Topical application of polyethylenimine as a candidate for novel prophylactic therapeutics against genital herpes caused by herpes simplex virus
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- Hayashi, K., Onoue, H., Sasaki, K. et al. Arch Virol (2014) 159: 425. doi:10.1007/s00705-013-1829-x
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Herpes simplex virus types 1 (HSV-1) and 2 (HSV-2) cause genital herpes, which can enhance the acquisition of human immunodeficiency virus. The development of anti-HSV agents with novel mechanisms of action is urgently required in the topical therapy of genital herpes. In this study, the in vitro and in vivo anti-HSV effects of Epomin SP-012®, a highly cationic polyethylenimine, were evaluated. When the in vitro antiviral effects of SP-012 were assessed, this compound showed potent activity against HSV-1 and HSV-2. It inhibited the attachment of HSV-2 to host cells and cell-to-cell spread of infection in a concentration-dependent manner and exerted a virucidal effect. No SP-012-resistant HSV-2 was found when the virus was successively passaged in the presence of SP-012. In a mouse genital herpes model, topically administered SP-012 inhibited the progression of the disease caused by HSV infection. These data illustrate that SP-012 may be a novel class of HSV inhibitor that would be acceptable for long-term topical application.
Herpes simplex virus type 2 (HSV-2) causes genital herpes, a common sexually transmitted lifelong infection in populations worldwide . After primary infection with HSV-2, sensory nerve endings become infected, and virus is transported via the axon to the sacral ganglia, where it establishes a latent infection . HSV-2 infection can recur, and the virus can be shed clinically or subclinically . Recently, HSV-2 infection has been shown to enhance human immunodeficiency virus (HIV) acquisition and transmission [1, 4, 5]. Genital herpes is mostly caused by HSV-2, but HSV-1 can also cause genital infections. Recent epidemiological studies have shown that the proportion of genital herpes due to HSV-1 has been increasing over time, particularly among women and younger age groups [6, 7]. Therefore, the development of simple and cost-effective therapeutic agents for preventing genital herpes has become very important in recent years.
Materials and methods
Epomin SP-012® (mean molecular weight, 3,610; determined by gel permeation chromatography) was obtained from Nippon Shokubai Co. Ltd. (Tokyo, Japan) . Acyclovir (ACV) was purchased from Sigma Aldrich Corp. (St. Louis, MO, USA).
Cell and viruses
African green monkey kidney (Vero) cells were grown in Eagle’s minimum essential medium (MEM) (Nissui Pharmaceutical Co. Ltd., Tokyo, Japan) supplemented with 5 % fetal bovine serum (FBS), 2 mM glutamine and 100 U/ml penicillin. HSV-1 (KOS strain) and HSV-2 (UW 268 strain), donated by the Toyama Institute of Health (Toyama, Japan), were propagated and titrated by a plaque assay on Vero cells as described previously .
For studies of inhibition of cell growth, Vero cells were seeded at a density of 1 × 103 cells/well in 96-well microplates and incubated for 3 days in medium containing different concentrations of SP-012. The cytotoxicity of SP-012 was determined by MTT test using Cell Count Reagent SF (NACALAI TESQUE, Inc., Kyoto Japan) according to the manufacturer’s instructions. The 50 % cytotoxic concentration (CC50) was determined using concentration-response curves.
In vitro antiviral assays
In anti-HSV assays, the plaque yield reduction assay was employed. Vero cell monolayers were infected for 1 h with virus at 0.1 plaque-forming unit (PFU) per cell and incubated at 37 °C. At 24 h postinfection (p.i.), the cells and media were harvested and then plaque-assayed on Vero cell monolayers. SP-012 was added to the medium at the same time as virus infection or immediately after infection, and the cultures were maintained in the medium containing the corresponding compound. The 50 % inhibitory concentration (IC50) was determined using concentration-response curves. Antiviral activities were estimated as selectivity indices (SIs) calculated from CC50 and IC50 values .
Vero cell monolayers were infected with HSV-2 at 10 PFU/cell for 1 h at room temperature. SP-012 was added to the cells at the concentrations of 1, 2.5, 5 and 10 μg/ml 3 h or 6 h before infection (pretreatment), at 0 h p.i. or at 3 h p.i. After a 20-h incubation at 37 °C, the cell cultures were subjected to a plaque assay. The number of plaques produced by no-drug control wells was set to 100 %.
