Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/745—Blood coagulation or fibrinolysis factors
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/5308—Immunoassay; Biospecific binding assay; Materials therefor for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/12—Antivirals
  • A61P31/14—Antivirals for RNA viruses
  • A61P31/16—Antivirals for RNA viruses for influenza or rhinoviruses
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
  • A61P37/02—Immunomodulators
  • A61P37/04—Immunostimulants
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/36—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against blood coagulation factors
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2400/00—Assays, e.g. immunoassays or enzyme assays, involving carbohydrates
  • G01N2400/10—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters

Definitions

• Glycomics an integrated approach to structure-function relationships of complex carbohydrates or glycans, is emerging as an important paradigm in post-genomics cellular and molecular biology.
• Glycans are primary components of the cell surface and the interface between cell and its extracellular environment.
• glycans interact with numerous proteins such as growth factors, cytokines, immune receptors, and enzymes, which modulate their activity and thus impinge on the above biological processes.
• the present invention provides a system for analyzing glycans and their interaction partners.
• the inventive system is particularly useful in the identification and analysis of glycoprotein binding interactions.
• the inventive system has been applied to several different glycoprotein analyses, in each case successfully identifying interaction characteristics.
• the principles of the inventive system are therefore widely applicable across glycan interactions.
• Figure 1 illustrates the data mining platform utilized herein. Shown in Panel A are the amin components of the data mining platform. The features are derived from the data objects which are extracted from the database. The features are prepared into datasets that are used by the classification methods to derive patterns or rules. Panel B shows certain software modules that enable the user to apply the data mining process to glycan array data.
• Figure 2 presents a schematic description of features. Shown in Panel A 9 is a representative high mannose motif to illustrate the definition of pairs, triplets and quadruplets. Shown in Panel B, is a representative O-linked glycan [Core 2] motif to illustrate the different classes of triplets. The following symbol nomenclature was used to represent monosaccharides: *Man *Gal ⁇ GlcNAc AFuc 4Neu5Ac ⁇ Neu5Gc ⁇ KDN.
• Figure 3 Figure 3 depicts the classification of high affinity binding.
• Figure 4 depicts the binding of Lewis" motif containing glycan structure Gal ⁇ 4 (Fuc ⁇ 3) GlcNAc ⁇ 3Gal to CRD of DC-SIGN (ribbon trace in gray). The monosaccharides and linkages are labeled on the glycan structure. Shown in red circle in the 3 -OH of the Gal which is close to the glycan binding site. Thus any substitution at this hydroxyl group would have a negative effect on binding consistent with the classifier rule obtained from data mining.
• Figure 5 Alignment of exemplary sequences of wild type HA. Sequences were obtained from the NCBI influenza virus sequence database (http://www.ncbi.nlm.nih.gov/genomes/FLU/FLU.html)
• Figure 6 Framework for understanding glycan receptor specificity.
• ⁇ 2-3- and/or ⁇ 2-6-linked glycans can adopt different topologies.
• the ability of an HA polypeptide to bind to certain of these topologies confers upon it the ability to mediate infection of different hosts, for example, humans.
• the present invention defines two particularly relevant topologies, a "cone" topology and an "umbrella" topology.
• the cone topology can be adopted by ⁇ 2-3- and/or ⁇ 2-6-linked glycans, and is typical of short oligosaccharides or branched oligosaccharides attached to a core (although this topology can be adopted by certain long oligosaccharides).
• the umbrella topology can only be adopted by ⁇ 2-6-linked glycans (presumably due to the increased conformational plurality afforded by the extra C5-C6 bond that is present in the ⁇ 2-6 linkage), and is predominantly adopted by long oligosaccharides or branched glycans with long oligosaccharide branches, particularly containing the motif Neu5Ac ⁇ 2-6Gal ⁇ l -3/4GIcNAc-.
• ability of HA polypeptides to bind the umbrella glycan topology confers binding to human receptors and/or ability to mediate infection of humans.
• FIG. 7 Interactions of HA residues with cone vs umbrella glycan topologies. Analysis of HA-glycan co-crystals reveals that the position of Neu5Ac relative to the HA binding site is almost invariant. Contacts with Neu5Ac involve highly conserved residues such as F98, S/T136, W153, H183 and L/I194. Contacts with other sugars involve different residues, depending on whether the sugar linkage is ⁇ 2-3 or ⁇ 2-6 and whether the glycan topology is cone or umbrella. For example, in the cone topology, the primary contacts are with Neu5Ac and with Gal sugars. El 90 and Q226 play particularly important roles in this binding.
• This Figure also illustrates other positions (e.g., 137, 145, 186, 187, 193, 222) that can participate in binding to cone structures.
• different residues can make different contacts with different glycan structures.
• the type of amino acid in these positions can influence ability of an HA polypeptide to bind to receptors with different modification and/or branching patterns in the glycan structures.
• contacts are made with sugars beyond Neu5Ac and Gal.
• residues e.g., 137, 145, 156, 159, 186, 187, 189, 190, 192, 193, 196, 222, 225, 226) that can participate in binding to umbrella structures.
• a D residue at position 190 and/or a D residue at position 225 contributes to binding to umbrella topologies.
• Figure 8. Exemplary cone topologies. This Figure illustrates certain exemplary (but not exhaustive) glycan structures that adopt cone topologies.
• Figure 9. Exemplary umbrella topologies. This Figure illustrates certain exemplary (but not exhaustive) glycan structures that adopt umbrella topologies.
• Figure 10. Sequence alignment of HA glycan binding domain.
• Gray conserved amino acids involved in binding to sialic acid. Red: particular amino acids involved in binding to Neu5Ac ⁇ 2-3/6Gal motifs. Yellow: amino acids that influence positioning of Q226 (137, 138) and E190 (186, 228). Green: amino acids involved in binding to other monosaccharides (or modifications) attached to Neu5Ac ⁇ 2-3/6Gal motif.
• the sequence for ASI30, APR34, ADU63, ADS97 and VietO4 were obtained from their respective crystal structures. The other sequences were obtained from SwissProt (http://us.expasy.org).
• ADA76 A/duck/ Alberta/35/76 (HlNl); ASI30, A/Swine/Iowa/30 (HlNl); APR34, A/Puerto Rico/8/34 (HlNl); ASC18, A/South Carolina/1/18 (HlNl), AT91, A/Texas/36/91 (HlNl); ANYl 8, A/New York/1/18 (HlNl); ADU63, A/Duck/Ukraine/1/63 (H3N8); AAI68, A/Aichi/2/68 (H3N2); AM99, A/Moscow/ 10/99 (H3N2); ADS97, A/Duck/Singapore/3/97 (H5N3); VietO4, A/Vietnam/I 203/2004 (H5N1). [0016] Figure 11. Sequence alignment illustrating conserved subsequences characteristic of Hl HA.
• Figure 12 Sequence alignment illustrating conserved subsequences characteristic ofH3 HA.
• Figure 13 Sequence alignment illustrating conserved subsequences characteristic ofH5 HA.
• FIG. 14 Conformational map and solvent accessibility ofNeu5Aco2-3Gal and Neu5Aca2-6Gal motifs.
• Panel A shows the conformational map of Neu5Ac ⁇ 2-3Gal linkage.
• the encircled region 2 is the trans conformation observed in the APR34_H1_23, ADU63_H3_23 and ADS97_H5_23 co-crystal structures.
