Safety, Formulation and In Vitro Antiviral Activity of the Antimicrobial Peptide Subtilosin Against Herpes Simplex Virus Type 1
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- Torres, N.I., Noll, K.S., Xu, S. et al. Probiotics & Antimicro. Prot. (2013) 5: 26. doi:10.1007/s12602-012-9123-x
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In the present study, the antiviral properties of the bacteriocin subtilosin against Herpes simplex virus type 1 (HSV-1) and the safety and efficacy of a subtilosin-based nanofiber formulation were determined. High concentrations of subtilosin, the cyclical antimicrobial peptide produced by Bacillus amyloliquefaciens, were virucidal against HSV-1. Interestingly, at non-virucidal concentrations, subtilosin inhibited wild type HSV-1 and aciclovir-resistant mutants in a dose-dependent manner. Although the exact antiviral mechanism is not fully understood, time of addition experiments and western blot analysis suggest that subtilosin does not affect viral multiplication steps prior to protein synthesis. Poly(vinyl alcohol)-based subtilosin nanofibers with a width of 278 nm were produced by the electrospinning process. The retained antimicrobial activity of the subtilosin-based fibers was determined via an agar well diffusion assay. The loading capacity of the fibers was 2.4 mg subtilosin/g fiber, and loading efficiency was 31.6 %. Furthermore, the nanofibers with and without incorporated subtilosin were shown to be non-toxic to human epidermal tissues using an in vitro human tissue model. Taking together these results, subtilosin-based nanofibers should be further studied as a novel alternative method for treatment and/or control of HSV-1 infection.
Herpes simplex virus types 1 and 2 (HSV-1 and HSV-2, respectively) are serious human pathogens. HSV-1 is often associated with orofacial infections and encephalitis, whereas HSV-2 has been found to be the most frequent cause of genital herpes, the major cause of genital ulcers worldwide [9, 40, 52]. However, in the past decade, several studies have identified HSV-1 as the causative agent of at least half of the new genital herpes episodes [4, 21]. HSV infections persist during the lifetime of the host, generally establishing latent infections [17, 18]. In addition, it has been shown that genital herpes represents a risk factor for the transmission of human immunodeficiency virus (HIV), increasing the risk of contagion by two- to threefold [26, 27, 31].
Nowadays, the agents most commonly used for the management of herpetic infections are the nucleoside analogs acyclovir (ACV), penciclovir (PCV) and their respective prodrugs valacyclovir and famciclovir. The triphosphate forms of these analogs selectively inhibit the viral DNA polymerase (DNA pol) activity. After more than three decades of ACV administration, drug-resistant HSV isolates are rarely found in immunocompetent subjects (0.1–0.7 %) but more frequently recovered from immunocompromised patients (4–14 %) [12, 34, 36, 47]. HSV resistance to ACV generally arises as a result of mutations in viral thymidine kinase and in a lesser number from mutations in the viral DNA pool [19, 38]. The pyrophosphate analog foscarnet (FOS) is often used in the management of resistant HSV infections. However, FOS resistance is rising rapidly and its use is reserved to the cases where other drugs fail because it is more toxic and less bioavailable [11, 33]. Considering the incidence of HSV infections, the reported emergence of ACV, PCV and FOS resistant mutants and that it has been recently shown that antiviral treatment of herpetic infection fail to reduce HSV and HIV transmission [10, 29], there is a need for new antiherpetic compounds with different mechanisms of action.
Antimicrobial peptides are produced by a variety of organisms including insects, fungi, Gram-positive and Gram-negative bacteria, and many of them have been reported as viral inhibitors [1, 28]. For example, cationic peptides like α-defensins, hecate and synthetic derivatives of magainins are active against HSV in vitro replication [6, 22] while melittin, a 26 amino acid amphipathic peptide isolated from the venom of the European honeybee Apis melliphera, inhibits the replication of both HIV and HSV [2, 6, 51]. Several antimicrobial peptides ribosomally produced by bacteria, collectively called bacteriocins, have been described for the members of the genus Enterococcus . Wachsman et al.  were the first to describe an antiviral peptide produced by E. faecium CRL35. The peptide, 3.5 kDa in size, inhibited late stages of the HSV-1 and HSV-2 multiplication cycle. In addition, Serkedjieva et al.  described a 5.0-kDa peptide, produced by Lactobacillus delbrueckii subsp. bulgaricus with activity against influenza virus.
