Alexander B. Tuzikov

Early Alterations of the Receptor-Binding Properties of H1, H2, and H3 Avian Influenza Virus Hemagglutinins after Their Introduction into Mammals

ABSTRACT Interspecies transmission of influenza A viruses circulating in wild aquatic birds occasionally results in influenza outbreaks in mammals, including humans. To identify early changes in the receptor binding properties of the avian virus hemagglutinin (HA) after interspecies transmission and to determine the amino acid substitutions responsible for these alterations, we studied the HAs of the initial isolates from the human pandemics of 1957 (H2N2) and 1968 (H3N2), the European swine epizootic of 1979 (H1N1), and the seal epizootic of 1992 (H3N3), all of which were caused by the introduction of avian virus HAs into these species. The viruses were assayed for their ability to bind the synthetic sialylglycopolymers 3′SL-PAA and 6′SLN-PAA, which contained, respectively, 3′-sialyllactose (the receptor determinant preferentially recognized by avian influenza viruses) and 6′-sialyl(N-acetyllactosamine) (the receptor determinant for human viruses). Avian and seal viruses bound 6′SLN-PAA very weakly, whereas the earliest available human and swine epidemic viruses bound this polymer with a higher affinity. For the H2 and H3 strains, a single mutation, 226Q→L, increased binding to 6′SLN-PAA, while among H1 swine viruses, the 190E→D and 225G→E mutations in the HA appeared important for the increased affinity of the viruses for 6′SLN-PAA. Amino acid substitutions at positions 190 and 225 with respect to the avian virus consensus sequence are also present in H1 human viruses, including those that circulated in 1918, suggesting that substitutions at these positions are important for the generation of H1 human pandemic strains. These results show that the receptor-binding specificity of the HA is altered early after the transmission of an avian virus to humans and pigs and, therefore, may be a prerequisite for the highly effective replication and spread which characterize epidemic strains.

Balanced Hemagglutinin and Neuraminidase Activities Are Critical for Efficient Replication of Influenza A Virus

ABSTRACT The SD0 mutant of influenza virus A/WSN/33 (WSN), characterized by a 24-amino-acid deletion in the neuraminidase (NA) stalk, does not grow in embryonated chicken eggs because of defective NA function. Continuous passage of SD0 in eggs yielded 10 independent clones that replicated efficiently. Characterization of these egg-adapted viruses showed that five of the viruses contained insertions in the NA gene from the PB1, PB2, or NP gene, in the region linking the transmembrane and catalytic head domains, demonstrating that recombination of influenza viral RNA segments occurs relatively frequently. The other five viruses did not contain insertions in this region but displayed decreased binding affinity toward sialylglycoconjugates, compared with the binding properties of the parental virus. Sequence analysis of one of the latter viruses revealed mutations in the hemagglutinin (HA) gene, at sites in close proximity to the sialic acid receptor-binding pocket. These mutations appear to compensate for reduced NA function due to stalk deletions. Thus, balanced HA-NA functions are necessary for efficient influenza virus replication.

6-sulfo sialyl Lewis X is the common receptor determinant recognized by H5, H6, H7 and H9 influenza viruses of terrestrial poultry

BackgroundInfluenza A viruses of domestic birds originate from the natural reservoir in aquatic birds as a result of interspecies transmission and adaptation to new host species. We previously noticed that influenza viruses isolated from distinct orders of aquatic and terrestrial birds may differ in their fine receptor-binding specificity by recognizing the structure of the inner parts of Neu5Acα2-3Gal-terminated sialyloligosaccharide receptors. To further characterize these differences, we studied receptor-binding properties of a large panel of influenza A viruses from wild aquatic birds, poultry, pigs and horses.ResultsUsing a competitive solid-phase binding assay, we determined viral binding to polymeric conjugates of sialyloligosaccharides differing by the type of Neu5Acα-Gal linkage and by the structure of the more distant parts of the oligosaccharide chain. Influenza viruses isolated from terrestrial poultry differed from duck viruses by an enhanced binding to sulfated and/or fucosylated Neu5Acα2-3Gal-containing sialyloligosaccharides. Most of the poultry viruses tested shared a high binding affinity for the 6-sulfo sialyl Lewis X (Su-SLex). Efficient binding of poultry viruses to Su-SLex was often accompanied by their ability to bind to Neu5Acα2-6Gal-terminated (human-type) receptors. Such a dual receptor-binding specificity was demonstrated for the North American and Eurasian H7 viruses, H9N2 Eurasian poultry viruses, and H1, H3 and H9 avian-like virus isolates from pigs.ConclusionInfluenza viruses of terrestrial poultry differ from ancestral duck viruses by enhanced binding to sulfated and/or fucosylated Neu5Acα2-3Gal-terminated receptors and, occasionally, by the ability to bind to Neu5Acα2-6Gal-terminated (human-type) receptors. These findings suggest that the adaptation to receptors in poultry can enhance the potential of an avian virus for avian-to-human transmission and pandemic spread.

