Anionic Polysaccharides From Phototrophic Microorganisms Exhibit Antiviral Activities to Vaccinia Virus

Abbreviations: CaSp: Calcium Spirulan; DENV: Denguevirus; DS: Dextran Sulfate ; EPS: Sulphur-Containing Exopolysaccharide from P. purpureum p; GFP: Green Fluorescent Protein; HCMV: Human Cytomegalovirus; HSV: Herpes Simplex Virus; MW: Molecular Weight; P. I.: Post Infectionem; RSV: Respiratory Syncytial Virus; OPV: Orthopoxvirus; sPS: Sulfated Polysaccharides; TK V3: Sulphur-Containing Exopolysaccharide from A. Platensis; VACV : Vaccinia Virus


Introduction
Vaccinia virus (VACV) belongs to the orthopoxvirus (OPV) genus and is closely related to variola virus, the causative agent of smallpox. The virions are generally enveloped, contain a double-stranded DNA and replicating within the cytoplasm. VACV represents the prototype of the OPV genus and had been successfully used as vaccine against smallpox which was declared eradicated in 1979 [1]. However, due to the severe adverse effects caused in a considerable proportion of vaccinees, vaccination was halted soon after eradication. Today, in the light of a decreasing immunity in the population, the potential abuse of variola virus for bioterrorist purposes is intensively discussed [2]. Therefore, and with regard to the increasing number of zoonotic infections, the development of strategies to counteract OPV infections by introducing new therapeutic and prophylactic measures is widely accepted [3].
Sulfated polysaccharides (sPS), in particular dextran sulfate (DS) and related polyanionic compounds, have been shown to be active against various viruses since the 1960ies [4]. Interest in these compounds increased when first studies on anti-human immunodeficiency virus (HIV) activities were published [5,6]. Most of the studies on the antiviral activity of polyanionic compounds, as well as their mode of action, were performed by De Clercq and co-workers [7]. Today it is known that the crucial factors for antiviral activitybeside the scaffold of a polymer (saccharides, alkyls, aminoacids etc.) and the type of anion (sulfate, sulphone, carboxylate, phosphate) -are mainly the molecule size, its conformation, charge frequency or charge spreading [9]. It is generally assumed that polyanionic compounds interact with positively charged viral membrane glycoproteins of enveloped viruses and thereby inhibit initial viral attachment to negatively charged heparan sulfate proteoglycans on the cellular membrane [8]. Referring to VACV, the interaction between A27L membrane gene product and heparan sulfate was revealed by Chung et al. [10]. Concerning HIV and influenza A virus, further specific interactions between sPS and the viral membrane have been elucidated in detail (reviewed by Witvrouw et al. [7] and Luscher-Mattli [9].
Aside from their favored antiviral activities, sPS exhibit several undesirable side effects, like anticoagulant activity and the induction of severe but reversible thrombocytopenia when applied intravenously [11]. Both effects depend on the compound structure. To overcome these side effects and isolate improved antiviral compounds, we aimed to isolate novel antiviral sPS from phototrophic microorganisms.

Antiviral substances
TK V2 (intracellular product from cyanobacterium A. platensis NIES 39 and presumably containing the already known CaSp), TK V3 (extracellular product from A. platensis) and EPS (extracellular product from the rhodophyt P. purpureum) were produced and structurally described as previously reported [31,32]. Briefly, TK V2 and TK V3 were isolated from culture supernatant by centrifugation of culture broth, lyophilization and dialysis against deionized ultrapure water. EPS extraction was performed using chloroform, methyl alcohol and Butanol, followed by cross-flow ultrafiltration with deionized ultrapure water, and the extract was finally lyophilised. Other extracts isolated according to the same procedure displayed no antiviral activity (data not shown). DS (MW 500,000) was used as antiviral reference.

Cells and viruses
HEp-2 cells (ATCC: CCL-23) were cultured in D-MEM (Gibco, Paisley, UK) supplemented with 5% foetal bovine serum (PAN Biotech GmbH, Aidenbach, Germany) and 1% L-Glutamine (PAA, Pasching, Austria). VERO C1008 cells (ATCC: CRL-1586) were cultured in D-MEM containing 10% foetal bovine serum and 1% L-Glutamine. All cells were maintained in a humidified environment with 5% CO 2 at 37°C, and confluent cells were seeded at the ratio of 1:4 into new culture flasks. VACV strain NYCBOH was obtained from ATCC (VR-1536). Ectromelia virus (ECTV) strain Nu-1 was obtained from Hermann Meyer, Institute of Microbiology, Bundeswehr (Federal Armed Forces), Munich, Germany. Recombinant VACV-expressing green fluorescent protein (VACV-GFP) was obtained from Rafael Blasco, Departamento de Sanidad Animal, Centro de Investigacion en Sanidad Animal-INIA, Madrid, Spain. Virus strains were propagated in HEp-2 cells at a multiplicity of infection (MOI) of 0.25. Infected cells were incubated for 4 days until a distinct cytopathic effect (CPE) was observed. Supernatants containing viral particles and infected cells were harvested by centrifugation (10 min at 1000 x g) after freeze thawing. Supernatants were titrated and stored at -75°C until use.

