Yutaro Kaneko

Synthesis of curdlan sulfates having inhibitory effects in vitro against AIDS viruses HIV-1 and HIV-2

Nucleoside analogues such as azidothymidine (AZT), dideoxyinosine (ddI), and dideoxycytidine (ddC) [1] inhibit the viral reverse transcriptase (RT) of human immunodeficiency virus (HIV) and terminate DNA chain synthesis from viral RNA inside the infected cells. However, these nucleosides manifest serious side-effects such as bonemarrow toxicity and the appearance of AZT-resistant viruses upon long-term treatment [2]. It is difficult to make an effective vaccine owing to the multiplicity of AIDS viruses. Therefore, anti-AIDS compounds having a different blocking mechanism are of interest. We have synthesized sulfated polysaccharides demonstrating high anti-AIDS virus activity by sulfation of synthetic and natural-occurring polysaccharides [3-6]. The mechanism of action of sulfated polysaccharides is assumed to be different from that of the nucleosides, that is, they bind to HIV virions and prevent them from penetrating into target cells [7-9]. However, sulfated polysaccharides such as dextran sulfate have high blood anticoagulant activity, leading to undesirable side-effects when used as anti-AIDS drugs [10].

Sulfation of the immunomodulating polysaccharide lentinan: A novel strategy for antivirals to human immunodeficiency virus (HIV)

Abstract It has been clearly established that human immunodeficiency virus (HIV) is a causative agent of the acquired immunodeficiency syndrome (AIDS). The virus preferentially infects and kills CD-4-positive (helper/inducer) T lymphocytes resulting in severe T4 lymphopenia as well as indirect suppression of multiple immune functions which are dependent on induction of T4 cells (1–2). Since AIDS is a disease in which the HIV destroys the T4 cells slowly but continuously, it is apparent that any attempt to treat AIDS must include anti-HIV therapy. Furthermore, attempts must be made to reconstitute immunologic disfunction caused by the virus. Recently, it has been reported that a sea algal extract (SAE) from Schizymenia pacifica inhibits viral adsorption to the cells and reverse transcriptase (RT) of HIV without interfering with cell growth in vitro (3–4). The active substance in the SAE was found to be a member of the carrageenan family, a sulfated polysaccharide. It has also been shown that a number of sulfated compounds such as Evans blue and suramin were anti-HIV substances in vitro (5). The following represents a continuation of this teams studies on the chemical modification of compounds for inhibitors against HIV. In this study, the immunomodulator lentinan and other nonsulfated polysaccharides were sulfated and their anti-HIV effects were studied in vitro .

Identification of multiple HIV-1 CTL epitopes presented by HLA-B*5101 molecules

We attempted to identify and characterize HIV-1 CTL epitopes presented by HLA-B51 which is associated with a slow progression to AIDS. HLA-B*5101 stabilization assay showed that 33 out of 172 HIV-1 peptides carrying HLA-B*5101 anchor residues bound to HLA-B*5101. Seven peptides were suggested as HIV-1 CTL epitopes presented by HLA-B*5101 because the specific CTL was induced for these peptides in PBMC from three HIV-1 seropositive individuals carrying HLA-B51 by stimulation with HLA-B*5101 binding peptides. Analysis of these epitopes using the specific CTL clones confirmed that six of seven HIV-1 peptides are epitopes presented by HLA-B*5101. Three epitopes presented by HLA-B*5101 are highly conserved among the clade B strain, suggesting that the specific CTL for these epitopes might play an important role in recognition of HIV-1 infected cells. These epitopes will be useful to analyze CTL responses in HIV-1 infected individuals.

Synthesis, structure and antiviral activity of sulfates of cellulose and its branched derivatives

Abstract Cellulose and branched cellulose having d -glucopyranose or d -galacto-pyranose as side chains were sulfated with piperidine N-sulfonic acid or SO3-dimethylformamide complex. The structures of the sulfated polysaccharides were analysed by elemental analysis and 13[C]-NMR spectroscopy. The 13[C]-NMR spectra of cellulose sulfate were assigned and degree of substitution values of each hydroxyl group were obtained. The reactivity was in the order C-6 ⪢ C-2> C-3. Sulfated polysaccharides with high degree of substitution values showed high anti-human-immuno-deficiency-virus activity.