Virus adsorption assay
The effect of the compound on HSV-2 adsorption to host cells was evaluated using an infectious center assay . Briefly, Vero cell monolayers, HSV-2 (1 PFU/cell), and the compound were pre-cooled on ice for 3 h before mixing at 4 °C. After 1 h of incubation at 4 °C in the presence of different concentrations of SP-012, the cell monolayers were washed three times with ice-cold MEM to remove unattached virus and free compound, overlaid with 0.8 % methylcellulose, and incubated for 2 days at 37 °C for plaques to develop. The number of plaques produced by no-drug control wells was set to 100 %. In order to confirm that incubation at 4 °C allowed viral attachment and not viral entry, Vero cells to which virus had been pre-attached at 4 °C for 1 h were treated with 40 mM citrate buffer (pH 3.0) for 1 min to inactivate virus that had attached to but not yet entered the cells before adding an overlay of 0.8 % methylcellulose. As a result, 100 % inhibition of plaque formation was observed, indicating that no virus had entered the cells during the viral attachment period.
Virus entry assay
Entry of HSV-2 into host cells was evaluated according to the method reported by Huang and Wagner  with some modifications. Vero cell monolayers pre-cooled for 3 h on ice were infected with pre-chilled HSV-2 (100 PFU) for 1 h at 4 °C in the absence of SP-012 to allow viral attachment. After washing three times with ice-cold MEM to remove unattached virus, the cell monolayers were treated with different concentrations of SP-012 at 37 °C. At 1, 3 and 6 h after a temperature shift to 37 °C, the cell monolayers were treated with the citrate buffer for 1 min at room temperature to inactivate extracellular virus, washed once with MEM to return the pH to neutral, and overlaid with 0.8 % methylcellulose for the plaque assay. As a positive control, the same amount of virus was allowed to attach and enter as described in the absence of SP-012, and the number of plaques produced was set to 100 %.
To determine the effect of SP-012 on direct inactivation of virus particles, HSV-2 (2 × 104 PFU/100 μl) was treated with an equal volume of the compound (final concentration, 1, 2.5, 5 and 10 μg/ml) at 37 °C. After incubation for 1, 3 and 6 h, the mixtures were diluted 100 times with medium to reduce the concentration of SP-012 to a level that was not active in an antiviral assay (0.1 μg/ml or less), and the diluents were then titrated on Vero cell monolayers. The number of plaques produced at 0 h (the time of mixing) was set to 100 %.
Plaque size-reduction assay
The effect of SP-012 on HSV-2 cell-to-cell spread was investigated. SP-012 was added to Vero cell monolayers in 0.8 % methylcellulose-containing medium immediately after a 1-h period of infection of cells with 200 PFU of HSV-2 in the absence of the compound. After 2 days of incubation at 37 °C in the presence of SP-012 (1, 2.5, 5 and 10 μg/ml), the plaques were detected by staining the cells with crystal violet. The size of plaques was determined by measuring the diameter of twenty plaques in each treatment.
SP-012 was conjugated with Alexa Fluor 488 NHS (Invitrogen, Eugene, Ore, USA) at a molar ratio of 1:1 by mixing the reagents for 1 h at room temperature according to the manufacturer’s instructions. Vero cells grown in glass base dishes (IWAKI, Asahi Glass Co., Ltd., Chiba, Japan) were treated with 10 μg/ml Alexa Fluor 488 NHS -conjugated SP-012 (Fl-SP-012) alone or in the presence of a tenfold excess of unlabeled SP-012 for 5 min, rinsed three times with PBS, and further incubated at 37 °C. The cells were fixed with 4 % paraformaldehyde at 5 min, 3 h, 6 h and 20 h after treatment with Fl-SP-012. Fl-SP-012 in the fixed cells was detected by fluorescence microscopy after counterstaining with 4’,6-diamidino-2-phenylindole (DAPI) (Wako Pure Chemical Industries, Ltd., Osaka, Japan). Images were recorded using a fluorescence microscope (HSBZ-9000, Keyence) and HSBZ-II analysis application (Keyence) with an exposure time of 1 s. In parallel, the anti-HSV-2 activity of Fl-SP-012 was compared with that of unlabeled SP-012 by a plaque assay.
Analysis of drug resistance
Virus was grown in the presence of 100 μg/ml SP-012 or 30 μg/ml ACV for three passages, and then in the presence of 200 μg/ml SP-012 or 60 μg/ml ACV for ten passages at 0.01–0.1 PFU/cell. SP-012- and ACV-treated virus clones were plaque-purified in Vero cell monolayers. The drug susceptibility of these clones was evaluated in the presence of increasing concentrations of SP-012 or ACV using a plaque assay on Vero cell monolayers and compared with that of challenge virus without drug treatment.