Based on the documented history of bacterially produced peptides, we chose to investigate the novel bacteriocin subtilosin, for such characteristics. Subtilosin is a 3.4-kDa, cyclical peptide  produced by both Bacillus subtilis  and B. amyloliquefaciens  with proven antimicrobial activity against a variety of human pathogens, including the bacterial vaginosis-associated Gardnerella vaginalis. Sutyak et al.  further established that subtilosin has potent spermicidal activity and is non-toxic to human vaginal tissues. Due to its range of activities and its safety for human use, subtilosin was an obvious target for investigation into its antiviral properties. Furthermore, we have chosen to examine the feasibility of subtilosin’s incorporation into a nanofiber-based delivery system, considering the successful use of this method with other bacteriocins [16, 24, 25]. Here, we report for the first time the antiviral activity of the anionic antimicrobial peptide subtilosin, and its safety for use in a novel nanofiber delivery system.
Materials and Methods
Subtilosin was isolated and purified from cultures of the producer strain, Bacillus amyloliquefaciens KATMIRA1933, according to the protocols previously described by Sutyak et al. . All subtilosin solutions were prepared in sterile ddH2O and maintained at 4 °C until use.
Acyclovir and foscarnet were purchased from Sigma-Aldrich Chemical Company (St. Louis, MO). Drug stock solutions were prepared in dimethyl sulfoxide (DMSO) with a final concentration of 0.1 % and diluted with maintenance medium (MM) consisting of minimum essential medium (MEM) (Gibco, Carlsbad, CA, USA) with 2 % inactivated fetal bovine serum.
Poly(vinyl alcohol) (PVOH, Mw = 61 kDa, Sigma-Aldrich) was chosen as a carrier polymer due to its high biocompatibility . Trypticase™ soy broth (TSB), trypticase™ soy agar (TSA), agar and yeast extract were purchased from Becton, Dickinson and Company (Sparks, MD, USA). Yeast extract was added into TSB and TSA in the amount of 0.6 % as a nutritional supplement to improve microbial growth. The indicator organism Micrococcus luteus ATCC 10240 was chosen due to its high sensitivity to bacteriocins and its widespread used as a bacteriocin-sensitive indicator strain by academia and industry .
Cells and Viruses
African green monkey kidney (Vero) cells were grown as monolayers in MEM (Gibco) supplemented with 5 % inactivated fetal bovine serum and 50 μg/mL of gentamycin.
HSV-1 strain F (tk+) was obtained from the American Type Culture Collection (Rockville, MD, USA). HSV-1 strain Field (tk deficient) was kindly provided by Dr. G. Andrei (Rega Institute, Leuven, Belgium). Virus stocks were prepared in Vero cells.
Cell Cytotoxicity Assays
To determine the cytotoxic concentrations of the compounds, confluent monolayers of Vero cells were grown in tissue culture plates for 48 h and exposed to various concentrations of the compounds. After 48 h of incubation, cell viability was examined by the ability of the cells to cleave the tetrazolium salt MTT (3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide) (Sigma-Aldrich) with the mitochondrial enzyme succinate dehydrogenase, yielding a quantifiable blue product (formazan) . The precise MTT procedure has been previously described [43, 50]. The CC50 was defined as the compound concentration that reduced Vero cells viability by 50 %, calculated by regression analysis.
HSV-1 tk+ and tk-deficient preparations were incubated with 200 μg/mL of subtilosin or with MM (control) for 0, 15, 45 and 90 min at 37 °C. Then, aliquots were taken and diluted in a serial fashion in MM in order to infect Vero cell monolayers grown in 24-well culture plates to test virus survival using a plaque assay. Plaques were counted exclusively in wells where subtilosin concentration was less than 0.2 μg/mL, a concentration 50-fold lower than the concentration needed to inhibit virus multiplication in 50 % compared to untreated infected cultures).
To assay the effect of subtilosin over the viral particles at antiviral concentrations, HSV-1 was incubated with subtilosin at concentrations ranging from 6 to 100 μg/mL or MM for 60 min at 37 °C. Then, aliquots were taken and Vero cells were infected to test virus survival using a plaque assay.
