Probiotics & Antimicro. Prot. (2013) 5:26–35 Safety, Formulation and In Vitro Antiviral Activityof the Antimicrobial Peptide Subtilosin AgainstHerpes Simplex Virus Type 1 Nicola´s I. Torres • Katia Sutyak Noll • Shiqi Xu •Ji Li • Qingrong Huang • Patrick J. Sinko •Mo´nica B. Wachsman • Michael L. Chikindas Published online: 13 January 2013Ó Springer Science+Business Media New York 2013 In the present study, the antiviral properties of an agar well diffusion assay. The loading capacity of the the bacteriocin subtilosin against Herpes simplex virus type fibers was 2.4 mg subtilosin/g fiber, and loading efficiency 1 (HSV-1) and the safety and efficacy of a subtilosin-based was 31.6 %. Furthermore, the nanofibers with and without nanofiber formulation were determined. High concentra- incorporated subtilosin were shown to be non-toxic to tions of subtilosin, the cyclical antimicrobial peptide pro- human epidermal tissues using an in vitro human tissue duced by Bacillus amyloliquefaciens, were virucidal model. Taking together these results, subtilosin-based against HSV-1. Interestingly, at non-virucidal concentra- nanofibers should be further studied as a novel alternative tions, subtilosin inhibited wild type HSV-1 and aciclovir- method for treatment and/or control of HSV-1 infection.
resistant mutants in a dose-dependent manner. Althoughthe exact antiviral mechanism is not fully understood, time Subtilosin  Bacteriocin  Antiviral  of addition experiments and western blot analysis suggest that subtilosin does not affect viral multiplication stepsprior to protein synthesis. Poly(vinyl alcohol)-based sub-tilosin nanofibers with a width of 278 nm were produced by the electrospinning process. The retained antimicrobialactivity of the subtilosin-based fibers was determined via Herpes simplex virus types 1 and 2 (HSV-1 and HSV-2,respectively) are serious human pathogens. HSV-1 is oftenassociated with orofacial infections and encephalitis, N. I. Torres  M. B. Wachsman whereas HSV-2 has been found to be the most frequent Laboratorio de Virologı´a, Departamento de Quı´mica Biolo´gica, cause of genital herpes, the major cause of genital ulcers Facultad de Ciencias Exactas y Naturales, Universidad worldwide [, ]. However, in the past decade, several de Buenos Aires, Ciudad Universitaria, Pabello´n 2, studies have identified HSV-1 as the causative agent of at Piso 4, 1428 Buenos Aires, Argentina least half of the new genital herpes episodes [, ]. HSV K. S. Noll  S. Xu  J. Li  Q. Huang  M. L. Chikindas (&) infections persist during the lifetime of the host, generally School of Environmental and Biological Sciences, Rutgers, establishing latent infections []. In addition, it has The State University of New Jersey, 65 Dudley Road, been shown that genital herpes represents a risk factor for New Brunswick, NJ 08901, USAe-mail: [email protected]; the transmission of human immunodeficiency virus (HIV), increasing the risk of contagion by two- to threefold, Nowadays, the agents most commonly used for the Kraft Foods, Inc., 555 South Broadway, Tarrytown,NY 10591, USA management of herpetic infections are the nucleosideanalogs acyclovir (ACV), penciclovir (PCV) and their respective prodrugs valacyclovir and famciclovir. The tri- Department of Pharmaceutics, Ernest Mario School phosphate forms of these analogs selectively inhibit the of Pharmacy, Rutgers, The State University of New Jersey,160 Frelinghuysen Road, Piscataway, NJ 08854, USA viral DNA polymerase (DNA pol) activity. After more than Probiotics & Antimicro. Prot. (2013) 5:26–35 three decades of ACV administration, drug-resistant HSV the antiviral activity of the anionic antimicrobial peptide isolates are rarely found in immunocompetent subjects subtilosin, and its safety for use in a novel nanofiber (0.1–0.7 %) but more frequently recovered from immu- delivery system.
nocompromised patients (4–14 %) , ]. HSVresistance to ACV generally arises as a result of mutationsin viral thymidine kinase and in a lesser number from Materials and Methods mutations in the viral DNA pool [The pyrophos-phate analog foscarnet (FOS) is often used in the man- agement of resistant HSV infections. However, FOSresistance is rising rapidly and its use is reserved to the Subtilosin was isolated and purified from cultures of the cases where other drugs fail because it is more toxic and producer strain, Bacillus amyloliquefaciens KATMIR- less bioavailable , Considering the incidence of A1933, according to the protocols previously described by HSV infections, the reported emergence of ACV, PCV and Sutyak et al. ]. All subtilosin solutions were prepared in FOS resistant mutants and that it has been recently shown sterile ddH2O and maintained at 4 °C until use.
