Gen. Pharmac. Vol. 29, No. 4, pp. 497511, 1997 Copyright 1997 Elsevier Science Inc.

. Printed in the USA.

ISSN 0306-3623/97 $17.00 .00 PII S0306-3623(96)00563-0 All rights reserved

REVIEW

Sulfated Polysaccharides Extracted from Sea Algae as Potential Antiviral Drugs

M. Witvrouw* and E. De Clercq
Rega Institute for Medical Research, Katholieke Universiteit Leuven, Minderbroedersstraat 10, B-3000 Leuven, Belgium ABSTRACT. The inhibitory effects of polyanionic substances on the replication of herpes simplex virus (HSV) and other viruses were reported almost four decades ago. However, these observations did not generate much interest, because the antiviral action of the compounds was considered to be largely nonspecific. Shortly after the identification of human immunodeficiency virus (HIV) as the causative agent of the acquired immune deficiency syndrome (AIDS) in 1984, heparin and other sulfated polysaccharides were found to be potent and selective inhibitors of HIV-1 replication in cell culture. Since 1988, the activity spectrum of the sulfated polysaccharides has been shown to extend to various enveloped viruses, including viruses that emerge as opportunistic pathogens (e.g., herpes simplex virus [HSV] and cytomegalovirus [CMV]) in immunosuppressed (e.g., AIDS) patients. As potential antiHIV drug candidates, sulfated polysaccharides offer a number of promising features. They are able to block HIV replication in cell culture at concentrations as low as 0.1 to 0.01 g ml1 without toxicity to the host cells at concentrations up to 2.5 mg ml1. We noted that some polysulfates show a differential inhibitory activity against different HIV strains, suggesting that marked differences exist in the target molecules with which polysulfates interact. They not only inhibit the cytopathic effect of HIV, but also prevent HIV-induced syncytium (giant cell) formation. Furthermore, experiments carried out with dextran sulfate samples of increasing molecular weight and with sulfated cyclodextrins of different degrees of sulfation have shown that antiviral activity increases with increasing molecular weight and degree of sulfation. A sugar backbone is not strictly needed for the anti-HIV activity of polysulfates because sulfated polymers composed of a carboncarbon backbone have also proved to be highly efficient anti-HIV agents in vitro. Other, yet to be defined, structural features may also play an important role. Sulfated polysaccharides may act synergistically with other anti-HIV drugs (e.g., azidothymidine [AZT]). They are known to lead very slowly to virus-drug resistance development and they show activity against HIV mutants that have become resistant to reverse transcriptase inhibitors, such as AZT, tetrahydro-imidazo [4,5,1-jk] [1,4]-benzodiazepin-2(1H)-thione (TIBO) and others. From studies on their mechanism of action we concluded that polysulfates exert their anti-HIV activity by shielding off the positively charged sites in the V3 loop of the viral envelope glycoprotein (gp120). The V3 loop is necessary for virus attachment to cell surface heparan sulfate, a primary binding site, before more specific binding occurs to the CD4 receptor of CD4 cells. This general mechanism also explains the broad antiviral activity of polysulfates against enveloped viruses. Variations in the viral envelope glycoprotein region may result in differences in the susceptibility of different enveloped viruses to compounds that interact with their envelope glycoproteins. The efficacy of polysulfates in the therapy and/or prophylaxis of retroviral infections and opportunistic infections remains to be demonstrated both in animal models and humans. It is important to consider not only treatment of patients who are already infected with HIV, but also prophylaxis and protection from HIV and/or other virus infections. Because (i) sexual transmission is responsible for the large majority of HIV infections worldwide; (ii) this transmission is mostly mediated via mononuclear cells that infect epithelial cells of the genital tract; and because (iii) polysulfates effectively inhibit cellcell adhesion, polysulfates may be considered as potentially effective in a vaginal formulation to protect against HIV infection. gen pharmac 29;4:497511, 1997. 1997 Elsevier Science Inc. INTRODUCTION Algae are difficult to define, as they include a wide variety of plants that range from microscopic, unicellular organisms to brown seaweeds, which may extend to 100 or 150 feet; their only common characteristic is their photosynthetic ability. They are grouped together for convenience into six main classes (Fogg, 1953) largely on the basis of their color. The principal photosynthetic pigment appears to be the same chlorophyll a as that found in higher plants,
*To whom correspondence should be addressed. Received 20 September 1996; accepted 5 November 1996.

but the accessory pigments differ quantitatively and qualitatively from one class to another. The morphology of the algae is considerably simpler than that of land plants: they are devoid of true roots, stem, and leaves, and they are not as high as land plants on an evolutionary scale. While a few of the Chrysophyta and Cyanophyta may be terrestrial, the majority of the algae are found in fresh or salt water, growing most abundantly at the intertidal level. In addition, vast quantities of free-floating Phaeophyceae (brown seaweeds) may be found in the open sea. The majority of algal polysaccharides are extractable from the tissue by hot water, dilute acid or alkali. While many of them are quite different from those of land plants, cellulose and starch-type poly-

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FIGURE 1. Structural formulae of dextran sulfate (DS) and pentosan polysulfate (PS). Structures are shown as if all hydroxyl groups are substituted by sulfates.

FIGURE 2. Structural formula of dermatan sulfate.

saccharides are synthesized by a number of algae, and all marine species metabolize in addition at least one sulfated polysaccharide. The precise function of the latter is not fully understood. Sulfated polymers such as carrageenan are often present in large quantities and appear to constitute the structural material of the cell. At the same time, evidence has been provided that these substances are involved in the ion-exchange mechanism (Eppley, 1958), whereby cations such as potassium and calcium are selectively absorbed from sea water. Their mucilaginous and hygroscopic nature also probably hinders the desiccation of the plant when it is exposed at low tide. The inhibitory effects of polysaccharides from marine algae on viral replication were reported almost four decades ago. In 1958, Gerber et al. (1958) reported that algal polysaccharides exhibited antiviral activity toward mumps virus and influenza B virus. In 1977, Ehresmann et al. (1977) associated the inhibition of herpes simplex and other viruses with polysaccharide fractions from extracts of ten red algae; similar observations were made by Richards et al. (1978). More recently, in 1987, Nakashima et al. (1987a,b) reported inhibition of HIV reverse transcriptase by sulfated polysaccharides from the red alga Schizymenia pacifica. In a comparative evaluation of diverse sulfated polysaccharides, Baba et al. (1988a,b) found that many, but not all, had anti-HIV activity. More recently, fucoidan, a complex sulfated polysaccharide from the alga Fucus vesiculosus, was found to inhibit HIV in vitro and was synergistic with AZT (Sugawara et al., 1989). This activity presumably resulted from a direct interaction of the polysaccharide with the HIV binding site of the target cells. The present study was aimed at isolating and characterizing sulfated polysaccharides from marine algae, determining their antiviral activity spectrum and resolving their mechanism of antiviral action. Dextran sulfate (MW 5000) and pentosan polysulfate, which have been extensively studied for their antiviral activity, were included as representative sulfated polysaccharides that consist of several repeating monosaccharide units substituted with sulfate groups. CHEMISTRY

units. Dextrans can differ in chain length and in degree of branching. The branching occurs via -(13) and -(14) branch points. Dextran fractions of different average molecular weight were obtained by partial hydrolysis. These different fractions were sulfated to obtain dextran sulfates of different molecular weight. Therefore, dextran of a certain molecular weight range was added in portions to a well-stirred solution of an excess of chlorosulfonic acid in anhydrous pyridine. The reaction occurred under effective stirring at 65C for 5 hr. After neutralization the sodium salt of the ester was precipitated with ethanol. After dialysis it was precipitated one more time with ethanol. The obtained sodium salt of dextran sulfate (DS) (Fig. 1) contains two to three sulfate groups per glucose unit.

