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Antimicrobial Agents and Chemotherapy, September 2008, p. 3078-3084, Vol. 52, No. 9
0066-4804/08/$08.00+0     doi:10.1128/AAC.00359-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Inhibition of Herpes Simplex Virus Types 1 and 2 In Vitro Infection by Sulfated Derivatives of Escherichia coli K5 Polysaccharide

Debora Pinna,¹ Pasqua Oreste,² Tiziana Coradin,¹ Anna Kajaste-Rudnitski,¹ Silvia Ghezzi,¹ Giorgio Zoppetti,² Antonella Rotola,³ Rafaela Argnani,³ Guido Poli,^4,5 Roberto Manservigi,³ and Elisa Vicenzi^1*

Viral Pathogens and Biosafety Unit,¹ AIDS Immunopathogenesis Unit, San Raffaele Scientific Institute,⁴ Glycores 2000 S.r.l,² Vita-Salute San Raffaele University, School of Medicine, Milan,⁵ Department of Experimental and Diagnostic Medicine, Section of Microbiology, University of Ferrara, Ferrara, Italy³

Received 14 March 2008/ Returned for modification 2 April 2008/ Accepted 13 June 2008

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Herpes simplex virus type 1 (HSV-1) and HSV-2 are neurotropic viruses and common human pathogens causing major public health problems such as genital herpes, a sexually transmitted disease also correlated with increased transmission and replication of human immunodeficiency virus type 1 (HIV-1). Therefore, compounds capable of blocking HIV-1, HSV-1, and HSV-2 transmission represent candidate microbicides with a potential added value over that of molecules acting selectively against either infection. We report here that sulfated derivatives of the Escherichia coli K5 polysaccharide, structurally highly similar to heparin and previously shown to inhibit HIV-1 entry and replication in vitro, also exert suppressive activities against both HSV-1 and HSV-2 infections. In particular, the N,O-sulfated [K5-N,OS(H)] and O-sulfated epimerized [Epi-K5-OS(H)] forms inhibited the infection of Vero cells by HSV-1 and -2, with 50% inhibitory concentrations (IC₅₀) between 3 ± 0.05 and 48 ± 27 nM, and were not toxic to the cells at concentrations as high as 5 µM. These compounds impaired the early steps of HSV-1 and HSV-2 virion attachment and entry into host cells and reduced the cell-to-cell spread of HSV-2. Since K5-N,OS(H) and Epi-K5-OS(H) also inhibit HIV-1 infection, they may represent valid candidates for development as topical microbicides preventing sexual transmission of HIV-1, HSV-1, and HSV-2.

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Herpes simplex viruses (HSV) cause various forms of disease: from lesions on the lips, eyes, or genitalia to encephalitis and even disseminated disease in immunocompromised individuals (38). In particular, genital herpes (generally caused by HSV type 2 [HSV-2], but also by HSV-1) is characterized by a high seroprevalence worldwide (8 to 40%), with an increasing trend in the last 20 years in nearly all countries (8, 16, 26). Among the high-risk population, HSV-2 infection, the most frequent cause of genital ulcer disease, is a major public health problem in young adults (13). In this regard, epidemiological studies have demonstrated that asymptomatic viral shedding episodes are a major cause of viral transmission (37). In addition, genital herpes has been shown to enhance the likelihood of sexual transmission of human immunodeficiency virus type 1 (HIV-1) (14). In fact, both HSV-1 and HSV-2 disrupt and/or activate epithelial cells, with production of chemokines and proinflammatory cytokines that recruit and activate HIV-1 target cells such as CD4⁺ T lymphocytes, macrophages, and dendritic cells (3). Thus, strategies aimed at preventing the transmission of HSV bear potential relevance also in terms of decreasing HIV-1 transmission (9). In this regard, several polyanions have shown potent inhibitory effects in vitro by inhibiting the attachment and entry of both HSV and HIV-1 (7, 28, 29), making them candidate microbicides to prevent mucosal (vaginal, rectal, and oral) HIV-1 transmission. After rigorous safety studies, PRO 2000, polystyrene sulfate, and polymethylenehydroquinone sulfonate are indeed in phase II/III clinical trials for efficacy as candidate microbicides for preventing HIV-1 transmission (21, 40), while other polyanions are either in discovery/early preclinical or advanced studies with similar goals (1, 20). Among others, derivatives of the Escherichia coli K5 polysaccharide have already shown potent, broad-spectrum anti-HIV activity in vitro (36).