that antiviral treatment of herpetic infection fail to reduce Acyclovir and foscarnet were purchased from Sigma- HSV and HIV transmission , ], there is a need for new Aldrich Chemical Company (St. Louis, MO). Drug stock antiherpetic compounds with different mechanisms of solutions were prepared in dimethyl sulfoxide (DMSO) with a final concentration of 0.1 % and diluted with maintenance Antimicrobial peptides are produced by a variety of medium (MM) consisting of minimum essential medium organisms including insects, fungi, Gram-positive and (MEM) (Gibco, Carlsbad, CA, USA) with 2 % inactivated Gram-negative bacteria, and many of them have been fetal bovine serum.
reported as viral inhibitors ]. For example, cationic Poly(vinyl alcohol) (PVOH, Mw = 61 kDa, Sigma- peptides like a-defensins, hecate and synthetic derivatives Aldrich) was chosen as a carrier polymer due to its high of magainins are active against HSV in vitro replication biocompatibility ]. TrypticaseTM soy broth (TSB), try- [, ] while melittin, a 26 amino acid amphipathic peptide pticaseTM soy agar (TSA), agar and yeast extract were isolated from the venom of the European honeybee Apis purchased from Becton, Dickinson and Company (Sparks, melliphera, inhibits the replication of both HIV and HSV MD, USA). Yeast extract was added into TSB and TSA in [, Several antimicrobial peptides ribosomally the amount of 0.6 % as a nutritional supplement to improve produced by bacteria, collectively called bacteriocins, have microbial growth. The indicator organism Micrococcus been described for the members of the genus Enterococcus luteus ATCC 10240 was chosen due to its high sensitivity []. Wachsman et al. [were the first to describe an to bacteriocins and its widespread used as a bacteriocin- antiviral peptide produced by E. faecium CRL35. The sensitive indicator strain by academia and industry ].
peptide, 3.5 kDa in size, inhibited late stages of the HSV-1and HSV-2 multiplication cycle. In addition, Serkedjieva Cells and Viruses et al. [described a 5.0-kDa peptide, produced by Lac-tobacillus delbrueckii subsp. bulgaricus with activity African green monkey kidney (Vero) cells were grown as against influenza virus.
monolayers in MEM (Gibco) supplemented with 5 % Based on the documented history of bacterially pro- inactivated fetal bovine serum and 50 lg/mL of gentamycin.
duced peptides, we chose to investigate the novel bacte- HSV-1 strain F (tk?) was obtained from the American riocin subtilosin, for such characteristics. Subtilosin is a Type Culture Collection (Rockville, MD, USA). HSV-1 3.4-kDa, cyclical peptide ] produced by both Bacillus strain Field (tk deficient) was kindly provided by subtilis and B. amyloliquefaciens [] with proven Dr. G. Andrei (Rega Institute, Leuven, Belgium). Virus antimicrobial activity against a variety of human patho- stocks were prepared in Vero cells.
gens, including the bacterial vaginosis-associated Gard-nerella vaginalis. Sutyak et al. [further established that Cell Cytotoxicity Assays subtilosin has potent spermicidal activity and is non-toxicto human vaginal tissues. Due to its range of activities and To determine the cytotoxic concentrations of the com- its safety for human use, subtilosin was an obvious target pounds, confluent monolayers of Vero cells were grown in for investigation into its antiviral properties. Furthermore, tissue culture plates for 48 h and exposed to various con- we have chosen to examine the feasibility of subtilosin's centrations of the compounds. After 48 h of incubation, incorporation into a nanofiber-based delivery system, cell viability was examined by the ability of the cells to considering the successful use of this method with other cleave the tetrazolium salt MTT (3-(4,5-dimethylthiazol- bacteriocins [, ]. Here, we report for the first time 2yl)-2,5-diphenyl tetrazolium bromide) (Sigma-Aldrich) Probiotics & Antimicro. Prot. (2013) 5:26–35 with the mitochondrial enzyme succinate dehydrogenase, extracellular virus infectivity was quantified by plaque yielding a quantifiable blue product (formazan) [The assay on Vero cells.
precise MTT procedure has been previously described, ]. The CC50 was defined as the compound concen- tration that reduced Vero cells viability by 50 %, calculatedby regression analysis.
