Chemoenzymatic synthesis and application of glycopolymers containing multivalent sialyloligosaccharides with a poly(L-glutamic acid) backbone for inhibition of infection by influenza viruses

Kazuhide Totani2, Takeshi Kubota2, Takao Kuroda2, Takeomi Murata2, Kazuya I.-P. Jwa Hidari3, Takashi Suzuki3, Yasuo Suzuki3, Kazukiyo Kobayashi4, Hisashi Ashida5, Kenji Yamamoto5 and Taichi Usui1,2

2 Department of Applied Biological Chemistry, Shizuoka University, Ohya 836, Shizuoka 422-8529, Japan
3 Department of Biochemistry, University of Shizuoka, School of Pharmaceutical Science, 52-1 Yada, Shizuoka 422-8526, Japan
4 Department of Molecular Design and Engineering, Graduate School of Engineering, Nagoya University, Chikusa-ku, Nagoya 464-8603, Japan
5 Department of Applied Molecular Biology, Division of Integrated Life Science, Graduate School of Biostudies, Kyoto University, Oiwake-cho, Kitashirakawa, Sakyo-ku, Kyoto 606-8502, Japan

Received on June 10, 2002; revised on November 18, 2002; accepted on November 23, 2002


    Abstract
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 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Highly water-soluble glycopolymers with poly({alpha}-L-glutamic acid) (PGA) backbones carrying multivalent sialyl oligosaccharides units were chemoenzymatically synthesized as polymeric inhibitors of infection by human influenza viruses. p-Aminophenyl disaccharide glycosides were coupled with {gamma}-carboxyl groups of PGA side chains and enzymatically converted to Neu5Ac{alpha}2-3Galß1-4GlcNAcß-, Neu5Ac{alpha}2-6Galß1-4GlcNAcß-, Neu5Ac{alpha}2-3Galß1-3GalNAc{alpha}-, and Neu5Ac{alpha}2-3Galß1-3GalNAcß- units, respectively, by {alpha}2,3- or {alpha}2,6-sialytransferases. The glycopolymers synthesized were used for neutralization of human influenza A and B virus infection as assessed by measurement of the degree of cytopathic inhibitory effect in virus-infected MDCK cells. Among the glycopolymers tested, {alpha}2,6-sialo-PGA with a high molecular weight (260 kDa) most significantly inhibited infection by an influenza A virus, strain A/Memphis/1/71 (H3N2), which predominantly binds to {alpha}2-6 Neu5Ac residue. The {alpha}2,6-sialo-PGA also inhibited infection by an influenza B virus, B/Lee/40. The binding preference of viruses to terminal sialic acids was affected by core determinants of the sugar chain, Galß1-4GlcNAcß- or Galß1-3GalNAc{alpha}/ß- units. Inhibition of infection by viruses was remarkably enhanced by increasing the molecular weight and sialic acid content of glycopolymers.

Key words: glycopolymers / influenza virus / inhibition / poly(L-glutamic acid) / sialyloligosaccharides


    Introduction
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 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Influenza A and B viruses infect host cells through the binding of viral hemagglutinins (HAs) to sialoglycoproteins or sialoglycolipids as receptors on the host cell surface. The viruses recognize not only sialic acids (Sia) in the receptors but also particular sugar chain structures, such as sialyllacto-series type I (Sia{alpha}2-3/6Galß1-3GlcNAcß1-) and type II (Sia{alpha}2-3/6Galß1-4GlcNAcß1-) (Suzuki, 1994Go). There are differences among influenza viruses in their recognition of types (Neu5Ac or Neu5Gc) and linkages ({alpha}2-3 or {alpha}2-6) of Sia residues (Suzuki et al., 2000Go). Human influenza A viruses, which have been isolated from humans for the last 30 years, preferentially recognize {alpha}2-6Neu5Ac residue (Suzuki, 1994Go). The host cell specificity of viruses is dependent on not only the linkage of Sia to the penultimate galactose but also the number of Sia residues and core structures. Multivalent interaction must occur between viral HAs and cell surface Sia, and they should be amplified by the so-called glycoside cluster effects (Sigal et al., 1996Go).

Various synthetic glycopolymers carrying multivalent Sia residues that target viral HAs have been prepared as influenza virus inhibitors using polyacrylamide (PA) (Gamian et al., 1991Go; Spaltenstein and Whitesides, 1991Go; Lees et al., 1994Go; Itoh et al., 1995Go; Sigal et al., 1996Go; Furuike et al., 2000Go; Wu et al., 2000Go), poly(acrylic acid) (PAA) (Mammen et al., 1995Go; Choi et al., 1997Go), and polystyrene (Tsuchida et al., 1998Go) as polymer backbones. These conventional glycopolymers inhibit the binding of influenza viruses to host cell receptors with high affinity (Ki < 10-5 M of Sia residue in solution). Monomeric Sia derivatives have shown inhibition with relatively lower affinity (Ki = 10-2 ~ 10-5 M) (Sauter et al., 1989Go; Toogood et al., 1991Go). Generally, synthetic glycopolymers could have several problems for in vivo use, such as low solubility, significant cytotoxicity (Reuter et al., 1999Go), and immunogenecity (Iurovskii et al., 1986Go; Tanaka et al., 2002Go). On the other hand, sialoglycoproteins (e.g., mucins) containing a number of Sia residues have shown inhibitory activities toward influenza virus infection comparable to those of conventional glycopolymers (Boat et al., 1978Go; Burness and Pardoe, 1983Go; Rogers et al., 1983Go; Hanaoka et al., 1989Go; Pritchett and Paulson, 1989Go; Ryan-Poirier and Kawaoka, 1991Go; Suzuki et al., 1994Go). {alpha}2,6-Sialyl-N-acetyllactosamine, a typical carbohydrate determinant, is expressed on these natural sialoglycoproteins. We planned to synthesize glycopolymers that mimic these sialoglycoproteins. In these glycopolymers, the sialyl sugar units were clustered in a pendant manner on a simple polypeptide backbone poly(L-glutamic acid) (PGA).