• the encircled region 1 is the conformation observed in the AAI68_H3_23 co-crystal structure.
• Panel B shows the conformational map of Neu5Ac ⁇ 2-6Gal where the the c/s-conformation (encircled region 3) is observed in all the HA- oc2-6 sialylated glycan co-crystal structures.
• Panel C shows difference between solvent accessible surface area (SASA) of Neu5Ac ⁇ 2-3 and cc2-6 sialylated oligosaccharides in the respective HA-glycan co-crystal structures.
• SASA solvent accessible surface area
• the red and cyan bars respectively indicate that Neu5Ac in ⁇ .2-6 (positive value) or ⁇ 2-3 (negative value) sialylated glycans makes more contact with glycan binding site.
• Panel D shows difference between SASA of NeuAc in ⁇ 2-3 sialylated glycans bound to swine and human Hl (Hl ⁇ 2 .3), avian and human H3 (H3 ⁇ 2-3), and of NeuAc in ⁇ 2-6 sialylated glycans bound to swine and human Hl (Hl ⁇ 2-6).
• the negative bar in cyan for H3 ⁇ 2 - 3 indicates lesser contact of the human H3 HA with Neu5Ac ⁇ 2-3Gal compared to that of avian H3.
• Torsion angles - ⁇ C2-C1-O-C3 (for Neu5 Ac ⁇ 2-3/6 linkage); ⁇ : C1-O-C3-H3 (for Neu5 Ac ⁇ 2- 3GaI) or C1-O-C6-C5 (for Neu5Ac ⁇ 2-6Gal); ⁇ : O-C6-C5-H5 (for Neu5Ac ⁇ 2- 6GaI) linkages.
• the ⁇ , ⁇ maps were obtained from GlycoMaps DB
• FIG. 15 Residues involved in binding of Hl, H3 and H5 HA to c ⁇ -3/6 sialylated glycans.
• Panels A-D show the difference ( ⁇ in the abscissa) in solvent accessible surface area (SASA) of residues interacting with ⁇ 2-3 and ⁇ 2-6 sialylated glycans, respectively, in ASI30_Hl, APR34_H1, ADU63_H3 and ADS97_H5 co-crystal structures.
• Green bars correspond to residues that directly interact with the glycan and light orange bars correspond to residues proximal to Glu/Aspl90 and Gln/Leu226.
• Panel E summarizes in tabular form the residues involved in binding to oc2-3/6 sialylated glycans in Hl, H3 and H5 HA. Certain key residues involved in binding to cc2-3 sialylated glycans are colored blue and certain key residues involved in binding to ⁇ 2-6 sialylated glycans are colored red.
• FIG. 16 Binding of VietO4_H5 HA to biantennary a2-6 sialylated glycan ( cone topology). Stereo view of surface rendered VietO4_H5 glycan binding site with Neu5Acct2-6Gal linkage in the extended conformation (obtained from the pertussis toxin co-crystal structure; PDB ID: IPTO). Lysl93 (orange) does not have any contacts with the glycan in this conformation. The additional amino acids potentially involved in binding to the glycan in this conformation are Asnl86, Lys222 and Ser227.
• FIG. 1 shows the structure with this branch attached to Man ⁇ l-3Man of the core (shown in figure where trimannose core is colored in purple) has steric overlaps with Lysl93 in the cw-conformation but can bind without any contact with Lysl93 in the extended conformation, albeit less optimally.
• Figure 17 Production of WT Hl, HS and H5 HA.
• Panel A shows the soluble form of HA protein from HlNl (A/South Carolina/1/1918), H3N2 (A/Moscow/10/1999) and H5N1 (A/Vietnam/I 203/2004), run on a 4-12% SDS-polyacrylaminde gel and blotted onto nitrocellulose membranes.
• HlNl HA was probed using goat anti-Influenza A antibody and anti-goat IgG-HRP.
• H3N2 was prodes using ferret anti-H3N2 HA antisera and anti- ferret-HRP.
• H5N1 was probed using anti-avian H5N1 HA antibody and anti-rabbit IgG- HRP.
• HlNl HA and H3N2 HA are present as HAO, while H5N1 HA is present as both HAO and HAl.
• Panel B shows full length H5N1 HA and two variants (Glul90Asp, Lysl93Ser, Gly225Asp, Gln226Leu, "DSDL” and GLul90Asp Lysl93Ser Gln223Leu Gly228Ser “DSLS”) run on an SDS-polyacrylamide gel and blotted onto a nitrocellulose membrane.
• the HA was probed with anti-avian H5N1 antibody and anti-rabbit IgG-HRP.
• Figure 18 Lectin staining of upper respiratory tissue sections.
• a co-stain of the tracheal tissue with Jacalin (green) and ConA (red) reveals a preferential binding of Jacalin (binds specifically to O-linked glycans) to goblet cells on the apical surface of the trachea and conA (binds specifically to N-linked glycans) to the ciliated tracheal epithelial cells.
• Jacalin binding specifically to O-linked glycans
• conA binding specifically to N-linked glycans
• Co-staining of trachea with Jacalin and SNA shows binding of SNA to both goblet and ciliated cells.
• co-staining of Jacalin (green) and MAL (red) which specifically binds to ⁇ 2-3 sialylated glycans, shows weak minimal to no binding of MAL to the pseudostratified tracheal epithelium but extensive binding to the underlying regions of the tissue.
• the lectin staining data indicated predominant expression and extensive distribution of ⁇ 2-6 sialylated glycans as a part of both N-linked and O-linked glycans respectigely in ciliated and goblet cells on the apical side of the tracheal epithelium.
• FIG. 19 Binding of recombinant wild type and mutant HA to tissue sections. Shown are wild type (WT) 3 DSLS 5 and DSDL binding to trachea, bronchus and alveolus tissue sections. For WT, the white arrow shows HA binding (green) to the alveolar tissue section. For DSLS mutant, the white arrow for the tracheal and bronchial tissue sections shows this mutant HA binding (green) to the apical side of the tissues. Note that the DSDL mutant does not bind to any tissue sections.
• affinity is a measure of the tightness with a particular ligand (e.g., an HA polypeptide) binds to its partner (e.g., and HA receptor). Affinities can be measured in different ways.
• biologically active refers to a characteristic of any agent that has activity in a biological system, and particularly in an organism. For instance, an agent that, when administered to an organism, has a biological effect on that organism, is considered to be biologically active.
• an agent that, when administered to an organism, has a biological effect on that organism is considered to be biologically active.
• a portion of that protein or polypeptide that shares at least one biological activity of the protein or polypeptide is typically referred to as a "biologically active" portion.
• Broad spectrum human-binding H5 HA polypeptides As used herein, the phrase "broad spectrum human-binding H5 HA” refers to a version of an H5 HA polypeptide that binds to HA receptors found in human epithelial tissues, and particularly to human HA receptors having ⁇ 2-6 sialylated glycans. Moreover, inventive BSHB H5 HAs bind to a plurality of different ⁇ 2-6 sialylated glycans.
• BSHB H5 HAs bind to a sufficient number of different ⁇ 2-6 sialylated glycans found in human samples that viruses containing them have a broad ability to infect human populations, and particularly to bind to upper respiratory tract receptors in those populations.
• BSHB H5 HA bind to umbrella glycans (e.g., long ⁇ 2-6 sialylated glycans) as described herein.
