the surface of many cell types, HSPG consist of a core protein and glycosaminoglycan chains of unbranched sulfated polysaccharides, known as heparan sulfates (HS), and are structurally related to heparin. Accordingly, heparin and other sulfated polysaccharides prevent the binding of HPV to the cell surface by mimicking HS (5, 9, 15, 20). The capsular K5 polysaccharide from Escherichia coli has the same structure, [-> 4)-β-D-GlcA-(1 -> 4)-{alpha}-D-GlcNAc-1(1->]n, as the heparin precursor N-acetyl heparosan. The possibility to generate K5 derivatives by chemical sulfation in the N and/or O positions along the polysaccharide has led to the synthesis of various K5 derivatives with different degrees of sulfation and charge distribution. With these tailored modifications a variety of chemically defined compounds have been generated that are devoid of anticoagulant activity and toxicity (29). Specifically, K5 derivatives have recently attracted attention as candidate microbicides since they potently inhibit a broad spectrum of human immunodeficiency virus type 1 (HIV-1) strains (32). In the present study, we tested a series of N-sulfated (K5-NS), O-sulfated (K5-OS), and N,O-sulfated (K5-N,OS) derivatives with different degrees of sulfation as potential inhibitors of HPV infection by integrating PsV-based neutralization assays with the high-throughput surface plasmon resonance (SPR) technology (Biacore) that allows the study of biomolecular interactions.


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MATERIALS AND METHODS
 
Cell culture. The human cervical carcinoma cell lines SiHa (ATCC HTB-35), CaSki (ATCC CRL 1550), and C33A (ATCC HTB31) and the human maxillary sinus carcinoma cell line HNO136 (a kind gift from Massimo Tommasino, IARC, Lyon, France) were grown as monolayers in Dulbecco's modified Eagle's medium (DMEM) (Gibco/BRL, Gaithersburg, MD) supplemented with heat-inactivated 10% bovine serum (Gibco/BRL) and Glutamax-I (Invitrogen, Carlsbad, CA). The 293TT cell line derived from human embryonal kidney cells transformed with the simian virus 40 (SV40) large T antigen was cultured in the medium previously described supplemented with nonessential amino acids. This cell line allows high levels of protein to be expressed from vectors containing the SV40 origin due to overreplication of the expression plasmid (7).

Heparin and K5 polysaccharide derivatives. Unmodified unfractionated beef mucosa heparin (average molecular weight [MW], 13,700; sulfate/carboxyl [SO3–/COO–] ratio, 2.14) was obtained from Laboratori Derivati Organici, Milan, Italy. K5 polysaccharide derivatives were obtained by N-deacetylation/N-sulfation and/or O-sulfation of a single batch of K5 polysaccharides as previously described (22). The chemical characterization of the compounds is shown in Table 1.


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  		TABLE 1. Chemical characterization of K5 derivatives

PsV production. Plasmids and 293TT cells used for PsV production were kindly provided by John Schiller (National Cancer Institute, Bethesda, MD). Detailed protocols and plasmid maps for this study can be seen at http://home.ccr.cancer.gov/lco/default.asp. HPV-16, HPV-18, HPV-6, and bovine papillomavirus type 1 (BPV-1) PsV were produced according to previously described methods (8). Briefly, 293TT cells were transfected with plasmids expressing the papillomavirus major and minor capsid proteins (L1 and L2, respectively) together with a reporter plasmid expressing the secreted alkaline phosphatase (SEAP) or the green fluorescent protein (GFP), named pYSEAP or pfwB, respectively. HPV-16, HPV-6, and BPV-1 PsV were produced using the bicistronic L1/L2 expression plasmids (p16sheLL, p6sheLL, and pSheLL, respectively), and HPV-18 PsV was produced using peL1fB and peL2bhb plasmids. Capsids were allowed to mature overnight in cell lysate; the clarified supernatant was then loaded on top of a density gradient of 27 to 33 to 39% Optiprep (Sigma-Aldrich, St. Louis, MO) at room temperature for 4 h. The material was centrifuged at 234,000 x g for 3.30 h at 16°C in an SW50.1 rotor (Beckman Coulter, Inc. Fullerton, CA) and collected by bottom puncture of the tubes. Fractions were inspected for purity on 10% sodium dodecyl sulfate (SDS)-Tris-glycine gels, titrated on 293TT cells to test for infectivity by SEAP or GFP detection, and then pooled and frozen at –80°C until needed. The L1 protein content of PsV stocks was determined by comparison with bovine serum albumin standards in Coomassie-stained SDS-polyacrylamide gel electrophoresis (PAGE) gels.