In the present study, enzymatically synthesized p-nitrophenyl ( pNP) disaccharides were introduced to various lengths of PGAs and subsequently sialylated to highly water-soluble glycopolymers carrying clustered identical sialyldisaccharide segments. Using these glycopolymers, the inhibition of infection by human influenza viruses was investigated by measurement of the degree of the cytopathic effect in virus-infected Madine-Darby canine kidney (MDCK) cells. The physiological merits of PGA as a backbone of glycopolymers in terms of cytotoxicity and immunogenicity are discussed in this article.


    Results
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 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Synthesis of glycopolymers
A series of asialoglycopolymers (1a1c, 4a4f, and 6) with different molecular weights and degrees of substitution (DSs) of sugar units was synthesized. The DS (%) and molecular weight were controlled by changing the coupling conditions of enzymatically synthesized p-aminophenyl ( pAP) disaccharides to PGAs with different degrees of polymerization (DPs). The data for the coupling reactions are summarized in Table I. Glycopolymers 1a1c were sialylated to 2a2c and 3a3c by {alpha}2,3- and {alpha}2,6-sialyltransferases, respectively (Scheme 1) (Weinstein et al., 1982Go; Wlasichuk et al., 1993Go). The structures of synthesized glycopolymers were confirmed by 1H- and 13C-nuclear magnetic resonance (NMR) analyses with reference to previous reports (Sabesan and Paulson, 1986Go; Furuike et al., 2000Go). In the 1H-NMR spectrum of 3a, characteristic signals at {delta} 1.71 (dd, 1H, J33,34 12.2 Hz, H-3''ax) due to H-3'' and {delta} 2.68 (dd, 1H, J33 12.5 and J34 4.6 Hz, H-3''eq) due to another H-3'' were assigned. In 2a, {delta} 1.81 (dd, 1H, J33,34 11.6 Hz, H-3''ax) and {delta} 2.75 (d, 1H, J33 8.0 Hz, H-3''eq) were assigned, respectively. The 1H-NMR spectrum also showed that the sialylation was almost quantitative from the integration data of these proton signals. In the 13C-NMR chemical shift data of 2a and 3a, the respective C-3' and C-6' signals were distinct in the more downfield with chemical shifts at {delta} 78.1 and {delta} 66.1. This indicates that galactosyl residues of sugar chains are regiospecifically sialylated in {alpha}2–3 and {alpha}2-6 linkages. In a similar manner, 5a5f and 7 were prepared from 4a4f and 6, respectively, through sialylation (Scheme 2).


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Table I. Synthesis of asialoglycopolymers with different degrees of sugar substitution

 


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Scheme 1. Synthesis of glycopolymers with PGA backbones carrying Neu5Ac{alpha}2-3 or {alpha}2-6Galß1-4GlcNAcß- units.

 


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Scheme 2. Synthesis of glycopolymers with PGA backbones carrying Neu5Ac{alpha}2-3Galß1-3GalNAc{alpha}- or ß- units.

 
By a different procedure, conventional glycopolymers with PA backbones carrying the same sialyl disaccharides segments were prepared as controls to the glycopolymers with PGA backbones. First, an asialo-PA (8) carrying the LacNAc unit as a homopolymer was prepared by our previously reported method (Kobayashi et al., 1994Go). The glycopolymer has an O-linked ß-anomeric phenyl aglycon along a PA backbone. Next, 8 was converted to 9 and 10 using sialyltransferases as mentioned. The ratio of sialylation in 9 was as high as 90%, despite the high density of sugar chains in 8 as side chains. In 10, the ratio was as low as 50%. The {alpha}2,6-sialylation was much more susceptible to the elongation of sugar chains in 8 than was the {alpha}2,3-sialylation. All of the glycopolymers synthesized are summarized in Table II.


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Table II. Glycopolymers for influenza virus infection inhibition

 
Binding of influenza viruses to glycopolymers
Influenza viruses A/PR/8/34 (H1N1) and A/Memphis/1/71 (H3N2) predominantly bind to {alpha}2-3 and {alpha}2-6Neu5Ac residues, respectively (Suzuki et al., 1992Go; Furuike et al., 2000Go). To confirm the binding of the viruses to the synthesized glycopolymers, 1b, 2b, and 3b immobilized on the plate were used for testing of the virus binding specificity by the enzyme-linked immunosorbent assay (ELISA) method. As expected, A/PR/8/34 preferentially bound to 2b (Figure 1A). In contrast, A/Memphis/1/71 bound to 3b (Figure 1B). Both virus strains showed almost no binding to 1b. The binding to 2a and 3a was similar to those to 2b and 3b (data not shown). These results indicate that the synthetic glycopolymers bind to the viruses based on the viral binding specificity for Sia-linkages.



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Fig. 1. Binding of influenza A viruses to glycopolymers immobilized on a microtiter plate. A/PR/8/34 (A) and A/Memphis/1/71 (B) were twofold serially diluted with PBS to 20–27 HAU, added to the glycopolymer-immobilized wells, and then incubated at 4°C for 12 h. The virions that had bound to fetuin on the wells were reacted with rabbit anti-influenza virus antiserum (anti-P-50), and bound antibodies were detected with HRP-conjugated protein A as described previously. The absorbance was measured at 492 nm with 630 nm as a reference wavelength. Each assay was carried out in duplicate. Immobilized glycopolymers were 1b ({blacktriangleup}), 2b ({blacksquare}), and 3b ({bullet}). See Materials and methods.

