Antiviral Effects of ABMA Against Herpes Simplex Virus Type 2 in Vitro and in Vivo

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Antiviral Effects of ABMA against Herpes Simplex Virus Type 2 In Vitro and In Vivo Wenwen Dai, Yu Wu, Jinpeng Bi, Shuai Wang, Fang Li, Wei Kong, Julien Barbier, Jean-Christophe Cintrat, Feng Gao, Daniel Gillet, et al.

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Wenwen Dai, Yu Wu, Jinpeng Bi, Shuai Wang, Fang Li, et al.. Antiviral Effects of ABMA against Herpes Simplex Virus Type 2 In Vitro and In Vivo. Viruses, MDPI, 2018, 10 (3), pp.119. 10.3390/v10030119. cea-02432376

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Article Antiviral Effects of ABMA against Herpes Simplex Virus Type 2 In Vitro and In Vivo

Wenwen Dai 1, Yu Wu 2, Jinpeng Bi 1, Shuai Wang 1, Fang Li 1, Wei Kong 1,3, Julien Barbier 2, Jean-Christophe Cintrat 2, Feng Gao 1,3, Daniel Gillet 2,*, Weiheng Su 1,3,* and Chunlai Jiang 1,3,*

1 National Engineering Laboratory for AIDS Vaccine, School of Life Sciences, Jilin University, Changchun 130012, Jilin, China; [email protected] (W.D.); [email protected] (Ji.B.); [email protected] (S.W.); [email protected] (F.L.); [email protected] (W.K.); [email protected] (F.G.) 2 SIMOPRO, CEA, Université Paris-Saclay, F-91191 Gif Sur Yvette, France; [email protected] (Y.W.); [email protected] (Ju.B.); [email protected] (J.-C.C.) 3 Key Laboratory for Molecular Enzymology and Engineering of the Ministry of Education, School of Life Sciences, Jilin University, Changchun 130012, Jilin, China * Correspondence: [email protected] (D.G.); [email protected] (W.S.); [email protected] (C.J.); Tel.: +33-(0)1-69-08-76-46 (D.G.); +86-0431-8516-7751 (W.S. & C.J.)

Received: 27 January 2018; Accepted: 8 March 2018; Published: 9 March 2018

Abstract: Herpes simplex virus type 2 (HSV-2) is the causative pathogen of genital herpes and is closely associated with the occurrence of cervical cancer and human immunodeficiency virus (HIV) infection. The absence of an effective vaccine and the emergence of drug resistance to commonly used nucleoside analogs emphasize the urgent need for alternative antivirals against HSV-2. Recently, ABMA [1-adamantyl (5-bromo-2-methoxybenzyl) amine] has been demonstrated to be an inhibitor of several pathogens exploiting host-vesicle transport, which also participates in the HSV-2 lifecycle. Here, we showed that ABMA inhibited HSV-2-induced cytopathic effects and plaque formation with 50% effective concentrations of 1.66 and 1.08 µM, respectively. We also preliminarily demonstrated in a time of compound addition assay that ABMA exerted a dual antiviral mechanism by impairing virus entry, as well as the late stages of the HSV-2 lifecycle. Furthermore, in vivo studies showed that ABMA protected BALB/c mice from intravaginal HSV-2 challenge with an improved survival rate of 50% at 5 mg/kg (8.33% for the untreated virus infected control). Consequently, our study has identified ABMA as an effective inhibitor of HSV-2, both in vitro and in vivo, for the first time and presents an alternative to nucleoside analogs for HSV-2 infection treatment.

Keywords: herpes simplex virus type 2; ABMA; antiviral effect; entry; late stage; vesicle transport

1. Introduction Genital herpes is one of the world’s most prevalent sexually transmitted diseases [1], and manifests as ulcerative and vesicular lesions on the genitals with lifelong latency [2]. Herpes simplex virus type 2 (HSV-2)—a single, large double-stranded DNA, enveloped virus belonging to the Herpesviridae family—is the major cause of genital herpes [3], and significantly increases the risk of developing cervical cancer and human immunodeficiency virus (HIV) infection [4–6]. HSV-2 infection is a global concern with estimates of 536 million people infected worldwide and an annual incidence of 23.6 million cases [7]. Despite the prevalence of infection in the global population, no vaccine has been developed and antiviral chemotherapy is standard practice in the management of HSV-2 infection [8,9]. However, long-term therapy with acyclovir and penciclovir as well as their prodrugs valaciclovir and famciclovir, respectively, has led to the emergence of drug resistance, especially in immune-compromised patients [10]. Additionally, various cases of toxicity have been encountered as a result of increasing

Viruses 2018, 10, 119; doi:10.3390/v10030119 www.mdpi.com/journal/viruses Viruses 2018, 10, 119 2 of 15 use of traditional antivirals [11,12]. Although some non-nucleoside inhibitors have been developed, few are currently approved for HSV-2 infection treatment [13]. Foscarnet is approved as a second-line drug for HSV-2 infection treatment only when the patient has failed first-line treatment with acyclovir or there is a proven resistance mutation, and the use of foscarnet is limited by its toxicity and the fact that it is available only as an intravenous formulation [14]. Therefore, alternative antivirals against HSV-2 are needed. The small molecule ABMA [1-adamantyl (5-bromo-2-methoxybenzyl) amine], was first identified from a cell-based high throughput screening, as an inhibitor of ricin, both in cell cultures and in mice, selectively acting on host-endosomal trafficking [15]. Subsequently, ABMA has been reported to be active against other infectious pathogens, including bacterial toxins (diphtheria toxin from Corynebacterium diphtheriae, lethal toxin from Bacillus anthracis, toxin B from Clostridium difficile and lethal toxin from Clostridium sordellii), viruses (Ebola virus, Rabies virus and Dengue-4 virus), bacteria (Simkaniaceae and Chlamydiaceae) and Leishmania parasite [15]. Each of these pathogens relies on host–endosomal trafficking for pathogenicity, indicating that the inhibitory effect of ABMA is related to host–vesicle transport [16–18]. HSV-2 initiates infection with attachment to host cells, followed by membrane fusion or endocytosis to enter the cells. Subsequently, de-enveloped tegument-capsids are transported to the nuclei, where genome transcription, DNA replication and new capsid assembly occur. Filled capsids then bud into vesicles derived from the trans-Golgi network to obtain an envelope and an outer membrane after release from the nuclei. Finally, viruses exit the host cells by fusion of enveloped virus-containing vesicles with the cell membranes. Several processes including endocytic virus entry, virus capsid envelopment and virus egress in the HSV-2 lifecycle depend on host–vesicle transport, providing a rationale for testing ABMA as an inhibitor of HSV-2. In this study, we evaluated the antiviral activity of ABMA against HSV-2, in vitro and in vivo, and provided data to address possible mechanisms of action. Chloroquine, which was reported to inhibit herpes simplex virus infection by interacting with endocytic virus entry and the late stages of infection, as well as acyclovir, which is commonly used as the drug for HSV-2 infection treatment, were chosen as positive control drugs in this study [19–24]. ABMA was demonstrated to be an effective inhibitor of HSV-2 by a dual mechanism of action, acting on virus entry as well as the late stages of infection.