Inhibition assays. For the SEAP-based assays 293TT cells were preplated 3 to 4 h in advance in 96-well tissue culture-treated flat bottom plates at a density of 30,000 cells/well in 100 µl of neutralization buffer (DMEM without phenol red, 10% heat-inactivated fetal bovine serum, 1% glutamate, 1% nonessential amino acids, 1% penicillin-streptomycin-fungizone, and 10 mM HEPES). To generate dose-response curves, diluted PsV stocks (80 µl/well) were placed on 96-well nontreated sterile, polystyrene plates (Nalge-Nunc, Roskilde, Denmark), combined with 20 µl of serially diluted heparin or K5 derivatives, and placed on ice for 1 h. The 100-µl PsV-compound mixture was transferred onto the preplated cells and incubated for 68 to 72 h. The final concentration of PsV was approximately 1 ng/ml L1. After incubation, 50 µl of supernatant was harvested and clarified at 1,500 x g for 5 min. The SEAP content in the clarified supernatant was determined using a Great Escape SEAP Chemiluminescence Kit (BD Clontech, Mountain View, CA) as directed by the manufacturer. Ten minutes after the substrate was added, samples were read using a Lumino luminometer (Stratec Biomedical System, Birkenfeld, Germany).

The 50% inhibition concentration (IC50) values and the 95% confidence intervals (CIs) were determined using the Prism program (GraphPad Software, San Diego, CA).

Preattachment assays were performed on 96-well plates by incubating a fixed dose of K5 derivatives (3 µg/ml) with 293TT cell monolayers for 2 h at 37°C. The monolayers were washed gently to avoid cell dislodgement, HPV-16 PsV containing the SEAP reporter plasmid (HPV-16-SEAP PsV) were added to the cells (1 ng/ml L1), and SEAP activity was measured in the cell culture supernatants 72 h after PsV inoculation.

Attachment assays were done by preincubating 3 µg/ml of each compound with HPV-16-SEAP PsV (1 ng/ml L1) at 4°C. The mixtures were added to the cooled 293TT cells, which were then incubated at 4°C for 2 h to ensure PsV attachment but not entry. After two gentle washes, the cells were shifted to 37°C, and SEAP activity was measured in the cell culture supernatants 72 h after PsV inoculation.

Postattachment assays were performed on 96-well plates by incubating HPV-16-SEAP PsV (1 ng/ml L1) with preplated 293TT cells for 2 h at 37°C, followed by two gentle washes to remove unbound virus. A fixed dose of K5 derivatives (3 µg/ml) was then added to cultures at set time points. For the 2-h time point, the compounds were added immediately after washout of the inoculum.

For the GFP-based assays, cells were plated at a density of 1 x 105 cells/well in 400 µl of DMEM supplemented with 10% fetal bovine serum (Life Technologies, Inc., Gaithersburg, MD) in 24-well plates. A fixed dose of K5 derivatives (3 µg/ml) was added to preplated cells, followed by 2 to 5 µl of PsV stock, and cultures were incubated for 44 to 52 h at 37°C. The flow cytometric analysis was fixed to count 15,000 live cells; only cells expressing high levels of GFP were included in the analysis. PsV doses were calibrated such that between 5% and 25% of cells were scored as GFP positive when no inhibitors were added. Percent inhibition was calculated by using the following formula: 100 x [1 –(net percentage of GFP+ cells in the test sample/net percentage of GFP+ cells in the mock sample)].