 
Inhibition of binding of influenza viruses by glycopolymers
Next we tested whether the glycopolymers inhibit the binding of influenza viruses to bovine fetuin containing {alpha}2-3 and {alpha}2-6Neu5Ac residues (Baenziger and Fiete, 1979Go; Edge and Spiro, 1987Go). The binding of A/PR/8/34 to immobilized fetuin was inhibited by 2a and 5b (Figure 2A). The binding of A/Memphis/1/71 to fetuin was inhibited by 3a (Figure 2B). These observation were in good agreement with the viral binding specificity for Sia-linkage as described previously (Suzuki et al., 1992Go; Furuike et al., 2000Go).



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Fig. 2. Inhibition of binding of influenza A viruses to fetuin. A/PR/8/34 (A) and A/Memphis/1/71 (B) diluted with PBS were incubated in fetuin-coated wells at 4°C in the presence or absence of glycopolymers 1a, 2a, 3a, or 5b (0.5–500 µg/ml) or fetuin. Bound viruses were detected as described previously. The data are expressed as means ± SE. Each assay was carried out in triplicate. See Materials and methods.

 
Glycopolymers (2a and 5a) synthesized from a PGA with a low molecular weight showed no inhibitory effects on infection of MDCK cells by A/PR/8/34 in viral neutralization assays (data not shown). As shown in Figures 3A and 3B, the infection of MDCK cells by A/PR/8/34 was significantly inhibited by 2c carrying Neu5Ac{alpha}2-3Galß1-4GlcNAcß- (IC50 20 µg/ml), 5f carrying Neu5Ac{alpha}2-3Galß1-3GalNAc{alpha}- (IC50 20 µg/ml), and 7 carrying Neu5Ac{alpha}2-3Galß1-3GalNAcß-. No inhibition was observed by 3c carrying Neu5Ac{alpha}2-6Galß1-4GlcNAcß-. Among the synthetic glycopolymers tested, the infection of MDCK cells by A/Memphis/1/71 was most strongly inhibited by 3c (IC50 3 µg/ml) (Figure 3D). Infection by A/Memphis/1/71 was also inhibited to a lesser extent by 2c (IC50 30 µg/ml) but not by 5f or 7 (Figure 3E). In type B viruses, B/Lee/40 predominantly binds to {alpha}2-6Neu5Ac residues, and B/Gifu/2/73 binds to both {alpha}2-3 and {alpha}2-6Neu5Ac residues (Xu et al., 1994Go). Infection by B/Lee/40 was inhibited by 3c (IC50 30 µg/ml) and by 10 (IC50 100 µg/ml) to a lesser extent (Figures 4A and 4B). Infection by B/Gifu/2/73 was inhibited equally by 2c and 3c (IC50 30 µg/ml) and by 5f (IC50 100 µg/ml) (Figure 4C).



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Fig. 3. Neutralization of infection of MDCK cells by influenza A virus. One hundred microliters of TCID50 of A/PR/8/34 (A, B, C) or A/Memphis/1/71 (D, E, F) were preincubated at 4°C for 1 h in the presence or absence of glycopolymers (0.01–1000 µg/ml) in PBS and inoculated at 34.5°C for 5 h on MDCK cell monolayers. The cells were washed three times with EMEM and incubated at 34.5°C for 18 h in 100 µl of EMEM supplemented with 5% FBS. The activity of LDH released from MDCK cells was determined as the viral-induced cytopathic effect using a slightly modified calorimetric assay as previously described. The data are expressed as means ± SE of three independent experiments. Each experiment was carried out in duplicate. See Materials and methods.

 


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Fig. 4. Neutralization of infection of MDCK cells by influenza B virus. All assays were performed in a manner similar to that described in the legend to Figure 3 using B/Lee/40 (A, B) and B/Gifu/2/73 (C) strains.

 
It was also found that PA-based glycopolymers inhibited infection by the viruses in an Sia-linkage-dependent manner, as described previously (Furuike et al., 2000Go). As shown in Figure 3C, infection by A/PR/8/34 was inhibited by 9 carrying Neu5Ac{alpha}2-3Galß1-4GlcNAcß- (IC50 20 µg/ml) but not by 10 carrying Neu5Ac{alpha}2–6Galß1–4GlcNAcß-. Infection by A/Memphis/1/71 was inhibited by 10 (IC50 20 µg/ml) but not by 9 (Figure 3F). This result was different from the case of 2c in Figure 3D. Infection by B/Lee/40 was also inhibited by 10 (IC50 100 µg/ml). Desialylated glycopolymers (1c, 4f, 6, and 8) and PGAs showed no inhibitory effects on infection by either virus strain. pNP sialyl oligosaccarides (Neu5Ac{alpha}2-3 or {alpha}2-6Galß1-4GlcNAcß-pNP) showed no inhibitory effects at concentration as high as 1000 µg/ml, which is equivalent to approximately 1.2 mM of Sia residue in solution (Table III).


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Table III. IC50 of glycopolymers in neutralization assays of influenza A and B viruses

 
Effects of molecular weights and Sia contents of glycopolymers
In order to determine the effects of molecular weights or DPs of PGAs on inhibition of viral infection of cells, neutralization assays were carried out using 5a, 5b, and 5e with different molecular weights (30, 80, and 180 kDa, respectively) or DPs (95, 238, and 475, respectively). These glycopolymers have almost equivalent DSs and Sia contents (around 25%). The inhibitory effects of the glycopolymers on infection by A/PR/8/34 increased in a molecular weight-dependent manner (Figure 5A). To determine the effects of Sia contents in glycopolymers, neutralization assays using 4f, 5d, 5e, and 5f with different Sia contents (0%, 13%, 27%, and 44%, respectively) were carried out. The inhibitory effect on infection by A/PR/8/34 was enhanced in a Sia content-dependent manner (Figure 5B). Taken together, the results indicate that the ability of glycopolymers to neutralize influenza viruses is dependent on the molecular weights and Sia contents of the glycopolymers.