Female BALB/c mice (5–6 weeks old) were obtained from Japan SLC (Shizuoka, Japan). All experiments were conducted in accordance with the animal experimentation guidelines of the University of Toyama and approved by the Animal Care Committee at the University of Toyama. To determine how long SP-012 would have a protective effect on the mice, 0.2 mg of the compound was administered intravaginally to mice (n = 5) once in a volume of 20 μl of saline. After 1, 3, 6, 9, 15, 20 and 24 h, the vaginal cavity of the mice was washed with 100 μl of saline and the washes, diluted 10 times with medium, were added to Vero cell monolayers infected with HSV-2 (0.1 PFU/cell) from 1 h prior to infection. At 24 h p.i., the cell cultures were harvested and subjected to a plaque assay. In the control wells, the same amounts of medium were added to the wells. The number of plaques in the control wells was set to 100 %. In virus infection experiments, mice were subcutaneously injected with 3 mg of medroxyprogesterone 17-acetate (Sigma) at 6 days and 1 day before virus infection to increase their susceptibility to vaginal HSV infection  and were then inoculated vaginally with 1 × 104 PFU HSV-1 or HSV-2. SP-012 and ACV at a dose of 10 mg/kg/day in 20 μl were administered intravaginally to mice (n = 10 per group) twice a day at 9:00 and 18:00 starting from 1 h or 24 h until 7 days after virus inoculation. For the control treatments, mice inoculated with the same amounts of virus were treated with 20 μl of saline using the same schedule that was used for drug treatment. Clinical signs of infection were scored based on disease severity as follows: 1 = slight genital erythema and edema; 2 = moderate genital inflammation; 3 = severe exudative genital lesions; 4 = hind limb paralysis; and 5 = death. On day 3 p.i., vaginal washes were collected by washing the vaginal cavity with 100 μl of saline at 15 h after the last administration of compounds, when the vaginal washes themselves should have no protective effect on the mice, as confirmed by the experiments described above. The infectivity of the washes was determined by a plaque assay on Vero cell monolayers.
Detection of DNA encoding HSV-2-specific thymidine kinase in dorsal root ganglia
For extraction of viral DNA, lumbosacral dorsal root ganglia were dissected from the surviving mice of the HSV-2-infected SP-012 group on day 28 p.i. Each sample was treated with 0.2 % SDS and 0.8 mg of proteinase K at 50 °C for 4 h. DNA was extracted with phenol-chloroform-isoamyl alcohol (25:24:1) and precipitated with ethanol with Dr. GenTLE precipitation carrier (Takara Bio.Inc, Ohtsu, Japan). PCR reactions were carried out using a Go Taq Flexi DNA Polymerase Kit (Promega KK, Tokyo, Japan) with forward primer (5’-tgtaaaacgacggccagtTGGCGTKRAACTCCCGCACCT-3’) and reverse primer (5’-caggaaacagctatgaccGGCCTTCCGTTCGGGCTTCC-3’) to amplify the tk gene. The amplification conditions included denaturing at 96 °C for 2 min; 30 cycles of denaturing at 96 °C for 30 s, annealing at 60 °C for 30 s, and extension at 72 °C for 2 min, with a final extension at 72 °C for 5 min. PCR products were subjected to electrophoresis on a 2 % agarose gel and visualized by ethidium bromide staining.
The differences between groups were analyzed by one-way ANOVA, and correction for multiple comparisons was made using Dunnett’s test.
In vitro antiviral efficacy of SP-012
Anti-HSV-1 and -HSV-2 activities of SP-012
Cytotoxicity (CC50, μg/mL)
Antiviral activity (IC50, μg/mL)
Selectivity index (CC50 / IC50)
To investigate the effects of SP-012 on the early events in HSV-2 replication, the assays for attachment and entry to host cells were performed separately. Inhibition of viral adsorption to host-cell membranes by the compound showed concentration dependence at the concentrations ranging from 1 to 10 μg/ml (Fig. 3b). When the numbers of penetrated viruses were determined at 1, 3 and 6 h after a temperature shift from 4 °C to 37 °C, no marked concentration-dependent inhibitory action was observed in the presence of 1-10 μg/ml of SP-012 (Fig. 3c). These results showed that the virus-binding step might be the antiviral target of SP-012.