Monolayers of confluent Vero cells were infected withHSV-1 (m.o.i. = 1) or left non-infected (control), and cultures were incubated at 37 °C for 1 h to allow virusinternalization. After removing the inoculum, monolayers HSV-1 tk? and tk-deficient preparations were incubated were covered with MM alone or containing different con- with 200 lg/mL of subtilosin or with MM (control) for 0, centrations of subtilosin, and incubated at 37 °C for 24 h.
15, 45 and 90 min at 37 °C. Then, aliquots were taken and Immediately after, cells in duplicate wells were lysed with diluted in a serial fashion in MM in order to infect Vero Laemmli sample buffer (Bio-Rad, CA, USA) with 5 % cell monolayers grown in 24-well culture plates to test b-mercaptoethanol. Samples were heated for 2 min in virus survival using a plaque assay. Plaques were counted boiling water before loading onto 10 % acrylamide gels.
exclusively in wells where subtilosin concentration was Following electrophoresis, the resolved proteins were less than 0.2 lg/mL, a concentration 50-fold lower than the transferred to a PVDF membrane (Perkin Elmer Life Sci- concentration needed to inhibit virus multiplication in ences, Inc., Waltham, MA, USA) in a dry system (LKB 50 % compared to untreated infected cultures).
Multiphor II, Pharmacia, Sweden). Glycoprotein gD was To assay the effect of subtilosin over the viral particles revealed with mouse anti-gD (Santa Cruz Biotechnology at antiviral concentrations, HSV-1 was incubated with Inc., Santa Cruz, CA, USA) and a peroxidase antimouse subtilosin at concentrations ranging from 6 to 100 lg/mL immunoglobulin G (Promega, Madison, WI, USA) as or MM for 60 min at 37 °C. Then, aliquots were taken and secondary antibody. ERK1 used as a loading control was Vero cells were infected to test virus survival using a revealed with rabbit anti-ERK1 (Santa Cruz Biotechnology plaque assay.
Inc.) and a peroxidase antirabbit immunoglobulin G (Pro-mega) as secondary antibody. Chemiluminescence was Virus Yield Reduction Assay visualized using a chemiluminescence kit (Perkin ElmerLife Sciences).
Antiviral activity was evaluated by the virus yield reduc-tion assay. For that purpose, Vero cells grown in 24-well Preparation of Antimicrobial Fibers culture plates for 48 h were individually infected with thetwo strains of HSV-1 at a multiplicity of infection (m.o.i.) PVOH (1.5 g) was dissolved in 10 mL Millipore water and of 1 PFU/cell. After 1 h of adsorption at 37 °C, the heated at 80 °C for 8 h. After cooling, the PVOH solution infective viral particles were removed and cells were was blended with a purified 4.6 mg/mL subtilosin solution covered with MM (control) or MM containing various for a final concentration of 0.12 g/mL PVOH and 0.9 mg/ concentrations of subtilosin. After 24 h of incubation at mL subtilosin. The PVOH? subtilosin solution was then 37 °C, the supernatants were harvested and cell-free virus transferred into a 10-mL plastic syringe with a blunt-end yields were determined by a plaque assay on Vero cell metal needle (O.D. 1.27 mm). The syringe filled with stock monolayers. The antiviral activity was expressed as the solution was mounted onto a syringe pump (New Era Pump EC50 (50 % effective concentration), that is, the compound Systems Inc., Farmingdale, NY, USA). Figure displays concentration required to reduce plaque formation by 50 % the schematic diagram of the electrospinning facility. A after 72 h compared with the untreated infected cultures.
high-voltage external electric field was applied to the The EC50 values were calculated by plotting percentages of polymer solution under which a Taylor cone [formed at the needle end. As electrostatic repulsion on the liquid surface surpassed the surface tension, a polymer liquid jetwas then erupted from the Taylor cone. After experiencing Effect of Time of Addition of Subtilosin on HSV-1 jet whipping ] and solidification, polymeric fibers were deposited onto the grounded aluminum foil collector. Thewhole process was maintained at the condition of 15 kV Subtilosin at a concentration of 50 lg/mL was added to for voltage supply, 0.2 mL/h for feeding rate and 10 cm confluent monolayers of Vero cells either at 1, 3, 5 or 8 h for needle-to-collector distance. As a negative control, post-infection (p.i.) with HSV-1 (m.o.i. = 1). Cultures 0.12 g/mL PVOH solution without subtilosin was electrospun were further incubated at 37 °C until 24 h p.i. and by the same procedure as above. After 5 h of processing, the

Probiotics & Antimicro. Prot. (2013) 5:26–35 fibrous mat was directly peeled off from the grounded milligrams of PVOH fibers was also dissolved as a nega- collector covered with aluminum foil. Fibers were then tive control to eliminate the possibility of PVOH inhibi- dried under vacuum for 4 h prior to further use.
