Evaluation of Cynanchum otophyllum glucan sulfate against human immunodeficiency virus and herpes simplex virus as a microbicide agent.

OBJECTIVE: The root of Cynanchum otophyllum-also known as Qing Yang Sheng-is a traditional ethnical Chinese medicine. The objective of this study was to evaluate in vitro activities and safety of C. otophyllum glucan sulfate (PS20) against Human Immunodeficiency Virus (HIV) and Herpes Simplex Virus (HSV).
MATERIALS AND METHODS: Anti-HIV activity was detected with syncytial formation assay and quantitative P24 Enzyme-Linked Immunosorbent Assay (ELISA). Anti-HSV activity was detected with plaque reduction assay; cytotoxicity was tested with MTT colorimetric assay; and anti-bacterial activity was tested with microdilution method. Anti-HIV mechanism was investigated with fusion inhibition, time of addition, and pretreatment.
RESULTS: The 50% Inhibition Concentration (IC(50)) of PS20 for HIV-1(IIIB), HIV-(Ada-M), HIV-1(Bal), HSV-I, and -II were 0.26 ± 0.02 mM, 0.46 ± 0.02 mM, 0.90 ± 0.04 mM, 3.45 ± 0.85 μM, and 0.70 ± 0.22 mM, respectively. Selectivity Indices (SI) were 653, 50, 39, 85, and 362, respectively. Studies on anti-HIV mechanism of PS20 showed that the target molecule should be the envelope protein. The 50% Cytotoxicity Concentrations (CC(50)) of PS20 for HeLa and ME-180 cell lines and human foreskin fibroblast cells was more than 70 μM. The Minimum Inhibitory Concentration (MIC) for vaginal lactobacilli was more than 1000 μM.
CONCLUSION: PS20 possesses anti-HIV and HSV effect and low cytotoxicity to epithelium cells and vaginal lactobacilli. It may be considered as a potential microbicide agent for further investigation.

Introduction

Globally more than 85% of new Human Immunodeficiency Virus (HIV) infections spread through unprotected intercourse. The comprehensive approaches to reduce HIV infection have been intensively promoted. Male and female condoms are the powerful prophylaxis methods for preventing sexual transmission of HIV. However, their usage depends on the consent of male sexual partner, and is not always a feasible option for most women. Development of a new prevention method that women can control is urgently needed. Microbicides that can be applied inside the vagina and rectum to protect against HIV has been recognized as an attractive method similar to the anti-HIV vaccine for preventing HIV-1 transmission.[1] Natural products are an important resource of leading compounds in development of anti-HIV drugs.[23] Most polysaccharides derived from numerous species have been found to exert a potent in vitro inhibitory effect against HIV-1.[4]

The roots of Cynanchum otophyllum, belonging to family Asclepiadaceae and named Qing Yang Sheng, is a traditional ethnical Chinese medicine used for treating viral infections (viral hepatitis, herpes, and influenza), epilepsy, and rheumatism in South-West China. C. otophyllum glucan sulfate (PS20) has been reported to possess anti-HIV and anti-Herpes Simplex Virus (anti-HSV) activities.[56] In this study, we report in vitro anti-HIV and HSV activities and safety of PS20 as a microbicide agent.

Materials and Methods

Cell Lines, Viruses, Chemicals, and Reagents

The human T-lymphocytic cell lines C8166, H9 and H9/HIV-1IIIB were obtained from the Centralized Facility for AIDS Reagents, National Institute for Biological Standards and Control (NIBSC), UK. The human epithelial carcinoma cell lines ME-180 and HeLa cells were from American Type Culture Collection (ATCC) (Rockville, MD). The primary human foreskin fibroblast cells were prepared as described previously.[7] HIV-1IIIB (CXCR4-tropic, X4), HIV-1Ada-M, and HIV-1Bal (CCR5-tropic, R5) were obtained from the Centralized Facility for AIDS Reagents, NIBSC, UK. All chemicals and reagents except those indicated below were purchased from Sigma (St Louis, MO).

Plant Material

The rhizome of C. otophyllum was bought from Lijiang Pharmaceutical Co, Yunnan Baiyao Group, Yunnan, China. It was identified by Professor Rong-hua Zhao and a voucher specimen (YUTCM, No. 0306151) was deposited in the Herbarium of Yunnan University of Traditional Chinese Medicine, Kunming, China.