We investigated whether SP-012 could reduce the infectivity of HSV-2 as a result of binding of SP-012 to virus particles. In the absence of SP-012, incubation of HSV-2 at 37 °C resulted in spontaneous inactivation over time, but no marked inactivation was observed at 4 °C (Fig. 3d). SP-012 caused a time-dependent reduction of viral infectivity at both 37 °C and 4 °C.
The effect of SP-012 on the cell-to-cell spread of HSV-2 was evaluated by plaque-size reduction assay. As shown in Fig. 3e, SP-012 reduced cell-to-cell spread of the virus at a concentration equivalent to its IC50 value (2.9 μg/ml in experiment A) as judged from a significant reduction in plaque size compared to the untreated control.
Incorporation of SP-012 into cells
Effect of successive treatment of virus with SP-012 on the drug sensitivity of progeny viruses
Therapeutic efficacy of SP-012 in vivo
The duration of anti-HSV-2 activity of SP-012 in the vaginal cavity of mice was evaluated by intravaginal administration of 0.2 mg of the compound. Approximately 50 % reduction of antiviral activity in vaginal washes was achieved at 9 h, and the activity had almost disappeared at 15 h after administration of SP-012 (data not shown). On the basis of these results, the vaginal washes for virus titration were collected after 15 h of treatment with of SP-012 to avoid the effect of its remaining activity.
Inoculation with HSV-2 at the same titer of virus as was used for HSV-1 produced more severe genital herpes than that observed in HSV-1-inoculated mice. In the control groups where the treatment was started at 1 h or 24 h p.i, no mice survived by 8 days p.i. (Fig. 6e, g). Administration of SP-012 starting from 1 h p.i. suppressed intravaginal virus production (Fig. 6d) and herpetic lesions (Fig. 6e), resulting in a higher survival rate of 80 % (Fig. 6g). Late SP-012 administration starting from 24 h p.i., however, resulted in less therapeutic efficacy judging from the intravaginal virus yields, severity of herpetic lesions, and survival rates (Fig. 6d, f, g).
To study murine sensory ganglia for evidence of HSV-2 infection in the surviving mice of the SP-012 group, the HSV-specific TK gene in the DNA preparations from the dorsal root ganglia was amplified by the PCR method. No bands were detected on agarose gels, suggesting that no viruses reached the ganglia.
In this study, a branched PEI (SP-012) was shown to have a selectivity index (SI) of 119 and 107 for enveloped HSV-1 and HSV-2 replication, respectively, when added to the cells at the time of infection. Spoden et al.  reported that the SI of a linear 25-kDa PEI for enveloped human cytomegalovirus was 32.4, and that for nonenveloped human papillomavirus was 212. They also showed a similar mode of antiviral action to that of SP-012, where the compound blocked virus attachment to host cells. Since polycations including PEIs are generally accepted to bind to heparan sulfate proteoglycans (HSPGs) on the cell surface, the interaction between PEI and HSPG molecules should play a critical role for antiviral action of the PEI . This might explain the much lower SIs of SP-012 when added to the cells after virus attachment (Table 1, experiment B), and also why there was no inhibitory effect on virus entry into the cells (Fig. 3c).
Because HSV-2 infection causes symptomatic and asymptomatic genital herpes, which promotes persistent inflammation, immune cell infiltration in the genital tract may explain the enhancement of HIV acquisition and transmission [4, 5]. In fact, administration of an antiherpetic agent decreased the plasma HIV-1 load and the frequency and mean quantity of HIV-1 shed in the genital tract . However, large-scale randomized controlled trials in Africa and in Peru showed no decrease in HIV-1 acquisition and transmission when ACV, an antiherpetic drug, was given to HSV-2-seropositive individuals [22–24]. These disappointing results may be at least in part due to incomplete blocking of HSV-2 infection by ACV in the vaginal cavity because the agent does not inactivate the virus particles shed into the cavity. The idea of microbicides, agents that inactivate viruses, is highly important and has great appeal in the therapy of sexually transmitted diseases, including HSV-2 infection . SP-012 exerted virucidal activity against HSV-2, as shown in this study (Fig. 3d). At a higher temperature of 37 °C, spontaneous inactivation of SP-012-untreated viruses was observed over 6 h. The modification of viral proteins and nucleic acids might lead to the inactivation of the pathogen . HSV inactivation occurred more rapidly after treatment with SP-012 than it did without drug treatment. This characteristic of SP-012 may allow it to be used for topical application against the pathogen.