K5 derivatives were originally synthesized in order to generate glycosaminoglycans of nonanimal origin, since the capsular K5 polysaccharide from E. coli was recognized as the best starting compound for the synthesis of heparin/heparan sulfate structures (4). The K5 structure is [4)-D-GlcA-(14)-D-GlcNAc-1(1]_n (GlcA is glucuronic acid, and GlcNAc is N-acetylglucosamine), the same as the structure of the heparin/heparan sulfate biosynthetic precursor N-acetyl heparosan (35). Previous studies have shown the possibility of generating K5 derivatives that lack the anticoagulant activity of heparin, and therefore could have a safe and broader medical use, by chemical sulfation in N and/or O positions (32). Among the sulfated K5 derivatives tested, two compounds in particular, the highly O-sulfated and N,O-sulfated compounds, exhibited inhibitory effects against the replication of both tissue culture laboratory-adapted viruses and primary HIV-1 isolates from infected individuals in cell lines, primary activated CD4⁺ T lymphocytes, and human monocyte-derived macrophages (36).

Since a number of sulfated or sulfonated polysaccharides have demonstrated anti-HSV activities (7), we hypothesized that various N-, O-, and N,O-sulfated K5 derivatives could be potential inhibitors of HSV infection. In this study, we also obtained and tested N-, O-, and N,O-sulfated K5 epimers (Epi-K5) (25), generated by extractive and/or recombinant C5 epimerase, which converts GlcA residues into iduronic acid (IdoA), in order to improve the flexibility of the K5 polysaccharide chain (5, 17). Indeed, these K5 derivatives are here shown to possess anti-HSV-1 and anti-HSV-2 activities that, combined with the activity against HIV-1, make them suitable for future development as candidate microbicides to prevent the sexual transmission of different enveloped viruses.

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K5 derivatives. Sulfated nonepimerized K5 derivatives were prepared from the K5 polysaccharide as previously described (4, 11). In order to obtain the epimerized K5 derivatives, part of the N-deacetylated, re-N-sulfated K5 polysaccharide starting material was incubated with bovine C5 epimerase immobilized onto a CNBr-activated Sepharose 4B column (GE Healthcare Italia, Milan, Italy) in the presence of 25 mM HEPES containing 50 mM CaCl₂ (pH 7.0) at 37°C for 24 h after complete N deacetylation and re-N sulfation. The molecular weights and sulfate/carboxyl ratios of the K5 derivatives are shown in Table 1.

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TABLE 1. Chemical features of K5 and Epi-K5 polysaccharide derivatives

Cell lines. African green monkey kidney epithelial Vero cells were obtained from the Istituto Zooprofilatto, Brescia, Italy, and maintained in Dulbecco's modified Eagle medium (DMEM; BioWhittaker, Verviers, Belgium) supplemented with 10% fetal bovine serum (FBS; HyClone, Perbio Science, Erembodegem [Aalst], Belgium), penicillin-streptomycin (BioWhittaker) and 2 mM L-glutamine (BioWhittaker) (complete medium). Cell cultures were maintained at 37°C under a 5% CO₂ atmosphere.

Human glioblastoma cell lines expressing human CD4 and either CCR5 (U373MG-R5) or CXCR4 (U373MG-X4) and containing the HIV-1 long terminal repeat (LTR)-LacZ cassette were obtained from Marc Alizon (Institut Cochin, Paris, France) and cultivated in DMEM supplemented with 10% FBS, 500 µg/ml Geneticin, 1 µg/ml puromycin, and 100 µg/ml hygromycin B (Sigma Chemical Co., St. Louis, MO) (22).

HSV strains. HSV-1 and HSV-2 recombinant viruses, containing E. coli LacZ under the transcriptional control of the human cytomegalovirus immediate-early promoter (HCMV-LacZ), were obtained as previously described (23). Briefly, mutant HSV-1 viruses were constructed for marker transfer by using Lipofectamine (Invitrogen Life Technologies, Carlsbad, CA); mutant and recombinant viruses were plaque-purified by limiting dilution prior to characterization. An HSV-1 recombinant virus (KCZ), in which 1,622 bp of the glycoprotein (gp) gC coding sequence was replaced with the HCMV-LacZ cassette, was selected by complement-dependent neutralization with a pool of gC-specific monoclonal antibodies (23). The viral plaques formed by the neutralization escape mutants were further screened for a "blue-plaque" phenotype in the presence of 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-Gal) substrate (Invitrogen Life Technologies). An HSV-1 ribonucleotide reductase (RR)-negative mutant (hrR3) with the HCMV-LacZ cassette inserted into the RR large-subunit (ICP6) gene of HSV-1 KOS (15), an HSV-2 gC virus containing the HCMV-LacZ cassette in place of the gC coding sequence (30), and the HSV-2 isolate strain 333 (10) were also used.