Synthesis of polymethacrylate derivatives having sulfated maltoheptaose side chains with anti‐HIV activities

Anti-HIV (human immunodeficiency virus) active polymethacrylates having pendant sulfated oligosaccharides were synthesized, and the relationship between structures and biological activities of the polymethacrylates was examined. Acetylated 1-O-methacryloyl maltoheptaoside (MA-AcM7) was polymerized with AIBN as an initiator to give polymethacrylates having a pendant acetylated maltoheptaose in every repeating unit, poly(MA-AcM7)s. After hydroxyl groups were recovered by deacetylation, the polymethacrylates having maltoheptaose units, poly(MA-M7)s, were sulfated to give polymethacrylates having sulfated maltoheptaose side-chains, poly(MA-SM7)s, with degrees of sulfation of 1.1 to 2.7 (maximum, 3.0). These polymethacrylates including sulfated oligosaccharides exhibited low anti-HIV activities represented by the 50% protecting concentration (EC 50 ) in the range of 15-62 μg/mL and low blood anticoagulant activities around 10 unit/mg (standard dextran sulfate, 22.7 unit/mg). The anti-HIV activity increased with increasing degree of sulfation to reach EC 50 of 15-16 μg/mL. In addition, copolymerization of MA-AcM7 with methyl methacrylate (MMA) and subsequent sulfation gave polymethacrylates consisting of various proportions of highly sulfated maltoheptaose and MMA units. It was revealed that the anti-HIV activity increased with decreasing proportion of the sulfated oligosaccharide moiety and that a copolymethacrylate having 22 mol % of sulfated maltoheptaose units (DS = 3.0) had a high anti-HIV activity in the EC 50 of 0.3 μg/mL. The blood anticoagulant activity increased slightly from 9 to 18 unit/mg with decreasing proportion of the sulfated maltoheptaose units. These results suggested that the biological activities were influenced strongly by the spatial distance between sulfated oligosaccharide substituents in the polymethacrylate main chain. Distinction and conformation of the oligosaccharide side chains also played an important role.

Immunization strategies to augment oral vaccination with DNA and viral vectors expressing HIV envelope glycoprotein

Induction of mucosal immunity to the human immunodeficiency virus (HIV) envelope (env; gp160) glycoprotein has been demonstrated with orally administered recombinant vaccinia virus (rVV) vectors and poly(DL-lactide-co-glycolide) (PLG)-encapsulated plasmid DNA expressing gp160. In this study, we investigated the effect of an oral DNA-prime/rVV-boost vaccine regimen in conjunction with adjuvants on the level of gp160-specific cellular and humoral responses in BALB/c mice. We demonstrated that DNA priming followed by a booster with rVV expressing gp160 (vPE16) significantly augmented env-specific immunity in systemic and mucosal tissues of the immunized mice. Association of vPE16 with liposomes and coadministration of liposome-associated beta-glucan lentinan or IL-2/Ig-encoded plasmid DNA-encapsulated in PLG microparticles triggered the optimal cell-mediated immune (CMI) responses. Lentinan was found to increase env-specific type 1 cytokine production and cytotoxic T-lymphocyte (CTL) activities but had no effect on humoral responses. On the other hand, IL-2/Ig-mediated increases in both type 1 and 2 activities were associated with higher levels of env-specific CTL and antibody responses. Results of these studies demonstrated the effectiveness of oral vaccines with DNA and rVV vectors in conjunction with immunomodulators in inducing specific immune responses in systemic and mucosal tissues.

Role of strong anchor residues in the effective binding of 10-mer and 11-mer peptides to HLA-A^*2402 molecules.

The binding capacity of one-hundred-and-seventy-two 8-mer to 11-mer peptides carrying HLA-A24 anchor residues to HLA-A*2402 molecules was analyzed by using a HLA class I stabilization assay. Most (76.2%) of these peptides bound to HLA-A*2402 molecules. These results confirmed previous findings that Tyr and Phe at P2 as well as Phe, Trp, Ile, and Leu at the C-terminus were main anchor residues for HLA-A*2402. Tyr at P2 was a stronger anchor residue than Phe, while bulky aromatic hydrophobic residues Phe and Trp at the C-terminus are stronger anchors than aliphatic hydrophobic residues Ile and Leu. These results were also supported by an analysis using a panel of mutated 9-mer peptides at P2 and P9. Taken together, these results suggest that HLA-A*2402 molecules have deep B- and F-pockets because they favor peptides carrying bulky aromatic hydrophobic residues at P2 and the C-terminus. The affinity of 8-mer peptides was significantly lower than that of 9-mer to 11-mer peptides, while there was no difference in affinity between 9-mer, 10-mer, and 11-mer peptides. The affinity of peptides carrying bulky aromatic hydrophobic residues at the C-terminus was higher than that of peptides carrying aliphatic hydrophobic residues in each of the 8-mer to 11-mer peptides, though the greatest difference in affinity was observed in 11-mer peptides. The strong interaction of side chains of these anchor residues with the corresponding pockets may permit the effective binding of 10-mer and 11-mer peptides to HLA-A*2402 molecules.