Pentosan sulfate
In the search for synthetic polysulfated analogs of heparin, xylan sulfuric esters were synthesized. Synthetic procedures were adapted to obtain xylan sulfuric esters of lower molecular weight (Swiss patent, 1955). Xylans are hemicelluloses which consist of chains of -(14) linked d-xylopyranosyl units. The semisynthetic sulfated polysaccharide was obtained by adding xylan to a vigorously stirred solution of an excess of chlorosulfonic acid in anhydrous pyridine at 65C. The reaction mixture was kept at 80C for 2.5 hr. The pyridinium salt of the ester was precipitated with methanol. The product was bleached and further partly hydrolyzed with hydrogen peroxide in acidic conditions at 97C. After neutralization the pentosan sulfate was bleached and dialyzed. The sodium salt was obtained by crystallization from a mixture of water, ethanol and acetone (2:4:4 v:v:v) by addition of sodium acetate. Approximately 100% of the hydroxyl groups of the pentosan sulfate (PS) (Fig. 1) used in our antiviral experiments were sulfated.

Dermatan sulfate
Dermatan sulfate (Fig. 2) was isolated in 1941 by Meyer and Chaffee. As this product was first isolated from pig skin (dermos) the name dermatan sulfate was introduced. Dermatan sulfate has also been isolated from beef lung after removal of heparin. Later it was detected in many other organs such as heart, brain, and spleen. As it also displayed anticoagulant activity it was called -heparin (Marbet and Winterstein, 1951). Dermatan sulfate is built up from alternating 1,3-linked iduronic acid and galactosamine (Table 1), the repeating sequence in dermatan sulfate being (13)-O-(2-acetamido-2-deoxy--d-galactopyra-

Dextran sulfate
In the early 1940s, dextran sulfuric acid esters were synthesized to investigate their inhibitory action on coagulation and their toxicity (Gro nwall et al., 1945). Dextran is a high-molecular-weight polysaccharide produced from sucrose by several bacteria, but only Leuconostoc mesenteroides and Leuconostoc dextranicum are used for commercial purposes. Its backbone consists predominantly of -(16) linked d-glucose

Polysaccharides Extracted from Sea Algae as Antiviral Drugs TABLE 1. Percentage of sulfation, sugar content and configuration of the sugar oligomers (i.e., nature and type of interglycosidic linkage) of polysaccharides Percentage of sulfation (%) 81a 100a 6b 25a 35a 25b 28b

499

Compound 1. Chemical synthesized DS PS 2. Extracted from sea algae Dermatan sulfate -Carrageenan -Carrageenan Xylomannan sulfate F6 Galactan sulfate
a

Sugar content Glucose Xylose

Configuration -1,6 -1,4

Iduronic acid N-acetyl-1,3 galactosamine Galactopyranosyl (14)1,3 anhydrogalactose Galactopyranosyl (14)1,3 galactose Xylose-mannose -1,3-linked mannose (98%) having single stubs of -1,2-xylose Galactose -1,3

Percentage of sulfation of hydroxyl groups. b Sulfate content on dry weight.

nosyl-4-sulfate)-(14)-O--l-idopyranuronosyl (Jeanloz et al., 1957). The chain is usually 20 to 100 monosaccharides long.

Carrageenan
- and -carrageenans were purchased from Sigma Chemical Co. (St. Louis, MO). Carrageenans are a mixture of sulfated polysaccharides extracted from red seaweed (Rhodophyceae). The source of carrageenan is Eucheuma cottonii. -carrageenan is isolated from two species, Gigartina aciculaire and Gigartina pistillata, which grow together in the sea. Gigartinaceae are found most abundantly in North Atlantic coastal regions from Norway to North Africa. The name carrageenan is derived from the Irish coastal town of Carragheen. The and structural families are identified based on position of sulfate and the presence or absence of anhydrogalactose. The form is characterized by a repeating unit of 4-sulfate--d-galactopyranosyl (14)-3,6-anhydro--d-galactose-linked (13). The form is characterized by a repeating (13)-linked disaccharide of 2-sulfate-d-galactopyranosyl (14)--d-galactose-2,6-sulfated (Morris et al., 1977). The degree of sulfation for the and form are 25% and 35%, respectively.

continued overnight. Then the precipitate was centrifuged and discarded and the supernatant was extracted with 1-pentanol, dialyzed, concentrated and freeze-dried. The upper limit of sodium chloride concentration was 4.0 M and the residual precipitate was suspended in water. The suspension was dialyzed and freeze-dried. The range of the sodium chloride concentrations and analysis of the seven fractions obtained (F1F7) are given in Table 2. The fraction that shows antiviral activity (F6) consists of 2.1% -d-xylose and 97.9% -d-mannose. The structure of xylomannan fraction F6 consists of an -d-(1,3)-linked mannan chain, which is sulfated at the 2 and 6 positions, and has single stubs of -(1,2)-linked d-xylose (Table 1) (Matulewicz and Cerezo, 1987).

Galactan sulfate
The polysaccharide, galactan sulfate, was extracted and purified from the Venezuelan red seaweed Agardhiella tenera, isolated from the Mochima National Park on the eastern coast of Venezuela. After harvesting, the alga was air-dried for transport to the laboratory, then washed in cold running water (30 min), drained, and heated (2 hr) in boiling water, before blending (Waring blender) and further heating (2 hr). After filtration, the polysaccharide was precipitated by addition of propan-2-ol (23 volumes), washed (2 volumes, 90% v/v water propan-2-ol), dried at 60C, pulverized, and converted into the potassium salt by stirring the recovered powder at 25C for 12 hr in 25 parts (by weight) of waterpropan-2-ol (50% v/v) containing 14% (by weight) potassium chloride (DiNinno and McCandless, 1978; Witvrouw et al., 1994a). The product was recovered on a Buchner filter and treated again three times, using a fresh solution each time, before it was redissolved in water; the solution was clarified by filtration, and the polysaccharide was reprecipitated with propan-2-ol as before. Finally, the polysaccharide was washed repeatedly with waterpropan-2-ol (three times in 60% v/v and twice in 90% v/v) until the washing gave a negative test for chloride, and then dried at 60C and ground to powder. This galactan contains various ratios of d- and l-galactose, 3,6-anhydro-d- and l-galactose and half-ester sulfate (Percival

Xylomannan
The red seaweed Nothogenia fastigiata grows attached to the rocks, by means of a disk, in the middle littorial floor of the shores of Patagonia and Tierra del Fuego, Argentina. The sample was collected in water near the Estacion Algologica of Puerto Deseado in southern Patagonia and was dried in the open air under strong winds. The dry seaweed was extracted with boiling water using the method described by Matulewicz and Cerezo (1987). The water-soluble polysaccharides were fractionated with cetrimide. To a solution of the polysaccharides (4.95 g) in water (500 ml) a 10% (w/v) aqueous solution of cetrimide (50 ml) was added slowly with stirring. The complexed material was removed by centrifugation, suspended in water and subjected to fractional solubilization in sodium chloride solutions of increasing concentration. Finely divided sodium chloride was added with constant stirring so that its concentration was increased by 0.51.0 M each time. After each addition, stirring was

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TABLE 2. Yields and analyses of the fractions obtained through redissolution in sodium chloride of the cetrimide salts of the sulfated polysaccharides from the red alga Nothogenia fastigiataa Range of redissolution (molar NaCl) 00.5 0.51.0 1.01.5 1.52.0 2.03.0 3.04.0 4.0 c Yieldb (%) 22.9 11.8 22.9 11.6 13.9 6.6 10.3 Sulfate (% SO3 Na) 12.9 19.2 23.4 27.6 27.3 24.7 14.0 Sugar composition Rha 3.0 2.6 1.1 Ara 2.6 2.5 1.7 1.9 2.4 4.4 Xyl 18.6 23.7 23.2 10.6 2.2 2.1 10.3 Man 12.1 57.1 68.4 77.8 82.8 97.9 17.0 Gal 54.4 11.9 6.7 9.8 12.6 64.5 Glc 9.3 2.2 2.7 Mannose:sulfate molar ratio 0.4 1.5 1.5 1.3 1.4 1.7 0.6

Fraction F1 F2 F3 F4 F5 F6 F7
a

Data taken from Matulewicz and Cerezo (1987). b Yields for fractions 17 are given as percentages of the recovered water-soluble polysaccharides (38% of the total polysaccharides). c Insoluble in 4.0 M sodium chloride.

and McDowel, 1967; Rees, 1965). The sulfate content of our sample was 28% of the dry weight (Table 1). INHIBITORY EFFECT ON VIRUS-INDUCED CYTOPATHIC EFFECT IN VITRO: STRUCTUREACTIVITY RELATIONSHIP