Infection with recombinant HSV. Vero cells were seeded at 1.6 x 10⁴/well in 96-well flat-bottom plastic plates (Falcon; Becton Dickinson Labware, Lincoln Park, NJ) and inoculated in triplicate with 1,600 PFU of recombinant viruses in the presence or absence of serial dilutions (ranging from 4.8 to 10,000 nM) of K5 derivatives in a total volume of 100 µl. The efficiency of infection was determined 18 h later by measurement of β-galactosidase (β-Gal) activity in cell lysates, using a colorimetric assay based on the enzymatic cleavage of chlorophenol red-D-galactopyranoside (CPRG; Sigma Chemical Co.) (12). Briefly, following elimination of the supernatant, target cells were lysed in 100 µl of lysis buffer (5 mM MgCl₂, 0.1% NP-40 in phosphate-buffered saline). After a 5-min incubation at room temperature, 100 µl of reaction buffer (6 mM CPRG in lysis buffer) was added to the cell lysates and incubated for 2 h at 37°C; the optical densities were read at 570 nm. The concentration that inhibited virus growth by 50% (IC₅₀) was determined for each virus by using GraphPad Prism, version 4.0b (GraphPad Software, San Diego, CA).

Infection by HSV-2 strain 333. Vero cells were seeded at 1 x 10⁶/well in 6-well plates in 2 ml of complete medium. After 24 h, confluent cultures were preincubated with serial dilutions (from 4.8 to 5,000 nM) of K5 derivatives in duplicate and were then infected with 200 PFU of the HSV-2 strain 333 in 1 ml of DMEM and 1% FBS. Cell cultures incubated with virus in the absence of a K5 derivative were included as a control. The viral inoculum was removed from cell cultures after 1 h, and 2 ml of DMEM supplemented with 1% FBS and 0.2% polyvalent human serum immunoglobulin G (IgG) (Endobulin; Baxter AG, Vienna, Austria) was added to the cell cultures. After 2 days of incubation, the cells were stained with 1% crystal violet (Sigma) in 70% methanol. The viral plaques were counted after examination with a stereoscopic microscope (SMZ-1500; Nikon Instruments, Florence, Italy).

HSV early step infection assay. Vero cells were seeded at 2 x 10⁴/well in 96-well flat-bottom plastic plates in 200 µl of complete medium. After 24 h, confluent Vero cells were precooled to 4°C, and each well was inoculated with 2,000 PFU of HSV-1 KCZ for 3 h. The unbound virus was removed by washing the cell cultures with cold medium, and the cell cultures were then warmed up to 37°C for 15 min in order to allow virus penetration. Then the cell monolayers were exposed to citrate buffer (pH 3.0) for 1 min to inactivate any virus that did not penetrate into the target cells. Next, the cells were washed and overlaid with medium. Serial dilutions (from 4.8 to 4,800 nM) of K5 derivatives were added in triplicate either during the 4°C incubation period or when the cells were transferred to 37°C in order to determine whether the K5 derivatives acted at either the level of binding or the level of penetration, respectively.

In order to examine whether the compounds interacted with HSV or with the epithelial cells, or both, K5 derivatives were preincubated with 10⁴ PFU of HSV-2 strain 333 for 1 h at 37°C, and the mixture was diluted 50-fold to yield 200 PFU/well and inoculated in duplicate onto a monolayer of Vero cells in 6-well plastic plates. For comparison, a viral suspension (200 PFU) was preincubated for 1 h with serial dilutions (from 4.8 to 4,800 nM) of the K5 derivatives, and the mixture was seeded undiluted onto the cells. In parallel, the compounds or an equivalent volume of medium (control) was preincubated with the cells for 1 h at 37°C and then either washed extensively or left unwashed prior to inoculation with 200 PFU of HSV-2 strain 333.

HSV postentry infection assay. Vero cells were seeded at 1 x 10⁶/well in 6-well plates in 2 ml of complete medium. After 24 h, cells were infected with 200 PFU of HSV-2 strain 333 in 1 ml of DMEM and 1% FBS. The residual viral inoculum was removed by citrate buffer washing after 1 and 4 h of incubation, and the cells were overlaid with 2 ml of DMEM supplemented with 1% FBS and with the K5 derivatives in the presence of 0.2% polyvalent human serum IgG (Endobulin; Baxter AG). The number of viral plaques was evaluated after 2 days of incubation.