Conversion of Complex Sialooligosaccharides into Polymeric Conjugates and their Anti-Influenza Virus Inhibitory Potency

ABSTRACT To investigate the specificity of various influenza virus strains we have prepared polyacrylic type conjugates of undecasaccharide (Neu5Acα2-6Galβ1-4GlcNAcβ1-2Manα1)2-3,6Manβ1-4GlcNAcβ1-4GlcNAc (YDS), and trisaccharides 6‵-sialyl-N-acetyllactosamine (6‵SLN), 6‵-sialyllactose (6‵SL), and 3‵-sialyllactose (3‵SL). Free oligosaccharides were transformed to glycosylamine-1-N-glycyl derivatives by sequential action of NH4HCO3, chloroacetic anhydride, and aqueous NH3. The known derivatization protocol has been optimized for these sialooligosaccharides. Coupling of obtained amino-spacered derivatives with poly(4-nitrophenyl acrylate) gave rise to two types of conjugates, namely with polyacrylic acid and polyacrylamide backbones; the conversion proceeded quantitatively and without destruction of the oligosaccharides. The content of oligosaccharides in the conjugates was 10, 20, and 30% mol for 3‵SL, 6‵SL, 6‵SLN, and 2, 5 and 10% mol for YDS. Free oligosaccharides and the glycoconjugates were tested as inhibitors of influenza virus adhesion, and also as blockers of virus infectivity in MDCK cell culture. Biantennary YDS demonstrated similar activity to trisaccharide 6‵SLN both as the free form and neoglycoconjugate.

Polymeric inhibitor of influenza virus attachment protects mice from experimental influenza infection.

Synthetic sialic acid-containing macromolecules inhibit influenza virus attachment to target cells and suppress the virus-mediated hemagglutination and neutralize virus infectivity in cell culture. To test the protective effects of attachment inhibitors in vivo, mice were infected with mouse-adapted influenza virus A/Aichi/2/68 (H3N2) and treated with synthetic polyacrylamide-based sialylglycopolymer PAA-YDS bearing moieties of (Neu5Acalpha2-6Galbeta1-4GlcNAcbeta1-2Manalpha1)2-3,6Manbeta1-4GlcNAcbeta1-4GlcNAc. Single intranasal inoculations with PAA-YDS 30 min before or 10 min after infection increased the survival of mice (P<0.01). Multiple treatments with aerosolized PAA-YDS on days 2-5 post infection also increased survival (P<0.01), alleviated disease symptoms, and decreased lesions in the mouse lungs. These data suggest that synthetic polyvalent inhibitors of virus attachment can be used for prevention and treatment of influenza.

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.

Polyglycine II Nanosheets: Supramolecular Antivirals?

Tetraantennary peptides [glycinen‐NHCH2]4C can form stable noncovalent structures by self‐assembly through intermolecular hydrogen bonding. The oligopeptide chains assemble as polyglycine II to yield submicron‐sized, flat, one‐molecule‐thick sheets. Attachment of α‐N‐acetylneuraminic acid (Neu5Acα) to the terminal glycine residues gives rise to water‐soluble assembled glycopeptides that are able to bind influenza virus multivalently and inhibit adhesion of the virus to cells 103‐fold more effectively than a monomeric glycoside of Neu5Acα. Another antiviral strategy based on virus‐promoted assembly of the glycopeptides was also demonstrated. Consequently, the self‐assembly principle offers new perspectives on the design of multivalent antivirals.