Activity against HIV

Various sulfated polysaccharides were examined for their inhibitory effect on HIV replication in cell culture. The sulfated polysaccharides showed marked differences in their in vitro anti-HIV activity, depending on the virus strain used (Busso and Resnick, 1990; Schols et al., 1992; Witvrouw et al., 1990, 1991). The sulfated polysaccharides DS and PS are significantly more inhibitory to HIV-2(ROD) than to HIV-1(IIIB), HIV-1(RF) or HIV-2(EHO) (Table 3). These findings reflect differences in the interaction of these polyanions with the different envelope glycoproteins of different HIV strains (see Mechanism of antiviral action subsection). DS (molecular weight [MW] 3000500,000) and PS are highly potent and selective inhibitors of HIV-1 replication in cell culture (Table 4). These compounds protect MT-4 cells against HIV-1induced cytopathicity at a concentration of 0.20.8 g ml 1. Neither DS nor PS reduce the viability of mock-infected MT-4 cells at concentrations up to 2.5 mg ml1 (Schols et al., 1992). Their selectivity index (SI) is approximately 10,000. To determine the importance of molecular size in the anti-HIV activity of sulfated polysaccharides, various DS samples prepared from dextran fractions with MW ranging from 1000 to 500,000, but with an identical sulfate content (81%), were examined for their inhibitory effect on HIV-1-induced cytopathicity (Table 4). A TABLE 3. Comparative antiviral activity of DS, PS and dermatan sulfate against various strains of HIV-1 and HIV-2 EC50a (g ml 1) HIV-1 Compound DS (MW 5000) PS (MW 3000) Dermatan sulfate IIIB 0.9 0.8 625 RF 0.4 0.9 625 HIV-2 ROD 0.08 0.01 625 EHO 3.8 2.7 625

marked increase in anti-HIV-1 activity was observed when the MW increased from 1000 to 5000 or 10,000. At higher MW (10,000 500,000), the antiviral activity of DS tended to level off. Against HIV-2-induced cytopathicity, the 1000 MW DS sample proved more inhibitory than the higher MW samples. In contrast to DS and PS, dermatan sulfate (Fig. 2 and Table 1) did not demonstrate any anti-HIV activity (Table 3). Because dermatan sulfate contains less than one sulfate group per disaccharide unit, this result indicates that a certain number of sulfate groups (at least two per monosaccharide unit) are required for anti-HIV activity. If, however, dermatan sulfate is supersulfated, it becomes as active as DS and PS (Jurkiewicz et al., 1989). The anti-HIV potential has also been studied for four sulfated polysaccharides (- and -carrageenan, xylomannan sulfate F6 and galactan sulfate) extracted from seaweeds (Baba et al., 1988a,b; Damonte et al., 1994; Witvrouw et al., 1994a). When we tested - and -carrageenan for their inhibitory effect on HIV-1 replication in MT-4 cells, -carrageenan was at least tenfold less active than penTABLE 4. Inhibitory effects of sulfated polysaccharides on HIVinduced cytopathicity EC50a (g ml1) Compound 1. Chemically synthesized DS (MW 1000) (MW 1500) (MW 3400) (MW 5000) (MW 10,000) (MW 40,000) (MW 70,000) (MW 110,000) (MW 500,000) PS (MW 3000) 2. Extracted from sea algae -Carrageenan -Carrageenan Xylomannan sulfate F6 Galactan sulfate HIV-1(IIIB) 7.1 2.9 0.7 0.5 0.2 0.4 0.6 0.6 0.6 0.8 12 1.9 13.7 0.6 HIV-2(ROD) 0.1 0.1 0.3 0.3 1.2 2.3 3.9 4.1 4.1 0.01 13.4 0.5

Source: Baba et al. (1988a) and Schols et al. (1992). a Fifty percent effective concentration, or concentration required to inhibit, by 50%, the cytopathicity of HIV in MT-4 cells.

Sources: Rees (1965); Baba et al. (1988a,b); Witvrouw et al. (1991, 1994a); Schols et al. (1992); Damonte et al. (1994). a Fifty percent effective concentration, or concentration required to inhibit, by 50%, the cytopathicity of HIV-1(III B) or HIV-2(ROD) in MT-4 cells.

Polysaccharides Extracted from Sea Algae as Antiviral Drugs TABLE 5. Inhibitory effects of sulfated polysaccharides on the replication of enveloped RNA viruses other than HIV EC50a (g ml1) Compound 1. Chemical synthesized DS (MW 1000) (MW 1500) (MW 3400) (MW 5000) (MW 10,000) (MW 40,000) (MW 70,000) (MW 110,000) (MW 500,000) PS (MW 3000) 2. Extracted from sea algae -Carrageenan -Carrageenan Xlyomarnan sulfate F6 Galactan sulfate Sindbis Semliki Junin Tacaribe Influenza A virus Forest virus virus virus VSV virus RSV 300 400 185 150 15 2 14 2 7 20 7 2 11 400 400 100 120 14 14 14 20 20 400 85 0.2 0.2 0.3 0.2 0.04 0.1 1.6 10 100 0.2 0.4 0.4 0.3 0.2 0.2 2.8 7.8 100 70 70 4.5 2 0.7 0.2 0.5 0.7 0.7 4 7 4 14 200 200 42 5.8 1.4 1.0 1.4 20 0.2 0.4 12 1.4 0.4 0.2 0.1 0.1 0.1 0.8 0.9 0.4

501

Sources: Baba et al. (1988a,b); Witvrouw et al. (1991, 1994a); Schols et al. (1992); Damonte et al. (1994). a Fifty percent effective concentration, or concentration required to inhibit, by 50%, the cytopathicity of Sindbis virus, Semliki Forest virus, Junin virus or Tacaribe virus in Vero cells, VSV or RSV in HeLa cells and influenza A virus in MDCK cells.

tosan polysulfate, whereas -carrageenan inhibited HIV-1 replication at lower concentrations, its EC50 being 1.9 g ml1 (Table 4). The carrageenans did not show any in vitro toxicity for the host cells at 625 g ml1 (data not shown) (Baba et al., 1988a). The xylomannan sulfate F6 was only weakly active against HIV-1 and HIV-2 (EC50: 13.7 and 13.4 g ml1, respectively) (Table 4), and it was rather toxic (EC50: 33 g ml1) (data not shown) (Damonte et al., 1994). When galactan sulfate was examined for its inhibitory effect on the cytopathicity of HIV-1(IIIB) and HIV-2(ROD), the compound provided protection at a much lower concentration (EC50: 0.5 g ml1) than xylomannan sulfate (Table 4). No reduced viability of mock-infected MT-4 cells at concentrations up to 2.5 mg ml1 was noted (data not shown) (Damonte et al., 1994). Besides the linear sulfated polysaccharides, we have also evaluated a series of sulfated cyclic oligosaccharides (cyclodextrins) containing six (-cyclodextrin), seven (-cyclodextrin) or eight (cyclodextrin) -(14)-linked glucopyranose units for their activity against HIV. The -, - and -cyclodextrins containing two sulfate groups per glucose unit were more active than the corresponding cyclodextrins containing only one sulfate group (data not shown) (Schols et al., 1991). A sugar backbone is not strictly needed for the anti-HIV activity of polysulfates. A simple carboncarbon backbone, such as that present in polyvinyl alcohol sulfate (PVAS) and its copolymer with acrylic acid (PAVAS), have also proved to be highly efficient antiHIV agents in vitro (data not shown) (Baba et al., 1990a; Schols et al., 1990a,b). Polysulfates are able to block HIV replication in cell culture at concentrations as low as 0.01 g ml1 without toxicity for the host cells, thus achieving in vitro selectivity indices up to 250,000. The fact that some polysulfates show a different inhibitory potency against HIV-1 and HIV-2 suggests that there exist marked differences in the target molecules of HIV-1 and HIV-2 with which the polysulfates interact (see Mechanism of antiviral action subsection).