Cell viability assay. Cells were seeded at a density of 5 x 104/well in 24-well plates; the next day they were treated with serially diluted K5 derivatives to generate dose-response curves. At 48 or 72 h after treatment, cell viability was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide method, as previously described (25); 50% cytotoxic concentration (CC50) values and 95% CIs were determined using Prism software.

Electron microscopy. An aliquot of diluted HPV PsV preparations was placed on a grid and air dried prior to examination. Microscopy was performed using a Philips CM10 transmission electron microscope; micrographs were taken of random sections at different powers of magnification.

Biacore binding assay. Heparin was biotinylated on its reducing end, and a flow cell of an F1 sensor chip was activated with streptavidin. Biotinylated heparin was then allowed to react with the streptavidin-coated sensor chip as previously described (28). A streptavidin-coated sensor chip was used for blank subtraction. HPV-16 PsV (33 nM in 10 mmol/liter HEPES, 150 mmol/liter NaCl, 3.4 mmol/liter EDTA, 0.005% surfactant P20, pH 7.4) were injected over a heparin-coated surface for 5 min to allow the association of the capsid proteins with heparin, and the sample was then washed until dissociation was observed. The SPR signal was then measured and expressed as resonance units after blank subtraction. A Biacore X apparatus (Biacore Inc., Piscataway, NJ) was used.


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RESULTS
 
Characterization of purified HPV-16 PsV. HPV-16 was chosen as a model for initial experiments since it depends on HSPG for its infectivity (15) and is the most oncogenic genital HPV type (2). To check the quality of the HPV-16-SEAP PsV preparation used in the subsequent Biacore and cell-based inhibition assays, an aliquot was subjected to SDS-PAGE. As shown in Fig. 1A, a major band migrating at 55 kDa was detected by Coomassie brilliant blue staining (lane 1) and was confirmed to be the L1 major capsid protein by Western blotting (lane 2). No L1-reactive proteolytic degradation products were observed at molecular masses below 55 kDa, indicating the good quality of the preparation. Figure 1B shows an electron micrograph of the same PsV stock. PsV routinely exhibited an average diameter of 50 to 60 nm, which is similar to that of an authentic HPV capsid, and appeared as individual, well-defined particles with minimal aggregation. Similar results were obtained with the other PsV types used in this study (data not shown).


Figure 1
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  		FIG. 1. Characterization of purified HPV-16-SEAP PsV. (A) An aliquot of purified PsV preparation was analyzed by SDS-PAGE with Coomassie brilliant blue staining (lane 1) or immunoblotting (lane 2) with an anti-L1 antibody (B0580; Dako Corporation, Carpinteria, CA). (B) Electron micrograph of a purified PV preparation. Scale bar, 100 nm. MW values are in thousands.

Biacore binding assay. HSPG expressed on the surface of target cells act as receptors for many HPV types during infection. Accordingly, the L1 major capsid protein of HPV-11 recombinant virus-like particles binds heparin immobilized to a Biacore sensor chip (20) (an experimental condition that resembles its binding to cellular HSPG). We evaluated the capability of K5 derivatives to prevent free HPV-16-SEAP PsV from interacting with sensor chip-immobilized heparin. Preliminary experiments demonstrated that in our hands HPV-16 PsV also binds specifically to the heparin surface (up to 80 resonance units after blank subtraction). Increasing concentrations of K5 derivatives or heparin (here used as a positive control) were then preincubated with HPV-16-SEAP PsV and injected onto the heparin-coated sensor chip. The results (Fig. 2) demonstrate that, at the doses tested, unsulfated K5 did not affect HPV-16-heparin interaction while K5-OS with a low degree of sulfation [K5-OS(L)] and K5-NS were weak inhibitors (IC50s of >1,400 ng/ml and 1,200 ng/ml, respectively). By contrast, K5-N,OS(L) inhibited the interaction with a potency similar to that of free unmodified heparin (IC50 of 170 ng/ml) while K5-N,OS with a high level of sulfation [K5-N,OS(H)] and K5-OS(H) turned out to be significantly more efficient inhibitors (IC50s of 51 and 22 ng/ml, respectively). These data indicate that selected K5 derivatives are able to prevent an HPV-16-SEAP PsV-heparin interaction. To test whether this class of compounds was also able to disrupt already established PsV-heparin complexes, HPV-16-SEAP PsV were injected and allowed to reach an equilibrium binding with the heparin surface, and only then was K5-OS(H) injected. Under these experimental conditions, K5-OS(H) retained its capability to inhibit the binding of PsV to heparin (Fig. 2), suggesting that it is able not only to prevent the binding of the viral protein to heparin/HSPG but also to reverse already established complexes.