tion. An overnight culture of M. luteus in TSB (c. 109CFU/mL) was diluted 100-fold by blending with *55 °Csoft agar (TSB supplemented with 7 g/L agar). Then, 4 mL Morphology Characterization of Antimicrobial of the soft agar containing M. luteus was transferred onto the surface of a TSA plate. After 30 min of solidification, asterile glass pipette (approx. 5 mm diameter) was used to Surface images of the antimicrobial PVOH fibers with and create wells in the agar plate. Fifty microliters of purified without subtilosin were collected with a commercial subtilosin, PVOH? subtilosin fibers (24 h water solution), Nanoscope IIIa Multi-Mode AFM (Veeco Instruments, negative control and their double dilutions were injected Plainview, NY, USA) equipped with a J scanner, which into the wells in duplicate. After 24 h incubation at 37 °C, was operated in tapping mode using a silicon cantilever.
minimum inhibitory concentration (MIC) was defined as The scanned images were obtained at the scan size of the lowest concentration to form a visible inhibition circle 5 9 5 lm and 50 9 50 lm. The scan frequency was set at on the M. luteus growth layer. Tests were repeated three 0.1 Hz. The section analysis embedded in the software Nanoscope 3.0 was utilized to calculate the fiber diameterdistribution.
Skin Cytotoxicity Assays Assessment of Subtilosin Loading Capacity The safety of nanofiber mats with and without incorporated and Efficiency Via Well Diffusion Assay subtilosin for use on human skin was tested on a cultured,reconstituted human epidermal model. The EpiDerm The evaluation of subtilosin loading capacity and effi- (NHEK) tissue model (MatTek Corporation, Ashland, MA, ciency was conducted by agar plate well diffusion inhibi- USA) consists of normal, human-derived epidermal kerat- tion assays [All nanofiber mats were sterilized by inocytes that are cultured to form a highly differentiated direct exposure to UV light (257 nm) in a biosafety cabinet model of the epidermis, and allows for in vitro testing of a (Forma Class II, A2 Biological Safety Cabinet, Thermo compound's irritancy and cytotoxicity. All EpiDerm tis- Fisher Scientific, Pittsburgh, PA, USA) for 10 min per side sues were maintained at 4 °C upon receiving and prior to prior to use. Twenty milligrams of PVOH? subtilosin use. The standard method developed by MatTek, and later fibers was cut with sterile tweezers, immersed in 200 lL outlined by Frasch et al. ], was followed. Briefly, all sterile ddH2O and kept at 4 °C for 24 h. Twenty tissues were preincubated in room temperature culturemedium 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 ina biosafety cabinet for 10 min per side, and then cut to sizeusing a sterilized size 5 cork borer (Thermo Fisher Scien-tific). These nanofiber ‘‘discs'' were applied onto the sur-face of the pre-conditioned tissues with light pressure toensure that the disc came in full contact with the tissue.
Positive (50 ll Triton-X) and negative (50 ll sterileddH2O) controls and all test compounds were assayed intriplicate. The tissues were exposed to the test compounds Fig. 1 Schematic diagram of nanofiber formation through the process for 24 h at 37 °C with 5 % CO2. After exposure, the tissues of electrospinning. A PVOH and subtilosin mixed solution (final were washed with PBS, transferred into MTT assay buffer concentrations: 0.12 g/mL PVOH and 0.9 mg/mL subtilosin) wasloaded into a plastic syringe, which was then mounted onto a syringe and further incubated for 3 h at 37 °C with 5 % CO2. After pump. During the electrospinning process, a liquid polymer jet is a second wash with PBS, the tissues were submerged in ejected at the end of the needle when a high-voltage electric field is isopropanol and held at room temperature overnight to applied to the polymer solution. The liquid polymer undergoes jet allow for extraction of the formazan dye from the tissues.
whipping and solidification, and the resultant polymer fibers are thendeposited onto the grounded aluminum collector surface. The whole The following day, the optical density of the formazan– process was maintained at the condition of 15 kV for voltage supply, isopropanol mixture from each tissue was measured in 0.2 mL/h for feeding rate and 10 cm for needle-to-collector distance.
triplicate at 570 nm. Cell viability was then calculated as a As a negative control, a 0.12-g/mL PVOH solution without subtilosin percentage of the average negative control tissues. Tests was electrospun by the same procedure as above. Fibers werecollected from the grounded surface after 5 h processing were performed at least twice.