Activity against enveloped viruses other than HIV

Polysulfates, in general, and sulfated polysaccharides, in particular, were found to be active against a wide variety of enveloped viruses (Andrei and De Clercq, 1990, 1993; Andrei et al., 1991; Baba et al., 1988a,b; Damonte et al., 1994; Ehresmann et al., 1977; Gerber et al., 1958; Hosoya et al., 1991; Lu schler-Mattli and Glu ck, 1990; Lu scher-Mattli et al., 1993; Mastromarino et al., 1991a,b; Nakashima et al., 1987a,b; Richards et al., 1978; Schols et al., 1990, 1991; Sugawara et al., 1989; Witvrouw et al., 1991, 1992, 1994a,b) such as herpes viruses (herpes simplex virus type 1 [HSV-1], thymidine kinase-deficient [TK] HSV-1, herpes simplex virus type 2 [HSV-2], cytomegalovirus [CMV]), togaviruses (Sindbis virus, Semliki Forest virus), arenaviruses (Junin virus, Tacaribe virus), rhabdoviruses (vesicular stomatitis virus [VSV]), orthomyxoviruses (influenza A virus) and paramyxoviruses (respiratory syncytial virus [RSV]). The polysulfates also showed some inhibitory activity against poxviruses (vaccinia virus [VV]), but are not inhibitory to nonenveloped viruses such as picorna-, reo- and adenovirus and some enveloped myxoviruses (influenza B virus and parainfluenza virus type 3). When DS samples derived from dextran with a MW ranging from 1000 to 500,000 were examined for their inhibitory effects on the replication of RNA viruses other than HIV and DNA viruses, the 1000 and 1500 MW DS samples were much less active than the higher MW samples against Sindbis virus, Semliki Forest virus, VSV, influenza A virus, RSV, HSV-1, TK HSV-1, HSV-2 and VV. As a rule, the antiviral activity increased with increasing MW, with an optimum at approximately MW 40,000. However, against Junin virus, Tacaribe virus and CMV (Tables 5 and 6), the 1000 and 1500 MW DS samples were almost as active as the higher MW samples. PS (MW 3000) was a more potent inhibitor of Sindbis virus, HSV and CMV replication than DS (MW 3400). Semliki Forest virus, Junin virus and Tacaribe virus proved to be less sensitive to PS than DS, whereas VSV, influenza A virus and RSV were equally sensitive to PS and DS (MW 3400).

502 TABLE 6. Inhibitory effects of sulfated polysaccharides on the replication of enveloped DNA viruses EC50a (g ml1) Compound 1. Chemical synthesized DS (MW 1000) (MW 1500) (MW 3400) (MW 5000) (MW 10,000) (MW 40,000) (MW 70,000) (MW 110,000) (MW 500,000) PS (MW 3000) 2. Extracted from sea algae -Carrageenan -Carrageenan Xylomannan sulfate F6 Galactan sulfate HSV-1 (KOS) 110 10 4.5 2 2 2 2 1 2 0.7 3.7 1.6 0.6 0.7 TK HSV-1 (B2006) 100 20 2 2 1.4 0.7 0.9 1.4 2 1.1 15 4.5 0.7

M. Witvrouw et al.

HSV-2 (G) 95 7 2 1.4 1.4 0.3 0.6 0.7 0.7 0.5 2 1.5 2.5 2.5

CMV (AD-169) 2.5 0.3 0.7 0.7 0.6 0.3 0.6 0.1 0.1 0.4 2.8 0.3 10

CMV (Davis) 3 0.8 0.8 0.7 0.5 0.3 0.6 0.3 0.3 0.1 2.8 4.0

VV 275 400 35 80 10 4.5 20 20 40 20 36 16 7

Sources: Baba et al. (1988a,b); Witvrouw et al. (1991, 1994a); Schols et al. (1992); Damonte et al. (1994). a Fifty percent effective concentration, or concentration required to inhibit, by 50%, the cytopathicity of HSV-1, TK HSV-1, HSV-2 or VV in E6SM cells and CMV in HEL cells.

When - and -carrageenan were examined for their inhibitory effects on the replication of DNA and RNA viruses other than HIV, the compounds were as equally active as DS (MW 5000) against all viruses tested, with HCMV being the most sensitive to the inhibitory effect of these compounds. The -carrageenan was slightly more active against all viruses tested than the -carrageenan. All seven polysaccharide fractions obtained from the red alga Nothogenia fastigiata were initially evaluated for their activity against HSV-1. Only the xylomannan sulfate fraction F6 elicited marked anti-HSV-1 activity, with its IC50 being 0.6 g ml 1 (Table 6). The anti-HSV-1 active fraction was then tested for its inhibitory effect on the replication of several RNA and DNA viruses (Tables 5 and 6), and showed a clearly different antiviral activity spectrum in comparison with other sulfated polysaccharides, such as DS. The red seaweed-derived xylomannan sulfate F6 was more active than DS (MW 5000) against influenza A virus and HSV-1, but less active against Junin virus, Tacaribe virus, RSV, HSV-2 and CMV. When galactan sulfate, extracted from Aghardhiella tenera, was examined for its inhibitory effect on RNA viruses other than HIV, the compound was found to be particularly active against Sindbis virus. It inhibited Sindbis virus at a concentration that was at least tenfold lower than the concentration at which DS (MW 5000) inhibited this virus. Galactan sulfate was not active against Junin virus and Tacaribe virus, was slightly active against VSV, but was equally as active as DS (MW 5000) against Semliki Forest virus, influenza A virus and RSV. When galactan sulfate was examined for its inhibitory effect on the replication of DNA viruses, the compound proved more inhibitory than DS (MW 5000) against HSV-1, TK HSV-1 and VV, although HSV-2 and CMV proved to be less sensitive. When the sulfated cyclodextrins were examined for their inhibitory effect on the replication of DNA viruses, the compounds with two sulfate groups per glucose unit were more active than the compounds with one sulfate group per glucose unit, and this difference was particularly striking for CMV (data not shown) (Schols et al., 1991). Carbohydrate polysulfates must fulfill certain structural requirements to be effective against HIV and other enveloped viruses. A

similar structureactivity relationship was found for all enveloped viruses with regard to the MW and degree of sulfation of the sulfated polysaccharides: the antiviral activity increased with increasing MW or degree of sulfation. While the MW and degree of sulfation certainly play an important role in the antiviral activity of polysulfates, still other structural features such as charge density and distribution must be involved as well. For instance, an acrylic polymer of polyvinyl alcohol sulfate (PAVAS # 3) is a more potent inhibitor of CMV infection than is DS (MW 5000), although they have a similar MW and comparable degree of sulfation (Schols et al., 1990a). The importance of the carbohydrate backbone has been previously demonstrated for the plant lectins (Balzarini et al., 1992; Stig Hansen et al., 1991). Only N-acetylglucosamine-specific and mannose-specific plant lectins were active against HIV, HSV and CMV. N-acetylgalactosamine-specific plant lectins were only active against HSV. This differential activity may depend on the nature of the sugars present in the HIV, HSV and CMV envelope glycoproteins. The oligosaccharides of HSV and CMV envelope glycoproteins have not been described in the same detail as for HIV, but HSV is believed to contain additional O-linked oligosaccharides (acetyl-galactosamine), known to be present in a localized cluster in gC (the heparin-binding moiety) of HSV. This coincides with the finding that enzymatic digestion of heparan sulfate (containing N-acetylglucosamine residues), but not chondroitin sulfate (containing N-acetylgalactosamine residues), influences HIV infectivity (Patel et al., 1993) (see Mechanism of antiviral action subsection).