Preparations of Extracts

The dried powder of the rhizome of C. otophyllum was extracted with ×10 boiling purified H2O for 1 h, and the aqueous extract was centrifuged at ×2,000 g for 15 min. The supernatant was evaporated. The crude polysaccharide was then extracted by selective ethanol precipitation, ie, discarding the material precipitated at 50% (v/v) ethanol and harvesting the material precipitated at 70% (v/v) ethanol. The product was further deproteinized using a Sevage reagent [CHCl3 :1-butanol = 4:1(v:v)], and applied to Sephadex G75 column and thereafter to an anion exchange DEAE-Sephadex A 50 column for further purification. The purified polysaccharide was finally lyophilized. The polysaccharide content of the purified product was 94% and detected by phenol-sulfuric acid method. For generating polysaccharide sulfate (PS20), 1 g of the lyophilized polysaccharide was suspended in 10 ml anhydrous pyridine at room temperature, and mixed with 15 ml of pyridine-chlorosulfonic acid complex. After 2 h of reaction at 65°C, the PS20 solution was brought to room temperature, and the pH was adjusted to 6.5-7.0 with 2.0 M NaOH. The PS20 solution was isolated from salts by dialysis, and lyophilized. The total polysaccharide content of PS20 was 62.3%, detected by phenol-sulfuric acid method; and SO 3 Na content was 34.2%, measured by indirect spectrophotometry. The chemical structure of PS20 was defined as P-D-(1-4)-glucan sulfate by Professor Mu [Figure 1a].[5]

Figure 1

Chemical structure and anti-HIV-1IIIB mechanism of PS20 (Cynanchum otophyllum glucan sulfate). (a) Chemical structure of Cynanchum otophyllum beta-D-(1-4)-glucan sulfate. (b) Time-of-addition experiment. (c) Inhibition of cell-cell fusion. C8166 cells and H9/HIV-1IIIB cells were co-cultured for 1 day in the presence of serially diluted PS20 or AZT. (d) Dose-response curves of PS20 anti-HIV-1IIIB activity in different pretreatments

Measurement of Anti-HIV Activity Analysis of Syncytial Formation

Anti-HIV-1IIIB activity was detected as described previously.[89] Briefly, serially diluted compounds in RPMI 1640 medium were added to a 96-well plate in triplicate, and then 3 × 104 C8166 cells and 200 50% Cell Culture Infectious Dose (CCID50) of HIV-lIIIB were added to each well. After incubation at 37°C for 72 h, syncytial cells from five different fields of each well were scored under an inverted microscope (×100). The percent inhibition of syncytial formation was calculated by percentage of syncytial number in compound treated culture to that in viral infected control culture.

P24 ELISA

Anti-HIV-lAda-M and HIV-lBal activities were analyzed by measuring supernatant p24 antigen according to the manufacturer's protocol (Perkin-Elmer). Briefly, Peripheral Blood Mononuclear Cells (PBMC) from normal donor were isolated by Ficoll-Hypaque gradient centrifugation, and cultured in the presence of 5 mg/ml Phytohemagglutinin (PHA) and Interleukin-2 (IL-2) (20 U/ml) (Sigma, USA) for 72 h. The non-adherent PBMCs were then removed, and remaining adherent cells were infected with HIV-1Ada-M or HIV-1Bal in the presence and absence of serially diluted PS20. After 7-14 days of culture in IL-2- and PHA-containing complete medium, the virus replicates in supernatant of each well were detected with p24 antigen ELISA. The percent inhibition of p24 antigen production was calculated by percentage of p24 level in PS20-treated culture to that in viral-infected culture without PS20.

Time-of-Addition Experiment

Time-of-addition experiment was carried out as described previously by Tao et al.[8] In brief, C8166 cells were preincubated with HIV-1IIIB [Multiplicity Of Infection (MOI) = 1.0] for 1 h at 4°C to allow HIV-1 to attach to cells without fusion, and subsequently washed three times with ice-cold medium to remove unbound viruses. Cells were then rapidly warmed to 37°C to allow the viral replication cycle to proceed. Compounds at a concentration of 100-fold IC50 were added at different time points (0, 10, 30, 45, 60, 120, 240, 360, 480, and 600 min post infection). Viral p24 antigen production was determined 72 h post infection using P24 ELISA assay.

Cell Fusion Assay

Cell fusion assay was performed as described previously.[810] In the assay, 3 × 104 C8166 cells and 1 × 104 H9/HIV-1IIIB cells per well were mixed and co-cultured in a 96-well plate in the presence or absence of serially diluted compounds PS20 or Azidothymidine (AZT) for 24 h. The number of syncytial cells in each well was counted under a microscope.