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Fig. 5. Effects of molecular weights (or DPs of PGA) (A) and sialic acid contents (B) of glycopolymers on A/PR/8/34 infection. The virus-induced cytopathic effect was determined as described in the legend to Figure 3.

 
Cytotoxicity and immunogenicity of glycopolymers
PGA, PA, and the glycopolymers tested (4d and 8) showed no cytotoxicity to MDCK cells in 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays at the concentrations tested. PAA-Na and acrylamide monomer showed cytotoxicity at concentrations of 1000–10,000 µg/ml (Figure 6). When 4b was injected into three mice by a conventional immunization method, no visible elevation in the titer of the antibody in sera reacting to the glycopolymer immobilized on the ELISA plate was observed (Figure 7).



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Fig. 6. MDCK cellular toxicity assay. MDCK cell monolayers were incubated with glycopolymers 4d, 8, PGA-Na, PA, PAA-Na, or acrylamide monomer (1–10,000 µg/ml) at 37°C for 24 h. The cell viability was quantified using the MTT spectrophotometric assay. The data are expressed as means ± SE. Two independent experiments were carried out in triplicate. See Materials and methods.

 


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Fig. 7. Change of titer of antibody reacting to a glycopolymer in sera from glycopolymer-immunized mice. Glycopolymer 4b (2 mg/ml) was injected into mice according to conventional immunization methods. Serially diluted sera (x20, x200) from the immune and control mice (N = 3) were reacted with 4b immobilized on microtiter plate and detected with HRP-conjugated anti-mouse immunoglobulins. The data from three mice are expressed as means ± SE. The immobilization of 4b on the plate was confirmed by binding of HRP-conjugated PNA to the plate (inset). See Materials and methods.

 

    Discussion
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 Abstract
 Introduction
 Results
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 Materials and methods
 References
 
PGA was used as a backbone of each glycopolymer because it was nontoxic and easy to couple with pAP disaccharides. DS (%) of pAP disaccharides in the resulting asialoglycopolymers increased to more than 60% by controlling the coupling reaction (Table I). {alpha}2,6-Sialylation by sialyltransferase occurred at low efficiency, around 50%, in the case of 8 with a PA backbone, possibly due to steric hindrance caused by the high density of pAP disaccharide units in the homopolymer (Table II). When LacNAc disaccharide was aminated at the C1 position and directly introduced to {gamma}-carboxy groups of PGA instead of LacNAcß-pAP, the resulting glycopolymers were hardly sialylated (data not shown). This indicates that sialylation in such glycopolymers requires some spacers, such as a pAP group between the sugar unit and PGA backbone.

Among the glycopolymers tested for viral neutralization assays, 3c carrying multivalent Neu5Ac{alpha}2-6Galß1-4GlcNAcß- units most significantly inhibited (IC50 3 µg/ml) the infection by A/Memphis/1/71. This glycopolymer also inhibited (IC50 30 µg/ml) infection by B/Lee/40. Therefore, the {alpha}2,6-sialoglycopolymer with a high-molecular-weight PGA backbone is possibly an effective inhibitor of infection by both influenza A and B viruses. To date, infections by influenza viruses have been successfully inhibited by Sia-conjugated PA (Gamian et al., 1991Go; Spaltenstein and Whitesides, 1991Go; Sparks et al., 1993Go; Lees et al., 1994Go; Itoh et al., 1995Go; Sigal et al., 1996Go; Wu et al., 2000Go), PAA (Mammen et al., 1995Go; Choi et al., 1997Go), dendrimers (Reuter et al., 1999Go), sialylphosphatidylethanolamine derivatives (Guo et al., 1998Go), sialyl lactose-conjugated polystyrene (Tsuchida et al., 1998Go), lyso-GM3-conjugated PGA (Kamitakahara et al., 1998Go), GM3 lactone (Sato et al., 1999Go), and sialyl LacNAc-conjugated PA (Furuike et al., 2000Go). Among these inhibitors, IC50 of glycopolymer 3c used in the present study is almost equal to that of polymers synthesized by Tuchida et al. (1998) or Furuike et al. (2000)Go (Table III).

The relationship between viral inhibitory activities and core sugar chain structure in glycopolymers is not clear. In our study, the contribution of asialo-portions in sugar chains was examined. Infection by A/PR/8/34 was inhibited equally by {alpha}2,3-sialoPGAs 2c, 5f, and 7 (IC50 ~ 20 µg/ml), suggesting that there is no contribution by the asialo-portion to inhibitory activity of the glycopolymers. Infection by A/Memphis/1/71 was inhibited by 2c carrying Neu5Ac{alpha}2-3Galß1-4GlcNAcß- but not by 5f with Neu5Ac{alpha}2-3Galß1-3GalNAc{alpha}- or 7 with Neu5Ac{alpha}2-3Galß1-3GalNAcß- units (Figure 3D and E), indicating that the asialo-portion (LacNAc) in sugar chain is important for the binding of A/Memphis/1/71 (H3N2) to terminal Neu5Ac residues. {alpha}- or ß-GalNAc residues in 5f and 7 may present unfavorable steric interactions to the virus HAs to decrease their affinity to the terminal Neu5Ac residues. Paulson and colleagues have demonstrated that the binding of influenza virus to Sia residues is influenced by the asialo-portion of the carbohydrate structure based on the inhibition of adsorption of A/Memphis/102/72 (H3) to erythrocytes using natural and synthetic monovalent sialosides (Rogers and Paulson, 1983Go; Pritchett et al., 1987Go; Matrosovich et al., 1993Go). B/Gifu/2/73 is known to have binding characteristics that are different to those of other influenza B viruses (Xu et al., 1994Go). The fact that the inhibitory activities of 5f and 7 were less than that of 2c indicates that the asialo-portion in sugar chains also affected the binding of B/Gifu/2/73 (Figure 4C).