Cytotoxicity assay. Vero cell viability was measured by the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; Sigma] assay. Confluent Vero cell cultures seeded in 96-well plates were incubated with different concentrations of the compounds in triplicate under the same experimental conditions described above for the antiviral assays. Then 10 µl of complete medium containing MTT (0.5 mg/ml) was added to each well. After 24 h of incubation at 37°C, the supernatant was removed and 200 µl of ethanol was added to each well to solubilize the formazan crystals. After vigorous shaking, absorbance was measured in a microplate reader at 490 nm.

HIV-1 infection of U373MG-R5 and U373MG-X4 astroglioma cell lines. The glioblastoma cell lines, expressing human CD4 together with either CCR5 (U373MG-R5) or CXCR4 (U373MG-X4), contain an HIV-1 LTR-LacZ cassette. Thus, interference with a single cycle of HIV-1 infection can be investigated within 30 h postinfection by a colorimetric assay based on the expression of β-Gal induced by the newly synthesized HIV-1 Tat (22). Cells were seeded into 96-well flat-bottom plastic plates (Falcon) at 4 x 10³ cells/well and were incubated in the presence or absence of compounds at 37°C for 30 min in a total volume of 180 µl. Aliquots of NL4-3 (CXCR4-dependent [X4]) or NL(AD8) (CCR5-dependent [R5]) viral stocks, containing 250,000 or 17,000 cpm equivalents of reverse transcriptase (RT) activity, respectively, were then added in a 20-µl volume. The efficiency of virus infection was determined 30 h later by measurement of β-Gal activity in cell lysates using a colorimetric assay based on the cleavage of CPRG (Sigma Chemical Co.) by the enzyme, as described above. The concentration that inhibited virus replication by 50% (IC₅₀) was determined for each virus by using GraphPad Prism.

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Chemical features of K5 derivatives. The backbone structures of the K5 derivatives are shown in the left panel of Fig. 1. Among the nonepimerized compounds, one N-sulfated derivative (K5-NS), two O-sulfated derivatives with low and high degrees of sulfation [K5-OS(L) and K5-OS(H), respectively], and two N,O-sulfated derivatives with low and high degrees of sulfation [K5-N,OS(L) and K5-N,OS(H), respectively] were obtained from a single batch of purified K5 polysaccharide. A new class of compounds, characterized by an epimerized backbone in which 50% of glucuronic acid residues were converted into iduronic acid through the inversion of the configuration of the carboxyl group, was synthesized and compared with the nonepimerized compound previously shown to inhibit HIV-1 entry into CD4⁺ target cells (36). The epimerized derivatives included two epimerized O-sulfated derivatives with low and high degrees of sulfation [Epi-K5-OS(L) and Epi-K5-OS(H), respectively] and two epimerized N,O-sulfated derivatives with low and high degrees of sulfation [Epi-K5-N,OS(L) and Epi-K5-N,OS(H), respectively] (Fig. 1, right).

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FIG. 1. Chemical structures of the K5 polysaccharide and its derivatives. (Left) The major disaccharide repeating units of the non-Epi and Epi K5 derivatives are composed of GlcA and GlcNAc and of IdoA and GlcNAc, respectively. (Right) The flow of chemical modifications, including both sulfation and epimerization, is indicated by the arrows. Sulfation (S) is present either on the oxygen (O), on the nitrogen (N), or on both the O and the N. The L and H in parentheses indicate low and high sulfation, respectively.

Inhibition of HSV-1 and HSV-2 infection by K5 derivatives. Infection of epithelial Vero cells by the recombinant HSV-1 gC-LacZ infectious virus KCZ was used as first line screening of the potential inhibitory effects of K5 derivatives on HSV infection. The inhibition curves of HSV-1 KCZ in the presence of 10-fold serial dilutions of the N-, O-, and N,O-sulfated K5 derivatives and their epimers are shown in Fig. 2. The low-sulfated molecules K5-OS(L) (Fig. 2A), Epi-K5 (Fig. 2B), and N-sulfated K5 (K5-NS) (Fig. 2B) did not inhibit HSV-1 KCZ infection even at the highest concentration tested (5 µM). However, both the epimerized low-O-sulfated [Epi-K5-OS(L)] (Fig. 2A) and the low-N,O-sulfated [K5-N,OS(L)] (Fig. 2B) compound impaired HSV-1 KCZ infection at the highest concentration, whereas the corresponding epimerized low-N,O-sulfated derivative [Epi-K5-N,OS(L)] showed improved anti-HSV activity (Fig. 2B).