PS20 Pretreatment

Pretreatment experiments were carried out as previously described.[811] In the treatment 1, 3 × 104 C8166 cells, 200 CCID50 of HIV-1IIIB and serially diluted PS20 were co-cultured in a 96-well plate at 37°C for 1.5 h, washed three times with RPMI 1640 medium at ×400 g centrifugation, and cultured for another 72 h before syncytia were counted under a microscope. In treatment 2, HIV-1IIIB was pretreated with serially diluted PS20 at 37°C for 1.5 h, washed three times and filtered with Microcon YM-100 (Millipore). PS20-pretreated HIV-1IIIB and 3 × 104 C8166 cells per well in a 96-well plate were co-cultured for 1.5 h, washed three times, and cultured for another 72 h before syncytia were counted. In treatment 3, C8166 cells were pretreated with serially diluted PS20 at 37°C for 3 h, and washed three times. About 200 CCID50 of HIV-1IIIB and 3 × 104 PS20-pretreated C8166 cells per well in a 96-well plate were co-cultured at 37°C for 1.5 h, washed three times, and cultured for another 72 h before syncytia were counted.

Anti-HSV Assay

Anti-HSV activity was detected by plaque reduction assay as described previously.[81213] Briefly, Vero cells were incubated at 37°C for 24 h in a 24-well plate. Confluent Vero cell monolayers were infected with 200 CCID50 of HSV-I or HSV-II in the presence or absence of serially diluted compounds. After adsorption at 37°C for 1 h, the inoculum was aspirated and washed with 1 ml Phosphate Buffered Saline (PBS). Unsolid 1 ml of 0.4% agar in RPMI 1640 complete medium was added. The cultures was incubated for 3 days until plaques appeared, fixed with 2% formalin, and stained with 0.4% crystal violet for 20 min. The plaques were counted under a microscope after removal of the agar overlay. The IC50 was calculated as the compound concentration required reducing virus plaques by 50%.

Measurement of Cytotoxicity

Compound cytotoxicity to cervical epithelial cells, C8166 cells, PBMC, and primary cultured foreskin fibroblast cells was determined by 3,(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay as described previously.[14] Briefly, cells were cultured at 37°C for 44 h in the presence or absence of serially diluted compounds in triplicate. MTT stock solution was added to each well. After 4 h of incubation, cell lysis buffer (20% sodium dodecyl sulfate-N,N-dimethyl formamide (SDS-DMF) were pipetted into each well. After 4 h of incubation, the plates were read on a microplate ELISA reader.

Toxicity Analysis to Human Vaginal Lactobacilli

Lactobacillus delbrueckii was isolated from the vagina of a healthy woman and grown in Lactobacilli MRS broth.[15] To assess the toxicity of the compound to L. delbrueckii growth, a microdilution assay was used. Briefly, lactobacilli were added into a 96-well plate at 106 colony-forming units (CFU)/ml per well in the presence of the serially diluted compound. The Minimum Inhibitory Concentration (MIC) of a compound was defined as the highest concentration of a compound in the well with clear culture. After cultured for 24 h at 37°C in an anaerobic Gas Pakpouch (BioMerieux, Marcy, France), the MIC was determined.

Statistical Analysis

Statistical calculations and curve fitting were performed using GraphPad Prism (GraphPad Software, Inc, San Diego, CA). Data are expressed as mean ± standard deviation (SD) of three independent experiments.

Results

PS20 has Strong Anti-HIV-1 Activity

To determine the anti-HIV-1 activity of PS20, a panel of HIV-1 strains, including R5 HIV-1Ba-L, R5 HIV-1Ada-M, and X4 HIV-1IIIB, was used in this study, and its IC50s are 0.90 ± 0.04 mM, 0.46 ± 0.02 mM, and 0.26 ± 0.02 mM, respectively [Table 1]. These results showed that PS20 potently inhibited the infection of R5 and X4 viruses.

Table 1

In vitro anti-HIV-1 activities of PS20 and AZT

PS20 Demonstrates Anti-HSV Activity

As sexually transmitted HSV may enhance the likelihood of acquiring and/or transmitting HIV infection, plaque reduction assay was applied to test whether PS20 can also suppress the replication of HSV. As shown in Table 2, our data indicated that PS20 can indeed inhibit both HSV-I and HSV-II replication; moreover, its IC50 against HSV-I and HSV-II was 3.45 + 0.85 μM and 0.70 + 0.22 μM, respectively.