• Characteristic portion As used herein, the phrase a "characteristic portion" of a protein or polypeptide is one that contains a continuous stretch of amino acids, or a collection of continuous stretches of amino acids, that together are characteristic of a protein or polypeptide. Each such continuous stretch generally will contain at least two amino acids. Furthermore, those of ordinary skill in the art will appreciate that typically at least 5, 10, 15, 20 or more amino acids are required to be characteristic of a protein. In general, a characteristic portion is one that, in addition to the sequence identity specified above, shares at least one functional characteristic with the relevant intact protein.
• Characteristic sequence is a sequence that is found in all members of a family of polypeptides or nucleic acids, and therefore can be used by those of ordinary skill in the art to define members of the family.
• Cone topology The phrase “cone topology” is used herein to refer to a 3- dimensional arrangement adopted by certain glycans and in particular by glycans on HA receptors. As illustrated in Figure 6, the cone topology can be adopted by ⁇ 2-3 sialylated glycans or by ⁇ 2-6 sialylated glycans, and is typical of short oligonucleotide chains, though some long oligonucleotides can also adopt this conformation.
• the cone topology is characterized by the glycosidic torsion angles of Neu5Ac ⁇ 2-3Gal linkage which samples three regions of minimum energy conformations given by ⁇ (C1-C2-O-C3/C6) value of around -60, 60 or 180 and ⁇ (C2-O-C3/C6-H3/C5) samples -60 to 60 ( Figure 14).
• Figure 8 presents certain representative (though not exhaustive) examples of glycans that adopt a cone topology.
• corresponding to is often used to designate the position/identity of an amino acid residue in an HA polypeptide.
• a canonical numbering system (based on wild type H3 HA) is utilized herein (as illustrated, for example, in Figures 5 and 10-13), so that an amino acid "corresponding to" a residue at position 190, for example, need not actually be the 190 th amino acid in a particular amino acid chain but rather corresponds to the residue found at 190 in wild type H3 HA; those of ordinary skill in the art readily appreciate how to identify corresponding amino acids.
• amino acids that are a "degree of separation removed” are HA amino acids that have indirect effects on glycan binding.
• one-degree-of-separation-removed amino acids may either: (1) interact with the direct-binding amino acids; and/or (2) otherwise affect the ability of direct-binding amino acids to interact with glycan that is associated with host cell HA receptors; such one-degree- of-separation-removed amino acids may or may not directly bind to glycan themselves.
• Two-degree-of-separation-removed amino acids either (1) interact with one-degree-of- separation-removed amino acids; and/or (2) otherwise affect the ability of the one-degree-of- separation-removed amino acids to interact with direct-binding amino acids, etc.
• Direct-binding amino acids refers to HA polypeptide amino acids which interact directly with one or more glycans that is associated with host cell HA receptors.
• Engineered describes a polypeptide whose amino acid sequence has been selected by man.
• an engineered HA polypeptide has an amino acid sequence that differs from the amino acid sequences of HA polypeptides found in natural influenza isolates.
• an engineered HA polypeptide has an amino acid sequence that differs from the amino acid sequence of HA polypeptides included in the NCBI database.
• Hl polypeptide An "Hl polypeptide", as that term is used herein, is an HA polypeptide whose amino acid sequence includes at least one sequence element that is characteristic of Hl and distinguishes Hl from other HA subtypes. Representative such sequence elements can be determined by alignments such as, for example, those illustrated in Figures 5 and 10-11 and include, for example, those described herein with regard to Hl- specific embodiments of HA Sequence Elements.
• H3 polypeptide An "H3 polypeptide", as that term is used herein, is an HA polypeptide whose amino acid sequence includes at least one sequence element that is characteristic of H3 and distinguishes H3 from other HA subtypes. Representative such sequence elements can be determined by alignments such as, for example, those illustrated in Figures 5, 10 and 12 and include, for example, those described herein with regard to H3- specific embodiments of HA Sequence Elements.
• H5 polypeptide An "H5 polypeptide", as that term is used herein, is an HA polypeptide whose amino acid sequence includes at least one sequence element that is characteristic of H5 and distinguishes H5 from other HA subtypes. Representative such sequence elements can be determined by alignments such as, for example, those illustrated in Figures 5, 10, and 13, and include, for example, those described herein with regard to
• Hemagglutinin (HA) polypeptide As used herein, the term “hemagglutinin polypeptide” (or “HA polypeptide') refers to a polypeptide whose amino acid sequence includes at least one characteristic sequence of HA.
• HA sequences from influenza isolates are known in the art; indeed, the National Center for Biotechnology Information (NCBI) maintains a database (www.ncbi.nlm.nih.gov/genomes/FLU/flu.html) that, as of the filing of the present application included 9796 HA sequences.
• HA polypeptides generally, and/or of particular HA polypeptides (e.g., Hl, H2, H3, H4, H5 5 H6 5 H7, H8, H9, HlO, Hl 1, H12, H13, H14, H15, or H16 polypeptides; or of HAs that mediate infection of particular hosts, e.g., avian, camel, canine, cat, civet, environment, equine, human, leopard, mink, mouse, seal, stone martin, swine, tiger, whale, etc.
• an HA polypeptide includes one or more characteristic sequence elements found between about residues 97 and 185, 324 and 340, 96 and 100, and/or 130-230 of an HA protein found in a natural isolate of an influenza virus.
• an HA polypeptide has an amino acid sequence comprising at least one of HA Sequence Elements 1 and 2, as defined herein.
• an HA polypeptide has an amino acid sequence comprising HA Sequence Elements 1 and 2, in some embodiments separated from one another by about 100-200, or by about 125-175, or about 125-160, or about 125-150, or about 129-139, or about 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, or 139 amino acids.
• an HA polypeptide has an amino acid sequence that includes residues at positions within the regions 96-100 and/or 130-230 that participate in glycan binding.
• HA polypeptides include one or more of the following residues: Tyr98, Ser/Thrl36 5 Trpl53, Hisl83, and Leu/Ilel94. In some embodiments, an HA polypeptide includes at least 2, 3, 4, or all 5 of these residues.
• Isolated refers to an agent or entity that has either (i) been separated from at least some of the components with which it was associated when initially produced (whether in nature or in an experimental setting); or (ii) produced by the hand of man. Isolated agents or entities may be separated from at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more of the other components with which they were initially associated. In some embodiments, isolated agents are more than 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% pure.
• oligosaccharide For purposes of the present disclosure, an oligosaccharide is typically considered to be “long” if it includes at least one linear chain that has at least four saccharide residues.
• Non-natural amino acid The phrase "n ⁇ -natural amino acid" refers to an entity
• non-natural amino acids may also have a second R group rather than a hydrogen, and/or may have one or more other substitutions on the amino or carboxylic acid moieties.
• Polypeptide A "polypeptide”, generally speaking, is a string of at least two amino acids attached to one another by a peptide bond.
• a polypeptide may include at least 3-5 amino acids, each of which is attached to others by way of at least one peptide bond.
• polypeptides sometimes include "non-natural" amino acids or other entities that nonetheless are capable of integrating into a polypeptide chain, optionally.
• an agent or entity is "pure” if it is substantially free of other components.
• a preparation that contains more than about 90% of a particular agent or entity is typically considered to be a pure preparation.