Virus Yield Reduction Assay
Antiviral activity was evaluated by the virus yield reduction assay. For that purpose, Vero cells grown in 24-well culture plates for 48 h were individually infected with the two strains of HSV-1 at a multiplicity of infection (m.o.i.) of 1 PFU/cell. After 1 h of adsorption at 37 °C, the infective viral particles were removed and cells were covered with MM (control) or MM containing various concentrations of subtilosin. After 24 h of incubation at 37 °C, the supernatants were harvested and cell-free virus yields were determined by a plaque assay on Vero cell monolayers. The antiviral activity was expressed as the EC50 (50 % effective concentration), that is, the compound concentration required to reduce plaque formation by 50 % after 72 h compared with the untreated infected cultures. The EC50 values were calculated by plotting percentages of inhibition versus different concentrations of each compound.
Effect of Time of Addition of Subtilosin on HSV-1 Production
Subtilosin at a concentration of 50 μg/mL was added to confluent monolayers of Vero cells either at 1, 3, 5 or 8 h post-infection (p.i.) with HSV-1 (m.o.i. = 1). Cultures were further incubated at 37 °C until 24 h p.i. and extracellular virus infectivity was quantified by plaque assay on Vero cells.
Monolayers of confluent Vero cells were infected with HSV-1 (m.o.i. = 1) or left non-infected (control), and cultures were incubated at 37 °C for 1 h to allow virus internalization. After removing the inoculum, monolayers were covered with MM alone or containing different concentrations of subtilosin, and incubated at 37 °C for 24 h. Immediately after, cells in duplicate wells were lysed with Laemmli sample buffer (Bio-Rad, CA, USA) with 5 % β-mercaptoethanol. Samples were heated for 2 min in boiling water before loading onto 10 % acrylamide gels. Following electrophoresis, the resolved proteins were transferred to a PVDF membrane (Perkin Elmer Life Sciences, Inc., Waltham, MA, USA) in a dry system (LKB Multiphor II, Pharmacia, Sweden). Glycoprotein gD was revealed with mouse anti-gD (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) and a peroxidase antimouse immunoglobulin G (Promega, Madison, WI, USA) as secondary antibody. ERK1 used as a loading control was revealed with rabbit anti-ERK1 (Santa Cruz Biotechnology Inc.) and a peroxidase antirabbit immunoglobulin G (Promega) as secondary antibody. Chemiluminescence was visualized using a chemiluminescence kit (Perkin Elmer Life Sciences).
Preparation of Antimicrobial Fibers
Morphology Characterization of Antimicrobial Nanofibers
Surface images of the antimicrobial PVOH fibers with and without subtilosin were collected with a commercial Nanoscope IIIa Multi-Mode AFM (Veeco Instruments, Plainview, NY, USA) equipped with a J scanner, which was operated in tapping mode using a silicon cantilever. The scanned images were obtained at the scan size of 5 × 5 μm and 50 × 50 μm. The scan frequency was set at 0.1 Hz. The section analysis embedded in the software Nanoscope 3.0 was utilized to calculate the fiber diameter distribution.
Assessment of Subtilosin Loading Capacity and Efficiency Via Well Diffusion Assay
The evaluation of subtilosin loading capacity and efficiency was conducted by agar plate well diffusion inhibition assays . All nanofiber mats were sterilized by direct exposure to UV light (257 nm) in a biosafety cabinet (Forma Class II, A2 Biological Safety Cabinet, Thermo Fisher Scientific, Pittsburgh, PA, USA) for 10 min per side prior to use. Twenty milligrams of PVOH+ subtilosin fibers was cut with sterile tweezers, immersed in 200 μL sterile ddH2O and kept at 4 °C for 24 h. Twenty milligrams of PVOH fibers was also dissolved as a negative control to eliminate the possibility of PVOH inhibition. An overnight culture of M. luteus in TSB (c. 109 CFU/mL) was diluted 100-fold by blending with ~55 °C soft agar (TSB supplemented with 7 g/L agar). Then, 4 mL of the soft agar containing M. luteus was transferred onto the surface of a TSA plate. After 30 min of solidification, a sterile glass pipette (approx. 5 mm diameter) was used to create wells in the agar plate. Fifty microliters of purified subtilosin, PVOH+ subtilosin fibers (24 h water solution), negative control and their double dilutions were injected into the wells in duplicate. After 24 h incubation at 37 °C, minimum inhibitory concentration (MIC) was defined as the lowest concentration to form a visible inhibition circle on the M. luteus growth layer. Tests were repeated three times.
