Monovalent and polymeric 5N-thioacetamido sialosides as tightly-bound receptor analogs of influenza viruses.

A possible approach to the development of synthetic inhibitors of influenza virus attachment to host cells is based on the anchoring of the minimum receptor determinant of influenza virus, sialic acid, to a polymeric carrier. In this study, the effect of substitution of oxygen by sulphur in the 5N-acetyl moiety of sialic acid on the binding of monovalent and polymeric sialosides by A and B influenza virus strains was investigated. The polymeric inhibitor with pendant 5N-thioacetylneuraminic acid residues was found to be more broadly active against different virus stains that the one prepared from the Neu5Ac ligand.

Monoclonal antibodies directed to the synthetic carbohydrate antigen Ley.

Tetrasaccharide Fuc alpha 1-2Gal beta 1-4(Fuc alpha 1-3)GlcNAc is known as carbohydrate determinant of cancer- and AIDS-associated antigen Lewisy (Ley). Synthetic antigen to generate mouse monoclonal antibodies (MAbs) directed to Ley was prepared and constructed as a spacer-armed tetrasaccharide coupled with lipophilized polymer, Ley-PAA-PE, where PAA is a 30-kD polyacrylamide and PE is phosphatidylethanolamine. An efficient immune response was provided by using Ley-PAA-PE adsorbed on Salmonella minnesota. Positive hybridomas were screened by enzyme-linked immunosorbent assay (ELISA) using Ley-PAA as a coating agent. An inhibitory version of the same test system showed absolute specificity of two MAbs: only hapten Ley and Ley-PAA were strong inhibitors, in contrast to Leb, tri- and disaccharidic fragments of the mentioned tetrasaccharides, as well as their PAA-conjugates. MAbs obtained against synthetic antigen specifically stained the Ley (+) cell line A431.

Synthetic polymeric inhibitors of influenza virus receptor-binding activity suppress virus replication.

A new approach to anti-influenza chemotherapy is based on the development of synthetic inhibitors of virus attachment to host cells. These inhibitors are prepared by anchoring the minimum receptor determinant of influenza virus, sialic acid, to polymeric or liposomal carriers. In this study, a series of poly(acrylic acid-co-acrylamides) and dextrans bearing pendant glycylamidobenzylsialoside groups were synthesized and evaluated for their binding to a panel of influenza A and B virus strains and for their ability to inhibit virus infectivity in cell culture. Significant type-, subtype-, and strain-specific variation in virus susceptibility to the synthetic inhibitors was observed. Among the viruses tested, H3 subtype strains evolved in humans since 1975 were the most sensitive, while the earlier H3 viruses and the type B strains were resistant. The virus-inhibitory potency of the polymeric sialosides correlated with their bindings to the virus, and was dependent on the virus affinity for the ligand, the density of the ligand, and the nature and molecular mass of the polymeric carrier. In embryonated eggs, the antiviral effect of poly(acryloyl-glycylamidobenzylsialoside-co-acrylic acid) was comparable to that of equine alpha 2-macroglobulin.

Probing of the receptor-binding sites of the H1 and H3 influenza A and influenza B virus hemagglutinins by synthetic and natural sialosides.

To compare features of the receptor-binding sites (RBSs) of different influenza virus hemagglutinins (HA), binding of a number of synthetic sialic acid (SA) analogs and natural sialosides by a panel of about 30 human influenza A and B virus strains was studied in a competitive ligand binding assay. For all the viruses tested, the N-acetyl group of Neu5Ac, as well as the natural orientation of the carboxylic group at C2 and the hydroxylic group at C4, was essential for binding. Significant type- and subtype-specific differences were observed in virus recognition of asialic parts of sialosides. H1 strains, unlike H3 and type B viruses, were found to bind alpha 2-6-sialyl-N-acetyllactosamine with about an order of magnitude higher affinity than alpha 2-6-sialyllactose (6'SL). The H1 viruses and the H3 strains with Gln in position 226 of HA, but not the H3 strains with Leu-226, bound 6'SL with a lower affinity than alpha 2-3-sialyllactose; this effect correlated clearly with the preferential binding by the former viruses of unsubstituted alpha Neu5Ac compared to methyl alpha-glycoside of Neu5Ac. Thus, differentiation between the types of the SA-Gal linkage by the A viruses appeared to depend, at least partially, upon the recognition by the HA of the first hydrocarbon group of the aglycon. Type B virus strains were distinct in having a lower affinity for the Neu5Ac moiety and in providing a higher contribution of the asialic portions of sialosides to the HA-ligand interactions. The last effects are presumably due to the amino acid insertions in the type B HA surrounding the RBS, which makes the receptor-binding pocket deeper. The results obtained in the present investigation indicate that while the functional groups of Neu5Ac studied are recognized by the RBSs of all influenza viruses, the magnitude of their contribution to the binding energy, as well as the contribution of the asialic portion of the receptor, may vary in dependence upon the virus type, subtype, and strain.

Synthesis of polymeric neoglycoconjugates based on N-substituted polyacrylamides.

Several types of polymeric glycoconjugates, N-substituted polyacrylamides, have been synthesized by the reaction of activated polymers with omega-aminoalkylglycosides: (i) (carbohydrate-spacer)n-polyacrylamide, 'pseudopolysaccharides'; (ii) (carbohydrate-spacer)n-phosphatidylethanolaminem-polyacrylamide, neoglycolipids, derivatives of phosphatidylethanolamine; (iii) (carbohydrate-spacer)n-biotin-polyacrylamide, biotinylated probes; (iv) (carbohydrate-spacer)n-polyacrylamide-(macroporous glass), affinity sorbents based on macroporous glass, covalently coated with polyacrylamide. An almost quantitative yield in the conjunction reaction makes it possible to insert in the conjugate a predetermined quantity of the ligand(s). Pseudopolysaccharides proved to be a suitable form of antigen for activation of polystyrene and poly(vinyl chloride) plates (ELISA) and nitrocellulose membranes (dot blot), being advantageous over traditional neoglycoproteins. Polyvalent glycolipids insert well in biological membranes: their physical properties, particularly solubility, can be changed in a desired direction. Biotinylated derivatives were used as probes for detection and analysis of lectins.

Synthetic polymeric sialoside inhibitors of influenza virus receptor-binding activity.

Anomeric aminobenzylglycosides of Neu5Ac were coupled with the polyacrylate carrier and a number of synthetic polyvalent sialosides obtained were investigated as inhibitors of influenza virus attachment. The inhibitory activity of polymeric sialosides is highly dependent upon the Neu5Ac residue content and the nature of the carrier. The polyacrylic acid based polymer bearing 10 mol% of Neu5Ac is 3 orders of magnitude more potent inhibitor than the corresponding monovalent benzylsialoside and considerably more active than fetuin.