Probiotics & Antimicro. Prot. (2013) 5:26–35 and because we found an unusually high inhibition at200 lg/mL, a virucidal assay was performed to determine Antiviral Activity: Subtilosin Inhibits HSV-1 whether subtilosin at this concentration produces a direct effect on the viral particle. To that purpose, viral suspen-sions were incubated with subtilosin 200 lg/mL for 15, 45 To assess the antiviral activity of subtilosin, we first and 90 min. Then, aliquots were taken, diluted in MM and determined the subtilosin's concentration that reduced cell used to infect Vero cell monolayers to test virus survival viability to 50 % as compared to the control (CC50) by using a plaque assay. The incubation of viral suspension using the MTT method. A virus yield inhibition assay was with subtilosin for 15 min at 37 °C resulted in an inhibition then performed to determine cell-free infectivity and the of the viral titer of 99 %. After 45 min of treatment, the selectivity index (SI), that is, the relationship between CC50 reduction in viral infectivity was higher than 99.99 %, and EC50 values. For comparative purposes, the inhibitory indicating that subtilosin is a potent virucidal agent active effect of ACV and FOS was also assayed. Between 12 and against tk-positive and tk-deficient (data not shown) HSV-1 100 lg/mL, subtilosin inhibited HSV-1 replication in a dose-dependent manner (Fig. ) and a remarkably strong To establish if subtilosin's inhibitory activity at low concentration was due to its direct effect on the viral par- 200 lg/mL subtilosin. At a concentration of 100 lg/mL, ticle, we performed a similar experiment at concentrations subtilosin inhibited replication in a 99 % and at 12 lg/mL, below 200 lg/mL. Treatment of viral suspensions for 1 h a concentration 26-fold lower than the CC50, subtilosin still at 37 °C with 6 to 100 lg/mL of subtilosin did not reduce inhibited approximately 90 % of HSV-1. A 50 % of inhi- viral infectivity (Fig. ), indicating that the reduction in the bition was found with subtilosin at a concentration of viral titers at these concentrations (as seen in Fig. ) were 9.6 lg/mL. Lower concentrations of the compound did not only due to antiviral activity.
display considerable antiviral activity. The SI value for In order to determine whether subtilosin interferes with subtilosin against HSV-1 tk? was 33, while the values for the initial steps of the viral multiplication cycle at a non- ACV and FOS were 957 and 14, respectively. On the other virucidal concentration, a time of addition experiment was hand, subtilosin rendered a SI value of 31 against tk-defi- performed. For that purpose, 50 lg/mL of subtilosin was cient HSV-1, while the SI value for ACV was only 10.3 added to HSV-1 infected Vero cells at different times after infection and at 24 h p.i., cell-free infectivity was deter- Since it has been reported that microbicidal peptides mined. As it is depicted in Fig. , when subtilosin was such as defensins may inactivate HSV on direct contact present starting from 1, 3, 5 or 8 h p.i., virus yields werereduced by approximately 2 log, suggesting that subtilo-sin's antiviral activity is not due to an inhibition of earlystages of viral multiplication.
To further characterize the inhibitory action of subtilo- sin, Vero cells were infected with HSV-1 and treated withdifferent concentrations of the peptide ranging from 25 to100 lg/mL. Afterward, a polyacrylamide gel electropho-resis followed by a Western blot analysis was performed todetermine whether subtilosin reduces viral protein synthe-sis. As shown in Fig. viral protein gD synthesis was notaffected by the different concentrations used in thisexperiment, suggesting that the antiviral mechanism ofsubtilosin does not interfere with this or previous steps inthe viral multiplication cycle.