Skin Cytotoxicity Assays
The safety of nanofiber mats with and without incorporated subtilosin for use on human skin was tested on a cultured, reconstituted human epidermal model. The EpiDerm (NHEK) tissue model (MatTek Corporation, Ashland, MA, USA) consists of normal, human-derived epidermal keratinocytes that are cultured to form a highly differentiated model of the epidermis, and allows for in vitro testing of a compound’s irritancy and cytotoxicity. All EpiDerm tissues were maintained at 4 °C upon receiving and prior to use. The standard method developed by MatTek, and later outlined by Frasch et al. , was followed. Briefly, all tissues were preincubated in room temperature culture medium at 37 °C with 5 % CO2 for 1 h prior to use. Nanofiber mats containing 12 % PVOH or 12 % PVOH+ subtilosin were sterilized by direct exposure to UV light in a biosafety cabinet for 10 min per side, and then cut to size using a sterilized size 5 cork borer (Thermo Fisher Scientific). These nanofiber “discs” were applied onto the surface of the pre-conditioned tissues with light pressure to ensure that the disc came in full contact with the tissue. Positive (50 μl Triton-X) and negative (50 μl sterile ddH2O) controls and all test compounds were assayed in triplicate. The tissues were exposed to the test compounds for 24 h at 37 °C with 5 % CO2. After exposure, the tissues were washed with PBS, transferred into MTT assay buffer and further incubated for 3 h at 37 °C with 5 % CO2. After a second wash with PBS, the tissues were submerged in isopropanol and held at room temperature overnight to allow for extraction of the formazan dye from the tissues. The following day, the optical density of the formazan–isopropanol mixture from each tissue was measured in triplicate at 570 nm. Cell viability was then calculated as a percentage of the average negative control tissues. Tests were performed at least twice.
Antiviral Activity: Subtilosin Inhibits HSV-1 Multiplication
Cytotoxicity and antiviral activity of subtilosin, aciclovir (ACV) and foscarnet (FOS) against HSV-1
HSV-1 tk deficient
Phosphonoformic acid trisodium salt hexahydrate
HSV-1 tk deficient
HSV-1 tk deficient
Loading Capacity and Loading Efficiency
Loading capacity was defined as the amount of subtilosin released from a gram of subtilosin + PVOH nanofiber. Loading efficiency was defined as the ratio of loading capacity to the amount of subtilosin used in the blended solution to produce 1 g of nanofiber.
First, the MIC of subtilosin against M. luteus was determined by well diffusion assay as 60 mg/L. The fourfold dilution of a 20-mg nanofiber per 200 μL ddH2O solution, equivalent to 25 mg fiber/mL ddH2O, was the lowest dilution that retained antimicrobial activity, establishing it as the MIC. The concentration of subtilosin released from 25 mg of fiber in 1 mL of sterile ddH2O was equal to 60 μg/mL. Thus, the loading capacity was calculated to be 2.4 mg subtilosin per gram of fiber.
Since the water acting as a solvent in the subtilosin + PVOH solution was evaporated and dried during the electrospinning and vacuum processes, 1 g of nanofiber was assumed to be primarily composed of PVOH. Since 8.3 mL of 0.12 g/mL PVOH solution was required to form 1 g of fiber, the amount of subtilosin used in the blended solution was 7.6 mg. Thus, loading efficiency was calculated to be 31.6 %. The loss of subtilosin may be the result of the high voltage applied  or the difficulty of subtilosin release from encapsulation.
The safety of subtilosin-based nanofibers for use on human skin tissues was tested using the in vitro EpiDerm model. After 24 h of exposure, the tissues exposed to the negative control (ddH2O) and PVOH fibers retained 100 % viability. Tissues exposed to the PVOH+ subtilosin fibers retained an average of 98.5 % viability in comparison to the negative control. There was zero viability for the tissues exposed to the positive control, Triton-X.