PA-based glycopolymers (homopolymers) gave somewhat different results compared to those obtained using PGA-based glycopolymers. Glycopolymer 9 with a PA backbone showed no inhibitory activity toward A/Memphis/1/71 infection (Figure 3F), whereas 2c with a PGA backbone inhibited the infection by this strain, as previously mentioned. These findings suggest that the backbone of a glycopolymer (PA or PGA) affects virus inhibitory activity. Roy and colleagues found that the type of carrier molecules of Sia in neoglycoconjugates also affected the inhibitory activity toward an influenza virus (Gamian et al., 1991Go). A similar effect by carriers of sugar chains was reported by Kojima et al. (2002)Go in adhesion of Helicobactor pylori to neoglycoconjugates carrying Leb saccharides. Our data on virus inhibition by 2c with a PGA backbone are in accordance with the results using a copolymer with a PA backbone (Furuike et al., 2000Go). The density of sugar chains in glycopolymers, which is higher in homopolymers and lower in copolymers, may affect the inhibitory activities of glycopolymers toward viruses.

Whitesides and colleagues reported that the crucial factors for influenza virus inhibition by conventional Sia-conjugated glycopolymers are the cluster effect of Sia, molecular weights, charges, bulky and hydrophobic groups, and steric stabilization of the glycopolymer (Spaltenstein and Whitesides, 1991Go; Sparks et al., 1993Go; Lees et al., 1994Go; Mammen et al., 1995Go; Sigal et al., 1996Go). The effect of molecular weight (length) of backbone was also found to be critical in our glycopolymers. Glycopolymers 2c and 3c were roughly 10-fold more effective than were 2b and 3b in viral inhibition (Figure 3). As shown in Figure 5, the inhibitory activity toward virus infections increased with increases in molecular weights (or DPs) of PGAs and Sia contents (%) in glycopolymers. This indicates that much stronger inhibition can be expected if a longer PGA is used in a sufficient Sia content.

Generally, conventional glycopolymers have a problem of toxicity caused by a backbone such as PA (Ikeda et al., 1994Go; Reuter et al., 1999Go). In our study, acrylamide monomer, which is known to be a potent neurotoxin (Spencer and Schaumburg, 1975Go), and PAA-Na were cytotoxic to MDCK cells in the conditions used for the tests (Figure 6). These are materials for the synthesis of conventional glycopolymers (Lees et al., 1994Go; Choi et al., 1997Go), and this would limit its utility in vivo. It has also been shown that sugar–bovine serum albumin(BSA) or PA conjugates act as antigens or immunogens. Sharp elevations (e.g., by 10,000-fold) of the titers in sera were observed after immunization of mice with a synthetic Galß1-3GalNAc{alpha}-BSA (Rittenhouse-Diakun et al., 1998Go), PA-carrying Lea saccharides (Iurovskii et al., 1986Go), or PA-carrying sulfated saccharides (Tanaka et al., 2002Go). We tested this point with our PGA-based glycopolymer. When BALB/c mice were immunized with a PGA-carrying Galß1-3GalNAc{alpha}-(4b), no visible elevation of the titer of the antibody in sera reacting to the glycopolymer was observed (Figure 7). This might be because PGA has a relatively low immunogenicity (Hoes et al., 1993Go). It should be emphasized that our PGA-based glycopolymers have extremely high solubility in water, more than 10% (w/v), compared to that of PA-based glycopolymers (less than 1%) and that they are remarkably heat-stable with no aggregation even in boiling water. Therefore, PGA-based glycopolymers have ideal characteristics as in vivo polymeric inhibitors.

Recently, noncytotoxic Sia-conjugated dendritic polymers have been successfully synthesized (Reuter et al., 1999Go). However, there have only been a few studies on in vivo inhibition of influenza virus infection by synthetic glycopolymers (Ikeda et al., 1994Go; Gambaryan et al., 2002Go). PGA has been extensively studied as a drug protectant in a drug delivery system (Jackman et al., 1993Go; Lescure et al., 1995Go; Akamatsu et al., 1997Go, 1999Go; Li et al., 1998Go; Zou et al., 2001Go) or as a biocompatible medical material (Honde et al., 1994Go; Lescure et al., 1995Go; Otani et al., 1996Go). In vivo application of PGA-based glycopolymers is expected in the near future.


    Materials and methods
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 Abstract
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 Results
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 Materials and methods
 References
 
Materials
PGA-Na (MW 14,300, 35,900, 54,800, and 71,700), PA (MW 10,000), PAA-Na (MW 30,000), MTT, fetal calf serum fetuin, fetal bovine serum (FBS), and Eagle's minimum essential medium (EMEM) were purchased from Sigma-Aldrich (St. Louis, MO). EMEM was used as a mixture with 2 mM L-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin (Life Technologies, GibcoBRL, Tokyo). CMP-ß-D-N-acetylneuraminic acid disodium salt (CMP-ß-Neu5Ac, 2Na) was a kind gift from Yamasa Corporation (Chiba, Japan). {alpha}2,3-(N)-sialyltransferase (rat recombinant, Spodoptera frugiperda), {alpha}2,6-(N )-sialyltransferase (rat recombinant, S. frugiperda), and {alpha}2,3-(O)-sialyltransferase (rat recombinant, S. frugiperda) were purchased from Calbiochem-Novabiochem (San Diego, CA). A kit for virus neutralization assay (lactate dehydrogenase [LDH] linear neo) was purchased from Shino-test (Tokyo). Rabbit anti-influenza A virus antiserum (anti-P-50) was raised against an influenza A virus carrying the HA gene from strains A/PR/8/34 (H1N1) and A/Aichi/2/68 (H3N2) (Suzuki et al., 1996Go). Human influenza A and B virus strains, A/PR/8/34 (H1N1), A/Memphis/1/71 (H3N2), B/Lee/40, and B/Gifu/2/73, were grown in the allantoic sacs of 10-day-old embryonated eggs and purified by sucrose-density gradient centrifugation. Viral hemagglutination units (HAUs) of purified viruses were determined as described previously (Suzuki et al., 1983Go). MDCK cells were propagated in EMEM supplemented with 10% FBS. All other reagents were of the highest quality commercially available and were used without further purification.