2. Materials and Methods

2.1. Cells, Virus, Compounds and Mice African green monkey kidney cells (Vero cells) obtained from American type culture collection (ATCC) (cat # CCL-81) were cultured in Dulbecco’s modiﬁed Eagle’s medium (DMEM, Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS, Invitrogen) (DMEM-10% FBS) ◦ at 37 C with 5% CO2. HSV-2 strain G obtained from ATCC (cat # VR-734) (KU310668) was propagated in Vero cells. Virus titration was performed by endpoint dilution and plaque assays. ABMA [1-adamantyl (5-bromo-2-methoxybenzyl) amine] (Figure1) was synthesized in-house. The positive control drugs, chloroquine and acyclovir, were purchased from Meilun Biotech Co., Ltd. (Dalian, China) [19–24]. The purities of the compounds were higher than 98%, as determined by High Performance Liquid Chromatography (HPLC). The compounds were dissolved in dimethyl sulfoxide (DMSO) as stock solutions. Speciﬁc-pathogen-free female BALB/c mice (6–8 weeks old) were obtained from the Changchun Institute of Biological Products and maintained under the guidelines for animal experiments at Jilin University, China. Viruses 2018, 10, 119 3 of 15 Viruses 2018, 10, x 3 of 15

Figure 1. Chemical structure of ABMA.

2.2. Cytotoxicity Assay ® Cytotoxicity was measured by the CellCell Titer-GloTiter-Glo® Luminescent cell viability assay, as reportedreported previouslypreviously [[25].25]. Serially diluted compounds were added to 90% confluent conﬂuent Vero Vero cells in 96-well plates. plates. ® AfterAfter incubationincubation for for 72 72 h, cellh, cell viability viability was assayedwas assayed with thewith Cell the Titer-Glo Cell Titer-Glo® reagent (Promega,reagent (Promega, Madison, WI,Madison, USA) andWI, quantiﬁedUSA) and by quantified the PerkinElmer by the VICTORTMPerkinElmer X2 VICTORTM (Waltham, MA, X2 USA).(Waltham, Cytotoxicity MA, USA). was measuredCytotoxicity by was the percentagemeasured ofby thethe luminescence percentage of intensity the luminescence of compound-treated intensity ofcells compound-treated relative to that cells relative to that of the untreated cell control. The 50% cytotoxicity concentration (CC50) was of the untreated cell control. The 50% cytotoxicity concentration (CC50) was calculated by regression calculated by regression analysis of the dose–response curves [26]. analysis of the dose–response curves [26].

2.3. Antiviral Activity Assay of ABMA against HSV-2 In Vitro The antiviralantiviral activity activity of ABMAof ABMA against against HSV-2 HSV- was2 measured was measured by cytopathic by cytopathic effect (CPE) effect inhibition (CPE) andinhibition plaque and reduction plaque assays.reduction In theassays. CPE In inhibition the CPE assay, inhibition serially assay, diluted serially ABMA diluted or chloroquine ABMA or (positivechloroquine control (positive drug), control was added drug), towas 90% added confluent to 90% Vero confluent cells in Vero 96-well cells platesin 96-well 5 h beforeplates 5 infection h before withinfection HSV-2 with (MOI HSV-2 = 0.04, (MOI which = 0.04, was which determined was determined to cause appropriateto cause appropriate CPE (100%) CPE and (100%) cell viabilityand cell reductionviability reduction (60%) on (60%) Vero on cells Vero after cells infection after infectio for 72n h),for while72 h), while acyclovir acyclovir (positive (positive control control drug) drug) was addedwas added at the at same the same time time as infection. as infection. After After incubation incubation for 72for h 72 post-infection h post-infection in the in presencethe presence of the of compounds,the compounds, cell viabilitycell viability was was measured measured as described as described in Section in Section 2.2. 2.2. InIn thethe plaqueplaque reductionreduction assay, seriallyserially diluteddiluted ABMAABMA or chloroquinechloroquine (positive control drug), waswas addedadded to Vero cellcell monolayersmonolayers inin 12-well12-well plates 5 h before infection with HSV-2 (50–100 PFU, whichwhich waswas determineddetermined to ensure thatthat appropriateappropriate numbers of plaques form in the plates and are counted accurately), while acyclovir (positive control drug) was added at the same time as infection. AfterAfter infection for 1 1 h, h, DMEM-2% DMEM-2% FBS-1% FBS-1% low-melting low-melting agarose agarose containing containing the the above above compounds compounds at atcorresponding corresponding concentrations concentrations was was overlaid overlaid in in pl placeace of of infected infected medium. Plaque Plaque numbers werewere counted after the cells werewere fixedfixed withwith 4%4% paraformaldehydeparaformaldehyde andand stainedstained withwith 0.5%0.5% crystalcrystal violetviolet whenwhen plaquesplaques formed.formed. CPE inhibition and plaque reduction rates werewere measured by the following equations: T−V ( ) = (T − V) (C − V) × CPE inhibition inhibition %% = /C−V ×100% 100%,, where where C, C, V V and and TT areare thethe luminescenceluminescence intensitiesintensities ofof thethe untreateduntreated cell cell control, control, the untreatedthe untreated virus infectedvirus infected control andcontrol compound-treated and compound-treated cells, respectively. cells, (plaque number) ( ) Plaque reduction (%) = [1 − T/plaqueplaque numbernumberV]× 100%, where (plaque number)T and respectively. Plaque reduction % =1− × 100% , where (plaque number)V are plaque numbers of compound-treated cellsplaque and number the untreated virus-infected control,(plaque respectively.number)T and The (plaque 50% effectivenumber) concentrationV are plaque numbers (EC50), which of compound-treated refers to the concentration cells and the of auntreated drug that virus-infected induces a response control, halfwayrespectively. between The 50% the baselineeffective andconcentration the maximum (EC50), after which a specified refers to exposurethe concentration time, was of calculated a drug that by induces regression a respon analysisse halfway of the dose–response between the baseline curves [and26]. the maximum after a specified exposure time, was calculated by regression analysis of the dose–response curves [26].

Viruses 2018, 10, 119 4 of 15

2.4. Western Blotting Cell samples were lysed in RIPA buffer (Beyotime Biotech Co., Ltd., Shanghai, China) and the lysates were cleaned by centrifugation at 12,000 rpm. The proteins were separated by SDS-PAGE and transferred onto nitrocellulose membranes. After being blocked with 3% non-fat milk for 1 h, the membranes were incubated with an anti-HSV-2 VP5 (major capsid protein of HSV-2 [27]) mouse monoclonal antibody (EastCoast Bio, North Berwick, ME, USA) or an anti-β-tubulin mouse monoclonal antibody (Covance, Emeryville, CA, USA) for 2 h. Blots were subsequently incubated with an alkaline phosphatase (AP)-conjugated anti-mouse IgG antibody (SouthernBiotech, Birmingham, AL, USA) for 1 h and developed by the interaction between AP and AP substrates, followed by termination with the exposure to light.

2.5. Quantitative Polymerase Chain Reaction (qPCR) Assay The Ezup Column Virus DNA Purification Kit (Sangon Biotech Co., Ltd., Shanghai, China) was used to extract HSV-2 DNA. TransStart® Top Green qPCR SuperMix (TransGen Biotech Co., Ltd., Beijing, China) and gG specific primers (forward primer: 50-CCCACACCCCAACACATC-30, reverse primer: 50-CCAAGGCGACCAGACAAAC-30) were used for amplification and subsequent quantification with the Bio-Rad CFX96 system (Hercules, CA, USA).