Down-regulation of tumor necrosis factor-α, moderate reduction of interleukin-1β, but not interleukin-6 or interleukin-10, by glucan immunomodulators curdlan sulfate and lentinan

The effects of glucan-based immunomodulators curdlan sulfate (CRDS) and lentinan on cytokine production stimulated by lipopolysaccharide (LPS) in bacillus Calmette-Guerin (BCG)-primed mice were investigated. Pretreatment with CRDS or lentinan before LPS administration induced a striking inhibition of up to 89% of circulating tumor necrosis factor-alpha (TNF), a moderate reduction of 25% of interleukin (IL)-1beta, no significant differences in IL-6 or IL-10 levels, and a marked depression of chemiluminescence activity. Animals receiving CRDS prior to infection with alpha-hemolysin positive Escherichia coli inhibited measurable TNF production by 63%. The ability of CRDS and lentinan to significantly reduce the TNF production in vivo indicates the potential of glucans in possible therapeutic strategies that are based on down-regulation of TNF.

SYNTHESIS OF SULFONATED AMINO-POLYSACCHARIDES HAVING ANTI-HIV AND BLOOD ANTICOAGULANT ACTIVITIES

Sulfonated 3-amino-3-deoxy-(1-->6)-alpha-D-allopyranans were synthesized to elucidate the relationship between structure and such specific biological activities as anti-HIV and blood anticoagulant activities. Ring-opening copolymerization of 1,6-anhydro-3-azido-2,4-di-O-benzyl-3-deoxy-beta-D-allopyranose 1 with 1,6-anhydro- 2,3,4-tri-O-benzyl-beta-D-allopyranose 2 with PF5 catalyst gave copolymers having various proportions of the 1 unit. 13C NMR spectroscopy showed the resulting copolymers to have alpha-(1-->6)-stereoregularity, and the monomer reactivity ratios were calculated to be r1 = 0.92 and r2 = 1.11 by the Kelen-Tüdös method. Reduction of the azido groups and subsequent debenzylation of the copolymers afforded new amino-polysaccharides consisting of 3-amino-3-deoxy-allose and allose units. Sulfonation of 3-amino-3-deoxy-(1-->6)-alpha-D-allopyranan and copolysaccharides consisting of 3-amino-allose and allose or glucose units was performed with piperidine-N-sulfonic acid to give polysaccharides containing sulfoamido and sulfonate groups in good yields. The sulfoamido-copolysaccharides containing of 3-amino-3-deoxy-allose and glucose units exhibited high anti-HIV activities manifested by the 50% protecting concentration (EC50) of 0.2-0.5 mg/mL and low cytotoxicity shown by a 50% cytotoxic concentration (CC50) of > 1000 micrograms/mL. The sulfoamido-copolysaccharides containing 3-amino-allose and allose units had somewhat lower anti-HIV activities (EC50 = 0.8-0.9 microgram/mL) and slightly higher cytotoxicities (CC50 = 740-797 micrograms/mL). In addition, it was found that the blood anti-coagulant activity increased, with increasing proportion of the amino-allose unit, from 30 to 58 unit/mg (standard dextran sulfate, 22.7 unit/mg). These results suggest that the sulfonated 3-amino-3-deoxyallose unit plays important roles on both anti-HIV and blood anticoagulant activities.

Stimulation of microbicidal host defence mechanisms against aerosol influenza virus infection by lentinan

The ability of polysaccharide immunomodulator lentinan to stimulate non-specific resistance against respiratory viral infections was investigated. Significant protection was conferred by lentinan administered intranasally before lethal influenza virus infection and could be corroborated by a reduction of the lung virus titres. Since the lung is the target organ of influenza virus infection, lentinan was also administered by the intravenous route. Lentinan conferred complete protection against a LD75 challenge dose of virulent influenza virus and significantly prolonged the survival time after a LD100 challenge. The effect on respiratory burst of broncho-alveolar macrophages was investigated by luminol-dependent chemiluminescence (CL) in response to stimulation by zymosan. Enhanced CL activity was present at an early stage in groups receiving lentinan. Significant nitric oxide activity could also be stimulated by culturing broncho-alveolar macrophages in the presence of lentinan. TNF activity could not be detected in lung lavage but measurable IL-6 was produced already after 6 h in animals administered lentinan alone and in lentinan-pretreated influenza virus-infected mice. Influenza virus alone did not induce measurable IL-6 at 6 h but high activity was present at later time periods.