• an agent or entity is at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% ⁇ Or
• Short oligosaccharide For purposes of the present disclosure, an oligosaccharide is typically considered to be “short” if it has fewer than 4, or certainly fewer than 3, residues in any linear chain.
• Specificity is a measure of the ability of a particular ligand (e.g., an HA polypeptide) to distinguish its binding partner (e.g., a human
• HA receptor and particularly a human upper respiratory tract HA receptor
• other potential binding partners e.g., an avian HA receptor
• Therapeutic agent refers to any agent that elicits a desired biological or pharmacological effect.
• treatment refers to any method used to alleviate, delay onset, reduce severity or incidence, or yield prophylaxis of one or more symptoms or aspects of a disease, disorder, or condition.
• treatment can be administered before, during, and/or after the onset of symptoms.
• Umbrella topology The phrase "umbrella topology" is used herein to refer to a 3-dimensional arrangement adopted by certain glycans and in particular by glycans on HA receptors. The present invention encompasses the recognition that binding to umbrella topology glycans is characteristic of HA proteins that mediate infection of human hosts.
• the umbrella topology is typically adopted only by ⁇ 2-6 sialylated glycans, and is typical of long (e.g., greater than tetrasaccharide) oligosaccharides.
• An example of umbrella topology is given by ⁇ angle of Neu5Ac ⁇ 2-6Gal linkage of around -60 (see, for example, Figure 14).
• Figure 9 presents certain representative (though not exhaustive) examples of glycans that adopt an umbrella topology.
• Vaccination refers to the administration of a composition intended to generate an immune response, for example to a disease-causing agent.
• vaccination can be administered before, during, and/or after exposure to a disease-causing agent, and in certain embodiments, before, during, and/or shortly after exposure to the agent.
• vaccination includes multiple administrations, appropriately spaced in time, of a vaccinating composition.
• Variant is a relative term that describes the relationship between a particular HA polypeptide of interest and a "parent" HA polypeptide to which its sequence is being compared.
• An HA polypeptide of interest is considered to be a "variant" of a parent HA polypeptide if the HA polypeptide of interest has an amino acid sequence that is identical to that of the parent but for a small number of sequence alterations at particular positions. Typically, fewer than 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% of the residues in the variant are substituted as compared with the parent.
• a variant has 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 substituted residue as compared with a parent.
• a variant has a very small number (e.g., fewer than 5, 4, 3, 2, or 1) number of substituted functional residues (i.e., residues that participate in a particular biological activity).
• a variant typically has not more than 5, 4, 3, 2, or 1 additions or deletions, and often has no additions or deletions, as compared with the parent.
• any additions or deletions are typically fewer than about 25, 20, 19, 181, 17, 16, 15, 14, 13, 10, 9, 8, 7, 6, and commonly are fewer than about 5, 4, 3, or 2 residues.
• the parent HA polypeptide is one found in a natural isolate of an influenza virus (e.g., a wild type HA).
• vector refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
• vectors are capable of extra-chromosomal replication and/or expression of nucleic acids to which they are linked in a host cell such as a eukaryotic or prokaryotic cell.
• Vectors capable of directing the expression of operatively linked genes are referred to herein as "expression vectors.”
• Wild type As is understood in the art, the phrase "wild type” generally refers to a normal form of a protein or nucleic acid, as is found in nature. For example, wild type HA polypeptides are found in natural isolates of influenza virus. A variety of different wild type HA sequences can be found in the NCBI influenza virus sequence database, http://www.ncbi.nlm.nih.gov/genomes/FLU/FLU.html.
• GBPs glycan binding proteins
• the main classes of GBPs include C-type lectins, galectins and siglecs. GBPs are typically either expressed as soluble or membrane bound proteins in the monomeric or multimeric forms with multiple glycan binding sites. Also, GBPs can be dispersed on the cell surface or localized in a microenvironment.
• the glycan binding site in a GBP is also known as a carbohydrate recognition domain (CRD).
• CRDs on GBPs typically accommodate mono — tetrasaccharide glycan ligand motifs.
• the interaction between a single CRD and a glycan motif is typically low affinity with values in ⁇ M range.
• most of the physiological glycan-GBP interactions are multivalent involving binding of an ensemble of glycan motifs to multimeric CRDs formed by association of GBPs.
• glycan-GBP interactions fine tune (analog modulation) protein function through avidity, graded affinity and multivalency.
• glycan arrays comprising several glycan structures that have enabled high throughput screening of GBPs for novel glycan ligand specificities. These glycan arrays are continuously being expanded to increase the diversity of glycan motifs to best mimic the physiological diversity of glycans. Most of the glycans on the CFG arrays were derived by chemical and chemoenzymatic synthesis.
• the CFG glycan arrays also comprise both monovalent and polyvalent glycan motifs (i.e. attached to polyacrylamide backbone), and are emerging as widely used resources for glycobiologists to discover new glycan ligands for their GBPs of interest.
• the CFG has also been developing state-of-the-art resources to generate diverse datasets ranging from gene expression of glycan biosynthetic enzymes and GBPs to whole organism glycome and phenome analysis.
• the public dissemination of CFG datasets via user-friendly interfaces has begun to motivate the development of data mining tools to find interesting patterns or make meaningful predictions by analyzing these complex data sets.
• the present invention therefore provides a system for understanding the structure-function relationships of glycan-GBP interactions.
• the invention provides a system for understanding how interactions between an ensemble of glycan structures and multivalent CRDs of GBPs modulate fundamental biological processes.
• the invention identifies features in glycans or their binding partners that determine the specificity of a given interaction.
• the invention also defined constraints provided by the features, for example based on analytical information (e.g., from X-ray crystallography, NMR, etc.) Such constraints can be used on their own or, optionally can be coupled with functional or other information. Appropriate functional information can, for example, be obtained from glycan binding studies. [0062 J
• the invention provides computational methods to analyze datasets obtained from glycan arrays such as those developed by the CFG, which are being increasingly utilized for the purpose of identifying novel candidate glycan ligands for different GBPs. As these glycan arrays continue to expand, the value of such computational methods for analyzing the datasets obtained from these arrays and understanding the basis for specificity in glycan- GBP interactions only increases.
• the present invention provides a novel approach to identifying patterns in glycans that have a positive and negative effect on binding to a GBP.
• One advantage of such a rule based approach is the presentation of the final patterns as a set of straightforward rules which can be easily applied to identify other potential glycans that satisfy these rules.
• DC-SIGN and SIGNR the rules gave three broad features for DC- SIGN viz. high mannose, Lewis x [Galb4(Fuca3)GlcNAc] and Fuca4GlcNAc containing motifs and only the high mannose feature for DC-SIGNR.
• the rules also captured features that were detrimental to binding such as absence of any 3-O substitution on the Gal for the Lewis x containing motifs.
• galectin-3 preferred linear repeat units of the Galb4GlcNAc rather than these units present in different branches in N-linked glycans. Since galectin-1 typically occurs as a homodimer with noncovalently associated CRDs, it is possible that the presence of Galb4GlcNAc on different branches would enhance high affinity multivalent binding. On the other hand, galectin-3 is a monomer with a N-terminus linker region and would most likely have a preference to linear repeats of the lactosamine unit iri comparison with the branched occurrence of these units.
• each of the rules comprise a primary glycan motif that is shared by a set of glycans with high affinity binding.
• the primary motifs are specified in conjunction with other constraints such as absence of other motifs or chain length requirements.