2.6. Time of ABMA Addition Assay In the antiviral activity assay against HSV-2 based on the measurement of viral protein and DNA content in the cell cultures, 3.13 µM ABMA or 15 µM chloroquine was added to 90% confluent Vero cells in 24-well plates 5 h before infection with HSV-2 (MOI = 1, which was determined to ensure synchronized infection in a single replicative lifecycle as reported [28]), while 1 µM acyclovir was added at the same time as infection. After infection for 1 h, DMEM-2% FBS containing the above compounds at their corresponding concentrations was overlaid in place of the medium. Proteins and HSV-2 DNA were extracted and quantified as described in Sections 2.4 and 2.5 at 18 h post-infection, when a single lifecycle had been completed without the occurrence of obvious CPE [29]. In the effective stage assay, ABMA (3.13 µM) and HSV-2 (MOI = 1) were added to 90% confluent Vero cells in 24-well plates following different treatment schemes, as reported with some modifications [29]. To study a prophylactic effect (pre: −5–0 h), the cells were pretreated with ABMA for 5 h, then infected with HSV-2 after removal of ABMA by washing. To study an inhibitory effect on virus binding or entry (simultaneous: 0–1 h), the cells were treated with ABMA and infected with HSV-2 at the same time, then overlaid with DMEM-2% FBS after removal of the medium by washing at 1 h post-infection. To study the effect on virus replication (early post: 1–6 h), the infected cells were treated with ABMA from 1 h to 6 h post-infection, then overlaid with DMEM-2% FBS after removal of ABMA by washing. To study the effect on late stage infection (late post: 6–18 h), the infected cells were cultured with DMEM-2% FBS for 5 h, then treated with ABMA from 6 h to 18 h post-infection. Besides, HSV-2 was pre-incubated with ABMA at 4 ◦C for 5 h before infection (direct) to study the direct interactions in a cell free system. HSV-2 infection was performed during 0–1 h for all procedures, except for direct procedure, in which the cells were infected with HSV-2 that had been pretreated with ABMA. HSV-2 DNA in the cell cultures was extracted and quantified as described in Section 2.5 at 18 h post-infection.

2.7. Binding and Entry Assays Binding and entry assays were performed as reported previously, with some modifications [30]. The 90% confluent Vero cells in 24-well plates were pretreated with 3.13 µM ABMA, 15 µM chloroquine or 1 µM acyclovir for 5 h before addition of HSV-2. In the binding assay, the cells were exposed to HSV-2 (MOI = 1) at 4 ◦C for 2 h. Unbound viruses were removed by washing twice with sterile PBS buffer Viruses 2018, 10, 119 5 of 15 at 4 ◦C, and HSV-2 DNA from the original virus inoculum and the unbound virus supernatant were extracted separately and quantified as described in Section 2.5 to calculate the amount of bound HSV-2. In the entry assay, the cells were further incubated at 37 ◦C for 1 h after the binding process. After two freeze–thaw cycles of the infected cells, HSV-2 DNA from the internalized virus was extracted and quantified to calculate the amount of HSV-2 that was able to enter the cells.

2.8. Late Stage Infection Assay To study the effects of ABMA on the late stages of the HSV-2 lifecycle, 90% conﬂuent Vero cells in 24-well plates were infected with HSV-2 (MOI = 1) for 1 h, then treated with 3.13 µM ABMA, 15 µM chloroquine or 1 µM acyclovir during 6–18 h post-infection. At 18 h post-infection, the supernatants and the infected cells were collected and subjected to direct extracellular virus titration and to intracellular virus titration after two freeze–thaw cycles. Virus titers were determined by the Reed and Muench dilution method and expressed as 50% tissue culture infectious doses per milliliter (TCID50/mL).

2.9. Antiviral Efficacy Assay of ABMA against HSV-2 In Vivo Female BALB/c mice (6–8 weeks old, n = 10–12 per group) were injected subcutaneously with 2 mg of Depo-Provera (XianJu Pharmaceutical Co., Ltd., Taizhou, China) per mouse to induce a diestrus phase in the genital tract. Seven days later, the mice were inoculated intravaginally with 50,000 PFU of HSV-2 in 10 µL of PBS after anesthesia. At 1 h post-inoculation, and subsequently once daily for seven consecutive days, 1.25 mg/kg or 5 mg/kg of ABMA (the doses were determined to ensure sufficient dissolution of ABMA in the injections), or 150 mg/kg of acyclovir (positive control) [31] was administered intraperitoneally. The compounds were all dissolved in PBS supplemented with 10% DMSO and PBS supplemented with 10% DMSO was administered as an untreated virus infected control. The mice were monitored daily for survival rate and clinical score. Signs of disease were evaluated as: 0, healthy; 1, genital erythema; 2, moderate genital inflammation; 3, genital lesion; 4, hind-limb paralysis; 5, death [32]. Vaginal swab samples were collected at day 5 and day 10 and transferred to 200 µL of Hank’s buffer. HSV-2 titers from the swab samples were determined by plaque assay in Vero cells as reported [33]. Protocols for animal experiments were approved by the Committee on Animal Experimental Ethics of School of Life Sciences at Jilin University [permission code: 2017-nsfc019, 15 January 2017].

2.10. Statistical Analysis In vitro experiments were conducted in technical triplicate and repeated three times independently. A one-way ANOVA test was used for statistical analysis to compare the differences between test groups and untreated virus infected control groups. A log-rank test (Mantel–Cox) was used for comparisons of the survival curves. Statistical significance is represented by asterisks and was marked correspondingly in the figures (* p < 0.05, ** p < 0.01, *** p < 0.001).

3. Results

3.1. Reductions of HSV-2-Induced Cytopathic Effects and Plaque Formation Were Detected in ABMA-Treated Cells ABMA was tested for cytotoxicity before assessing its antiviral activity. As shown in Figure2A and Table1, Vero cells responded to ABMA in a dose-dependent manner with a CC 50 value of 34.75 µM. No cytotoxicity was observed at the concentrations effective against HSV-2 infection. It is current practice in drug discovery processes to pretreat cells with the compound before infection, in order to better monitor a positive effect. ABMA was tested for anti-HSV-2 activity with treatment administered from 5 h before infection to the end of the assays, as reported previously [15]. As shown in Figure2B and Table1, ABMA inhibited HSV-2-induced CPE in a dose-dependent manner with an EC 50 value of 1.66 µM and a maximum inhibition rate of 93.36% at 3.13 µM. The selective index (SI) measuring Viruses 2018, 10, 119 6 of 15