Figure 2
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  		FIG. 2. K5 derivatives inhibit HPV-16-SEAP PsV-heparin interaction. A purified PsV dilution (33 nM) was injected over the Biacore sensor chip containing immobilized heparin in the presence of increasing concentrations of different K5 derivatives or of free heparin (here used as a control). Alternatively, HPV-16-SEAP PsV was injected over the Biacore sensor chip containing immobilized heparin and allowed to reach equilibrium binding. Then, K5-OS(H) (1.1 µg/ml) was injected and evaluated for its capability to destroy established HPV-16-SEAP PsV-heparin complexes. The response was recorded after the dissociation phase and plotted as a function of K5 derivative concentrations. The experiment is representative of two others that gave similar results.

Inhibitory effects of K5 derivatives against various papillomavirus types in a cell-based assay. The results of the Biacore assays revealed that some K5 derivatives have the potential to neutralize the infectivity of HPV-16. To prove this hypothesis, we tested the inhibitory activity of K5 derivatives using a HPV-16-SEAP PsV-based assay. The early events of a PsV infection resemble those of a natural HPV infection since the PsV consist of a SEAP reporter plasmid encapsidated by a capsid composed of the two viral capsid proteins (L1 and L2), which is like an authentic HPV capsid. After PsV binding to and entry into the cell, the reporter plasmid is transported to the nucleus for expression of the reporter gene (7). To generate dose-response curves, serial dilutions of compounds were preincubated with aliquots of HPV-16-SEAP PsV and then added to 293TT cell cultures. Inhibition of PsV-mediated delivery of the SEAP reporter plasmid 72 h postinfection was measured by chemiluminescence analysis of the cell supernatants. As shown in Table 2, three compounds, K5-OS(H), K5-N,OS(L), and K5-N,OS(H), strongly inhibited the HPV-16-SEAP PsV infection at IC50s in the nanogram/milliliter range (0.21 µg/ml, 0.18 µg/ml, and 0.65 µg/ml, respectively). As expected, heparin also inhibited the infection but with an efficiency that was an order of magnitude lower than the efficiency of K5-OS(H) and K5-N,OS(L) (IC50 of 2.88 µg/ml). By contrast, K5, K5-NS, and K5-OS(L) failed to display any significant inhibitory effect. The CC50s were >100 µg/ml for all compounds tested, indicating that the inhibitory activity was not a consequence of cytotoxicity. To assess whether K5 derivatives are HPV type specific in their inhibitory activity, we repeated the assay using two other HPV-SEAP PsV (HPV-18 and HPV-6), and the SEAP-BPV-1 PsV. The results shown in Table 3 demonstrate that the compounds that displayed an inhibitory effect on HPV-16 PsV were also active on the other PsV types tested. K5-OS(H), K5-N,OS(L), and K5-N,OS(H) were then subjected to further investigations.


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  		TABLE 2. Inhibition of HPV16-SEAP PsV infection of 293TT cells


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  		TABLE 3. Inhibition of HPV-18, HPV-6, BPV-1 PsV infection of 293TT cells