Cell proliferation tests
5000 HEp-2 cells were seeded as described above in 100µl medium per well in a 96-well plate and grown for 24 hours. Subsequently, medium was replaced by 100µl of antiviral substance dilution which was tested 8-fold in parallel. Substances were incubated at cell growth conditions for 48 hours. Following removal of supernatant and cell treatment with 10µl of trypsin/EDTA (0.08/0.13%) for 5 min, 100µl medium and 10µl WST-1 reagent (Roche, Penzberg, Germany) were added. Reaction was incubated for 4 hours and finally dye extinction was measured at 450 nm.
Infection in 96-well plates 5 x 10 3 cells in 100µl medium per well were incubated in a humidified environment with 5% CO 2 at 37°C. After 24 hours, supernatant was removed and cells were infected and treated with different concentrations of substances from phototrophic microorganisms. A final volume of 100µl per well was adjusted. 24 hours (VACV), 30 hours (ECTV) and 48 hours (VACV-GFP) p. i., respectively, medium was removed and stored at -20°C to perform a plaque titration assay later on. The proportion of infected cells was calculated after immunofluorescence staining.

Plaque assay
1.2-1.5 x 10 6 VERO C1008 cells were seeded in 200µl medium per well of a 24-well plate. Serial dilutions (10 -1 -10 -6 ) of 200µl viruscontaining cell culture supernatants were applied in quadruplicate. Infection occurred at 37°C for 4-5 hours and was terminated by overlaying of 400µl of carboxymethyl cellulose (BDH Ltd., Poole, UK). Cells were cultured for 4 further days, subsequently fixed with 4% formaldehyde for 20 min, stained with 0.1% naphthalene black solution (1.7% w/v Naphthol Blue Black [Sigma-Aldrich, Munich, Germany] and 22.7% w/v sodium acetate in absolute acetic acid) for 15 min and finally washed with water. Plaques within the naphthalene black cell layer were counted per well.

Immunofluorescence staining
Cells were fixed with 200µl formaldehyde (4%) for 20 min, subsequently treated with 70µl Triton X-100 (0.2% in PBS) for 15 min, afterwards incubated with 50µl human anti-pox antibody (Omrigam; Omrix. Biopharmaceutics Ltd, Givat Shmuel, Israel), 1:500 in diluent buffer [PBS, 2% BSA, 0.2% NaN 3 ] for 1 hour and eventually stained with 50µl FITC-conjugated goat anti-human IgG (Caltag Laboratories, Burlingame/CA, USA, 1:50 in diluent buffer containing 200 ng/ml Evan's Blue) for 1 hour. All incubations were performed at room temperature, and between each step cells were thoroughly, but carefully, washed with PBS. Volumes of reagents are indicated per well. Regarding VACV-GFP-infected cells, only counterstaining was performed using 50µl Evan's Blue (200 ng/ml) for 10 min. To determine the ratio of infected cells, representative pictures were taken, and infected and non-infected cells were quantified using the Wright cell imaging facility ImageJ software (National Institutes of Health, Toronto, Canada). The inhibitory substance concentration, causing a 50% decrease of viral replication (IC 50 ), was determined by plotting the ratio of infected cells per well against the substance concentration used and calculated using the equation of a quadratic smoothing function.

In ovo tests
Fertile eggs from hens of the breed White Leghorn were incubated at 37.7°C and a relative humidity of 50-60% under forced air circulation. Before inoculation on day 11, eggs were candled to eliminate infertile eggs and dead embryos. Approximately 50µl of VACV (100 PFU) and 50µl of antiviral compound (5µg/mL) were inoculated simultaneously onto the chorioallantoic membrane (CAM) [29,30]. Embryos were incubated at 37°C, examined daily for signs of mortality and sampled when the positive control (VACV injection only) caused embryos' death. Four days after infection, chick embryo livers and CAMs were removed and homogenized in 1 mL growth medium. Aliquots were processed for standard plaque assay.

Statistics
General calculations were performed with Microsoft Excel. Statistical analysis was performed with SPSS.