theViruses safety 2018 of, 10 a, compoundx to be developed as an antiviral agent was calculated to be 20.93 by6 of CC 15 50 relativeViruses to 2018 EC,50 10[, 34x ], which was higher than that of the positive control drug, chloroquine.6 Aof plaque15 reductionplaque reduction assay was assay performed was performed subsequently subsequently to conﬁrm to theconfirm anti-HSV-2 the anti-HSV-2 activity. activity. As shown As in shown Figure in2 C andFigure Tableplaque 2C1, reduction ABMAand Table inhibitedassay 1, wasABMA HSV-2-inducedperformed inhibited subsequently HSV-2- plaqueinduced to formation confirm plaque the in anti-HSV-2 formation a dose-dependent activity. in a dose-dependent As mannershown in with mannerFigure with 2C andan EC Table50 value 1, ABMA of 1.08 inhibitedμM, which HSV-2- was ininduced accordance plaque with formation the results in aobtained dose-dependent in the CPE an EC50 value of 1.08 µM, which was in accordance with the results obtained in the CPE inhibition inhibitionmanner withassay. an Morphological EC50 value of 1.08 changes μM, which of the was cells in accordance also confirmed with thethe results protective obtained effects in the of ABMACPE assay. Morphological changes of the cells also conﬁrmed the protective effects of ABMA against HSV-2 againstinhibition HSV-2 assay. infection. Morphological As shown changes in Figure of the 3, cellsuntreated also confirmed virus-infected the protective cells appeared effects ofall ABMA rounded infection. As shown in Figure3, untreated virus-infected cells appeared all rounded up and detached upagainst and detached HSV-2 infection. from the As plates, shown uninfected in Figure 3,cells untreated looked virus-infected all spread out, cells while appeared virus all infected rounded cells from the plates, uninfected cells looked all spread out, while virus infected cells treated with the drugs treatedup and with detached the drugs from were the plates,mostly uninfectedspread out cells and looked partially all roundedspread out, up. while Thus, virus ABMA infected is a safe cells and wereeffectivetreated mostly antiviralwith spread the drugs outagent and wereagainst partially mostly HSV-2 roundedspread in vitro, out up. an whichd Thus, partially protects ABMA rounded cells is a safefromup. Thus, and HSV-2 effectiveABMA infection is antivirala safe below and agent its effective antiviral agent against HSV-2 in vitro, which protects cells from HSV-2 infection below its againsttoxic HSV-2concentration. in vitro, which protects cells from HSV-2 infection below its toxic concentration. toxic concentration.

Figure 2. Cytotoxicity and antiviral activity of ABMA against HSV-2 in vitro. (A) Serially diluted FigureFigure 2. Cytotoxicity 2. Cytotoxicity and and antiviral antiviral activity activity of ABMA of ABMA against against HSV-2 HSV-2in vitroin vitro..(A )(A Serially) Serially diluted diluted ABMA ABMAABMA was was added added to to Vero Vero cells,cells, thenthen cell viabilityity waswas measuredmeasured and and compared compared to tothat that of ofthe the was added to Vero cells, then cell viability was measured and compared to that of the untreated cell control untreateduntreated cell cell control control after after incubation incubation forfor 72 h. (B)) SeriallySerially diluted diluted ABMA ABMA was was added added to toVero Vero cells cells 5 5 after incubation for 72 h. (B) Serially diluted ABMA was added to Vero cells 5 h before infection with h beforeh before infection infection with with HSV-2 HSV-2 (MOI(MOI == 0.04),0.04), then cellcell viabilityviability was was measured measured to to calculate calculate the the CPE CPE HSV-2 (MOI = 0.04), then cell viability was measured to calculate the CPE inhibition percentage after inhibitioninhibition percentage percentage after after infection infection forfor 7272 h in thethe presencepresence of of ABMA. ABMA. (C (C) Serially) Serially diluted diluted ABMA ABMA infection for 72 h in the presence of ABMA. (C) Serially diluted ABMA was added to Vero cells 5 h before waswas added added to to Vero Vero cells cells 5 5 h h before before infectioninfection with HSV-2HSV-2 (50–100 (50–100 PFU) PFU) for for 1 1h, h, then then DMEM-2% DMEM-2% FBS- FBS- infection with HSV-2 (50–100 PFU) for 1 h, then DMEM-2% FBS-1% low-melting agarose containing ABMA 1%1% low-melting low-melting agarose agarose containing containing ABMAABMA at correspondingonding concentrations concentrations was was overlaid overlaid in inplace place of of at corresponding concentrations was overlaid in place of infected medium, followed by counting of plaque infectedinfected medium, medium, followed followed byby countingcounting of plaque numbersnumbers toto calculatecalculate the the plaque plaque inhibition inhibition numberspercentagepercentage to calculate when when plaques theplaques plaque formed. formed. inhibition percentage when plaques formed.

Table 1. Cytotoxicity and antiviral activity of compound against HSV-2 in Vero cells. TableTable 1. 1.Cytotoxicity Cytotoxicity and and antiviral antiviral activityactivity of compound against against HSV-2 HSV-2 in in Vero Vero cells. cells. Cytotoxicity Antiviral Activity Cytotoxicity Antiviral Activity Compounds CytotoxicityCC50 (μM) EC50 a (μM) Antiviral EC50 b Activity(μM) SI c a b c Compounds CC50 (μM) EC50a (μM) EC50 b (μM) SI c CompoundsABMA CC 34.7550 (µ ±M) 0.28 EC 1.6650 ±(µ 0.14M) 1.08EC50 ± 0.25(µM) 20.93SI ChloroquineABMA 34.75 15.67 ±± 0.280.47 1.861.66 ±± 0.200.14 1.38 1.08 ± ±0.03 0.25 8.42 20.93 ABMA 34.75 ± 0.28 1.66 ± 0.14 1.08 ± 0.25 20.93 ChloroquineAcyclovir 15.67 >1000 ± 0.47 0.821.86 ±± 0.060.20 0.81 1.38 ± ±0.07 0.03 1219.51 8.42 Chloroquine 15.67 ± 0.47 1.86 ± 0.20 1.38 ± 0.03 8.42 Acyclovir >1000 0.82 ± 0.06 0.81 ± 0.07 1219.51 a ConcentrationAcyclovir at which the comp >1000ound CPE 0.82inhibition± 0.06 rate reaches 0.81 ± halfway0.07 between 1219.51 the baseline a Concentration at whichb the compound CPE inhibition rate reaches halfway between the baseline a Concentrationand the maximum; at which the concentration compound CPE atinhibition which the rate compound reaches halfway plaque between reduction the rate baseline reaches and halfway the maximum; b c a b concentrationandbetween the maximum; the at baseline which concentration the and compound the maximum; at plaque which selective reduction the compound index rate (SI) reaches value plaque halfwayrepresents reduction between the rateratio thereaches of baselineCC50/EC halfway50 and the c c a a maximum;betweenfor each selectivethe compound; baseline index and Results (SI) the value maximum;are represents presented theselective as ratiomean of indexva CClues50 /EC(SI) ± standard 50valuefor represents each deviations compound; the (SD) ratio Resultsobtained of CC are 50from presented/EC 50 as meanforthree each values independent compound;± standard experiments.Results deviations are presented (SD) obtained as mean from threevalues independent ± standard experiments. deviations (SD) obtained from three independent experiments.

FigureFigure 3. Effects 3. Effects of compounds of compounds on HSV-2-induced on HSV-2-induced CPE. CPE. Morphological Morphological changes changes of Vero of Vero cells cells were were recorded by phase-contrastrecorded by phase-contrast microscopy at microscopy 72 h post-infection at 72 h post-infection following the following CPE inhibition the CPE assay. inhibition Virus controlassay. and FigureVirus 3. control Effects andof compounds cell control on represented HSV-2-induced untreated CPE. virus Morphological infected cells changes and uninfectedof Vero cells cells, were cell control represented untreated virus infected cells and uninfected cells, respectively. Scale bar = 200 µm. recordedrespectively. by phase-contrast Scale bar = 200 microscopyμm. at 72 h post-infection following the CPE inhibition assay. Virus control and cell control represented untreated virus infected cells and uninfected cells, respectively. Scale bar = 200 μm.