• additional features based on these primary patterns can be defined and the roles of these features on glycan binding can be further investigated.
• the location of Galb4GlcNAc in terms of distance from reducing end or non-reducing end and occurrence as a part of a linear chain or branched chain can be defined as additional features to evaluate their effect on binding.
• additional glycan features that combine all modifications to each monosaccharide such as GaINAc, Gal[3-O-SO3], Gal[6-O-SO3] can be combined into a single feature to evaluate the importance of each of these modifications to the binding.
• the present invention allows detailed characterization of glycan-GBP binding interaction.
• the invention therefore provides definitions of sets of glycans that do (or do not) interact with a given GBP.
• the invention thus ⁇ llows the preparation of GBP-specific glycan arrays, i.e., of arrays containing a set of glycans sufficient to establish or define the presence or identity of a particular GBP.
• an array containing glycans that are bound, glycans that are not bound, and/or combinations thereof can be assembled and used, for example, to detect that particular GBP in samples and/or to characterize derivatives of the GBP.
• HA hemagglutinin
• HA on the surface of flu virus particles recognizes sialylated glycans that are associated with
• HA receptors on the surface of the cellular host. After recognition and binding, the host cell engulfs the viral cell and the virus is able to replicate and produce many more virus particles to be distributed to neighboring cells.
• HA receptors are modified by either ⁇ 2-3 or ⁇ 2-6 sialylated glycans near the receptor's HA-binding site, and the type of linkage of the receptor-bound glycan affects the conformation of the receptor's HA-binding site, thus affecting the receptor's specificity for different HA subtypes.
• the present inventors have determined that the topology of the linked glycans (umbrella-like or cone-like) influences the receptor's specificity for different Has.
• the glycan binding pocket of avian HA is narrow. According to the present invention, this pocket binds to the trans conformation of ⁇ 2-3 sialylated glycans, and/or to cone-topology glycans, whether ⁇ 2-3 or ⁇ 2-6 linked.
• HA receptors in avian tissues and also in human deep lung and gastrointestinal
• GI tract tissues are characterized by ⁇ 2-3 sialylated glycan linkages, and furthermore
• glycans including ⁇ 2-3 sialylated and/or ⁇ 2-6 sialylated glycans, which predominantly adopt cone topologies.
• human HA receptors in the bronchus and trachea of the upper respiratory tract are modified by a.2-6 sialylated glycans.
• the ct2-6 motif has an additional degree of conformational freedom due to the C6-C5 bond (Russell et ah, Glycoconj J 23:85, 2006).
• HAs that bind to such ⁇ 2-6 sialylated glycans have a more open binding pocket to accommodate the diversity of structures arising from this conformational freedom.
• HAs may need to bind to glycans (e.g., ⁇ 2-6 sialylated glycans) in an umbrella topology, and particularly may need to bind to such umbrella topology glycans with strong affinity and/or specificity, in order to effectively mediate infection of human upper respiratory tract tissues.
• glycans e.g., ⁇ 2-6 sialylated glycans
• umbrella topology e.g., ⁇ 2-6 sialylated glycans
• the portions of the human respiratory tract that are most likely to encounter virus i.e., the trachea and bronchi
• cone glycans e.g., ⁇ 2-3 sialylated glycans. and/or short glycans
• wild type avian HAs typically bind primarily or exclusively to receptors associated with cone glycans (e.g., ⁇ 2-3 sialylated glycans, and/or short glycans)
• humans rarely become infected with avian viruses. Only when in sufficiently close contact with virus that it can access the deep lung and/or gastrointestinal tract receptors having umbrella glycans (e.g., long ⁇ 2-6 sialylated glycans) do humans become infected.
• the present invention allows identification of a set of glycans that can be used to detect the H5 HA protein and/or to detect variants of the protein that might emerge with altered binding specificity.
• an inventive array can be used to detect any H5 variant or indeed any of HA protein or variant thereof, with an ability to bind to upper respiratory human HA receptors and/or with an ability to bind (optionally with high affinity and/or specificity, preferably with high affinity) to umbrella- topology glycans.
• inventive H5 HA variant proteins are tested on such arrays to assess their ability to bind to umbrella-topology (e.g., ⁇ 2-6 glycans, and particularly long ⁇ 2-6 glycans), and particularly to assess their ability to bind to multiple such glycans.
• umbrella-topology e.g., ⁇ 2-6 glycans, and particularly long ⁇ 2-6 glycans
• the present invention provides arrays of umbrella glycans (e.g., ⁇ 2-6 glycans, and particularly long a.2-6 glycans) and optionally cone-topology glycans (e.g., ⁇ 2- 3 sialylated glycans), that can be used to characterize HA binding capabilities and/or as a diagnostic to detect, for example, human-binding HAs.
• umbrella glycans e.g., ⁇ 2-6 glycans, and particularly long a.2-6 glycans
• cone-topology glycans e.g., ⁇ 2- 3 sialylated glycans
• the CFG has developed two kinds ofglycan arrays: (1) well based microarray and (2) solid phase printed array.
• the printed array was more recently developed, so most of the initial ligand screening was performed using the well based microarray.
• the first version of the well-based array developed by the CFG comprised around 60 differentglycans with triplicate representations of each glycan.
• Each successive version of the array incorporated additional glycans.
• the current version comprises 195 glycans with quadruplicate representation of each glycan (see http://vvrww.functionalglycomics.org/static/consortium/resources/resourcecoreh5.shtrnl).
• the array predominantly comprises synthetic glycans that capture the physiological diversity of N- and O-linked glycans.
• the array also comprises polyvalent glycan ligands attached to a polyacrylamide backbone.
• N-linked glycan mixtures derived from different mammalian glycoproteins are also represented on the array.
• Data objects are the raw data that are stored in the database.
• features Important properties of the data objects.
• the choice of features to describe a data object allows the rules or patterns to be obtained.
• Classifiers are the rules or patterns that are used to either cluster data objects into specific classes or determine relationships between features.
• the classifiers provide specific features that are satisfied by the glycans that bind with high affinity to a GBP. These rules are of two kinds: (1) features present on a set of high affinity glycan ligands, which can be considered to enhance binding, and (2) features that should not be present in the high affinity glycan ligands, which can be considered not favorable for binding.
• the data mining platform comprises software modules that interact with each other ( Figure 1) to perform the operations described above.
• One component is the feature extractor that will interface to the CFG database to extract features.
• the object based relational database used by CFG facilitates the flexible definition of features.
• features can be extracted from glycans and/or from their binding partners.
• certain features were extracted from glycans on the glycan array, as listed in Table 1:
• Table 1 Features extracted from the glycans on the glycan array.
• Higher order features Pairs Pair refers to a pair of monosaccharide, connected covalently by a linkage.
• the pairs are classified into two categories, regular [B] and terminal [Tj to distinguish between the pair with one monosaccharide that terminates in the non reducing end [Figure 2].
• the frequency of the pairs were extracted as features
• Triplets refers to a set of three monosaccharides connected covalently by two linkages. We classify them into three categories namely regular [B], terminal [T] and surface [S] [ Figure 2]. The compositions of each category of triplets were extracted as features
• Quadruplets Similar to the triplet features, quadruplets features are also extracted, with four monosaccharides and their linkages [Figure 2]. Quadruplets are classified into two varieties regular [B] and surface [S]. The frequencies of the different quadruplets were extracted as features
• Average Leaf Depth As an indicator of the effective length of the probes, average depth of the reducing end of the tree is extracted as a glycan feature.