Table 2

In vitro anti-HSV activities of PS20 and acyclovir

PS20 Inhibits HIV-1 Infection by Targeting Viral Entry

Few experiments were carried out to elucidate the anti-HIV mechanism of PS20. As shown in the time-of-addition experiments [Figure 1b], PS20 almost completely inhibited HIV-1 replication when added between 0 and 60 min post-HIV infection, and its anti-HIV-1 activity dramatically decreased when added at later time points. As a control, AZT, a reverse transcriptase inhibitor, showed anti-HIV-1 activity when added as late as at 8 h post infection [Figure 1b]. These data suggest that PS20 inhibits HIV-1 infection by targeting viral early lifecycle before reverse transcription.

The interaction between HIV-1 envelope glycoprotein and host cell receptor and co-receptor may induce syncytium formation or cell-cell fusion. A cell fusion assay was used to determine whether the inhibition of syncytium formation by PS20 was due to blockade of interaction between envelope glycoprotein and cellular receptor and co-receptor. As seen in Figure 1c, inhibition of syncytium formation by PS20 was dose-dependent, while a control compound AZT did not show any activity of inhibiting syncytium formation in cell-cell fusion, indicating that PS20 prevented the syncytium formation/cell fusion likely through blocking the interaction between envelope glycoprotein and cell receptor and co-receptor. In addition, to further understand whether PS20 interacts with viral envelope glycoprotein and/or cellular receptor or co-receptor, PS20 pretreatment experiments were subsequently performed [Figure 1d]. The pretreatment of virus plus cells or virus alone with PS20 potently inhibited HIV-1 infection, with an IC50 of 0.60 ± 0.1 μM and 22.41 ± 4.2 μM, respectively. Pretreatment of target cells alone had no anti-HIV-1 activity [Figure 1d]. Taken together, the above experiments provide strong evidences that PS20 inhibits HIV-1 infection likely by interacting with viral envelope glycoprotein but not with cellular receptors or co-receptors.

PS20 has Low Toxicity to Human Genital Cells and Vagina Lactobacilli

For preventing sexual transmission of HIV-1, microbicides should be mostly applied vaginally. Lactobacilli are the dominant member in vaginal flora, playing a key role in vaginal health, and often used for evaluating the safety of microbicide application. L. delbrueckii isolated from normal human vagina was used in the present study. The MIC of PS20 to the bacterium was > 1000 μM, the highest concentration in the culture medium [Table 3]. Human genital cells, including primary human foreskin fibroblast cells, cell lines ME-180 and HeLa were used for evaluating the safety profile of PS20. The CC50 values of PS20 for these cells were more than 70 μM [Table 3]. These results together demonstrate that PS20 has low toxicity to these cells.

Table 3

Toxicity of PS20 to various human cells and vaginal lactobacilli

Discussion

Carbohydrate-derived drugs (such as Tamiflu and zanamivir) have been approved and used in the prevention and treatment of human influenza; sodium hyaluronate, a type of polysaccharide is applied for therapy of osteoarthritis.[16] In the present study, a polysaccharide sulfate, C. otophyllum glucan sulfate (PS20) demonstrated anti-HIV activity against R5 and X4 viruses. Both the time-of-addition experiment and pretreatment experiment showed that PS20 blocked the entry of HIV-1 by binding to envelope glycoprotein. In addition, PS20 demonstrated a good anti-HSV activity. PS20 molecule carries a large amount of sulfate anionic ions and is structurally similar to microbicide candidate Pro2000 under phase 3 clinical trial.[17]

According to recommendations for nonclinical development of topical microbicides,[18] the cytotoxicity of PS20 to human genital cells and vaginal lactobacilli was evaluated. The CC50 of PS20 in ME180, HeLa, and primary foreskin fibroblast cells is more than 70 μM, and the MIC of PS20 to vaginal lactobacilli is more than 1000 μM, these data suggest that PS20 is safe for epithelium cells and vaginal bacterial flora. In conclusion, PS20 has good in vitro anti-HIV and anti-HSV activities and low toxicity to human genital cells and vaginal lactobacilli. As indicated by Lard-Whiteford et al.[18] and Microbicide Development Strategy Working Groups,[19] further investigation of PS20 on the rest of the preclinical assessment will be needed for developing a topical microbicide.