Inhibition of syncytium formation between HIV-infected cells and uninfected cells

Syncytium (giant cell) formation induced by the interaction between the gp120 glycoprotein expressed on the surface of cells infected with HIV and the CD4 receptor of uninfected CD4 cells may play an important role in the depletion of T4 lymphocytes in AIDS patients (Haseltine, 1988). Schols et al. (1989c) demonstrated, by using a flow cytometric method, that this syncytium formation

Polysaccharides Extracted from Sea Algae as Antiviral Drugs

503

FIGURE 3. Three-dimensional presentation of flow cytometric analysis of cocultures of HUT-78/HIV-1(IIIB) and MOLT-4 cells (upper panels) and microscopic appearance of these cocultures (lower panels). MOLT-4 cells were cultured with an equal number of HUT-78/HIV-1(IIIB) cells in the absence (left and middle panels) or presence of PS (100 g ml1) (right panels). Immediately after cocultivation (left panels), or at 24 hr after cocultivation (middle and right panels), the cells were harvested and stained with two different fluorescent MAbs (anti-HLA-DR PE and anti-Leu2a FITC). In the upper panels, the x and y axes indicate the intensity of green (FITC) and red (PE) fluorescence, respectively. The z-axis corresponds to the cell number. Therefore, the peaks along the x and y axes represent the cells that express Leu2a antigen (MOLT-4 cells) and HLA-DR antigen [HUT-78/HIV-1(IIIB) cells], respectively. leads to a selective destruction of the uninfected CD4 cells. In this assay the cells are stained with specific monoclonal antibodies (MAbs) that react with antigens expressed on either HIV-1-infected HUT-78 cells [HUT-78/HIV-1(IIIB) cells] or uninfected MOLT-4 cells (Fig. 3). In the presence of PS (100 g ml 1), no giant cell formation is observed and the uninfected MOLT-4 cells are protected against the destructive effect of the persistently HIV-1-infected HUT-78 cells. Except for the low MW DS samples and xylomannan sulfate, all polysulfates that are inhibitory to HIV-1 replication are able to block HIV-1-induced giant cell formation. Yet, higher concentrations are required for inhibition of giant cell formation than for inhibition of HIV-1 replication (compare data in Tables 4 and 7). A similar correlation was found between the MW of the DS samples and their inhibitory effect on HIV-1-induced giant cell formation as mentioned above for their inhibitory effect on HIV-1-induced cytopathicity. A marked increase in anti-HIV activity was observed when the MW increased from 1000 to 5000 or 10,000. At higher MW (10,000500,000), the activity of DS against HIV-1 tended to level off. For HIV-2-induced syncytium formation, the 1000 MW sample proved more inhibitory than the higher MW samples. Low MW DS samples are unable to inhibit HIV-1-induced syncytium formation (Table 7). Yet, they are active in inhibiting the cytopathic effect of HIV-1. This means that the MW requirements are more stringent for inhibition of HIV-1-induced giant cell formation than for inhibition of HIV-1-induced cytopathicity, but, in contrast, the MW requirements for inhibition of HIV-2-induced syncytium formation are less stringent than for inhibition of HIV-2-induced cytopathicity. Sulfated polysaccharides are known to interact with the V3 region of the viral envelope gp120 glycoprotein (see Mechanism of antiviral action subsection). This would explain why they inhibit fusion (syncytium formation) of HIV-infected cells with uninfected CD4 cells. The fact that sulfated cyclodextrins inhibit HIV-

2-induced, but not HIV-1-induced, syncytium formation, suggests that there exist marked differences in those parts of the HIV-1 and HIV-2 gp120 glycoproteins that are involved in viruscell fusion. In conclusion, most of the sulfated polysaccharides that have been found to inhibit HIV replication also inhibit syncytium formation, although the structural requirements with respect to MW and number of sulfate groups seem to be more stringent for inhibition of syncytium formation than for inhibition of HIV replication (cyto-

TABLE 7. Inhibitory effects of sulfated polysaccharides on HIVinduced syncytium formation EC50a (g ml1) Compound 1. Chemically synthesized DS (MW 1000) (MW 1500) (MW 3400) (MW 5000) (MW 10,000) (MW 40,000) (MW 70,000) (MW 110,000) 1. PS (MW 500,000) 2. Extracted from sea algae Xylomannan sulfate F6 Galactan sulfate HIV-1(IIIB) 500 200 3.4 2.2 1.2 1.2 2.0 3.0 3.0 200 5 HIV-2(ROD) 1.8 12 11 30 40 30 40 40 28 200 5

Sources: Witvrouw et al. (1991, 1994a); Schols et al. (1992); Damonte et al. (1994). a Fifty percent effective concentration, or concentration required to inhibit, by 50%, syncytium formation between MOLT-4 cells and HIV-1(IIIB )or HIV-2(ROD)-infected HUT-78 cells.

504

M. Witvrouw et al. TABLE 8. The inhibitory indexes of dextran sulfate (MW 5000) and galactan sulfate (GS) for HIV-1(IIIB) binding (IIIB) and cytopathic effect (IICPE ) in MT-4 cells IIVB Concentration (g ml1) 125 25 5 1 0.2 0.04 Immunofluorescence (FACS) DS 1.00 0.80 0.77 0.20 0.03 p24 content (ELISA) DS 0.97 0.75 0.69 0.57 0.37 GS 0.63 0.44 0.09 IICPE DS 1.00 0.86 0.64 0.40 0.29

FIGURE 4. Effect of various concentrations of DS on HIV1(IIIB) binding to MT-4 cells. The solid line histograms represent the nonspecific fluorescence in the absence of DS (A) or in the presence of DS at 25 g ml 1 (B); 5 g ml1 (C), 1 g ml1 (D), 0.2 g ml1 (E) and 0.04 g ml1 (F). The dotted line histograms represent the specific cellular HIV-1 fluorescence in the absence of DS (A) or in the presence of DS at 25 g ml1 (B), 5 g ml 1 (C), 1 g ml1 (D), 0.2 g ml1 (E) and 0.04 g ml1 (F). The mean fluorescence values were as follows: 22 (A), 25 (B), 22 (C), 23 (D), 21 (E) and 22 (F) for the nonspecific fluorescence; and 52 (A), 25 (B), 28 (C), 30 (D), 45 (E) and 51 (F) for the specific fluorescence.

The inhibitory effects of the test compounds on virus adsorption were measured in parallel using two techniques. First, an indirect immunofluorescence laser flow cytofluorographic method has been specifically designed for this purpose (Schols et al., 1989a). Briefly, MT-4 cells were exposed to HIV-1 virions, which had been concentrated from the supernatant of HIV-1-infected MT-4 cells in the presence or absence of the test compounds. The compounds were added 1020 sec before the virus was added. The cells were processed for indirect immunofluorescence using a polyclonal antibody to HIV-1, and analyzed for cell-bound HIV-1 virions by laser flow cytofluorography. The inhibitory index for virus binding (IIVB) determined by FACS analysis was calculated according to the following formula: 1 (MF VC MFCC)/MFV MFC), whereby MFVC is the mean fluorescence (MF) with a given concentration of the compound in HIV-1-inoculated cells, MFCC is the mean fluorescence for the control cells (not exposed to HIV-1) treated with compound, MFV is the mean fluorescence for the HIV-1-inoculated cells (not treated with any compound), and MFC is the mean fluorescence for the control cells (not exposed to HIV-1 and not treated with any compound). In a second assay, MT-4 cells (510 5 cells per tube) were incubated with HIV-1 (corresponding to 100 ng of p24) in the presence or absence of the test compound. After a 2-hr incubation period at 37C, cells were extensively washed with phosphate buffered saline (PBS) to remove unadsorbed virus particles. The cells were then lysed with PBS containing 0.5% Nonidet P40. The amount of antigen was determined by an enzyme-linked immunosorbent assay (ELISA) (DuPont). The inhibitory index for virus binding (IIVB) determined by p24 ELISA was calculated according to the following formula: 1 (ODT)HIV (ODC)mock /(ODC) HIV (ODC)mock , the inhibitory index for cytopathic effect (IICPE) was calculated according to the following formula: 1 (ODT)HIV (ODC )HIV/(ODC )mock (ODC )HIV,whereby (ODT)HIV is the optical density measured with a given concentration of the test compound in HIV-1-infected cells; (ODC)HIV is the optical density measured for the control untreated HIV-1-infected cells; (ODC)mock is the optical density measured for the control, untreated, mock-infected cells.

pathicity) (Tables 4 and 7) (Baba et al., 1990b; Montefiori et al., 1990). For all polysulfates tested the concentration required to block HIV replication (Table 4) was consistently lower than the concentration required to block syncytium formation (Table 7).