• the leaf depths are 3,4 and 3, and the average is 3.34 Number of Leaves
• the number of non reducing monosaccharides is extracted as a feature.
• the number of leaves is 3.
• glycan binding sites on GBPs typically accommodate di-tetra —saccharides.
• a tree-based representation was used to capture the information on monosaccharides and linkages in the glycan structures (root of the tree at the reducing end). This representation facilitated the abstraction of various features including higher order features such as connected set of monosaccharide triplets, etc ( Figure 2).
• the data preparation involved generating a column- wise listing of all the glycans in the latest version of the glycan array along with the abstracted features (Table 1) for each glycan. From this master table of glycans and their features, a subset was chosen for the rule based classification (see below) to determine specific patterns that govern the binding to a specific GBP or set of GBPs.
• IFA Glycan contains "Galb4GlcNAcb3Gal[B]” and DOES NOT contain "Fuca3GlcNAc[B]” ⁇ THEN the Glycan will bind with higher affinity to Galectin 3.
• the specific Rule Induction algorithm that was used in this case is the one developed by Weiss & Indurkya (Predictive data mining — A practical guide. Morgan Kaufmann, San Francisco, 1998).
• the CFG glycan arrays represent a much larger domain of glycan structures that have been used to screen ligand specificities of different galectins.
• the application of our methodology to the galectin datasets provides additional rules that govern the binding of different galectins to their glycan ligands.
• DC-SIGN and DC-SIGNR belong to the type II transmembrane receptor subfamily of C-type lectins which recognize and bind to glycan ligands in a Ca 2+ dependent manner.
• DC-SIGN is abundantly expressed in dendritic cells, and plays a key role in adhesion of T-cells to the antigen presenting dendritic cells via ICAM-3 molecule, thereby initiating an immune response.
• DC-SIGN has also been shown to play an important role in recognition of pathogens such as HIV, etc. by the dendritic cells.
• DC-SIGNR which shares a 77% sequence identity with DC-SIGN, is found on endothelial cells in liver, lymph nodes and placenta.
• Each of these proteins contains a single carbohydrate recognition domain (CRD) at the C-terminus.
• CRD carbohydrate recognition domain
• the extracellular alpha helical domain (adjacent to CRD) on both the proteins facilitates tetramerization of the CRDs, thus enabling multivalent interactions with glycan ligands.
• the rules are derived based on features [Table 1] where #[] is used to specify number of occurrences.
• #[] is used to specify number of occurrences.
• the last rule governing DC-SIGN binding is more complex involving a "&" combination of multiple features which implies that each of the individual rules must be satisfied.
• the glycans which could not be clustered into rules [false negatives] are also shown for DC-SIGN.
• DC-SIGN bound to an additional set of fucosylated ligands that were characterized by distinct features. These fucosylated ligands did not bind to DC-SIGNR.
• the Fuca4GlcNAc is a commonly observed motif in Lewis a [Fuca4(Galb3)GlcNAc] containing glycan structures.
• the Fuca3(Galb4)GlcNAc is another commonly observed Lewis x motif present on the non-reducing terminal of N- and O-linked glycans. Both these features were characteristic of high affinity binders to DC-SIGN.
• Galectins belong to a family of soluble GBPs that are known to bind ⁇ - galactosides which were earlier defined as S-type lectins due to their requirement for reducing thiols for their activity. Unlike the C-type lectins (such as DC-SIGN and - SIGNR), galectins do not require Ca 2+ for ligand binding. Galectins have been implicated in numerous biological roles viz. cell development, apoptosis, cancer, and immune response.
• galectins are generally known to bind to type I (Galb3 GIcNAc) and type II (Galb4GlcNAc) lactosamine units, their finer substrate specificity and its implications on their numerous biological roles is less understood.
• the data sets for human galectin-1 and - 3 were analyzed using the rule based data mining approach. These two galectins are fundamentally different in terms of organization of their CRDs. Both galectin -1 and -3 share a similar C-terminal F3 type CRD.
• Galectin-1 is typically a homodimer of CRDs whereas galectin-3 comprises of single CRD with a N-terminus linker domain.
• the rules are derived based on features [Table 1] where #[] is used to specify number of occurrences, & implies a combination of rules where each must be satisfied. ![] implies absence of the pattern. / in the a2/3 and a3/6 to represents a2 or a3 and a3 or a6 respectively.
• galectin -1 and -3 The rules that govern the high affinity ligand binding of galectin -1 and -3 are more complex that those derived for DC-SIGN and -SIGNR. Although it is known that galectins -1 and -3 bind with similar affinity to both type II and type I lactosamine units, the data from the glycan array did not reveal any type I (Galb3 GIcNAc) binders based on the threshold intensities that were used to distinguish high binders.
• the first rule (Table 3) that captured 8 out of the 9 high binders included the presence of at least one lactosamine unit in a chain length of at least 3 monosaccharides. Again it is significant to note that there were no false positives. Based on analysis of the low and high affinity binders, several patterns in rule 1 were implicated to have a negative effect on binding. Fucosylation of the GIcNAc, terminal fucosylation of the GaI 5 sialylation of Gal and also presence of Gala3Gal or Gala4Gal in conjunction with the type II lactosamine unit had negative effects on binding. Furthermore the - Galb4GlcNAcb6GalNAc- unit which comprises of the type II lactosamine on a Core 2 (or Core 4) O-linked core had a negative effect on binding.
• the second rule gave an interesting pattern which indicated that sialylation did not have an effect on high affinity binding if the glycan motif comprised of a type II polylactosamine repeat with at least two Galb4GlcNAc units.
• glycans with terminal sialylation are candidate ligands for galectin-1. Since the sialylated glycans used in this study comprised of at least two Galb4GlcNAc units, these results are consistent with our rules.
• our rules also indicate that galectin-1 binds to internal Galb4GlcNAc units and any other patterns that are farther way in the chain towards non-reducing end have no effect on high affinity binding. There was only one false negative which comprised of Gal[3-O-SO 3 ]b3GalNAc.
• Galectin-3 favors the linear repeat of lactoseamine
• Galectin-1 favors the lactoseamine found in a branched arrangement. This is consistent with the ligand binding propensity that was observed in Hirabayashi et al. (2002).
• This analysis provides important insights into the interactions of an HA glycan binding site with a variety of ⁇ 2-3/6 sialylated glycans, including glycans of either umbrella or cone topology.
• the second involves a data mining approach to analyze the glycan array data on the different Hl, H3 and H5 HAs. This data mining analysis correlates the strong, weak and non-binders of the different wild type and mutant HAs to the structural features of the glycans in the microarray (Table T).
• correlations capture the effect of subtle structural variations of the ⁇ 2-3/6 sialylated linkages and/or of different topologies on binding to the different HAs.
• the correlations of glycan features obtained from the data mining analysis are mapped onto the HA glycan binding site, providing a framework to systematically investigate the binding of Hl, H3 and H5 HAs to cc2-3 and ⁇ 2-6 sialylated glycans, including glycans of different topologies, as discussed below.