Analytical methods
High-performance liquid chromatography was done using a Jasco Gulliver system (Jasco Corp., Tokyo, Japan). 1H- and 13C-NMR spectra were recorded on a JEOL JNM-LA 500 spectrometer or a JEOL JNM-EX 270 spectrometer. Chemical shifts were expressed in {delta} relative to sodium 3- (tri-methylsilyl)-propionate as an external standard.

Synthesis of glycopolymers with PGA backbones
Glycopolymers 1a1c [Poly(Galß1-4GlcNAcß-pAP/Gln- co-Glu)], 4af [Poly(Galß1-3GalNAc{alpha}-pAP/Gln-co-Glu)], and 6 [Poly(Galß1-3GalNAcß-pAP/Gln-co-Glu)] were prepared by our previously reported methods (Zeng et al., 1998Go, 2000Go). The compositions of pAP disaccarides, PGAs, and reagents used for the coupling reactions are summarized in Table I. The DS in the mole fraction of substituted residues in glycopolymers as a percentage was calculated from the relative intensities of 1H-NMR signal areas of phenolic protons, H-1 proton, peptide {gamma}- and ß-methylene protons, and acetyl protons. Glycopolymers 5af [Poly(Neu5Ac{alpha}2-3Galß1-3GalNAc{alpha}-pNP/Gln-co-Glu)] and 7 [Poly(Neu5Ac{alpha}2-3Galß1-3GalNAcß-pNP/Gln-co-Glu)] were prepared by {alpha}2,3-(O)-sialyltransferase from 4af and 6, respectively, as previously described (Zeng et al., 2000Go).

Glycopolymers 2a2c [Poly(Neu5Ac{alpha}2-3Galß1-4Glc-NAcß-pAP/Gln-co-Glu)] were enzymatically synthesized from 1a1c as follows. A mixture containing 5 mg of 1a, 1b, or 1c, 7.5 mg CMP-ß-Neu5Ac, 15 mU of rat recombinant {alpha}2,3-(N )-sialyltransferase, 2.5 mM MnCl2, 0.1% BSA, and 10 U calf intestine alkaline phosphatase (Boehringer-Mannheim, Mannheim, Germany) in 50 mM sodium cacodylate buffer ( pH 6.0) was incubated at 37°C for 48 h in a total volume of 0.5 ml. After heating at 100°C and centrifugation, the supernatant from the reaction mixture was charged onto a Sephadex G-25M PD-10 column Amersham Biosciences Corp. (NJ, USA) in the presence of 0.15 M NaCl. The high-molecular-weight fraction collected was dialyzed against distilled water for 3 days and lyophilized to afford the respective glycopolymers 2a, 2b, and 2c (6.3, 6.7, and 3.3 mg). 1H-NMR data of 2a (500 MHz, D2O, 30°C): {delta} 7.26 (o-Ph), 6.97 (m-Ph), 5.04 (H-1), 4.57 (H-1'), 4.3 ({alpha}-methine, Gln-co-Glu), 3.5–4.0 (m, 19H, from sugar), 2.75 (d, 1H, J33 8.0 Hz, H-3''eq), 2.2–2.5 ({gamma}-methylene, Gln-co-Glu), 2.03 (ß-methylene, Gln-co-Glu), 2.01 (CH3CO, GlcNAc, Neu5Ac), and 1.81 (dd, 1H, J33,34 11.6 Hz, H-3''ax). 13C-NMR data of 2a (500 MHz, D2O, 30°C): {delta} 177.9 (CH3CO, Neu5Ac), 177.7 (COOH, Gln-co-Glu), 176.5 (CH3CO, GlcNAc), 173.5 (C''-1), 155.9 (c-Ph), 135.6 ( p-Ph), 127.8 (m-Ph), 120.2 (o-Ph), 106.3 (C-1'), 104.5 (C-2''), 103.2 (C-1), 82.6 (C-4), 78.5 (C-5'), 78.1 (C-3'), 76.6 (C-5), 75.9 (C-6''), 75.4 (C-3), 74.9 (C-2'), 74.7 (C-8''), 71.3 (C-4'), 71.2 (C-4''), 71.1 (C-7''), 65.5 (C-9''), 63.9 (C-6'), 62.9 (C-6), 57.8 (C-2), 56.4 ({alpha}-methine, Gln-co-Glu), 54.9 (C-5''), 43.1 (C-3''), 36.3 ({gamma}-methylene, Gln-co-Glu), 30.9 (ß-methylene, Gln-co-Glu), 25.2 (CH3CO, Neu5Ac), and 25.0 (CH3CO, GlcNAc). Glycopolymers 2b and 2c gave similar NMR data.