Substance-induced cytotoxicity
Initially, the in vitro cytotoxicities of TK V3, EPS as well as DS were determined. For this purpose, HEp-2 cells were treated with each substance for 48 hours and the mitochondrial dehydrogenase activity was quantified using WST-1 reagent which directly correlates to cellular proliferation. DS as antiviral reference did not exhibit any cytotoxic activity at concentrations of up to 50µg/ml. TK V3 from A. platensis and EPS from P. purpureum were not cytotoxic in concentrations of up to 500µg/ml (data not shown).

Antiviral in vitro activity
Antiviral activities of TK V3, TK V2 and EPS against VACV-GFP were determined by estimating the ratio of infected cells 48 hours p. i. Figure 1 shows the decrease of the viral load with the concentrations of TK V3, EPS and DS, respectively. The IC 50 were calculated to be 0.78µg/ml for TK V3, 0.65 µg/ml for EPS and 1.24µg/ ml for DS. TK V3 and EPS did not achieve complete viral inhibition up to concentrations of 10µg/ml, and DS did not achieve complete viral inhibition up to concentrations of 5µg/ml. TK V3 and EPS also highly and TK V2 weakly inhibited replication of unlabeled VACV as determined by plaque reduction assay 24 hours p. i. (data not shown). Table 1 demonstrates that the antiviral activities of the substances derived from phototrophic microorganisms are not restricted to the HEp-2 host cell type and VACV, but are also detectable in VERO C1008 host cells infected with a distant relative of VACV, the ectromelia virus. TK V3 acts comparably against VACV-GFP and ECTV in HEp-2 and VERO C1008 cells. Interestingly, EPS activity against VACV-GFP in VERO C1008 cells (20% vs. positive control of 68%) is reduced compared to HEp-2 cells (54% vs. positive control of 74%). Even if the overall infection rates for ectromelia are low, resulting in marginal differences for extract-treated cells, the data represent a trend that underlines the antiviral effect of the extracts used in VACV infection.
To determine the number of infectious virus particles produced by infected cells in the presence and absence of inhibitory substances, VERO C1008 cells were infected with VACV and the proportion of infected cells analysed 48 hours p.i. In addition, the respective supernatants were used for titre determination by plaque test. The number of primary infected cells as well as the number of infectious virus particles found in the supernatant were decreased by TK V3 and EPS, respectively, in a concentration-dependent manner (data not shown).
Furthermore, the antiviral activities of TK V3 and EPS against VACV-GFP were analysed in relation to the applied MOI. Figure 2 illustrates the strong antiviral activities of TK V3 and EPS even for high amounts of virus. Even using high MOI = 10, broad antiviral activities were observed, with a reduction of about 20% (TK V3) and 30% (EPS), respectively, at concentrations of 10µg/ml of both antiviral substances. Substance activity against ECTV in HEp-2 and VERO C1008 cells also correlated with MOI (data not shown).

Antiviral in ovo activity
The CAM of embryonated eggs was treated with EPS and TK V3 and was simultaneously infected with VACV. Four days p. i. embryo vitality (candling) and viral load of CAM and liver extracts (plaque assay) were determined, respectively. In contrast to untreated and infected embryos, the substance-treated and infected embryos were vital even after 4 days p. i. Figure 3 demonstrates the pronounced decrease of viral load (> 90%) within the CAM of TK V3-or EPS-treated eggs as determined by plaque titration. Viral hepatic replication was decreased by 50% and 70%, respectively. Except for non-infected controls treated with substance only, all embryos were dead after 7 days p. i. These data indicate low substance toxicities.
Of note, in contrast to non-infected and substance-treated embryonated eggs, infected and substance-treated deceased embryonated eggs smelled of hydrogen sulphide and developed green   discolorations. It is conceivable that these effects might occur due to the high sulphur content of substances derived from phototrophic microorganisms which might be reduced to hydrogen sulphide and further react to green iron sulphide in non-vital organisms.

Mode of action
As already mentioned, sPS were shown to act by inhibition of the viral entry process by blocking viral membrane glycoproteins. In order to reveal the mode of antiviral action of the substances derived from phototrophic microorganisms, we performed time-of-addition assays. As demonstrated in Figure 4, substances acted best when antiviral treatment and infection were performed simultaneously (bars B) compared to untreated infections (grey bars, A). Lowest antiviral activities were determined when antiviral treatment had occurred p. i. (bars C). Good antiviral activities were also detected when antiviral treatment was carried out prior to infection (bars D), but substances possessed less antiviral activities if they had been removed before infection (bars E). These results demonstrate that the investigated substances do not interact with cells but with free viral particles, which corresponds to the theory about the mode of antiviral sPS action. This theory is supported by the observation that the virus spread at MOI < 0.1 appeared not to be random via free virus in the cell culture supernatant, but occurred mainly from cell to cell.