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FIG. 2. Anti-HSV-1 effects of K5 derivatives. The dose dependency profiles were obtained by incubation of 10-fold serial dilutions of the K5 derivatives with Vero cells infected with 1,600 PFU of the recombinant HSV-1 strain KCZ. Virus infection was determined by a colorimetric assay based on the cleavage of CPRG by β-Gal 18 h postinfection. Each result is presented as the absorbance read in the presence of the compound as a percentage of the absorbance read in the presence of the medium only. The means ± standard deviations for three independent experiments performed in triplicate are shown.

The most active compounds were characterized by a high level of either O sulfation [K5-OS(H)] or N and O sulfation [K5-N,OS(H)], whereas among the epimerized compounds, the high-O-sulfated compound [Epi-K5-OS(H)] was more effective than the high-N,O-sulfated compound [Epi-K5-N,OS(H)], which showed an anti-HSV activity similar to that of K5-N,OS(H) (Fig. 2A and B, respectively).

Since K5-N,OS(H) has previously been shown to inhibit the entry and replication of HIV-1 in CD4⁺ T cells and macrophages (36), further studies were focused on this specific K5 derivative and the other most potent anti-HSV-1 compound [Epi-K5-OS(H)] that did not show cytotoxicity after 24 h of treatment at concentrations up to 5 µM (data not shown). Since the infectious recombinant HSV-1 strain KCZ lacks gC, which is known to mediate the binding and penetration of the virus (23), an HSV-1 recombinant expressing LacZ and maintaining gC but with the RR coding sequence deleted (hrR3) was used to determine the anti-HSV-1 activities of K5-N,OS(H) and Epi-K5-OS(H). In addition, an HSV-2 recombinant clone expressing LacZ (HSV-2 LacZ) was utilized to determine the capacities of the K5 derivatives to inhibit HSV-2 infection. As shown in Fig. 3, K5-N,OS(H) inhibited both HSV-1 and -2 infection, with IC₅₀s between 48 and 15 nM, and Epi-K5-OS(H) was also active against HSV-1 and -2, showing IC₅₀s of 22 and 3 nM, respectively, as summarized in Table 2. Of note is the fact that the IC₅₀s of both K5-N,OS(H) and Epi-K5-OS(H) were significantly lower than that of heparin in inhibiting HSV-1 (Table 2; P < 0.01 by one-way analysis of variance with Bonferroni's multiple-comparison test). In contrast, these differences were not present when K5-N,OS(H) was compared with heparin in the HSV-2 inhibition tests (Table 2); however, Epi-K5-OS(H) was more potent than K5-N,OS(H) at inhibiting HSV-2, showing an IC₅₀ significantly lower than that of heparin (Table 2; P < 0.05 by one-way analysis of variance with Bonferroni's multiple-comparison test). These findings were confirmed by a classic plaque formation assay on Vero cells infected with the primary HSV-2 isolate strain 333 (data not shown).

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FIG. 3. K5 derivatives inhibit both HSV-1 and HSV-2 infection. The dose dependency profiles were obtained by incubation of 10-fold serial dilutions of K5-N,OS(H) with Vero cells infected with 1,600 PFU of recombinant HSV-1 KCZ, HSV-1 hrR3, or HSV-2 LacZ. Each result is presented as the absorbance read in the presence of the compound as a percentage of the absorbance read in the presence of medium only. Data are means ± standard deviations for three independent experiments performed in triplicate.

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TABLE 2. Determination of the IC50s of the most effective K5 derivatives, K5-N,OS(H) and Epi-K5-N,OS(H), compared with that of heparin

K5-N,OS(H) inhibits the early steps of HSV infection. In order to investigate whether K5 derivatives interfered with HSV attachment/binding to specific receptors on target cells, Vero cells were precooled and incubated with the virus at 4°C. Under these conditions, the virus can still attach to target cells and bind to specific receptors, but the temperature-dependent step of viral entry is inhibited (6, 7, 28). K5-N,OS(H) was added to the cells immediately prior to infection (binding). After 3 h at 4°C, cells were first washed three times with complete medium and then transferred to 37°C to allow viral entry. As shown in Fig. 4, K5-N,OS(H) prevented the binding of both HSV-1 and HSV-2 to the Vero cells in a concentration-dependent manner; in agreement with the IC₅₀s shown in Table 2, HSV-2 binding was inhibited by 48 nM, while 10-fold-higher concentrations of the compound were required to inhibit the attachment of HSV-1. In order to evaluate the interference of K5-N,OS(H) with HSV entry, Vero cells were infected at 4°C for 3 h, and then the compound was added at room temperature immediately prior to incubation at 37°C. The penetration of both HSV-1 and HSV-2 was prevented by K5-N,OS(H) with similar concentration dependencies (Fig. 4). In addition, this compound showed the capacity of detaching virus already bound to the cell surface after 3 h of incubation at 4°C more efficiently than heparin (Fig. 5).