• IRVT, IRVX, IRVZ), H3 (PDB IDs: IMQL, IMQM, IMQN) and H5 (USN, USO, 2FK0) and their complexes with cc2-3 and/or ⁇ 2-6 sialylated oligosaccharides have provided molecular insights into residues involved in specific HA-glycan interactions. More recently, the glycan receptor specificity of avian and human Hl and H3 subtypes has been elaborated by screening the wild type and mutants on glycan arrays comprising of a variety of ⁇ 2-3 and ct2-6 sialylated glycans.
• the Aspl90Glu mutation in the HA of the 1918 human pandemic virus reversed its specificity from ⁇ 2-6 to ⁇ 2-3 sialylated glycans (Stevens et ah, J. MoI. Biol., 355:1143, 2006; Glaser et ah, J. Virol, 79: 11533, 2005).
• the double mutation GIu 190 Asp and Gly225Asp on an avian Hl (A/Duck/Alberta/35/1976) reversed its specificity from ⁇ 2-3 to cc2-6 sialylated glycans.
• the amino acid changes from Gln226 to Leu and Gly228 to Ser between the 1963 avian H3N8 strain and the 1967-68 pandemic human H3N2 strain correlate with the change in their preference from ⁇ 2-3 to ⁇ 2-6 sialylated glycans (Rogers et ah, Nature, 304:76, 1983).
• the relationship between the HA glycan binding specificity and transmission efficiency was demonstrated in a ferret model using the highly pathogenic and virulent 1918 HlNl viruses (Tumpey, T. M. et al. Science 315: 655, 2007).
• the conformation of the Neu5Ac ⁇ 2-3Gal linkage is such that the positioning of Gal and sugars beyond Gal in ⁇ 2-3 fall in a cone-like region governed by the glycosidic torsion angles at this linkage ( Figure 6).
• the typical region of minimum energy conformations is given by ⁇ values of around -60 or 60 or 180 where ⁇ samples -60 to 60 ( Figure 14). In these minimum energy regions, the sugars beyond Gal in oc2-3 are projected out of the HA glycan binding site. This is also evident from the co-crystal structures of HA with the ⁇ 2-3 motif (Neu5Ac ⁇ 2-3Gal ⁇ l -3/4GIcNAc-) where the ⁇ value is typically around 180 (referred to as trans conformation).
• the trans conformation causes the ⁇ 2-3 motif to project out of the pocket.
• This structural implication is consistent with the three distinct classifiers for HA binding to ⁇ 2-3 sialylated glycans obtained from the data mining analysis (Table 7).
• the common feature in all these three classes is that the Neu5Ac ⁇ 2-3Gal should not be present along with a GalNAc ⁇ / ⁇ l-4Gal. Analysis of the crystal structures showed that the GaINAc linked to Gal of Neu5Ac ⁇ 2-3Gal made unfavorable steric contacts with the protein, consistent with the classifiers.
• Gln226 and Glul90 are involved in binding to the Neu5Ac ⁇ 2-3Gal motif.
• Glul90, located on the opposite side of Gln226 interacts with Neu5Ac and Gal monosaccharides ( Figure 15, Panels C,D).
• APR34 a human Hl subtype, contains all the four amino acids Alal38, GIu 190, Gln226 and Gly228 and binds to ct2-3 sialylated glycans as observed in its crystal structure ( Figure 14, Panel B).
• the Neu5Ac ⁇ 2-3Gal motif in this conformation provides less optimal contacts with human H3 HA binding site compared to those provided by this motif in the trans conformation with the avian H3 HA ( Figure 14).
• the Gly228Ser mutation in human H3 HA makes its glycan binding site less favorable for interaction with ⁇ 2-3 sialylated glycans.
• Table 7 shows that the human H3 HA has only a moderate affinity for some of the ⁇ 2-3 sialylated glycans.
• Lysl93 which is highly conserved in the avian H5 ( Figure 5) is positioned to interact with 6-0 sulfated Gal and/or 6-0 sulfated GIcNAc in Neu5Ac ⁇ 2- 3Gal ⁇ l -4GIcNAc. This observation is validated by the data mining analysis wherein only the avian H5 binds with high affinity to ⁇ 2-3 sialylated glycans that are sulfated at the Gal or GIcNAc (Table 7).
• a basic amino acid at position 222 could interact with 4-0 sulfated GIcNAc in Neu5Ac ⁇ 2-3Gal ⁇ 1-3 GIcNAc motif or 6-O sulfated GIcNAc in Neu5Ac ⁇ 2-3Gal ⁇ l -4GIcNAc motif.
• a bulky side chain such as Lys222 in Hl and H5 and Trp222 in H3 potentially interferes with a fucosylated GIcNAc in Neu5Ac ⁇ 2-3Gal ⁇ l-4(Fuc ⁇ l-3) GIcNAc motif.
• amino acid residues in these positions are not conserved across the different HAs and this accounts for the different binding specificities Structural constraints imposed by glycan topology and substitutions on Hl and HS HA binding to a2-6 sialylated glycans
• the presence of a GIcNAc instead of GIc in the ⁇ 2-6 motif Neu5Ac ⁇ 2-6Gal ⁇ l -4GIcNAc- would potentially favor the umbrella topology which is stabilized by optimal van der Waals contact between the acetyl carbons of both GIcNAc and Neu5Ac.
• the ⁇ 2-6 motif can also adopt a cone topology such that additional factors such as branching and HA binding can compensate for the stability provided by the umbrella topology.
• the cone topology of the ⁇ 2-6 motif present as a part of multiple short oligosaccharide branches in an N-linked glycan could be stabilized by intra sugar interactions.
• the umbrella topology would be favored by the ⁇ 2-6 motif in a long oligosaccharide branch (at least a tetrasaccharide).
• the co-crystal structures of Hl and H3 HAs with the ⁇ 2-6 motif (Neu5Ac ⁇ 2-6Gal ⁇ 1 -4GIcNAc-) motif supports the above notion wherein the ⁇ — 60 (referred to as cis conformation) causes the sugars beyond Neu5Ac ⁇ 2-6Gal to bend towards the HA protein to make optimal contacts with the binding site ( Figure 7).
• Hl HA In Hl HA, superimposition of the glycan binding domain of HA from a human HlNl (A/South Carolina/1/1918) subtype with that of ASI3O_H1_26 and APR34_H1_26 provided insights into the amino acids involved in providing specificity to the ⁇ 2-6 sialylated glycan. Lys222 and Asp225 are positioned to interact with the oxygen atoms of the Gal in the Neu5Ac ⁇ 2-6Gal motif.
• Aspl90 and Ser/Asnl93 are positioned to interact with additional monosaccharides GlcNAc ⁇ l-3Gal of theNeu5Ac ⁇ 2-6Gal ⁇ l-4GlcNAc ⁇ l- 3GaI motif ( Figure 15, Panels A,B).
• Asp 190, Lys222 and Asp225 are highly conserved among the Hl HAs from the 1918 human pandemic strains. Although the amino acid Gln226 is highly conserved in all the avian and human Hl subtypes, it does not appear to be as involved in binding to ⁇ 2-6 sialylated glycans (in human Hl subtypes) compared to its role in binding to ⁇ 2-3 sialylated glycans (in the avian Hl subtypes).
• the data mining analysis of the glycan array results for wild type and mutant form of the avian and human Hl HAs further substantiates the role of the above amino acids in binding to ⁇ 2-6 sialylated glycans (Table 7).
• the Glul90Asp/Gly225Asp double mutant of the avian Hl HA reverses its binding to ⁇ 2-6 sialylated glycans (Table 7).