Viruses 2018, 10, 119 7 of 15

3.2. ReductionsViruses 2018, 10 of, x HSV-2 Protein and DNA Content Were Detected in ABMA-Treated Cells 7 of 15 The3.2. Reductions effects of of ABMA HSV-2 onProtein HSV-2 and DNA proliferation Content Were were Detected measured in ABMA-Treated by quantifying Cells HSV-2 protein and DNA contentThe effects in the of cellABMA cultures. on HSV-2 ABMA proliferation treatment were was measured administered by quantifying from HSV-2 5 h prior protein to infection,and throughDNA to content the end in of the the cell assays. cultures. HSV-2 ABMA protein treatment and DNAwas administered content were from assayed 5 h prior by Westernto infection, blot and qPCRthrough assays, to respectively, the end of the after assays. a single HSV-2 replicative protein and cycle, DNA before content the were occurrence assayed by of Western obvious blot CPE and at 18 h post-infectionqPCR assays, [29 ].respectively, As shown after in Figure a single4A, replicative a signiﬁcant cycle, reduction before the inoccurrence the content of obvious of HSV-2 CPE VP5 at 18 (main capsidh post-infection protein of HSV-2 [29]. [27As]) shown was observed in Figure in 4A, ABMA-treated a significant reduction cells, while in the that content of β-tubulin of HSV-2 (constitutive VP5 protein(main essential capsid forprotein cell function)of HSV-2 [27]) was notwas affected.observed Asin ABMA-treated shown in Figure cells,4B, while a signiﬁcant that of β reduction-tubulin in (constitutive protein essential for cell function) was not affected. As shown in Figure 4B, a significant HSV-2 DNA content was also detected in ABMA-treated cells. The positive control drugs, chloroquine reduction in HSV-2 DNA content was also detected in ABMA-treated cells. The positive control and acyclovir reduced HSV-2 protein synthesis and DNA replication as expected [19–24]. Based on drugs, chloroquine and acyclovir reduced HSV-2 protein synthesis and DNA replication as expected those[19–24]. data, ABMA Based on appears those data, to be ABMA an effective appears antiviralto be an effective agent againstantiviral HSV-2 agent againstin vitro HSV-2, which in vitro, can cause reducedwhich HSV-2 can cause protein reduced and DNAHSV-2 contentprotein an ind the DNA cell content cultures. in the cell cultures.

Figure 4. Effects of compounds on HSV-2 protein and DNA content in cell cultures. 3.13 μM ABMA Figure 4. Effects of compounds on HSV-2 protein and DNA content in cell cultures. 3.13 µM ABMA or 15 μM chloroquine was added to Vero cells 5 h before infection with HSV-2 (MOI = 1), while 1 μM or 15 µM chloroquine was added to Vero cells 5 h before infection with HSV-2 (MOI = 1), while 1 µM acyclovir was added at the same time as infection. Then DMEM-2% FBS containing the compounds acyclovirat their was corresponding added at the concentrations same time aswas infection. overlaid in Then place DMEM-2% of the medium FBS after containing infection the for compounds1 h. (A) at theirProteins corresponding were extracted concentrations from the cell cultures was overlaid at 18 h post-infection in place of, thethenmedium separated afterby SDS-PAGE infection and for 1 h. (A) Proteinsanalyzed were by Western extracted blot from using the an cellanti-HSV-2 cultures VP5 at 18antibody h post-infection, or an anti-β-tubulin then separated antibody. by “+” SDS-PAGE and and analyzed“−” represented by Western “with” blot and using “without” an anti-HSV-2 the additions, VP5 antibodyrespectively. or an (B anti-) HSV-2β-tubulin DNA was antibody. extracted “+” and “−” representedfrom the cell “with” cultures and at “without”18 h post-infection the additions, and quantified respectively. by qPCR. (B) HSV-2 Statistical DNA significance was extracted was from the cellcompared cultures between at 18 h test post-infection groups and the and untreated quantified virus by infected qPCR. Statisticalcontrol group significance and was represented was compared betweenby asterisks test groups marked and in thethe untreatedfigures, where virus *** infectedp < 0.001.control group and was represented by asterisks marked in the figures, where *** p < 0.001. 3.3. ABMA Blocks HSV-2 Entry into Cells

3.3. ABMAThe Blocks effects HSV-2 of ABMA Entry on into different Cells stages of the HSV-2 lifecycle were measured by a mode of action assay following different ABMA treatment schemes (Figure 5A). ABMA was added at time Thepoints effects corresponding of ABMA to on different different events stages in of the the HSV-2 HSV-2 lifecycle. lifecycle HSV-2 were measured DNA content by a in mode the ofcell action assaycultures following for all different assay conditions ABMA were treatment measured schemes at 18 h (Figure post-infection.5A). ABMA Significant was reductions added at in time HSV- points corresponding2 DNA content to different were detected events when in the the HSV-2cells were lifecycle. pre-treated HSV-2 with DNA ABMA content prior to in infection the cell (pre), cultures or for all assaywhen conditions ABMA was were introduced measured from at 6–18 18 h h post-infection. post-infection Significant(late post) (Figure reductions 5B). The in HSV-2results DNAstrongly content weresuggested detected whenthat ABMA the cells affected were the pre-treated early events with in ABMAthe HSV-2 prior lifecycle to infection by acting (pre), on the or whencells directly. ABMA was introducedABMA fromalso had 6–18 an h effect post-infection on the late (late stages post) of th (Figuree HSV-25B). lifecycle The results as a result strongly of treatment suggested during that ABMA6– 18 h post-infection (Figure 5B). As there was no loss in cell viability at the concentration of ABMA affected the early events in the HSV-2 lifecycle by acting on the cells directly. ABMA also had an effect used in the experiments (3.13 μM) (Figure 2A), the inhibitory effects of ABMA were not due to on the late stages of the HSV-2 lifecycle as a result of treatment during 6–18 h post-infection (Figure5B). cytotoxicity. Therefore, ABMA affects both early and late stages of the HSV-2 lifecycle. The latter As theremechanism was no was loss discussed in cell viability further in at Section the concentration 3.4. of ABMA used in the experiments (3.13 µM) (Figure2A), the inhibitory effects of ABMA were not due to cytotoxicity. Therefore, ABMA affects both early and late stages of the HSV-2 lifecycle. The latter mechanism was discussed further in Section 3.4.