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FIG. 4. Mechanism of anti-HSV action of sulfated K5 polysaccharides. The concentration dependency profiles of K5-N,OS(H) added to target cells maintained at 4°C (binding) (hatched bars) versus 37°C (penetration) (filled bars) were compared. Each result is presented as the absorbance read in the presence of the compound as a percentage of the absorbance read in the presence of medium only. Data are means ± standard deviations for three independent experiments performed in triplicate. K5-N,OS(H) inhibited the binding and penetration of both HSV-1 and HSV-2 in a concentration-dependent manner.

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FIG. 5. The K5 derivative K5-N,OS(H) removes virus bound to the cell surface. Confluent Vero cells were exposed to 2,000 PFU of HSV-1 KCZ for 3 h at 4°C. Cells were then washed three times in the presence of serial dilutions of the K5 derivative (from 0.24 to 24 µM) at 4°C and were transferred to 37°C. Each result is presented as the absorbance read in the presence of the compound as a percentage of the absorbance read in the presence of medium only. Data are means ± standard deviations for two independent experiments performed in triplicate. Heparin was tested in parallel as a control.

Next, serial concentrations of K5-N,OS(H) (from 4.8 to 4,800 nM) were preincubated for 1 h with a 50-fold excess of HSV-2 strain 333. When HSV-2 virions were preincubated with the K5 derivative, anti-HSV activity was enhanced over that observed when cells were pretreated for 1 h prior to addition of the virus. The anti-HSV effects persisted when the mixture of the 50-fold virus inoculum and the K5 derivative was diluted prior to addition to target cells. In addition, the antiviral activity was reduced but not abolished when the cells were exposed to the compound for 1 h and then washed prior to infection (Fig. 6), suggesting that the main mechanism of this K5 derivative is interference with virion binding to the target cells, as also suggested by the experiments for which results are shown in Fig. 4.

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FIG. 6. Viral infection following preincubation of either target cells or virus with K5-N,OS(H). Different concentrations of K5-N,OS(H) (or complete medium, used as a control) were preincubated with Vero cells for 1 h at 37°C and then either washed or not washed prior to infection with 200 PFU of HSV-2 strain 333. In parallel, K5-N,OS(H) was preincubated with a 50-fold excess of HSV-2 strain 333 for 1 h and then diluted 50-fold before being added to the target cells (1:50 dilution) in order to expose the cells to 200 PFU. As a control, K5-N,OS(H) was preincubated with HSV-2 strain 333 and added undiluted to the target cells (no dilution). Data are mean numbers of PFU/well ± standard deviations in the absence (Nil) or presence of the compound in two independent experiments performed in duplicate.

K5 derivatives inhibit cell-to-cell spread of HSV. To test whether the K5 derivatives had the potential to interfere with HSV replication and cell-to-cell virus spreading, confluent Vero cells were infected with 200 PFU of HSV-2 strain 333 in 1 ml of DMEM plus 1% FBS and 0.2% polyvalent human serum IgG. The viral inoculum was removed by two washes after 1 h postinfection, and the cells were overlaid with 2 ml of DMEM supplemented with 1% FBS and the K5 derivatives; after 2 days of incubation, the viral plaques were counted. As shown in Fig. 7A, both K5-N,OS(H) and Epi-K5-OS(H) significantly reduced the number of HSV plaques, three- and sevenfold (P < 0.001 by one-way analysis of variance with Bonferroni's multiple-comparison test), when added 1 h postinfection at concentrations of 480 and 500 nM, respectively. This effect persisted when the compounds were added 4 h postinfection (data not shown). In addition to the significant reduction in the number of viral plaques, a significant average reduction in the plaque size was observed in the presence of either K5-N,OS(H) or Epi-K5-OS(H) (P < 0.001 by one-way analysis of variance with Bonferroni's multiple-comparison test), as shown in Fig. 7B. In contrast, postinfection treatment with heparin at 730 nM did not diminish the number and size of the plaques.