Mechanism of antiviral action

Mechanism of anti-HIV action. As mentioned above, the mechanism by which the polysulfates inhibit HIV replication can be attributed to the inhibition of the binding of the virions to CD4 cells (Nakashima et al., 1989) and subsequent syncytium formation (Baba et al., 1990b; De Clercq, 1993; Mitsuya et al., 1988). The inhibitory effect of polysulfates on HIV attachment to CD4 cells can be demonstrated by monitoring viruscell binding with radiolabeled HIV particles (Baba et al., 1988c; Mitsuya et al., 1988), a radioimmunoassay (Nakashima et al., 1989), flow cytometry (Fig. 4) (Otake et al.,1993; Schols et al., 1989a, 1990b, 1991, 1992; Witvrouw et al.,

1991, 1992, 1994a) and a p24 ELISA assay (Table 8) (Witvrouw et al., 1994a). Dose-dependent inhibition of virus adsorption is observed with polysulfates, as demonstrated for DS (Fig. 4 and Table 8). The inhibitory effect on giant cell (syncytium) formation can be demonstrated following cocultivation of persistently HIV-1- or HIV-2-infected HUT-78 cells with MOLT-4 cells (see Inhibition of syncytium formation between HIV-infected cells and uninfected cells subsection). DS, galactan sulfate and other polysulfates do not interfere with the binding of OKT4A/Leu3a MAbs to the CD4 target cells (Table 9; Baba et al., 1988c; Bagasra and Lischner, 1988; Lederman et al., 1989; Mitsuya et al., 1988; Schols et al., 1989b; Witvrouw et al., 1994a) (OKT4A/Leu3a MAbs bind at the same site as HIV and can block HIV infection), although DS has been shown to interact with the CD4 molecule at an epitope distinct from the OKT4A epitope (Lederman et al., 1989). The latter investigators also developed a solid-phase assay to study the influence of DS on the interaction of rCD4 and gp120 in the absence of any other cellular or viral components. The interaction of rCD4 with rgp120 was inhibited by DS

Polysaccharides Extracted from Sea Algae as Antiviral Drugs TABLE 9. Effects of dextran sulfate (MW 5000) and galactan sulfate (GS) on the binding of OKT4A MAb to MT-4 cells, as detected by FACS analysis Compound DS GS Simultest control OKT4A-leu3a MAb Concentration (g ml1) 25 200 100 Mean channel fluorescence 89.2 89.8 89.2 Percentage of CD4 cells (%) 99.8 99.7 99.8 25.3 90.8 IICD4 0.02 0.02 0.02 0.3 99.9

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CD4 expression was determined by FACSTAR (Becton-Dickinson, Erembodegem, Belgium) analysis, as described previously (Schols et al., 1989b). Briefly, MT-4 cells were incubated for 1020 sec at room temperature in PBS in the absence of serum with or without test compound. The cells were then stained with optimal concentrations of the monclonal antibodies OKT4A-FITC (Ortho Diagnostics), and Simultest immune monitoring kit control (FITClabeled IgG1phycoerythrin [PE]-labeled IgG2a) (Becton-Dickinson) for 20 min at 4C, washed once in PBS, and fixed in 0.5 ml of 0.5% paraformaldehyde in PBS. Measurements were based on the percentage of cells showing fluorescence intensity greater than the control cells stained with normal mouse IgG1 FITCphycoerythrin (PE)-labeled IgG2a (Simultest Control) to monitor nonspecific immunoglobulin labeling. The inhibitory index for OKT4A MAb binding inhibition (IICD4 was calculated according to the following formula: IICD4 1 (MFCD4X MFC /MFCD4 MFC ), where MF CD4 is the mean channel fluorescence (MF) for the cells incubated only with OKT4A MAb; MFCD4X is the MF for the cells incubated with test compound; and OKT4A MAb and MFC are the MFs of the cells incubated with IgG1 FITC and IgG2PE (Simultest immune control). The MF was determined by the Consort 30 program (Becton-Dickinson).

but not dextran. Pretreatment experiments indicated that DS interferes predominantly with rCD4. This interaction seems to be relatively irreversible (Lederman et al., 1992). Thiele et al. (1989) and Parish et al. (1990) demonstrated that DS MW 500,000, but not DS MW 8000 or PS (MW 3000), seems to modulate detection of CD4 receptors (also the OKT4A epitope), but only at a very high concentration, on PBL cells. The modulation of CD4 by DS MW 500,000 is highly temperature-dependent suggesting an active, energy-dependent mode of action. Although the low MW sulfated polysaccharides DS MW 5000 and PS do not generate this cellular response, they retain their high antiviral activity even at a much lower concentration. A molecular weight of 5000 to 10,000 seems optimal for the inhibitory effects of DS on HIV-1 replication and giant cell formation (Baba et al., 1990; Witvrouw et al., 1991). Polysulfates interact with positively charged amino acids located in the principal neutralizing domain of gp120 (V3) (Batinic and Robey, 1992; Callahan et al., 1991; Mbemba et al., 1992). They interfere with the binding of a specific MAb (9284) to the V3 loop of gp120 of HIV-1 (Fig. 5 and Table 10) (Schols et al., 1990; Witvrouw et al., 1994a,b), but not with the binding of MAb 9305, a neutralizing anti-gp120/V3 MAb that binds to an adjacent epitope (Lederman et al., 1992). Taken together, these data suggest that polysulfates interact with the V3 loop of gp120, as has been previously demonstrated with heparin fractions (Cardin et al., 1989; Jackson et al., 1991), and inhibit the binding of certain anti-V3 loop MAbs, but also reveal that the portion of the V3 loop recognized by 9305 is distinct from the polysulfate binding site on gp120. The inhibitory effect of DS on gp120/V3 detection by flow activated cell sorter (FACS) analysis appears to be reversible (Lederman et al., 1992; Schols et al., 1990). When the treatment period with DS was extended, there was no further decrease of gp120 detection (Schols et al., 1990b). DS and PS are only slightly inhibitory to the binding of specific MAbs to gp41 and gp160/41, and to the binding of polyclonal antiHIV-1 serum to HIV-1 infected cells (inhibition obtained with DS at 100 g ml1 was less than 10% for the anti-gp41 MAb, 15% for the anti-gp160/41 MAb and 12% for the polyclonal anti-HIV-1 serum) (Schols et al., 1989a). Thus, the inhibitory effect of DS and

its cogeners on the interaction of HIV gp120 with the cellular CD4 receptor can be ascribed to a specific shielding of gp120/V3. These findings indicate that the mechanism by which polysulfates inhibit HIV replication appears to be related to an interaction

FIGURE 5. Inhibitory effect of DS (MW 5000) on the binding of anti-gp120 MAb, recognizing the V3 loop of gp120, to persistently HIV-1-infected HUT-78 cells (strain IIIB). The first histogram represents the control fluorescence of uninfected HUT-78 cells incubated with anti-gp120 MAb and RaMIgG(Fab)2PE. The second histogram represents the specific fluorescence of HIV-1-infected HUT-78 cells incubated with the anti-gp120 MAb and RaMIgG(Fab) 2PE. The third, fourth, fifth, sixth and seventh histograms represent the binding of anti-gp120 MAb, but, in these, the cells were pretreated for 15 min with DS at concentrations varying from 0.16 g ml1 (back) to 100 g ml 1 (front). The mean fluorescence values were (back to front) 81, 74, 63, 52, 47, 96 and 40, respectively.