Fig. 2 Subtilosin inhibits HSV-1 replication in a dose-dependentmanner. Vero cells infected with HSV-1 tk? (m.o.i. = 1) were Nanofiber Morphology incubated with different concentrations of subtilosin for 24 h. Then,supernatants were harvested and cell-free virus yields were deter-mined by a plaque assay. Values are mean ± SD of the log reduction Figure shows the tapping mode-atomic force microscopy in HSV-1 titers for each concentration compared to no drug. Data are (TP-AFM) images of antimicrobial PVOH fibers. Rela- from three independent experiments, where each titration was carried tively straight fibers were formed from the pure PVOH out in duplicate. Percentages of inhibition of the different treatmentsare expressed in each column solution (Fig. a), and an individual PVOH fiber's

Probiotics & Antimicro. Prot. (2013) 5:26–35 Table 1 Cytotoxicity and antiviral activity of subtilosin, aciclovir (ACV) and foscarnet (FOS) against HSV-1 Structural formula Phosphonoformic acid trisodium salt CC50: compound concentration required to reduce cell viability by 50 %, as determined by the MTT method EC50: compound concentration required to reduce virus yield by 50 % SI (selectivity index): ratio CC50/EC50 * Structural formula of subtilosin reproduced from Marx et al. [] Fig. 4 Low concentrations of subtilosin do not produce a virucidaleffect against HSV-1. HSV-1 tk? was incubated with subtilosin atconcentrations ranging from 6 to 100 lg/mL or MM for 60 min at37 °C to assay virucidal activity of subtilosin at concentrations thatdisplay antiviral activity. Then, aliquots were taken and Vero cells Fig. 3 Subtilosin inactivates HSV-1 particles at a concentration of were infected to test virus survival using a plaque assay. Values are 200 lg/mL. HSV-1 tk? and tk deficient (data not shown) were mean ± SD from three independent experiments, where each titration incubated with subtilosin at a concentration of 200 lg/mL or with was carried out in duplicate MM for 0, 15, 45 and 90 min at 37 °C. Then, aliquots were taken andVero cells were infected to test virus survival using a plaque assay.
Values are means of HSV-1 tk? infectivity ± SD from three An individual PVOH? subtilosin fiber diameter was independent experiments, where each titration was carried out in determined to be 278 nm (Fig. d). This phenomenon is likely due to subtilosin's interaction with PVOH, resulting diameter was 567 nm (Fig. as calculated by the section in weakened entanglement between the PVOH polymer analysis embedded in the Nanoscope software. Addition of chains, shifting the critical entanglement concentration of subtilosin affected the fiber's morphology, resulting in PVOH solution toward a higher value and subsequently thinner fibers from the electrospinning process (Fig. c).
reducing fiber diameter.

Probiotics & Antimicro. Prot. (2013) 5:26–35 equal to 60 lg/mL. Thus, the loading capacity was calcu-lated to be 2.4 mg subtilosin per gram of fiber.
Since the water acting as a solvent in the subtilo- sin ? PVOH solution was evaporated and dried during theelectrospinning and vacuum processes, 1 g of nanofiberwas assumed to be primarily composed of PVOH. Since8.3 mL of 0.12 g/mL PVOH solution was required to form1 g of fiber, the amount of subtilosin used in the blendedsolution was 7.6 mg. Thus, loading efficiency was calcu-lated to be 31.6 %. The loss of subtilosin may be the resultof the high voltage applied ] or the difficulty of sub-tilosin release from encapsulation.
Nanofibers Cytotoxicity Fig. 5 Effect of time of subtilosin addition on HSV-1 production.
Vero cells infected with HSV-1 (m.o.i. = 1) were incubated withMM (VC) or MM containing subtilosin 50 lg/mL added either at 1, 3, The safety of subtilosin-based nanofibers for use on human 5 or 8 h p.i. Cultures were further incubated at 37 °C until 24 h p.i skin tissues was tested using the in vitro EpiDerm model.
Then, cell-free virus yields were determined by plaque assay. Values After 24 h of exposure, the tissues exposed to the negative are mean ± SD from three independent experiments, where each titration was carried out in duplicate 2O) and PVOH fibers retained 100 % viability.
Tissues exposed to the PVOH? subtilosin fibers retainedan average of 98.5 % viability in comparison to the neg-ative control. There was zero viability for the tissuesexposed to the positive control, Triton-X.