Investigation of the inhibitory mechanism by K5-OS(H), K5-N,OS(L), and K5-N,OS(H). To determine at which step the active K5 derivatives interfere with the infectious process of PsV, three types of binding assays were performed employing HPV-16-SEAP PsV and 293TT cells as a model system and a fixed dose of compounds (3 µg/ml) that routinely inhibit ≥90% of PsV infectivity. In the first assay (preattachment assay), K5-OS(H), K5-N,OS(L), and K5-N,OS(H) were allowed to interact with the cell surface by incubating cell monolayers for 2 h at 37°C with the compounds. After removal of the medium containing the compounds, the cells were washed and then incubated with PsV for 72 h at 37°C. As shown in Fig. 3A, K5-OS(H), K5-N,OS(L), and K5-N,OS(H) failed to display any inhibitory activity in the preattachment assay. This finding demonstrates that the activity of the compounds is independent of interaction with cell surface components. The second assay (attachment assay) was carried out by preincubating K5-OS(H), K5-N,OS(L), and K5-N,OS(H) with PsV at 4°C before they were transferred to cold cells for 2 h of incubation at 4°C. When performed in the absence of infection inhibitors, this procedure ensures PsV attachment but not entry until the cells are washed twice to remove unbound PsV, and the infection is then allowed to proceed at 37°C. The results of this assay demonstrate that all the compounds tested exerted a strong inhibitory activity (Fig. 3B), suggesting that they inhibit PsV attachment to the cell surface.


Figure 3
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  		FIG. 3. Investigation of inhibitory mechanism used by K5-OS(H), K5-N,OS(L), and K5-N,OS(H) on HPV-16-SEAP PsV infectivity on 293TT cells. Three different assays were performed: preattachment assay (A), attachment assay (B), and postattachment assay (C). Results are given as the mean ± standard deviation of triplicates. The corresponding experimental scheme is illustrated next to the graphs. K5s, K5 derivatives.

Monoclonal antibodies and heparin have been shown to neutralize the infectivity of cell-bound virus for many hours after the addition of virus to susceptible cells (10, 15). K5-OS(H) is also able to disrupt established PsV-heparin complexes in the Biacore assay (Fig. 2). We tested whether K5-OS(H), K5-N,OS(L), and K5-N,OS(H) could also exert a postattachment inactivation of HPV-16-SEAP PsV. To this aim, PsV were added to cells, the cells were washed after 2 h to remove unbound virus, and the compounds were then added at different time points (2, 4, 6, and 8 h) after removal of the PsV inoculum. As shown in Fig. 3C, most of the cell-bound PsV remained susceptible to inhibition by K5-OS(H) and K5-N,OS(H) at the 2-h and 4-h time points. Six hours after PsV infection, K5-OS(H) was still able to inhibit about half of the PsV in the infectious titer while K5-N,OS(H) lost its activity. By contrast, K5-N,OS(L) showed only a minimal inhibitory activity at each time point tested.

These results demonstrate that, in addition to preventing PsV from attaching to cells, K5-OS(H) and K5-N,OS(H) also exert a postattachment inhibitory effect on infectivity, at least until the particles are internalized following binding to surface HSPG.

Inhibition of HPV-16 PsV by K5-OS(H), K5-N,OS(L), and K5-N,OS(H) in different cell lines. The 293TT cells are preferred indicator cells for neutralization assays because high levels of the SV40 large T antigen in these cells allow the overreplication of the SEAP reporter plasmid. However, to verify whether the inhibitory effect of K5-OS(H), K5-N,OS(L), and K5-N,OS(H) on HPV-16 PsV infection is not restricted to 293TT cells, we tested their activity on keratinocyte cell lines derived from the uterine cervix (CaSki, SiHa, and C33A) and the oropharynx (HNO136), two anatomical sites that are a major target for high-risk HPV infection. Unlike 293TT, these cell lines do not express the SV40 large T antigen, resulting in very low levels of SEAP protein expression. Therefore, we turned to GFP as a reporter gene since it allows reliable detection by flow cytometry of its product even in cell types where the reporter plasmid does not overreplicate. The results (Table 4) demonstrate that treatment with 3 µg/ml of each compound strongly inhibited the HPV-16-GFP PsV infection in all the keratinocyte cell types and that the effect was comparable to that on 293TT cells. The results of the cell viability assay of keratinocyte cell lines treated with each compound from 1 µg/ml up to 100 µg/ml revealed no evidence of cytotoxicity even at the maximum concentration tested (data not shown).