Discussion
Our data indicate that anionic exopolysaccharides (EPS) TK V3 from cyanobacterium A. platensis and EPS from rhodophyt P. purpureum possess anti-OPV activities. TK V3 had been shown to be also active against HCMV, HHV-6, HIV-1 and HSV-1 [31,32] and EPS had already been demonstrated to inhibit HCMV and HHV-6 replication [31]. Using recombinant GFP-expressing VACV, we determined IC 50 values against VACV of 0.78µg/ml (TK V3) and 0.65µg/ ml (EPS), respectively, compared to 1.24 µg/ml (DS, MW 500.000). Hence, compared to DS, the antiviral activity is slightly higher for the substances derived from phototrophic microorganisms. The IC 50 determined for DS is significantly lower than that observed by other workgroups, e.g. IC 50 = 40µg/ml [7], which might be explained by differently used virus strains (wild type/GFP-expressing strain) and host cell systems (primary rabbit kidney cells/HEp-2 cells). WST-1 tests did not reveal any inhibition of HEp-2 cell proliferation by TK V3 or EPS up to a concentration of 500µg/ml and up to 50µg/ml for DS. Thus, the selectivity indices (SI) for VACV in HEp-2 cells can be estimated to be SI>640 for TK V3, SI>770 for EPS and SI>40 for DS, respectively. Irrespective of the initial MOI, the substances were also active against VACV and ECTV in HEp-2 and VERO C1008 cells. Even using a high MOI of 10, substances were shown to inhibit the VACV-GFP replication in a concentration-dependent manner. Furthermore, TK V3 and EPS were marginally toxic for embryonated eggs and decreased VACV in ovo replication. Thus, the substances are suitable for in vivo application in further experiments.
As generally accepted, anionic polysaccharides show antiviral activity against enveloped viruses whose initial cellular receptors are anionic (sulfated) carbohydrates. They act via inhibition of viral membrane glycoproteins [7]. Accordingly, the mechanism of the anti-VACV action of TK V3 and EPS may be attributed to the inhibition of the binding of virus to cells (Figure 4). The results showed that the highest antiviral effect can be observed when performing antiviral treatment and infection at the same time. The second highest antiviral effect was detected by preincubation of virus with the TK V3 and EPS. This can be attributed to the concentration changes that occur when preincubated antivirals and virus are added to cells. Like all chemical processes, this is an equilibrium reaction. The concentration changes may lead to a decrease of the anionic polysaccharides on the virus membrane for a short time and may allow better virus interaction with the cells before the reaction reaches an equilibrium. This antiviral mode of action is completely different from that of other known anti-VACV drugs, e.g. Cidofovir, Ribavirin, ST-246 or Gleevec, which opens the possibility of combinatory therapies and makes further investigation into TK V3 and EPS anti-VACV activities advisable. Intracellular anionic polysaccharide TK V2 from A. platensis presumably contains the already known CaSp which was shown to be active against various enveloped viruses in vitro, for example HSV-1, HIV-1, HCMV, measles virus and influenza A virus [26,33]. Surprisingly, TK V2 which is highly active against HIV-1, HSV-1, HCMV and HHV-6 in vitro [32], is not active against VACV-GFP up to a concentration of 250µg/ml and only weakly inhibitory of VACV strain NYCBOH replication. Possibly, disparities in the activities against different viruses as well as different VACV strains might be caused by single specific variations within viral membrane glycoproteins.
Anionic polysaccharides derived from phototrophic microorganisms represent a novel substance group featuring various antiviral activities [7,34]. Here we demonstrated the antiviral activities of two sulphur-containing anionic exopolysaccharides from cyanobacterium A. platensis (TK V3) and from rhodophyt P. purpureum (EPS) against the OPV VACV and ECTV.So far, there have been few reports of anti-OPV polyanionic activities derived from phototrophic microorganisms [8,35].  Production of the compounds in monoseptic cultures of microalgae and cyanobacteria occurred in photo-bioreactors and can be scaled up successfully [36]. Ionic polysaccharides can easily be extracted from culture broth, avoiding extensive separation from and elimination of cellular components. From our experience with this method, the whole process is highly reproducible. These aspects favour further steps towards the development of the compounds as potential therapeutic agents.

Conclusion
Our data suggest that anionic polysaccharides TK V3 and EPS from phototrophic microorganisms are novel inhibitors of OPV and other enveloped viruses. Their in vivo efficacies and bio-availabilities as well as their toxicity profile upon systemic and topical application have to be studied further. However, anionic polysaccharides may be considered for combinatorial treatment with already established antiviral substances.