506 TABLE 10. Effect of dextran sulfate (MW 5000) and galactan sulfate (GS) on the binding of anti-gp120 MAb to persistently HIV-1-infected HUT-78 cells, as detected by FACS analysis Compound DS Concentration (g ml1) 25 5 1 0.2 25 5 1 Mean channel fluorescence 31.6 34.9 40.3 41.3 29.2 33.1 52.7 26.6 57.2 Percentage of gp120 cells (%) 8.7 16.7 30.1 35.1 2.7 10.7 70.3 1.3 80.7 IIgp120 0.84 0.73 0.55 0.52 0.96 0.79 0.15

M. Witvrouw et al.

GS

RaMIgG(ab)2FITC Anti-gp120 MAb RaMIgG F(ab)2FITC

Normal HUT-78 and HIV-1-infected HUT-78 (HUT-78/HIV-1) cells were grown in RPMI 1640 supplemented with 10% heat-inactivated FCS, 100 IU ml1 of penicillin G and 20 g ml 1 of gentamicin. HUT-78/HIV1 cells (200,000 cells) in 100 l of RPMI with 10% FCS were washed twice in RPMI with 10% FCS, incubated with the compounds at various concentrations at 20C for 1520 min in RPMI, washed twice with RPMI to remove residual compound, stained with anti-gp120 MAb (9284, DuPont de Nemours, Brussels, Belgium) for 50 min at 37C, washed twice in PBS, incubated with FITC-conjugated F(ab)2 fragments of rabbit anti-mouse immunoglobulin antibody [RaMIgGF(ab)2FITC] (Prosan, Ghent, Belgium) for 50 min at 37C, washed once in PBS, resuspended in 0.5 ml of 0.5% paraformaldehyde in PBS, and analyzed by flow cytometry as described previously (Schols et al., 1990b). The threshold of positivity for green fluorescence intensity was arbitrarily established on the basis of HIV-1-infected HUT-78 cells incubated solely with RaMIgGF(ab)2 FITC. The inhibitory index for anti-gp120 MAb binding inhibition (IIgp120) was calculated according to the formula: IIgp120 1 (MFgp120X MF C/MFgp120 MFC), where MFgp120 is the mean channel fluorescence (MF) for the cells incubated with anti-gp120 MAb at concentration X, MFgp120X is the MF for the cells incubated with test compound and anti-gp120 MAb, and MFC is the MF of the cells incubated with RaMIgGF(ab) 2FITC to determine nonspecific fluorescence. MF was determined by the Consort 30 program (Becton-Dickinson).

with the epitope of the V3 loop of gp120 recognized by the MAb 9284. This interaction is probably mediated by the binding of negatively charged sulfate groups to positively charged amino acids side chains in the V3 loop. We postulate that polysulfate binding to gp120 disrupts two (related) processes critical to HIV infection: HIV binding to the cell surface, and the subsequent viruscell fusion step. The initial virus-binding event depends on the interaction with the CD4 membrane receptor, but additionally has to overcome the electrostatic repulsive forces between the cell membrane and the virion envelope. The early inhibitory effect of polysulfates can be the result of a disruption of ionic interactions between charged regions of viral surface glycoproteins, including gp120, and membrane phospholipids. Enveloped viruses that bud from cells carry part of the cell membrane and therefore may interact more effectively with the negatively charged cell surface if the outer part of the envelope protein contains regions of high positive charge density (Stegmann et al., 1989). The V3 loop in HIV may provide this function and the binding of polysulfates to the loop not only abolishes its positive charge but also adds an additional negative potential, thereby electrostatically preventing the interaction between the virion and the cell. This mechanism may also explain the broad-spectrum antiviral activity of polysulfates against various enveloped viruses other than HIV. Enveloped viruses (e.g., HSV, CMV and pseudorabies virus [PRV]) have been shown to interact with cell surface heparan sulfate as the initial binding site (Lycke et al., 1991; Neyts et al., 1992; Sawitzky et al., 1990; WuDunn and Spear, 1989). Their adsorption to the cells is also inhibited by the polysulfates. By analogy, heparan sulfate has recently been reported to participate in HIV-cell attachment and virus entry (Patel et al., 1993). The importance of the V3 loop in the attachment to heparan sulfate molecules at the cell surface is in agreement with the findings previously reported for the heparin-binding moieties of, for instance, CMV gC-II and PRV gIII

(Kari and Gehrz, 1993; Robbins et al., 1986; Sawitzky et al., 1990). The binding of CMV gC-II to immobilized heparin depends on structures maintained by disulfide bonds. Moreover, the PRV gIII glycoprotein also contains an arginine-rich region as previously documented for the HIV V3 loop. The function of heparan sulfate depends on sulfation of these molecules, again suggesting that ionic interactions contribute to virus binding to heparan sulfate. Based on previous data showing a potent inhibitory effect of polysulfates on binding of MAb 9284 to the gp120 V3 loop, positively charged amino acids in the V3 domain may bind to the cell surface heparan sulfate. The antiviral activity of heparin (which is chemically very n and Lindahl, 1991) may be similar to heparan sulfate) (Kjelle achieved by occupying the sites on the virion envelope that are necessary for attachment of the virus particles to cell surface heparan sulfate. It is likely that molecules that are related to heparin, such as DS, PS, PVAS, PAVAS and polysulfates in general, achieve their antiviral activity in a similar way as heparin. The most likely role of the V3 loop is to assist in membrane fusion after gp120 binds to CD4. Once again, the high positive charge density could be essential to this function, presumably by bringing two negatively charged membranes together (Stegmann et al., 1989). Specifically, this could be accomplished by binding to a negatively charged region on the cell membrane, thereby orienting the fusiogenic portion of gp41 and the viral membrane so that fusion occurs. The V3 region may be physically close to gp41, because mutations in the loop have been reported to lead to gp41gp120 dissociation (Kowalski et al., 1987). Proteolysis has also been shown to occur within the V3 loop and could be involved in inducing fusion (Hattori et al., 1989). Binding of the polysulfate to the V3 region may neutralize the local positive charge density and thus disturb binding of the V3 region to the CD4 receptor, proteolysis and fusion. On the other hand, antibodies directed against the V3 domain may not inhibit virion binding (Skinner et al., 1988), because they

Polysaccharides Extracted from Sea Algae as Antiviral Drugs TABLE 11. Influence of various treatment periods on the antiHSV-1 activity of xylomannan sulfate fraction F6 Xylomannan sulfate F6 (g ml1) present Treatment None A B C During virus adsorption 0 3.3 0 3.3 After virus adsorption 0 0 3.3 3.3 Plaque number 56 6 54 8 Inhibition (%) 0 90 4 85

507

Vero cells were infected with 60 PFU of HSV-1. After 1 hr virus adsorption at 37C in MEM with or without xylomannan sulfate (3.3 g ml1 ), unadsorbed virus and the compound were removed. The cells were overlaid with medium with or without xylomannan sulfate, and further incubated for 3 days, whereafter the number of plaques was determined. Each value represents the mean of duplicate assays.

do not eliminate the positive charge density but still physically block an essential interaction between the V3 loop and either a cell surface protease or the cell surface membrane. Polysulfates have been reported to inhibit reverse transcriptase activity of various retroviruses (Baba et al., 1988c; Cronn et al., 1992; Moelling et al., 1989; Nakashima et al., 1987b; Sydow and Klo cking, 1987). However, this inhibitory effect cannot be considered specific, because the compounds also interfere with host cellular DNA polymerases (Mitsuya et al., 1988). Should reverse transcriptase act as a target for the anti-HIV activity of polysulfates, it would be mandatory that the compounds are taken up into the cells. Given their polyanionic character, such uptake may not readily occur. Also, inhibition of reverse transcriptase by polysulfates is achieved only at concentrations far in excess of those required for inhibition of virus replication, which, in turn, coincides quite well with those that have been found to be inhibitory to virus adsorption. It seems justified, therefore, to attribute the anti-HIV-1 activity of the polysulfates to their inhibitory effects on virus adsorption and fusion. Mechanism of anti-herpes action. The red-seaweed-extracted xylomannan sulfate F6 was found to exert its anti-HSV activity by interference with a very early stage of the HSV-1 replication cycle, because replication was blocked only when the compound was present during the virus adsorption period. Further experiments were performed to determine whether xylomannan sulfate F6 inhibited virus adsorption (Table 11). The compound was exposed to the cells either: (1) during the virus adsorption period only; (2) after virus adsorption only; or (3) both during and after virus adsorption. As can be concluded from the data presented in Table 11, xylomannan sulfate F6 lost virtually all antiviral activity when added after the virus adsorption period. The presence of xylomannan sulfate F6 during the virus adsorption period was only as effective as the presence of the compound during the whole incubation period (during and after virus adsorption). The inhibitory effect of xylomannan sulfate F6 on virus adsorption to the host cells was directly measured by monitoring the attachment of infectious HSV-1 to the cells in the presence of the compound. As shown in Figure 6, adsorption of HSV-1 to the cells was inhibited by the xylomannan sulfate F6 in a concentrationdependent manner, with an IC50 of 0.25 g ml1. Xylomannan sulfate F6 inhibited binding of radiolabeled HCMV to HEL cells with an IC50 of 16 g ml1 (Fig. 7). DS inhibited binding of radiolabeled HCMV to HEL cells with an IC50 of 0.8 g ml1 (data not shown). Also, when present at the time of infection at 100