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  		TABLE 4. Inhibition of HPV-16-GFP PsV infection of keratinocyte cell lines


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DISCUSSION
 
HPV infection requires primary interaction with cell surface HSPG (15, 20, 31). Accordingly, heparin inhibits HPV-16 infection by competing with cell surface HS for binding to the virus capsid (15, 20). K5 polysaccharide derivatives are formed by a backbone with the same structure as the biosynthetic precursor of heparin and HS. Nonetheless it is very well known that heparin and HS structures are different: heparin is more sulfated and rich in iduronic acid while HS has a more heterogeneous structure characterized by a lower degree of sulfation and lower iduronic acid content (11). Since K5 polysaccharide derivatives contain no iduronic acid residue, their structures are closer to the structure of HS. Owing to their structural similarities with HS, K5 derivatives act as efficient inhibitors of several HSPG-binding molecules, including viral proteins (29). In the present study we synthesized a number of K5 derivatives with different degrees of sulfation and tested them as potential anti-HPV agents.

By using the recently developed PsV-based neutralization assays, we found that K5-OS(H) and K5-N,OS(H) or (K5-N,OS(L) acted as efficient inhibitors of HPV infection, whereas unmodified K5, N-sulfated K5, and O-sulfated K5 with low levels of sulfation were devoid of any anti-HPV activity. These findings demonstrate that the antiviral activity depends on the degree of sulfation and on the sulfation position and not on the lower molecular weight of the K5 derivatives with respect to the parental K5 polysaccharide.

The anti-HPV potency of K5-N,OS(L) is 10-fold higher than that of commercial heparin, despite the similarity of the sulfate/carboxyl ratio of the two glycosaminoglycans. Taken together, these observations suggest that the binding to the virus critically depends on the presence of glucuronic acid instead of iduronic acid in the polysaccharide backbone. These data are consistent with those obtained by the SPR technology, highlighting this assay as a fast and reliable primary screening system to predict the HPV antagonist potential of candidate drugs for preventing HPV infection.

The results of the Biacore assay and the preattachment and attachment assays indicate that K5 derivatives prevent viral infection by binding the virus and sequestering it in the extracellular environment rather than through a direct effect exerted on the cells. However, K5 derivatives retain their HPV antagonist activity even when added to cell cultures after PsV exposure, thus exerting a postattachment inactivation similar to that previously observed with heparin and neutralizing monoclonal antibodies (10, 15). This is an important property for developing a topical microbicide that can prevent HPV infection during or immediately after sexual intercourse. Relevant to this point, K5-OS(H) turned out to be the derivative that better retains its anti-HPV capacity over time in the postattachment assay although its activity in the attachment assay was comparable to that of other bioactive K5 derivatives. A tentative hypothesis is that K5-OS(H), because of its higher affinity for HPV-16 PsV, efficiently competes with HSPG and even disrupts already established complexes. Accordingly, in the Biacore assay, K5-OS(H) was the most potent inhibitor of an HPV-16 PsV-heparin interaction and was able to detach HPV-16 PsV from the Biacore heparin surface. HPV receptors other than HSPG (i.e., the extracellular matrix protein laminin 5 and {alpha}6 integrins) have been proposed to mediate secondary binding events (11). So it is possible that K5 derivatives that retain their HPV antagonist activity over time may target the interaction of the virus with these secondary receptors.