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FIG. 7. K5 derivatives inhibit HSV at a postentry level. Monolayers of Vero cells were infected with 200 PFU of HSV-2 strain 333. After 1 h of incubation, the virus was removed by citrate buffer washing, and the cells were overlaid with 2 ml of DMEM supplemented with 1% FBS plus 480, 500, or 730 nM K5-N,OS(H), Epi-K5-OS(H), or heparin, respectively, and 0.2% polyvalent human serum IgG. (A) After 2 days of incubation, the number of viral plaques was examined by optical microscopy. Data are mean numbers of PFU/well ± standard deviations in the absence (Nil) or presence of the compounds in three independent experiments performed in duplicate. (B) Twenty representative plaques were photographed with a digital camera (Leica DFC280), and the plaque sizes were calculated using the ImageJ processing program (http://rsb.info.nih.gov/ij/). The P values were calculated by using a one-way analysis of variance followed by Bonferroni's multiple-comparison test.

Epi-K5-OS(H) inhibits HIV-1 infection. K5-N,OS(H) has previously been reported to inhibit the infection of primary CD4⁺ T cells and macrophages by both R5 and X4 HIV-1 (36). In order to investigate whether Epi-K5-OS(H) was also endowed with anti-HIV activity, U373MG-R5 and U373MG-X4 cells stably transfected with the LacZ gene under the control of the HIV-1 clade B LTR (22) were infected in the presence of serial dilutions of Epi-K5-OS(H), and β-Gal activity was determined 30 h after infection. Indeed, Epi-K5-OS(H) inhibited both R5 and X4 HIV-1 replication, with IC₅₀s of 32 ± 20 and 26 ± 30 nM, respectively. When the anti-HSV and anti-HIV activities were compared, both K5-N,OS(H) and Epi-K5-OS(H) were active in the same concentration range (Fig. 8). Epi-K5-OS(H) showed a relatively higher potency than K5-N,OS(H), a difference that reached statistical significance in the case of HSV (P = 0.04 by a one-tailed unpaired t test).

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FIG. 8. Anti-HSV and anti-HIV activities of K5 derivatives. The anti-HSV activities were determined in both HSV-1- and HSV-2-infected cells in the presence of log10 serial dilutions of K5-N,OS(H) or Epi-K5-OS(H). The anti-HIV-1 activities were determined in the U373MG-R5 and U373MG-X4 cell lines exposed to NL4-3 (CXCR4-dependent) or NL(AD8) (CCR5-dependent) virus. Virus expression was determined by β-Gal activity in cell lysates, by using a colorimetric assay based on the cleavage of CPRG. Data are the mean IC50 ± standard deviation of each compound for each virus as determined with GraphPad Prism software. Epi-K5-OS(H) was significantly more potent than K5-N,OS(H) for HSV inhibition (P = 0.04 by a one-tailed unpaired t test).

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
In the present study we have investigated the potential anti-HSV-1 and -2 activities of E. coli K5 derivatives previously characterized as potent, broad-spectrum anti-HIV-1 compounds (36). Indeed, these derivatives, as well as newly generated epimers (also showing anti-HIV-1 activity), inhibited both HSV-1 and HSV-2 infection by free virions as well as cell-to-cell virus spread in Vero cells.

It is well established that numerous polyanionic compounds are endowed with the capacity to inhibit HSV infection and replication in vitro, while the high affinity and stability of the sulfated-compound-virus complex are the bases of their broad antiviral activities (7, 19). In regard to HSV-1 and -2, negatively charged polyanions are known to bind to HSV envelope (Env) gp, thereby impeding virion binding and penetration into target cells (39). In particular, comparative studies of sulfated and sulfonated compounds such as PRO 2000, polystyrene sulfonate, polymethylenehydroquinone sulfonate, and cellulose sulfate have demonstrated their activities against HSV-2 (7). Their common mechanism of action appears to be the formation of complexes with the gp gB, thus inhibiting virion binding and entry as well as cell-to-cell spread of the infection (7). These properties, together with their demonstrated anti-HIV activities, make these compounds suitable for development as microbicides for preventing sexual transmission of both HIV and HSV (20).