FIGURE 6. Effect of xylomannan sulfate fraction F6 on HSV-1 adsorption. Vero cells were incubated for 30 min at 4C with HSV-1 in the presence of varying concentrations of xylomannan sulfate fraction F6. At the end of the incubation period, cellbound infectivity was determined. g ml1, xylomannan sulfate F6 reduced the expression of HCMV immediate early antigens at 24 hr postinfection by 96%, as compared with the control cultures. To rule out that xylomannan sulfate F6 also interfered with a postadsorption event, the effect of xylomannan sulfate F6 on HSV-1 penetration was examined. To this end, cells were first infected with HSV-1 at 4C to allow virus adsorption but not virus internalization. The cells were then incubated with xylomannan sulfate F6 and the temperature was immediately raised to 37C to initiate virus entry. Under these conditions, the amount of internalized virus, monitored by the number of infectious centers after incubation at 37C, was similar in xylomannan sulfate-treated and untreated cells (data not shown). Thus, xylomannan sulfate F6 did not appear to inhibit virus penetration. Moreover, the fact that the polysulfates are inhibitory to some enveloped viruses (see Activity against enveloped viruses other than HIV subsection) seems to depend on the composition of the amino

FIGURE 7. Inhibitory effect of xylomannan sulfate fraction F6 on the binding of radiolabeled HCMV (Davis strain) to HEL cells after a 30-min incubation period, as monitored by cell-associated radioactivity. Data are mean values for three separate experiments.

508 acid sequences of the viral envelope glycoproteins that are involved in viruscell fusion (Gallaher, 1987; Hosoya et al., 1991). The viruses sensitive to the polysulfates share a tripeptide segment (PheLeu-Gly). This tripeptide segment may be involved either directly (as a target sequence) or indirectly (via its influence on the overall conformation) in the inhibitory effects of these sulfated compounds (Hosoya et al., 1991). In conclusion, all the data obtained with sulfated polysaccharides clearly point to the attachment of the virus to the cell membrane as their target of antiviral action. The compounds may be assumed to block this attachment through formation of a shield between the viral envelope glycoproteins and the cellular membrane receptor.

M. Witvrouw et al.

Future perspectives
As a retrovirus, HIV characteristically inserts its genome into host cell chromosomes, leading to a persistently infected state. The problems encountered in developing agents that could specifically excise the integrated copies of the HIV genome from all infected host cells of a patient suggest that an absolute cure may be exceedingly difficult to achieve. Nonetheless, it is important to consider not only treatment of patients already infected with the virus, but also prophylaxis and protection from new infections. The awareness of the necessity for chemoprophylaxis is more recent. Because heterosexual transmission is now responsible for the large majority of infections worldwide (Nkowane, 1991), it is of paramount importance to develop effective prevention strategies to combat further spread of the epidemic via this route. Various groups and organizations have, based on epidemiological and sociological research, pointed to the importance of female-controlled methods to prevent or reduce the risk of sexually acquired HIV. The urgent need for methods to prevent sexual transmission of HIV that are under the control of women was addressed at a meeting convened by the World Health Organization (WHO) in Geneva on 1113 November 1993 (Lange et al., 1993). It led to the recommendation to encourage the development of vaginal microbicides active against HIVideally, preparations that women could use without the knowledge of their sexual partners. Although their use should not be seen as a substitute for, but as a complement to, other approaches to stop the sexual spread of HIV, it would provide women with a means of protection when they could not persuade their partner to use a condom. Considerable evidence suggests that sexual transmission of HIV is mediated via mononuclear cells that can infect epithelia of the genital tract. Pearce-Pratt and Phillips (1993) described and employed an in vitro model to examine the mechanism of cell-to-cell transmission of this virus and to identify agents that may be effective in a vaginal formulation to prevent HIV transmission via sexual contact. They found that seminal fluid significantly increases the number of lymphocytes adhering to epithelia. On the other hand, sulfated polysaccharides, or polysulfates in general, effectively inhibit cellcell adhesion. Polysulfates have yet other properties that make them an attractive candidate for a vaginal anti-HIV formulation. They are inexpensive, water-soluble and poorly absorbed (Lorentsen et al., 1989; Yarchoan et al., 1989), and, in addition to their anti-HIV activity, they are also endowed with activity against various other enveloped viruses (such as HSV). These results suggest that polysulfates may be effective in a vaginal formulation to reduce the probability of sexually transmitted HIV infection and other virus infections (such as genital herpes). Our recent data obtained with polyvinyl alcohol sulfate (PVAS) and its copolymer with acrylic acid (PAVAS) in the topical prevention of HSV-2 infection in mice (Neyts and De Clercq, 1995) point to the feasibility of such approach. References
Andrei G. and De Clercq E. (1990) Inhibitory effect of selected antiviral compounds on arenavirus replication in vitro. Antiviral Res. 14, 287 300. Andrei G., Snoeck R., Goubau P., Desmyter J. and De Clercq E. (1991) Comparative activity of selected antiviral compounds against clinical isolates of human cytomegalovirus. Eur. J. Microbiol. Infect. Dis. 10, 10261033. Andrei G. and De Clercq E. (1993) Molecular approaches for the treatment of hemorrhagic fever virus infections. Antiviral Res. 22, 4575. Baba M., Nakajima M., Schols D., Pauwels R., Balzarini J. and De Clercq E. (1988a) Pentosan polysulfate, a sulfated oligosaccharide, is a potent and selective anti-HIV agent in vitro. Antiviral Res. 9, 335343.

In vitro anti-HIV activity in combination with nucleoside analogs

Numerous antivirals with various mechanisms of action against HIV, the causative agent of AIDS, have been identified. The drugs that have been approved by the U.S. Food and Drug Administration (FDA) for the treatment of AIDS, are targeted at either the viral reverse transcriptase or protease. The nucleoside analog, 3-azido3-deoxythymidine (AZT), was the first one to be approved for clinical use. The active intracellular metabolite, AZT-triphosphate (AZT-TP), inhibits reverse transcriptase (RT) and acts as a chain terminator. The second nucleoside analog, 2,3-dideoxyinosine (ddI), which has been approved, acts at the RT level via its active metabolite, ddATP. Three other nucleoside analogs that have been approved are ddC, d4T and 3TC, which act at the RT level via their 5-triphosphates ddCTP, d4T-TP and 3TC-TP, respectively. The significant toxicity associated with administration of AZT, ddI, ddC and d4T (myelosuppression, neuropathy and/or pancreatitis) and the risk of emergence of HIV strains that are resistant to these drugs (including, in particular, 3TC) (Larder et al., 1989; St. Clair et al., 1991) emphasize the critical need to identify anti-HIV agents to be used in combination with the existing antiviral agents. Combination of different antiviral drugs has a triple aim: (i) it may prevent or delay the emergence of drug-resistant virus strains; (ii) it may lead to increased activity (synergism) and extend the antiviral spectrum to include viruses that would otherwise not be affected by the individual compounds; and (iii) it may allow reduction of the individual dosages and thus decrease drug toxicity. In 1987, Ueno and Kuno (1987) reported synergism between DS and AZT. This observation was confirmed by Hayashi et al. (1990) and Busso and Resnick (1995). Fucoidan, a complex-sulfated polysaccharide from the alga Fucus vesiculosus, was found to act synergistically with AZT (Sugawara et al., 1989). Various other drugs, for instance, soluble CD4 (Johnson et al., 1989a), castanospermine (Johnson et al., 1989b), PMEA (Smith et al., 1989), phosphonoformate (foscarnet) (Eriksson and Schinazi, 1989), TIBO derivatives (Buckheit et al., 1993) and -interferon (Hartshorn et al., 1987), have been previously reported to act synergistically with AZT in vitro. Optimal AIDS therapy may require the use of combinations of agents that exhibit synergistic antiviral effects (e.g., polysulfates and nucleoside analogs). Ideally, such combinations should target different sites in the HIV replicative cycle (e.g., virus attachment and reverse transcriptase), inhibit viral replication in a broad range of cell types, and yet not display accrued toxicity. Combination therapies may help prevent the emergence of drug-resistant HIV mutants and may also allow the use of the individual drugs below their toxicity threshold.

Polysaccharides Extracted from Sea Algae as Antiviral Drugs

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