Glycopolymers 3a3b [Poly(Neu5Ac{alpha}2-6Galß1-4Glc-NAcß-pAP/Gln-co-Glu)] were also synthesized from 1a1c using rat recombinant {alpha}2,6-(N )-sialyltransferase (15 mU) in a similar manner to afford the respective glycopolymers 3a, 3b, and 3c (5.0, 6.1, and 5.7 mg). 1H-NMR data of 3a (500 MHz, D2O, 30°C): {delta} 7.29 (o-Ph), 7.01 (m-Ph), 5.08 (H-1), 4.45 (H-1'), 4.28 ({alpha}-methine, Gln-co-Glu), 3.5–4.0 (m, 19H, from sugar), 2.68 (dd, 1H, J33 12.5 and J34 4.6 Hz, H-3''eq), 2.2–2.5 ({gamma}-methylene, Gln-co-Glu), 2.04 (ß-methylene, Gln-co-Glu), 2.01 (CH3CO, GlcNAc, Neu5Ac), and 1.71 (dd, 1H, J33,34 12.2 Hz, H-3''ax). 13C-NMR data of 3a (500 MHz, D2O, 30°C): {delta} 184.2 (COOH, Gln-co-Glu), 177.7 (CH3CO, Neu5Ac), 176.3 (CH3CO, GlcNAc), 174.1 (C''-1), 155.9 (c-Ph), 134.9 (p-Ph), 126.6 (m-Ph), 120.0 (o-Ph), 106.3 (C-1'), 103.0 (C-2''), 102.4 (C-1), 82.9 (C-4), 77.5 (C-5'), 76.4 (C-5), 75.4 (C-6''), 75.2 (C-3'), 75.1 (C-3), 74.5 (C-8''), 73.6 (C-2'), 71.2 (C-4'), 77.1 (C-4''), 71.0 (C-7''), 66.1 (C-6'), 65.5 (C-9''), 62.9 (C-6), 57.6 (C-2), 56.5 ({alpha}-methine, Gln-co-Glu), 54.3 (C-5''), 42.9 (C-3''), 36.4 ({gamma}-methylene, Gln-co-Glu), 30.7 (ß-methylene, Gln-co-Glu), 25.1 (CH3CO, Neu5Ac), and 24.9 (CH3CO, GlcNAc). Glycopolymers 3b and 3c gave similar NMR data.

Synthesis of glycopolymers with PA backbones
PA-based asialoglycopolymer 8 [poly(pAP ß-N-acetyllactosaminide-carrying acrylamide): PAP(LacNAcß-pAP)] was prepared by coupling LacNAcß-pAP to an acrylate monomer and its homopolymerization according to our previously reported method (Kobayashi et al., 1994Go). Glycopolymer 9 [PAP(Neu5Ac{alpha}2-3Galß1-4GlcNAcß-pAP), 5.7 mg] was enzymatically synthesized from 5 mg of 8 and 10 mg of CMP-ß-Neu5Ac using {alpha}2,3-(N )-sialyltransferase (20 mU) in the same manner as that for the syntheses of 2a2c. Glycopolymer 10 [PAP(Neu5Ac{alpha}2-6Galß1-4GlcNAcß-pAP), 5.6 mg] was synthesized using {alpha}2,6-(N )-sialyltransferase (20 mU) in a similar manner. The structures of 9 and 10 were confirmed with reference to NMR data of PA-based glycopolymers reported previously (Kojima et al., 2002Go).

Synthesis of pNP oligosaccharides
Neu5Ac{alpha}2-3Galß1-4GlcNAcß-pNP was enzymatically synthesized from LacNAcß-pNP prepared by our previously reported method (Usui et al., 1993Go) as follows: a mixture containing 12.5 mg LacNAcß-pNP, 16 mg CMP-ß-Neu5Ac, 30 mU rat recombinant {alpha}2,3-(N)-sialyltransferase, 2.5 mM MnCl2, 0.1% BSA, and 30 U alkaline phosphatase in 50 mM sodium cacodylate buffer (pH 6.0) was incubated at 37°C for 48 h in a total volume of 1.0 ml. After terminating the reaction, the supernatant from the reaction mixture was charged onto a Toyopearl HW-40 s column ({phi} 2.5x85 cm) (Tosoh Corp., Tokyo, Japan) and developed with 25% MeOH monitoring at 210 nm (carbonyl group), 300 nm ( pNP group), and 485 nm (sugar content, phenol–sulfuric acid method). The flow rate was 1.0 ml/min, and the fraction size was 6 ml/tube. The fractions (tubes 31–37) in which profiles of three absorbances agreed were lyophilized to afford Neu5Ac{alpha}2-3Galß1-4GlcNAcß-pNP (18.1 mg). Neu5Ac{alpha}2-6Galß1-4GlcNAcß-pNP (18.7 mg) was synthesized using {alpha}2,6-(N )-sialyltransferase (30 mU) in a similar manner. The structures of these compounds were confirmed with reference to NMR data reported previously (Sabesan and Paulson, 1986Go).

Binding of influenza A viruses to glycopolymers
Glycopolymers 1b, 2b, and 3b were covalently immobilized on a microtiter plate (Corning-Costar, Labcoat 2504, Cambridge, MA). The wells were treated with a glycopolymer solution (10 µg/ml) in 10 mM sodium acetate buffer (pH 4.0) at 25°C for 1 h and irradiated under UV light at 254 nm for 1 min. The wells were blocked with 100 µl phosphate buffered saline (PBS) containing 2% BSA at 25°C for 1 h and then washed with PBS five times. Influenza viruses (A/PR/8/34 and A/Memphis/1/71) were twofold serially diluted with PBS to 20–27 HAU, added to the glycopolymer-immobilized wells, and then incubated at 4°C for 12 h. The virions that had bound to fetuin on the wells were reacted with rabbit anti-influenza virus antiserum (anti-P-50) and detected with horseradish peroxidase (HRP)–conjugated protein A (Organon Teknika N. V. Cappel Products, Turnhout, Belgium) as described previously (Suzuki et al., 1992Go). The absorbance was measured at 492 nm with 630 nm as a reference wavelength. All assays were carried out in duplicate (Figure 1A and B).