The backbone of the compounds tested in the present study is the capsular K5 polysaccharide of E. coli, which has the same structure as the biosynthetic precursor of heparin/heparan sulfate, N-acetyl heparosan, on which sulfate groups are inserted in different positions and degrees (25). In the compounds tested in the present study, C5 epimerization was introduced to improve the flexibility of the K5 polysaccharide chain (5, 17). In agreement with findings for previously characterized sulfated polysaccharides (27), a gradient of electrostatic interactions (which are functions of relative charge densities) correlated with the anti-HSV activities of the compounds. However, comparison of the IC₅₀s of the epimerized compound Epi-K5-OS(H) and the nonepimerized compound K5-N,OS(H) to that of heparin indicated that both types of K5 derivatives were more efficient than heparin at inhibiting HSV-1, whereas no differences were observed between heparin and the K5 derivative K5-N,OS(H) with regard to inhibition of HSV-2 (Table 2) These findings support the notion that homologous gC and gB (known to bind to heparin) do not play identical roles in HSV-1 and HSV-2 infection, at least in Vero cells (2, 6, 34). Alternatively, it is possible that K5-N,OS(H) and Epi-K5-OS(H) bind more efficiently to gB than to gC. In this regard, the efficiency of inhibition of strain KCZ, lacking gC, was equal to the efficiency of inhibition of the hrR3 virus, which maintains gC. Epimerization and a high degree of O sulfation improved anti-HSV-2 activity in that Epi-K5-OS(H) was more active than heparin and K5-N,OS(H) by approximately 1 log₁₀ unit. This observation suggests that the conversion of GlcA into IdoA may improve the interactions with HSV-2 gp.

According to nuclear magnetic resonance studies, the GlcA-GlcNAc repeating units from E. coli K5 polysaccharide confer rigidity on the molecule, whereas the iduronate ring flexibility has been postulated to enhance the specificity of heparan sulfate glycosaminoglycan (HSGAG) oligosaccharides binding to both cell and viral proteins (18). In this regard, one of the best model systems for protein-HSGAG interactions is the fibroblast growth factor family of molecules. Although the sulfate and carboxylate groups of the HSGAG chain interact with the basic residues on fibroblast growth factor, local deviations in the helical axis of the oligosaccharide chain provide optimal contact with the protein (31). The improvement of Epi-K5-OS(H) in anti-HSV-2 activity compared with K5-N,OS(H) (Table 2) suggests that, due to their flexibility, the iduronate-containing chains are able to adapt their shape to the binding site of the HSV-2 gp more efficiently than the glucuronate-containing chains. By comparison of their antiviral activities, Epi-K5-OS(H) appeared more effective than K5-N,OS(H), particularly against HSV (Fig. 8).

With regard to the mechanism of antiviral action, heparin inhibited the entry of both HSV strains but was not effective at the postentry level, whereas K5-N,OS(H) blocked HSV infection both at the entry and postentry levels and limited the cell-to-cell spread of HSV-2. In addition, K5-N,OS(H) was as efficient as Epi-K5-OS(H) in reducing the cell-to-cell spread of HSV-2 (and of HSV-1 [data not shown]). The molecular basis of the inhibition by K5 derivatives of postentry HSV infection and of the cell-to-cell spread of the virus is currently unknown. The polymeric structure of K5 derivatives likely impedes their transport across the plasma membrane into the cell. In addition, it is unlikely that the K5 derivatives affect viral gene expression in cells upon initial infection. In support of this hypothesis, the K5 derivatives failed to suppress the expression of HIV-1 in the chronically infected promonocytic U1 cell line stimulated with phorbol esters (E. Vicenzi, unpublished data). However, K5 derivatives may bind to the Env gp of budding virions and thus inhibit cell-to-cell spread, a mechanism of virus propagation that is usually more efficient than cell-free virion-dependent infection (33). Worthy of note is the recent finding on the capacity of highly sulfated K5 derivatives to block human papillomavirus infection at both the entry and postentry levels (24).

In conclusion, the present study confirms our previous findings on the capacity of K5 derivatives to inhibit HIV-1 infection and spreading (36), and it extends the potential preventive effects of these compounds to HSV-1 and HSV-2 infections of epithelial cells. Therefore, these compounds could be developed as potential microbicides with the goal of preventing the sexual transmission not only of HIV-1 but also of HSV-1 and -2, and potentially of human papillomavirus.

 
This work was supported by the 6th Framework Programme of the European Commission Integrated Project European Microbicides Project (EMPRO) and Europrise (Network of Excellence), and by the Fondation Dormeur.

 
* Corresponding author. Mailing address: P2-P3 Laboratories DIBIT, Via Olgettina 58, 20132, Milan, Italy. Phone: 39-02-2643-4908. E-mail: vicenzi.elisa{at}hsr.it

Published ahead of print on 23 June 2008.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 

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Antimicrobial Agents and Chemotherapy, September 2008, p. 3078-3084, Vol. 52, No. 9
0066-4804/08/$08.00+0     doi:10.1128/AAC.00359-08
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