Antimicrobial peptides have shown to act against viruses in Fig. 6 Viral protein gD synthesis is not affected by subtilosin many different ways. Most of them exert their activity by treatment. Vero cells infected (Lanes 2–5) or not (Lane 1) with HSV- directly inactivating the viral particle or inhibiting early 1 tk? at an m.o.i. of 1 were incubated at 37 °C for 1 h to allow events in virus multiplication cycle For example, the internalization. Afterward, the inoculum was discarded and themonolayers were covered with MM or MM containing subtilosin at peptides MCP-1 and MCP-2, the human neutrophil peptide different concentrations (Lane 2: virus control; lane 3: 25 lg/mL; (HNP)-1 and brevinin-1 inactivate viral particles [, lane 4: 50 lg/mL; lane 5: 100 lg/mL) and incubated at 37 °C. At . On the other hand, dermaseptins, lactoferricin and the 24 h p.i. cell lysates were collected and subjected to Western blotting polyphemusin analog T22 seem to interfere at the level of and glycoprotein gD was analyzed with an antibody against gD. Toverify equal loading, Western blotting was performed with an virus–cell interface, thus blocking viral entry [, antibody to ERK1. Data are from one of two different experiments However, other antimicrobial peptides exert their antiviralactivity by preventing virus multiplication, as melittin andcecropin which were shown to inhibit the HIV replication by Loading Capacity and Loading Efficiency suppressing viral gene expression Other bacteriocinssuch as CRL35 and ST4 V, isolated from Enterococcus Loading capacity was defined as the amount of subtilosin faecium and Enterococcus mundii respectively, inhibited the released from a gram of subtilosin ? PVOH nanofiber.
HSV-1 and HSV-2 replication in a dose-dependent manner Loading efficiency was defined as the ratio of loading , ]. In this work, we studied subtilosin's activity capacity to the amount of subtilosin used in the blended against HSV-1. We found that subtilosin at a non-cytotoxic solution to produce 1 g of nanofiber.
concentration of 200 lg/mL inactivates HSV-1 viral parti- First, the MIC of subtilosin against M. luteus was cles. The incubation of viral suspension with 200 lg/mL of determined by well diffusion assay as 60 mg/L. The four- subtilosin for 15 min was enough to reduce infectivity by fold dilution of a 20-mg nanofiber per 200 lL ddH2O 99 % and after 45 min of treatment the reduction in viral solution, equivalent to 25 mg fiber/mL ddH2O, was the infectivity was higher than 99.99 %. Interestingly, at lower lowest dilution that retained antimicrobial activity, estab- non-virucidal doses, subtilosin inhibits HSV-1 multiplica- lishing it as the MIC. The concentration of subtilosin tion cycle in a dose-dependent manner. At subtilosin's released from 25 mg of fiber in 1 mL of sterile ddH2O was concentration of 100 lg/mL, 99 % inhibition of HSV-1

Probiotics & Antimicro. Prot. (2013) 5:26–35 Fig. 7 Surface morphology ofpolyvinyl alcohol-basednanofibers with and withoutincorporated subtilosin. Tappingmode-atomic force microscopy(AFM) images of PVOHelectrospun fibers includinga 50 lm 9 50 lm height imageof PVOH electrospun fibers;b 5 lm 9 5 lm height imageof PVOH electrospun fibersc 50 lm 9 50 lm height imageof PVOH ? subtilosinelectrospun fibers;d 5 lm 9 5 lm height imageof PVOH ? subtilosinelectrospun fiber multiplication was achieved. Remarkably, 12 lg/mL sub- reduce HSV and HIV transmission, because they do not tilosin, a concentration 26-fold lower than the CC50, caused prevent subclinical reactivations of the virus novel more than 90 % inhibition of HSV-1. In addition, subtilosin treatment methods are of critical importance. One possible was equally active against HSV-1 tk? and tk-deficient alternative is the use of subtilosin in a nanofiber-based for- strains and rendered higher SI values than FOS, raising the mulation. As we demonstrated, subtilosin is readily encap- possibility for using subtilosin against ACV-resistant sulated into PVOH fiber mats, which may be directly applied mutants. It was previously mentioned that antimicrobial onto areas of infection. Moreover, nanofibers both with and peptides generally display virucidal activity or interfere with without incorporated subtilosin were demonstrated to be fully early events in viral multiplication cycle. Interestingly, safe for human use. Heunis and Dicks have demonstrated subtilosin is able to inactivate viral particles and to inhibit the feasibility of incorporating a variety of different types of viral multiplication, although in different concentrations.
antimicrobials into nanofibers, and proven that the fibers Furthermore, it does not seem to affect any viral multipli- retain antimicrobial activity against bacterial pathogens such cation step up to viral protein synthesis as shown by the time as methicillin-resistant Staphylococcus aureus, Escherichia of addition of subtilosin experiment and Western blot coli, Pseudomonas aeruginosa and Klebsiella pneumoniae.