Viruses 2018, 10, 119 8 of 15 Viruses 2018, 10, x Viruses 2018, 10, x 8 of 15 8 of 15

Figure 5. Effects of Figure ABMA 5. Effectson different of ABMA stages on of different the HSV-2 stages lifecycle. of the ( AHSV-2) ABMA lifecycle. (3.13 μµ (AM)) ABMA treatment (3.13 μM) treatment and HSV-2 (MOI and = 1) HSV-2 infection (MOI schemes = 1) infection in the time schemes of ABMA in the addition time of assay. ABMA ( B addition) HSV-2 assay. DNA was(B) HSV-2 DNA was extracted from the extracted cell cultures from atthe 18 cell h post-infectionpostcultures-infection at 18 followingh post-infection the treatment following schemes the treatment in ( A) and schemes in (A) and quantifiedquantified byby qPCR.qPCR.quantified Statistical Statistical by significance qPCR.significance Statistical was was compared significancecompared between between was testcompared test groups groups andbetween theand untreated thetest untreated groups virus and the untreated virusinfected infected control control groupvirus group andinfected was and representedcontrol was represented group by and asterisks bywas asterisks represented marked marked in the by figures, asterisksin the wherefigures, marked *** wherep in< 0.001.the *** figures, p < where *** p < 0.001. 0.001. As antiviral agents targeting the essential early stages of the HSV-2 lifecycle (binding and As antiviral agentsAs antiviraltargeting agents the essential targeting early the st essentialages of the early HSV-2 stages lifecycle of the HSV-2(binding lifecycle and entry) (binding and entry) entry) may be more effective than those targeting the late stages, events in the early stages of the may be more effectivemay be thanmore those effective targeting than thosethe late targeting stages, theevents late in stages, the early events stages in the of theearly HSV-2 stages of the HSV-2 HSV-2 lifecycle that could be targeted by ABMA were further investigated [35]. HSV-2 binding and lifecycle that couldlifecycle be targeted that could by ABMAbe targeted were by further ABMA investigated were further [35]. investigated HSV-2 binding [35]. HSV-2and entry binding and entry entry assays were performed at 4 ◦C and 37 ◦C, respectively, after pretreatment of the cells with assays were performedassays were at 4 performed°C and 37 at°C, 4 respectively,°C and 37 °C, after respectively, pretreatment after of pretreatment the cells with of thethe cells with the the compounds. This was followed by quantification of bound and entered viruses by measuring compounds. Thiscompounds. was followed This by was quantification followed by of quantification bound and entered of bound viruses and enteredby measuring viruses HSV-2 by measuring HSV-2 HSV-2 DNA content in the original virus inoculum, the unbound virus supernatant from the binding DNA content inDNA the original content virus in the inoculum, original virus the unbound inoculum, virus the supernatantunbound virus from supernatant the binding from assay the binding assay assay (unbound virus) and the internalized virus after freeze–thaw cycles of the infected cells from (unbound virus)(unbound and the internalizedvirus) and the virus internalized after freeze–thaw virus after cycles freeze–thaw of the infected cycles cellsof the from infected the cells from the the entry assay (entered virus), respectively (Figure6A). As shown in Figure6B,C, HSV-2 entry entry assay (enteredentry virus),assay (enteredrespectively virus), (Figure respectively 6A). As (Figure shown 6A).in Figure As shown 6B,C, inHSV-2 Figure entry 6B,C, was HSV-2 entry was was significantly reduced by ABMA, similarly to chloroquine, which is known to affect HSV-2 significantly reducedsignificantly by ABMA, reduced similarly by ABMA, to chloroquine, similarly towhich chloroquine, is known which to affect is known HSV-2 to entry affect HSV-2 entry entry [20,21,24]. As expected, acyclovir that blocks virus replication had no effect on virus binding or [20,21,24]. As expected,[20,21,24]. acyclovir As expected, that blocks acyclovir virus that replication blocks virus had noreplication effect on had virus no binding effect on or virus entry binding or entry entry [19]. Therefore, ABMA blocks HSV-2 entry into cells. [19]. Therefore, [19].ABMA Therefore, blocks HSV-2 ABMA entry blocks into HSV-2 cells. entry into cells.

Figure 6. Cont.

Viruses 2018, 10, 119 9 of 15 Viruses 2018, 10, x 9 of 15

Figure 6. Effects ofof compoundscompounds onon HSV-2HSV-2 binding binding and and entry. entry. (A (A) Drug) Drug treatment treatment and and HSV-2 HSV-2 (MOI (MOI = 1)= incubation1) incubation schemes schemes in bindingin binding and and entry entry assays. assays. (B) HSV-2(B) HSV-2 DNA DNA was extractedwas extracted from from the original the original virus inoculumvirus inoculum and unbound and unbound virus virus supernatant supernatant at 2 h at post-incubation 2 h post-incubation of the of cells the withcells with HSV-2 HSV-2 at 4 ◦ atC 4 and °C quantifiedand quantified to calculate to calculate the amount the amount of HSV-2 of boundHSV-2 tobound the cells to the in the cells binding in the assay.binding (C) assay. HSV-2 ( DNAC) HSV-2 was extractedDNA was from extracted the internalized from the interna virus afterlized two virus freeze-thaw after two cycles freeze-thaw of the infected cycles cellsof the at infected 1 h post-infection cells at 1 withh post-infection HSV-2 at 37 with◦C following HSV-2 at the 37 binding °C following process th ande binding quantified process to calculate and quan thetified amount to ofcalculate HSV-2 thatthe wasamount internalized of HSV-2 in cellsthat inwas the internalized entry assay. Statisticalin cells in significance the entry wasassay. compared Statistical between significance test groups was andcompared the untreated between virus test infectedgroups and control the groupuntreated and wasvirus represented infected control by asterisks group markedand was in represented the figures, whereby asterisks *** p < marked 0.001. in the figures, where *** p < 0.001.

3.4. ABMA Inhibits the Late Stages of the HSV-2 Lifecycle The effects of ABMA on the late stages of the HSV-2HSV-2 lifecycle were further studied using the late stage infectioninfection assay atat 1818 hh post-infection.post-infection. HSV-2-infected cells were treatedtreated withwith thethe compoundscompounds during 6–18 h post-infection,post-infection, which corresponds to the late stages of thethe HSV-2HSV-2 lifecycle.lifecycle. FollowingFollowing this,this, intracellularintracellular and extracellular virus titers we werere measured (Figure 77A).A). AsAs shownshown inin FigureFigure7 B,7B, both intracellularintracellularand and extracellular extracellular HSV-2 HSV-2 titers weretiters significantlywere significantly reduced byreduced ABMA. by As chloroquineABMA. As waschloroquine reported was to interact reported with to the interact late stages with of the the HSVlate stages lifecycle, of virusthe HSV titers lifecy werecle, significantly virus titers reduced, were assignificantly expected [reduced,20,22,23]. as As expected the target [20,22,23]. of acyclovir As the was target demonstrated of acyclovir towas be demonstrated HSV-2 DNA replication,to be HSV- which2 DNA mainly replication, takes placewhich during mainly 3–6 takes h post-infection, place during virus3–6 h titerspost-infection, were reduced virus to titers a lesser were extent reduced [19]. Theseto a lesser results extent confirmed [19]. These the results inhibitory confirmed effects the of i ABMAnhibitory on effects the late of ABMA stages on of the late HSV-2 stages lifecycle, of the asHSV-2 described lifecycle,in Section as described 3.3. Additionally, in Section 3.3. these Additionally, results also these suggested results that also the suggested HSV-2 packaging that the HSV- and egress2 packaging process and was egress most process likely to was be blocked most likely by ABMA, to be blocked as capsid by formation ABMA, as and capsid progeny formation infectious and particleprogeny packaging infectious graduallyparticle packaging take place gradually from 5 h post-infectiontake place from onwards 5 h post-infection [36]. onwards [36].