Serious problems in the treatment of HSV infections with ACV are the emergence of drug-resistant viruses, especially in immunocompromised individuals . Because of the increase in the number of people who are immunocompromised due to aggressive anticancer chemotherapy and expanded organ transplantation, prolonged antiviral therapy may be necessary for management of the infection due to the lifelong possibility of reactivation of HSV, and this could lead to the selection of drug-resistant mutants. In these situations, the frequency of ACV-resistant mutants has been reported to be in the range of 3–6 % in immunocompromised people, and as high as 14–30 % in bone marrow transplant recipients [18, 28, 29]. In this study, we showed that ACV-resistant mutants could be obtained after as few as five passages of HSV-2 in the presence of ACV in in vitro systems. Therefore, development of antiviral agents that have a different mechanism of action from ACV or its related compounds and do not allow selection of drug-resistant virus is of intense interest in the therapy of HSV infection. In the present study, unlike ACV, SP-012 produced no drug-resistant mutants, and its antiviral actions differed from that of ACV.
SP-012 was found to target events outside the host cell or on the surface of the cell membrane, that is, inactivation of virus particles as well as inhibition of virus binding to host cells and cell-to-cell spread of virus infection. Heparan sulfate proteoglycans (HSPGs) act as the interaction factor for PEI on the cell surface . Glycoprotein D (gD) of HSV-2 is required for cell-to-cell spread . Thus, the interaction between SP-012 and HSPGs and/or gD might contribute to the inhibition of cell-to-cell spread of HSV-2 by the compound as found in the present study. Also, since the HSV-2 virion surface has regions of negative charge derived from the glycoproteins on the envelope, including gD, the electrostatic interaction between cationic SP-012 molecules and the viral glycoproteins might result in disturbing the binding of virus particles to the cell membrane via these glycoproteins, that is, in inactivation of virus particles. It has been reported that, upon contact with the polycationic coating, influenza virus irreversibly adhered to the compound, followed by structural damage, inactivation, and subsequently, release of viral RNA . These events might occur in HSV particles treated with SP-012. SP-012 was incorporated rapidly into the nucleus of Vero cells (Fig. 4). This phenomenon might reflect the characteristics of strong DNA-binding ability that are common to polyethylenimine [8, 9]. Sieb et al. revealed that a branched PEI was predominately internalized by cholesterol-dependent pathways, and there was no obvious exocytosis during the 60-min chase period . In the present study, SP-012 might have been internalized by this pathway, and it remained in the cells for more than 1 h. As shown by time-of-addition experiments (Fig. 3a) and experiment B in Table 1, SP-012 exhibited no marked inhibitory effects on viral replication in HSV-2-infected cells. Therefore, topical administration of this compound should be advantageous as compared with its oral administration. In addition, the high concentration of SP-012 that is required could be readily achieved for topical application.
There are several reports that describe the in vitro antiviral activity of PEIs [11, 13, 32, 33]. To the best of our knowledge, however, there is no report testing their therapeutic effects in animals. In future clinical studies, it will be important to investigate candidate compounds for antiviral efficiency in animal models. Therefore, in the present study, we performed in vivo studies to investigate topical application of SP-012 in a mouse model of genital herpes caused by HSV-1 and HSV-2. SP-012 significantly reduced topical virus yields and increased survival rates of HSV-infected mice when drug administration was started as early as 1 h postinfection at the same dose that was used for ACV, a first-line choice for HSV infection. Much less efficiency of SP-012 was observed in the mice treated with the PEI starting 24 h postinfection. This might have been due to the drug being administered too late to interfere with the early stage of viral infection, that is, virus binding to the mucosa in the vaginal cavity.
In this study, we demonstrated that topical application of a highly cationic polyethylenimine, SP-012, provided a protective effect against genital herpes caused by HSV-1 and HSV-2 in mice. The magnitude of the responses was comparable to that of acyclovir. In addition, the agent has a versatile chemical modification process, and the safety of this product has been confirmed as an indirect food additive according to the information from the manufacturer. Liquid SP-012, however, would need to be formulated in a vehicle that is acceptable for longer vaginal retention.
We appreciate Mr. O. Takahashi for high-magnification fluorescence microphotography. This work was supported by a University of Toyama Grant-in-Aid.
Conflict of interest
T.K. is an employee of Nippon Shokubai Co., Ltd., and does not own stock or options in the company. All other authors: nothing to declare.