Inhibition of influenza virus binding to fetuin by glycopolymers
Fetuin was adsorbed on a polystyrene-surface microtiter plate (Nunc-Immuno Plate, MaxiSorp, Nalge Nunc International, Roskilde, Denmark). The wells were treated at 37°C for 1 h with fetuin (10 µg/ml) in 50 µl PBS. The wells were washed with PBS three times and blocked at 25°C for 2 h with 100 µl PBS containing 1% BSA. The wells were then washed with PBS five times. Influenza A viruses (A/PR/8/34 and A/Memphis/1/71) were diluted with 50 µl PBS to 27–28 HAU and bound on the fetuin-coated surface at 4°C for 12 h in the presence or absence of glycopolymers 1a, 2a, 3a, or 5b (0.5–500 µg/ml) or fetuin. As negative controls, diluted viruses were added to wells that had not been coated with fetuin. The virions that had bound to fetuin on the wells were detected as described previously (Suzuki et al., 1992Go). All assays were carried out in triplicate.

Neutralization of infection of MDCK cells by influenza virus
The TCID50 (50% tissue-culture infectious dose) of each virus to MDCK cells was determined as described previously (Suzuki et al., 1996Go). One hundred microliters of TCID50 of each virus, which corresponds to 2-2–21 HAU, was preincubated at 4°C for 1 h in the presence or absence of glycopolymers (0.01–1000 µg/ml) in PBS. The preincubated mixtures were inoculated at 34.5°C for 5 h on MDCK cell monolayers in 96-well microtiter plates (Nunclon Delta Surface, Nalge Nunc International Corp., Roskilde, Denmark). After removal of the inoculums, the cells were washed three times with EMEM and incubated at 34.5°C for 18 h in 100 µl EMEM supplemented with 5% FBS. Viral-induced cytopathic effect was monitored by light microscopy. The activity of LDH released from MDCK cells was determined using a slightly modified calorimetric assay as previously described (Watanabe et al., 1995Go; Suzuki et al., 1996Go; Tsuchida et al., 1998Go). The culture medium (40 µl) was mixed with fivefold concentrated PBS (10 µl). The plates were preincubated at 37°C for 5 min. LDH reagent (50 µl) was added, and the mixture was incubated 37°C for 10 min. The reaction was stopped by the addition of 100 µl of 0.5 M HCl. The absorbance was measured at 550 nm with 630 nm as a reference wavelength. Each experiment was carried out in duplicate.

MDCK cellular toxicity assay
MDCK cell monolayers in 96-well microtiter plates (Delta Surface) were washed with 200 µl of serum-free EMEM three times and incubated with 100 µl (1–10,000 µg/ml) of 4d, 8, PA, PAA-Na, or acrylamide monomer at 37°C for 24 h. The cell viability was quantified using the MTT spectrophotometric assay (Watanabe et al., 1994Go; Reuter et al., 1999Go).

Immunization of mice with a glycopolymer
Glycopolymer 4b (2 mg/ml in saline) was mixed with an equal volume of Freund's complete adjuvant (Difco Laboratories, Detroit, MI) and ID injected into female BALB/c mice (4b, 200 µg/animal, N = 3). Three and five weeks later, the mice were boosted by intraperitoneal injection of 0.2 ml of the Freund's-antigen mixture (Rittenhouse-Diakun et al., 1998Go).

ELISA for antisera
Sera from the immune and control mice (N = 3) were diluted with PBS, pH 7.4, 0.05% Tween 20 containing 1% BSA, and 50 µl were added to each well of a 4b-immobilized microtiter plate (Labcoat 2504, Corning International K.K., Tokyo, Japan). The samples were analyzed in duplicate. The plates were incubated for 18 h at 4°C, and bound antibodies were detected by 1:1000-diluted HRP-conjugated anti-mouse immunoglobulins (IgG, IgA, and IgM) (Cappel, ICN Pharmaceuticals, Aurora, OH). The absorbance was measured at 492 nm as described earlier. To confirm the immobilization of 4b on the plate, HRP-conjugated PNA (from Arachis hypogaea, Honen, Honen Corp., Tokyo, Japan) was serially diluted with PBS, pH 7.4, 0.05% Tween 20 and shown to bind to the plate as described earlier. PNA is known to bind to 4b strongly (Zeng et al., 2000Go).


    Acknowledgements
 
We are grateful to the Yamasa Corporation for generously presenting the CMP-Fuc. This work was supported by a grant-in-aid for scientific research (nos. 13556017 and 13557207) from the Ministry of Education, Science, Sports, and Culture of Japan.

1 To whom correspondence should be addressed; e-mail: actusui{at}agr.shizuoka.ac.jp Back


    Abbreviations
 
BSA, bovine serum albumin; DP, degree of polymerization; DS, degree of substitution; ELISA, enzyme-linked immunosorbent assay; EMEM, Eagle's minimum essential medium; FBS, fetal bovine serum; HA, hemagglutinin; HAU, hemagglutination unit; HRP, horseradish peroxidase; LacNac, N-acetyllactosamine; LDH, lactate dehydrogenase; MDCK, Madine-Darby canine kidney; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; NMR, nuclear magnetic resonance; PA, polyacrylamide; PAA poly(acrylic acid); pAP, p-aminophenyl; PBS, phosphate buffered saline; PGA, poly(L-glutamic acid); PNA, peanut agglutinin; pNP, p-nitrophenol, p-nitrophenyl.


    References
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 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
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