• the Lys222Leu mutant of human ANYl 8_H1 removes its binding to all the sialylated glycans on the array consistent with the essential role of Lys222 in glycan binding.
• GIu 190 in human H3 HA is displaced slightly into the binding site by about 0.7 A in comparison with that of GIu 190 in avian H3 HA.
• These differences limit the ability of human H3 HA to bind to ⁇ 2-3 sialylated glycans and correlate with its preferential binding to oc2-6 sialylated glycans.
• the Gln226Leu and Gly228Ser mutations cause a reversal of the glycan receptor specificity of avian H3 to human H3 subtype during the 1967 pandemic.
• the HA binding to cc2-6 sialylated glycans has a more open binding pocket to accommodate this conformational freedom. While Leu226 in human H3 HA is positioned to provide optimal van der Waals contact with Neu5Ac ⁇ 2-6Gal, the ionic contacts provided by Gln226 in Hl HA to this motif are not as optimal. On the other hand in Hl , the amino acids Lys222 and Asp225 provide more optimal ionic contacts with ⁇ 2-6 sialylated glycans compared to Trp222 and Gly225 in H3.
• Glul90 and Gly225 in VietO4_H5 do not provide the necessary contacts with the Neu5Ac ⁇ 2-6Gal ⁇ l -4GIcNAc motif similar to Hl . Therefore Glul90Asp.and Gly225Asp mutations in H5 HA could potentially improve the contacts with oc2-6 sialylated glycans.
• Gln226 in Hl HA is also conserved in the avian H5 HA. Given that Gln226 plays a less active role in Hl HA binding to ⁇ 2-6 sialylated glycans (as discussed above), mutation of this amino acid to a hydrophobic amino acid such as Leu could potentially enhance its van der Waals contact with C6 atom of Gal in Neu5Accc2-6Gal motif.
• H3N2 pandemic H3N2
• Asp 190 in H5 HA would be positioned to provide better ionic contacts with Neu5Ac ⁇ 2-6Gal motif in longer oligosaccharides.
• the Neu5Ac ⁇ 2- 6GaI on the Mal ⁇ l-3Man branch binds more favorably compared to the same motif on the Man ⁇ l-6Man branch which has unfavorable steric contacts with the glycan binding site of H5 HA ( Figure 16).
• the narrow specificity of the Gln226Leu/Gly228Ser double mutant to a.2-6 sialylated glycans is consistent with Lysl93 interfering with the binding.
• the present inventors propose that a necessary condition for human adaptation of influenza A virus HAs is to gain the ability to bind to long a.2-6 (predominantly expressed in human upper airway) with high affinity.
• an aspect of glycan diversity is the length of the lactosamine branch that is capped with the sialic acid.
• This is captured by the two distinct features of cc2-6 sialylated glycans derived from the data mining analysis (Table 7).
• One feature is characterized by the Neu5Ac ⁇ 2-6Gal ⁇ l -4GIcNAc linked to the Man of the N-linked core and the other is characterized by this motif linked to another lactose amine unit forming a longer branch (which typically adopts umbrella topology).
• desirable binding patterns include binding to umbrella glycans depicted in Figure 9.
• modified H5 HA proteins containing Gly228Ser and Gln226Leu/Gly228Ser substitution
• binding to only a single biantennary a2-6 sualyl-lactosamine glycan structure on the glycan array (Stevens et ah, Science 312:404, 2006).
• modified H5 HA proteins are therefore not BSHB H5 HAs, as described herein.
• the present invention demonstrates that the current avian H5N1 HA can undergo mutations that would alter its specificity towards ⁇ 2-6 glycans based on interactions of human Hl or H3 HA with these glycans.
• the GIu 190 Asp, Lysl93Ser, Gly225Asp and Gln226Leu mutations Q 1 DSDL mutant”) could potentially make the H5 HA binding site similar to that of the human Hl HA, while the Glul90Asp, Lysl93Ser, Gln226Leu and Gly228Ser ("DSLS mutant") could potentially make it similar to that of the human H3 HA for optimal interactions with cc2-6 sialylated glycans.
• DSDL and DSLS H5 HA mutants were designed and tested based on the above framework. Wild type and mutant BSHB H5 HAs were expressed in baculovirus and purified as reported earlier ( Figure 10XXXY).
• tissue binding experiment tissue sections were deparaffinized, rehydrated and incubated with the WT and the mutant HA proteins (diluted in PBS) for 3hr. Based on the protein concentration for a given lot after purification, appropriate serial dilutions in the ranges of 1 : 10 - 1 : 100 were tested. After extensive washing with PBS, the sections were blocked with 2% BSA-PBS for 30 min and then incubated with rabbit anti avian H5N1 hemagglutinin antibody (Pro-Sci Inc, 1 : 1000 in 2% BSA-PBS) for 3hr.
• Sections were washed with PBS and then incubated with secondary goat- anti rabbit antibody (Invitrogen; 1 :500 in 2% BSA-PBS) for 90 min. Sections were counterstained with propidium iodide (in red; Invitrogen; 1 :200 in PBS) and then viewed under a confocal microscope (Zeiss LSM510 laser scanning confocal microscopy). All incubations were at room temperature.
• the lack of binding of the DiS mutant to the deep lung tissues is consistent with the loss of binding to ⁇ 2-3 sialylated glycans (due to Glul90Asp mutation).
• the lack of binding of this mutant to the upper airway tissues further supports the disruptive effect of Aspl87 on Aspl90 which could lower the binding of this mutant to ct2-6 sialylated glycans.
• the mutations in current avian H5N1 HA would prefer leading to a H3-like (as compared to an Hl -like) glycan binding site having a broad specificity for ⁇ 2-6 sialylated glycans.
• the glycans were further desalted and purified into neutral (25% acetonitrile fraction) and acidic (50% acetonitrile containing 0.05% trifluoroacetic acid) fractions using graphitized carbon solid— phase extraction columns (Supelco).
• the acidic fractions (containing sialylated glycans) were analyzed by MALDI-TOF MS in negative ion mode with soft ionization conditions (accelerating voltage 22 kV, grid voltage 93%, guide wire 0.3% and extraction delay time of 150 ns).
• This MALDI TOF-TOF fragmentation analysis of representative mass peaks illustrated the diversity in terms of branching pattern and increased branch length in the N-linked glycans. The longer branch length versus higher branching observed in the glycan profile can influence the binding of H5 HA to these glycans.
• an aspect of glycan diversity is the length of the lactosamine branch that is capped with the sialic acid. This is captured by the two distinct features of ⁇ 2-6 sialylated glycans derived from the data mining analysis (Table 7). One feature is characterized by the Neu5Ac ⁇ 2-6Gal ⁇ 1-4GIcNAc linked to the Man of the N-linked core and the other is characterized by this motif linked to another lactose amine unit forming a longer branch. Thus, the extensive binding of the mutant H5 HAs to the upper airways is only possible if these mutants have a broad binding specificity to ⁇ 2-6 sialylated glycans.
• desirable binding patterns include those depicted in Figure 9 and/or: ) and combinations thereof:
• modified H5 HA proteins containing Gly228Ser and Gln226Leu/Gly228Ser substitution
• binding to only a single biantennary a2-6 sualyl-lactosamine glycan structure on the glycan array (Stevens et al., Science 312:404, 2006).
• modified H5 HA proteins are therefore not BSHB H5 HAs, as described herein.

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