Antimicrobial peptides have shown to act against viruses in many different ways. Most of them exert their activity by directly inactivating the viral particle or inhibiting early events in virus multiplication cycle . For example, the peptides MCP-1 and MCP-2, the human neutrophil peptide (HNP)-1 and brevinin-1 inactivate viral particles [14, 30, 54]. On the other hand, dermaseptins, lactoferricin and the polyphemusin analog T22 seem to interfere at the level of virus–cell interface, thus blocking viral entry [3, 8, 44]. However, other antimicrobial peptides exert their antiviral activity by preventing virus multiplication, as melittin and cecropin which were shown to inhibit the HIV replication by suppressing viral gene expression . Other bacteriocins such as CRL35 and ST4 V, isolated from Enterococcus faecium and Enterococcus mundii respectively, inhibited the HSV-1 and HSV-2 replication in a dose-dependent manner [46, 49, 51]. In this work, we studied subtilosin’s activity against HSV-1. We found that subtilosin at a non-cytotoxic concentration of 200 μg/mL inactivates HSV-1 viral particles. The incubation of viral suspension with 200 μg/mL of subtilosin for 15 min was enough to reduce infectivity by 99 % and after 45 min of treatment the reduction in viral infectivity was higher than 99.99 %. Interestingly, at lower non-virucidal doses, subtilosin inhibits HSV-1 multiplication cycle in a dose-dependent manner. At subtilosin’s concentration of 100 μg/mL, 99 % inhibition of HSV-1 multiplication was achieved. Remarkably, 12 μg/mL subtilosin, a concentration 26-fold lower than the CC50, caused more than 90 % inhibition of HSV-1. In addition, subtilosin was equally active against HSV-1 tk+ and tk-deficient strains and rendered higher SI values than FOS, raising the possibility for using subtilosin against ACV-resistant mutants. It was previously mentioned that antimicrobial peptides generally display virucidal activity or interfere with early events in viral multiplication cycle. Interestingly, subtilosin is able to inactivate viral particles and to inhibit viral multiplication, although in different concentrations. Furthermore, it does not seem to affect any viral multiplication step up to viral protein synthesis as shown by the time of addition of subtilosin experiment and Western blot analysis, suggesting that it may interfere with late stages such as viral formation and release. Taking into account that some studies suggest that subtilosin binds to lipid membranes, that high concentrations of the compound may result in membrane permeabilization  and that viral formation and release are viral steps particularly sensible to membrane structure; future analysis will be conducted to elucidate the precise mechanism underlying the antiviral activity of subtilosin.
Considering the incidence of HSV infections and the issues regarding the clinical approved antiherpetic drugs, such as the reported emergence of ACV, PCV and FOS resistant mutants and that it has been recently shown that the available antiviral treatment of herpetic infections fail to reduce HSV and HIV transmission, because they do not prevent subclinical reactivations of the virus [10, 29], novel treatment methods are of critical importance. One possible alternative is the use of subtilosin in a nanofiber-based formulation. As we demonstrated, subtilosin is readily encapsulated into PVOH fiber mats, which may be directly applied onto areas of infection. Moreover, nanofibers both with and without incorporated subtilosin were demonstrated to be fully safe for human use. Heunis and Dicks  have demonstrated the feasibility of incorporating a variety of different types of antimicrobials into nanofibers, and proven that the fibers retain antimicrobial activity against bacterial pathogens such as methicillin-resistant Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa and Klebsiella pneumoniae.
In this report, the natural, safe antimicrobial peptide subtilosin was shown to have potent virucidal and antiviral activity against HSV-1. Preliminary research on the safety of subtilosin in a novel nanofiber formulation indicates that the formulation has potential for use in human applications. Further research will be conducted to elucidate the molecular mechanism underlying the antiviral activity of subtilosin, to optimize the nanofiber formulation and to assay the activity of subtilosin nanofibers against this and other Herpesviridae family members.
This work was supported by the Universidad de Buenos Aires (UBACYT 20020090200271 and UBACYT 20020110100076 to N.I.T. and M.B.W.); the Agencia Nacional de Promoción Científica y Técnica (ANPCYT) PICT (00985/07 to N.I.T. and M.B.W.); the Bill and Melinda Gates Foundation Grand Challenges Exploration (Round 5, Phase I Grant OPP1025200 to M.L.C., S.X., P.J.S., and K.S.N.); the United States Department of Agriculture National Research Initiative (2009-35603-05071 to J.L. and Q.H.), and the National Institutes of Health/National Institute of Allergy and Infectious Diseases (1R01AI084137 to K.S.N., M.L.C. and P.J.S).