Viruses 2018, 10, 119 10 of 15 Viruses 2018, 10, x 10 of 15

FigureFigure 7. 7. EffectsEffects of of compounds compounds on the late stages of the HSV-2 lifecycle. (A)) Drug treatmenttreatment andand HSV-2HSV- 2(MOI (MOI = = 1) 1) incubation incubation schemesschemes inin thethe latelate stage infection assay. ( B) Intracellular Intracellular viruses viruses were were collected collected fromfrom the the infected infected cells cells after after two two freeze-thaw freeze-thaw cycles cycles at at 18 18 h h post-i post-infectionnfection following the the treatment schemesschemes in in ( (AA)) and and subjected subjected to to virus virus titration. titration. ( (CC)) Extracellular Extracellular viruses viruses were were collected collected from from the the cell cell supernatantssupernatants at at 18 18 h h post-infection post-infection following following the the treatm treatmentent schemes schemes in in ( (AA)) and and subjected subjected to to virus virus titration.titration. Statistical Statistical significance signiﬁcance was was compared compared be betweentween test test groups groups and and the the untreated untreated virus virus infected infected controlcontrol groups groups and was represented by as asterisksterisks marked in the ﬁgures,figures, where **** p < 0.01, *** p < 0.001. 0.001.

3.5.3.5. ABMA ABMA Protects Protects BALB/c BALB/c Mice Mice from from Intravaginal HSV-2 Challenge HavingHaving identifiedidentified the the anti-HSV-2 anti-HSV-2 potency potency of ABMAof ABMAin vitro in vitro,, we next we evaluatednext evaluated the protective the protective efficacy efficacyof ABMA of againstABMA intravaginalagainst intravaginal challenge challenge of HSV-2 inof BALB/cHSV-2 in mice BALB/c using mice a reported using a mouse reported model mouse [37]. modelAs shown [37]. in As Figure shown8A, in ABMA Figure significantly 8A, ABMA significan improvedtly the improved survival rates the survival of drug-treated rates of infecteddrug-treated mice infectedcompared mice to the compared untreated to virus the untreated infected control, virus whoseinfected survival control, rate whose was 8.33%.survival ABMA rate was given 8.33%. daily ABMAintraperitoneally given daily at 5intraperitoneally mg/kg provided theat 5 best mg/kg survival provided rate of the 50%. best As survival shown in rate Figure of 50%.8B, ABMA As shown at both in Figuredoses tested 8B, ABMA reduced at the both clinical doses score tested in the reduced same trend the clinical as the survival score in rate. the Micesame treated trend withas the 5 mg/kgsurvival of rate.ABMA Mice showed treated the with lowest 5 mg/kg clinical of score, ABMA below showed 3, while the thelowest value clinical of the score, untreated below virus 3, while infected the control value ofreached the untreated 4.58. Several virus mice infected with clinical control score reached below 2 (moderate4.58. Several genital mice inflammation) with clinical recovered score below from the 2 (moderateinfection along genital with inflammation) time. However, recovered mice with from clinical the score infection higher along than 2 with did not time. recover. However, As clinical mice scores with clinicalin Figure score8B were higher presented than 2 asdid the not mean recover. value As of 10–12clinical mice, scores there in wasFigure no reduction8B were presented in clinical as scores the mean along valuewith time of 10–12 as a result.mice, there Despite was that, no reduction ABMA significantly in clinical scores reduced along the with severity time of as the a result. disease Despite and slowed that, ABMAthe progress significantly time course reduced of mice the compared severity of to the the dise untreatedase and virus slowed infected the mice.progress As acyclovirtime course is normally of mice comparedused as the to standard the untreated treatment virus of HSV-2 infected infection, mice. it As was acyclovir used as the is positivenormally control used drug as the in thestandardin vivo treatmentexperiment of [ 31HSV-2]. The infection, protective it ratewas of used acyclovir as the atposi 150tive mg/kg control was drug shown in the to bein 100%,vivo experiment as expected [31]. [31]. TheBody protective weight changes rate of ofacyclovir mice were at 150 also mg/kg recorded was over shown 20 days,to be but100%, there as wasexpected no significant [31]. Body difference weight changesamong these of mice groups were (dataalso recorded no shown). over HSV-2 20 days titers, but from there the was vaginal no significant swabs at difference day 5, when among viral these load groups (data no shown). HSV-2 titers from the vaginal swabs at day 5, when viral load reached its peak, and at day 10, for a later time point, were also detected to confirm the protective efficacy of

Viruses 2018, 10, 119 11 of 15

reachedViruses its 2018 peak,, 10, andx at day 10, for a later time point, were also detected to confirm the protective11 of efficacy15 of ABMA against HSV-2 infection in vivo [38]. As shown in Figure8C, HSV-2 was detected in all groups indicatingABMA a against successful HSV-2 HSV-2 infection challenge. in vivo ABMA [38]. As at shown 5 mg/kg in Figure significantly 8C, HSV-2 reduced was detected HSV-2 in titers all groups by 0.55 log and 0.60indicating log at daya successful 5 and day HSV-2 10, challenge. respectively. ABMA Acyclovir at 5 mg/kg at 150 significantly mg/kg significantly reduced HSV-2 reduced titers HSV-2by 0.55 titers log and 0.60 log at day 5 and day 10, respectively. Acyclovir at 150 mg/kg significantly reduced HSV- by 0.97 log and 1.20 log at day 5 and day 10, respectively. Based on these data, ABMA effectively protects 2 titers by 0.97 log and 1.20 log at day 5 and day 10, respectively. Based on these data, ABMA BALB/c mice from intravaginal HSV-2 challenge. effectively protects BALB/c mice from intravaginal HSV-2 challenge. Overall,Overall, ABMA ABMA is an is effective an effective antiviral antiviral agent agent against against HSV-2 HSV-2in vitro in vitroand andin vivo in vivo,, with with two two putative modesputative of action, modes affecting of action, both affecting virus both entry virus and en thetry late and stagesthe late of stages the HSV-2 of the HSV-2 lifecycle. lifecycle.

Figure 8. Antiviral efficacy of ABMA against intravaginal HSV-2 challenge in BALB/c mice. Female Figure 8. Antiviral efficacy of ABMA against intravaginal HSV-2 challenge in BALB/c mice. Female BALB/c mice (6–8 weeks old, n = 10–12 per group) were injected subcutaneously with 2 mg of Depo- BALB/c mice (6–8 weeks old, n = 10–12 per group) were injected subcutaneously with 2 mg of Provera per mouse seven days before intravaginal inoculation with 50,000 PFU of HSV-2 in 10 μL of Depo-Provera per mouse seven days before intravaginal inoculation with 50,000 PFU of HSV-2 in PBS. At 1 h post-inoculation and once daily for seven consecutive days, 1.25 mg/kg or 5 mg/kg of 10 µLABMA, of PBS. 150 At mg/kg 1 h post-inoculation of acyclovir, or andPBS oncesuppl dailyemented for sevenwith 10% consecutive DMSO was days, administered 1.25 mg/kg or 5 mg/kgintraperitoneally. of ABMA, 150 (A)mg/kg Survival of rate acyclovir, and (B) clinical or PBS score supplemented of the mice we withre monitored 10% DMSO daily was for administered 20 days. intraperitoneally.(C) Vaginal swab (A) samples Survival were rate collected and (B) at clinical day 5 and score day of 10, the respectively, mice were and monitored transferred daily to 200 for 20μL days. (C) Vaginalof Hank’s swab buffer, samples followed were by collected virus titer at day determination 5 and day 10,by respectively,plaque assay andin Vero transferred cells. Statistical to 200 µ L of Hank’ssignificance buffer, followed was compared by virus between titer determination test groups and by plaquethe untreated assay invirus Vero infected cells. Statistical control and significance was was comparedrepresented between by asterisks test mark groupsed in and the figures, the untreated where * virusp < 0.05, infected *** p < 0.001. control and was represented by asterisks marked in the figures, where * p < 0.05, *** p < 0.001. 4. Discussion 4. DiscussionThe prevalence, severity of complications and the close association with cervical cancer and HIV infection make HSV-2 infection a global health concern [39]. The lack of an available vaccine and the Theemergence prevalence, of drug severity resistance of complicationshighlight the importan and thece close of developing association alternative with cervical antivirals cancer against and HIV infectionHSV-2 make with HSV-2 distinct infection modes of a globalaction [10]. health ABMA concern has [been39]. Thedemonstrated lack of an previously available to vaccine be active and the emergenceagainst ofseveral drug intracellular resistance pathogens highlight exploiting the importance host–vesicle of developing transport [15]. alternative Here we demonstrated antivirals against HSV-2that with ABMA distinct is an effective modes ofinhibitor action of [ 10HSV-2,]. ABMA in vitro has and been in vivo. demonstrated previously to be active against several intracellular pathogens exploiting host–vesicle transport [15]. Here we demonstrated that ABMA is an effective inhibitor of HSV-2, in vitro and in vivo. Viruses 2018, 10, 119 12 of 15