analysis, suggesting that it may interfere with late stagessuch as viral formation and release. Taking into account thatsome studies suggest that subtilosin binds to lipid mem- branes, that high concentrations of the compound may resultin membrane permeabilization [] and that viral formation In this report, the natural, safe antimicrobial peptide sub- and release are viral steps particularly sensible to membrane tilosin was shown to have potent virucidal and antiviral structure; future analysis will be conducted to elucidate the activity against HSV-1. Preliminary research on the safety precise mechanism underlying the antiviral activity of of subtilosin in a novel nanofiber formulation indicates that the formulation has potential for use in human applications.
Considering the incidence of HSV infections and the Further research will be conducted to elucidate the issues regarding the clinical approved antiherpetic drugs, molecular mechanism underlying the antiviral activity of such as the reported emergence of ACV, PCV and FOS subtilosin, to optimize the nanofiber formulation and to resistant mutants and that it has been recently shown that the assay the activity of subtilosin nanofibers against this and available antiviral treatment of herpetic infections fail to other Herpesviridae family members.
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ST4 V isolated from soybeans. Int J Antimicrob Agents 25: J Appl Phys 90:4836–4846 54. Yasin B, Pang M, Turner J et al (2000) Evaluation of the inac- 47. van Velzen M, van Loenen F, Meesters R et al (2012) Latent tivation of infectious herpes simplex virus by host-defense pep- acyclovir-resistant herpes simplex virus type 1 in trigeminal tides. Eur J Clin Microbiol Dis 19:187–194 Probiotics & Antimicro. Prot. (2013) 5:36 Erratum to: Safety, Formulation and In Vitro Antiviral Activityof the Antimicrobial Peptide Subtilosin Against Herpes SimplexVirus Type 1 Nicola´s I. Torres • Katia Sutyak Noll • Shiqi Xu •Ji Li • Qingrong Huang • Patrick J. Sinko •Mo´nica B. Wachsman • Michael L. Chikindas Published online: 16 February 2013Ó Springer Science+Business Media New York 2013 Erratum to: Probiotics & Antimicro. Prot.
Transparency declaration: Nicola´s I. Torres, Katia Sut- yak Noll and Shiqi Xu equally contributed to the researchand to this manuscript. Mo´nica B. Wachsman, Michael L.
In the original version of this paper, the article note Chikindas and Qingrong Huang are the manuscript's senior (Transparency declaration) was missed out unfortunately, authors in the areas of virology, bacteriology and formu- which should have come in the front page of the article.
lation chemistry, correspondingly.
The missed article note is as follows.
The online version of the original article can be found under doi: N. I. Torres  M. B. WachsmanLaboratorio de Virologı´a, Departamento de Quı´mica Biolo´gica,Facultad de Ciencias Exactas y Naturales, Universidad deBuenos Aires, Ciudad Universitaria, Pabello´n 2, Piso 4, 1428Buenos Aires, Argentina K. Sutyak Noll  S. Xu  J. Li  Q. Huang M. L. Chikindas (&)School of Environmental and Biological Sciences,Rutgers, The State University of New Jersey,65 Dudley Road, New Brunswick, NJ 08901, USAe-mail: [email protected];[email protected] K. Sutyak NollKraft Foods, Inc., 555 South Broadway, Tarrytown, NY 10591,USA P. J. SinkoDepartment of Pharmaceutics, Ernest Mario School ofPharmacy, Rutgers, The State University of New Jersey,160 Frelinghuysen Road, Piscataway, NJ 08854, USA


Importancia del agua

Elementalwatson "la" revista ………………. Revista cuatrimestral de divulgación "En el conocimiento y la cultura no Año 4, número 11 sólo hay esfuerzo sino también placer. Llega un punto donde estudiar, o investigar, o Universidad de Buenos Aires Ciclo Básico Común (CBC) aprender, ya no es un esfuerzo y es puro


The diabetic foot infection can lead to tissue necrosis and amputation. Diabetes is the leading non-traumatic cause of major amputation of the lower limbs. Miles J levyJonathan ValabhjiQ2 NeuropathyNerve damage due to disease of the vasa nervorum results in a ‘glove and stocking' sensorimotor peripheral neuropathy that may progress proximally. The motor component results in dener-vation of the small muscles of the foot, leading to: • hyperextension at the metatarsophalangeal joints