Very recently Knappe et al. (21) studied the structural requirements of the HPV-16 major capsid protein L1 and heparin sequence and discovered that their interaction depends on sulfated groups present in defined positions of the polysaccharide backbone. Accordingly, we found that when the K5 derivative is highly O sulfated, the N sulfate group is not essential, but it does become necessary when the number of O sulfate groups is less, as in the case of K5-N,OS(L), demonstrating that a certain number of sulfate groups per disaccharide must be present in the molecule. To have a maximum of activity, however, almost all the hydroxyl groups must be sulfated. The involvement of the negatively charged sulfated groups of K5 derivatives in their binding to HPV raises the question of the functional domain(s) of the viral proteins that mediate binding. Positively charged sequences present at the C terminus of the L1 HPV-16 capsid protein were reported to bind HSPG receptors and to mediate gene transfer into target cells (3), whereas the corresponding synthetic peptides abrogate infectivity (4). Moreover, it was recently demonstrated that lysine residues 278, 356, or 361 of the HPV-16 major capsid protein L1 are critical for cell binding and infectivity of PsV (21), further extending the notion that linear or conformational "basic domains" present within a wide array of heparin-binding proteins are involved in binding to HSPG (27) and can be considered as targets for the development of efficient polyanionic inhibitors. The basic amino acids critical for cell binding are conserved in most genital HPV types (3, 4, 20, 21). This suggests that K5 derivatives may inhibit a wide range of genital HPV types by interacting with the well-conserved basic amino acid stretches within the capsid protein. The finding that K5 derivatives are active even against BPV-1, which is phylogenetically distant from the genital HPV (12), further supports the idea of K5 derivatives as broad-spectrum inhibitors of papillomavirus infection.

Several competitive advantages recommend K5 derivatives as ingredients of a topical microbicide: their activity is not papillomavirus type restricted since they potently inhibited the infection by three common sexually transmitted HPV types causing cervical cancer and genital warts (HPV-16, HPV-18, and HPV-6). Since the novel prophylactic vaccines are HPV type restricted, a microbicide against all genital HPV types could be a useful adjuvant to vaccination programs, especially in underdeveloped countries.

Compared with other inhibitors of HPV infection (5, 9), K5 derivatives can be tailored in structure and molecular weight to closely mimic cell surface HSPG to reach high and specific affinities for the HPV capsid. Unlike heparin, they are devoid of anticoagulant activity (29), and since their structure is very similar to natural HS, they are metabolically recognized and easily catabolized; therefore, no toxicity is expected. Moreover, recent results from whole-blood assays to detect proinflammatory activity showed that neither cytokines nor chemokines were mobilized in the presence of K5 derivatives (E. Vincenzi, personal communication).

K5-OS(H) and K5-N,OS(H) have also shown good anti-HIV-1 activity in vitro on HIV-1 strains that use CCR5, CXCR4, or both coreceptors in different CD4 target cells, including cell lines, primary activated T lymphocytes, and monocyte-derived macrophages (32). K5-N,OS(H) was also tested in an ex vivo model of mucosal HIV infection using ectocervical tissue explants, and no cytotoxicity was detected up to 4 µg/ml (E. Vincenzi, personal communication). Therefore, K5 derivatives fulfill the criteria for the development of a safe, broad-spectrum microbicide that is effective in preventing two major sexually transmitted infections that raise the risk of cancer in seropositive individuals (1), a risk that will probably become an increasingly important complication of long-term HIV infection (16).

Finally, we expect the K5 derivatives to retain antiviral activity in the acidic vaginal environment since the linkages of the most active compounds are very stable at low pH (P. Oreste, unpublished results).

Further investigation is required to determine the effectiveness of K5 derivatives as topical microbicides against HPV in vivo. In relation to this point, a recently developed PsV-based murine genital model has proven useful for testing in vivo the activity of candidate microbicides against HPV (26).


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ACKNOWLEDGMENTS
 
This work was supported by grants from MIUR (Fondi di Ateneo ex 60% and PRIN 2005) and from the Regione Piemonte (Bando CIPE 2004) to D.L. and S.L., from AIRC and ISS (AIDS project) to M.R., and from Compagnia di San Paolo, special project "Oncology" to M.G.

We are grateful to John Schiller and to Susana Pang (National Cancer Institute, Bethesda, MD) for providing plasmids, cells, and technical advice for PsV production.


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Clinical and Biological Sciences, University of Turin, San Luigi Gonzaga Hospital, Regione Gonzole 10, 10043 Orbassano, Turin, Italy. Phone: 39 011 6705484. Fax: 39 011 2365484. E-mail: david.lembo{at}unito.it Back

{triangledown} Published ahead of print on 4 February 2008. Back


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Antimicrobial Agents and Chemotherapy, April 2008, p. 1374-1381, Vol. 52, No. 4
0066-4804/08/$08.00+0     doi:10.1128/AAC.01467-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.



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