The anti-HSV-2 potential of ABMA was first identified in vitro with EC50 values below 2 µM and an SI value of 20.93, which is suitable for an antiviral agent [40] (Figure2 and Table1). Treatment with ABMA also resulted in significant reductions of HSV-2 protein and DNA content in the cell cultures (Figure4). Subsequently, ABMA was found to target both early and late stages of the HSV-2 lifecycle in the time of compound addition assay, which was similar to the effects of SPL-2999 (a dendrimer with the active surface group of naphthyl 3,6-disulfonic acid sodium salts to interact with biological surfaces) on HSV-2 [41] (Figure5). ABMA was found to affect the early stages of HSV-2 infection by acting on cells directly, as reported in previous studies on the effects of polysaccharide extracts from algal species and SPL-2999 on HSV-2 [41,42]. The specific event targeted by ABMA in the early stages of HSV-2 infection was found to be virus entry into cells, while binding was unaffected by ABMA treatment, which was similar to the effect of Dynasore (a small-molecule inhibitor of dynamin, which is a GTPase that controls multiple endocytic pathways and also plays a role in actin assembly and reorganization) on HSV-2 entry [30] (Figure6). Although herpes simplex virus (HSV) may enter cells by direct fusion between the virus envelope with the cell membranes, substantial evidences for HSV entry through an endocytic-dependent mechanism have come to light [43]. HSV begins endocytic entry by using the host membrane machinery to envelop viruses. The viruses trapped in endocytic vesicles can then be released to the host cytoplasm by fusion of the virus envelope with the vesicle membranes [44]. The inhibitory effect of ABMA on HSV-2 entry might be related to its effect on host–vesicle transport [15], which is involved in the endocytic entry pathway of HSV-2. Antivirals targeting early stage infection have attracted significant attention because reduced entry of viruses into cells translates to decreased replication and spread to other cells [35]. Most early-stage prevention drugs that show promise in treating HSV-2 infection are binding inhibitors, targeting the host cell receptor or the viral glycoprotein required for binding [43,44]. Agents preventing HSV-2 entry into cells, including SPL-2999, PM-19 (a keggin-type heteropolyoxotungstate K7[PTi2W10O40]·6H2O), Dynasore and ABMA reported in our study may provide alternative early stage inhibitors [30,41,45]. In addition to virus entry, ABMA was also found to inhibit the late stages of HSV-2 infection, which was confirmed by significantly reduced intracellular and extracellular virus titers when ABMA was applied during 6–18 h post-infection (Figure7). Additionally, because HSV-2 DNA replicates rapidly between 3–6 h and the formation of both the capsids and the progeny infectious particles of HSV-2 gradually takes place from approximately 5 h post-infection onwards [36], the results also suggested that ABMA was most likely to hinder the HSV-2 packaging and egress process, as reported in a study on the effects of Nelfinavir on HSV-1 [46]. The HSV-2 packaging and egress process begins with capsid assembly in the nuclei, then infectious particles are packaged by budding the capsids into specialized vesicles derived from the trans-Golgi network to gain an envelope and an outer vesicular membrane. Finally, the virus-containing vesicles move to and fuse with the cell membranes to release the viruses into the extracellular medium [47–49]. The fact that ABMA was likely to block the HSV-2 packaging and egress process suggested two possible explanations: the target of ABMA present on host–endosomal transport is also present on the HSV-2 packaging and egress process. Alternatively, host–endosomal transport, which can be affected by ABMA, participates in the HSV-2 packaging and egress process. Significant protective efficacy of ABMA against intravaginal HSV-2 challenge in female BALB/c mice was demonstrated by an improved survival rate, reduced clinical score and reduced vaginal virus load compared to the untreated virus infected control (Figure8). ABMA administered at the highest tested dose of 5 mg/kg showed the best survival rate of 50%, while that of the untreated virus infected control was 8.33%. Several recently developed inhibitors of HSV-2 have undergone, or are currently undergoing clinical trials, but most of them have yet to gain licensure due to adverse effects on the host [13]. Based on our study, ABMA might provide an alternative. As ABMA is an initial hit from a high throughput screening, further medicinal chemistry and pharmaceutical optimizations are still required and may lead to candidates with higher anti-HSV-2 activities. Viruses 2018, 10, 119 13 of 15

Multi-drug therapy, combining drugs with different modes of action to limit the manifestation of drug resistance and to increase the selectivity by reduced dose, is common practice in the treatment of viral infections, including HIV and HCV (hepatitis C virus), but not HSV, for which currently available drugs have the same target, the viral DNA polymerase [50,51]. ABMA was demonstrated to be an effective inhibitor of HSV-2 with the targets of virus entry as well as the late stages of viral lifecycle, which were different from commonly used acyclovir. It might well be possible to develop combinations of ABMA with acyclovir, or other drugs targeting stages different from ABMA in the HSV-2 lifecycle, such as virus replication. These novel combinations might be more effective and might provide an alternative to HSV-2 infection treatment. In conclusion, AMBA has been identiﬁed as an effective inhibitor of HSV-2 in vitro and in vivo, by inhibiting virus entry, as well as the late stages of the HSV-2 lifecycle. Our study expands the list of pathogens against which ABMA is active and exempliﬁes the potential of ABMA to be developed as a broad-spectrum inhibitor. As the target of ABMA is a host component rather than the pathogens themselves, drug resistance may be less likely to arise [52].

Acknowledgments: This work was supported by grants from Natural Science Foundation of China [grant number 31770184]; a grant from the Agence Nationale de la Recherche and the LERMIT LabEx [grant number ANR-10-LABX-33], Ile de France Region grant from the DIM Malinf initiative 140101 and CEA. Author Contributions: Wenwen Dai, Wei Kong, Feng Gao, Daniel Gillet, Weiheng Su and Chunlai Jiang conceived and designed the experiments; Wenwen Dai, Jinpeng Bi, Shuai Wang and Fang Li performed the experiments; Yu Wu analyzed the data; Wenwen Dai, Julien Barbier and Jean-Christophe Cintrat wrote the paper. All authors listed have approved the submission. Conﬂicts of Interest: The authors declare no conﬂict of interest.

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