Human influenza virus recognition of sialyloligosaccharides

Sialic acids are essential components of cell‐surface receptors utilized by influenza viruses. To evaluate the recognition of asialic sugar parts of the receptor, three representative strains of human influenza A and B viruses were tested for their binding of a panel of sialyloligosaccharides. The highest affinity binding carbohydrate determinants recognized by the viruses in a context of different core structures were Neu5Acα2‐3Gal for the type B virus, Neu5Acα2‐6Gal for the H3 subtype virus, and Neu5Acα2‐6Ga/β1‐4GlcNAc for the H1 subtype virus. Penultimate to these determinants parts of sialyloligosaccharides studied either contributed less significantly to the binding affinity, or interfered with the binding.

A simple procedure for the synthesis of the methyl and benzyl glycosides of Neu5Ac and 4-epi-Neu5Ac. Conversion of the benzyl and methyl glycosides of Neu5Ac into N-trifluoroacetylneuraminic acid benzyl glycosides*

Abstract From the reaction products of the Kuhn-Bashang synthesis of Neu5Ac, 5-acetamido-3,5-dideoxy- α -and - β - d - glycero - d - talo -2-nonulopyranosonic acid (4- epi -Neu5Ac) were isolated as the acetylated methyl esters ( 1 and 2 ). Treatment of methyl (5-acetamido-4,7,8,9-tetra- O -acetyl-3,5-dideoxy- d - glycero - d - galacto -2-nonulopyranosyl bromide)onate ( 5 ) with an excess of methanol gave a high yield of a 9:1 α,β -mixture of the methyl glycosides ( 13 and 14 ). Likewise, with benzyl alcohol, 5 gave a 63:32 α,β -mixture of the benzyl glycosides ( 17 and 18 ). Treatment of methyl (5-acetamido-4,7,8,9-tetra- O -acetyl-3,5-dideoxy- β - d - glycero -pd- talo -2-nonulopyranosyl bromide)onate ( 7 ) with an excess of benzyl alcohol gave a 3:1 α,β -mixture of the benzyl glycosides ( 21 and 22 ) together with methyl 5-acetamido-4,7,8,9-tetra- O -acetyl-2,6-anhydro-3,5-dideoxy- d - glycero - d - talo -non-2-enonate ( 9 ). Condensation of bromide 7 in chloroform with benzyl alcohol in the presence of silver carbonate afforded a 7:1 α,β -mixture of benzyl glycosides together with 9 . The benzyl glycosides 17 and 18 were converted into their respective N -trifluoroacetyl derivatives 27 and 28 by saponification and then N -trifluoroacetylation. Methanolysis of Neu5Ac followed by N -trifluoroacetylation and O -acetylation afforded methyl (methyl 4,7,8,9-tetra- O -acetyl-3,5-dideoxy-5-trifluoroacetamido- β - d - glycero - d - galacto -2-nonulopyrano-sid)onate ( 30 ), which was converted into the benzyl glycosides ( 32 and 33 ) via the 2-bromide ( 31 ). A simplified preparation of the protected 2-halogeno derivatives of Neu5Ac and 4- epi -Neu5Ac is described. The conversion of neuraminic acid methyl glycoside into the corresponding 2-bromide derivative by the action of hydrogen bromide is demonstrated.

Multimeric glycotherapeutics: New paradigm

The general principle of anti-adhesion therapy is the inhibition of microorganism adhesion to the host cell with the help of a soluble receptor analog. Despite an evident attractiveness of the concept and its long existence, the therapeutics of the ‘post-antibiotic era’ have not yet appeared. This can be explained by the contradictoriness of requirements for anti-adhesion drugs: to be efficient a drug must be multivalent, i.e. large molecule, but to obtain FDA approval it should be a small molecule. A way to overcome this contradiction is self-assembly of glycopeptides. The carbohydrate part of glycopeptide is responsible for binding with the lectin of microorganisms, whereas a simple peptide part is responsible for an association to the so-called tectomers. Depending on the structure, tectomers are formed either spontaneously or upon promotion of a microorganism. In particular, sialopeptide, which is capable of converting to a tectomer only in the presence of the influenza virus, has been obtained. Thus, the new strategy of anti-adhesion therapy can be formulated as follows: (1) identification of oligosaccharide-receptor for a particular virus (bacteria); (2) optimization of the peptide part; (3) conventional trials. The expected advantages of this strategy are the following: (i) no polymer; (ii) a virion completely covered with a tectomer, i.e. blocking is both complete and irreversible; (iii) rapid and rational lead identification and optimization; (iv) minimum side effects; (v) potential for microorganism resistance to natural receptor is lower than in the case of mimetics. Published in 2004.