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EP2238146A2 - Polypeptides d'hémagglutinine, réactifs, et procédés s'y rapportant - Google Patents

Polypeptides d'hémagglutinine, réactifs, et procédés s'y rapportant

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Publication number
EP2238146A2
EP2238146A2 EP09701014A EP09701014A EP2238146A2 EP 2238146 A2 EP2238146 A2 EP 2238146A2 EP 09701014 A EP09701014 A EP 09701014A EP 09701014 A EP09701014 A EP 09701014A EP 2238146 A2 EP2238146 A2 EP 2238146A2
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EP
European Patent Office
Prior art keywords
glycans
umbrella
binding agent
binding
topology
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP09701014A
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German (de)
English (en)
Other versions
EP2238146A4 (fr
Inventor
Ram Sasisekharan
Karthik Viswanathan
Aarthi Chandrasekaran
Rahul Raman
Aravind Srinivasan
S. Raguram
Viswanathan Sasisekharan
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Massachusetts Institute of Technology
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Massachusetts Institute of Technology
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Priority claimed from US11/969,040 external-priority patent/US20090081193A1/en
Application filed by Massachusetts Institute of Technology filed Critical Massachusetts Institute of Technology
Publication of EP2238146A2 publication Critical patent/EP2238146A2/fr
Publication of EP2238146A4 publication Critical patent/EP2238146A4/fr
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • 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
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B40/00—Libraries per se, e.g. arrays, mixtures
    • C40B40/04—Libraries containing only organic compounds
    • C40B40/12—Libraries containing saccharides or polysaccharides, or derivatives thereof
    • 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
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/16011—Orthomyxoviridae
    • C12N2760/16111—Influenzavirus A, i.e. influenza A virus
    • C12N2760/16122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/08—RNA viruses
    • G01N2333/11—Orthomyxoviridae, e.g. influenza virus
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

  • HEMAGGLUTININ POLYPEPTIDES AND REAGENTS AND METHODS RELATING
  • Influenza has a long history of pandemics, epidemics, resurgences and outbreaks.
  • Avian influenza including the H5N1 strain, is a highly contagious and potentially fatal pathogen, but it currently has only a limited ability to infect humans.
  • avian flu viruses have historically observed to accumulate mutations that alter its host specificity and allow it to readily infect humans.
  • two of the major flu pandemics of the last century originated from avian flu viruses that changed their genetic makeup to allow for human infection.
  • H5N1 , H7N7, H9N2 and H2N2 avian influenza strains might accumulate mutations that alter their host specificity and allow them to readily infect humans.
  • the present invention binding agents with particular glycan binding characteristics.
  • the present invention provides binding agents that bind to sialylated glycans having an umbrella-like topology.
  • inventive binding agents bind to umbrella- topology glycans with high affinity and/or specificity.
  • inventive binding agents show a binding preference for umbrella-topology glycans as compared with cone- topology glycans.
  • inventive binding agents compete with hemagglutinin for binding to glycans on hemagglutinin receptors.
  • inventive binding agents compete with hemagglutinin for binding to umbrella-topology glycans.
  • the present invention also provides diagnostic and therapeutic reagents and methods associated with provided binding agents, including vaccines.
  • Figure 1 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)
  • FIG. 1 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 E 190 (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). Abbreviations: ADA76,
  • A/duck/Alberta/35/76 HlNl
  • ASBO A/Swine/Iowa/30
  • APR34 A/Puerto Rico/8/34
  • ASC18 A/South Carolina/1/18 (HlNl), AT91, A/Texas/36/91 (HlNl); ANY18, 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/1203/2004 (H5N1).
  • Figure 3 Sequence alignment illustrating conserved subsequences characteristic of Hl HA.
  • Figure 4 Sequence alignment illustrating conserved subsequences characteristic of H3 HA.
  • Figure 5 Sequence alignment illustrating conserved subsequences characteristic of H5 HA.
  • 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 Neu5 Ac ⁇ 2-6Gal ⁇ 1 -3/4GIcNAc-.
  • Panel B of this Figure specifically shows the topology of ⁇ 2-3 and ⁇ 2-6 as governed by the glycosidic torsion angles of the trisaccharide motifs - Neu5Ac ⁇ 2-3Gal ⁇ l -3/4GIcNAc and Neu5Ac ⁇ 2-6Gal ⁇ l- 4GIcNAc respectively.
  • a parameter ( ⁇ ) - angle between C2 atom of Neu5Ac and Cl atoms of the subsequent Gal and GIcNAc sugars in these trisaccharide motifs was defined to characterize the topology.
  • umbrella-like topology which is unique to ⁇ 2-6, ⁇ GIcNAc and subsequent sugars bend towards the HA binding site (as observed in HA- ⁇ 2-6 co-crystal structures).
  • Longer ⁇ 2-6 oligosaccharides e.g. at least a tetrasaccharide
  • HA interactions with umbrella-like topology involve contacts of amino acids at the numbered positions (based on H3 HA numbering) with GIcNAc and subsequent sugars in addition to contacts with Neu5Ac and Gal sugars.
  • Panel C of this Figure depicts conformational sampling of cone- and umbrella-like topology by ⁇ 2-3 and ⁇ 2-6.
  • Sections (A) - (D) show the conformational ( ⁇ , ⁇ ) maps of Neu5Ac ⁇ 2-3Gal, Neu5Ac ⁇ 2-6Gal, Gal ⁇ 1-3 GIcNAc, and Gal ⁇ l- 4GIcNAc linkages, respectively.
  • GlycoMaps DB http://www.glycosciences.de/modeling/glycomapsdb/
  • Energy distribution is color coded starting from red (representing highest energy) to green representing lowest energy.
  • Encircled regions 1 - 5 represent ( ⁇ , ⁇ ) values observed for the ⁇ 2-3 and ⁇ 2-6 oligosaccharides in the HA-glycan co- crystal structures.
  • the trans conformation (encircled region 1) of Neu5Ac ⁇ 2-3Gal predominates in HA binding pocket with the exception of the co-crystal structure of A/Aichi/2/68 H3N2 HA with ⁇ 2-3 where this conformation is gauche (encircled region 2).
  • the cis conformation of Neu5Ac ⁇ 2-6Gal predominates in HA binding pocket.
  • Sections (E) - (F) show sampling of cone- like and umbrella-like topologies by ⁇ 2-3 and ⁇ 2-6 motifs, respectively. Regions marked in red in the conformational maps were used as the outer boundaries to calculate the ⁇ parameter (angle between C2 atom of Neu5Ac and Cl atoms of subsequent Gal and GIcNAc sugars) for a given set of ( ⁇ , ⁇ ) values. Based on the energy cutoff, the value of ⁇ > 110° was used to characterize cone-like topology and ⁇ ⁇ 100° was used to characterize umbrella-like topology.
  • Contacts with Neu5 Ac 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 Neu5 Ac and with Gal sugars. E 190 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. In some cases, 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.
  • This Figure illustrates 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.
  • 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.
  • a D residue at position 190 and/or a D residue at position 225 contribute(s) to binding to umbrella topologies.
  • Figure 8 Exemplary cone topologies. This Figure illustrates certain exemplary (but not exhaustive) glycan structures that adopt cone topologies.
  • FIG. 9 Exemplary umbrella topologies.
  • A Certain exemplary (but not exhaustive) N- and 0-linked glycan structures that can adopt umbrella topologies.
  • (B) Certain exemplary (but not exhaustive) O-linked glycan structures that can adopt umbrella topologies.
  • Figure 10. Glycan profile of human bronchial epithelial cells and human colonic epithelial cells. To further investigate the glycan diversity in the upper respiratory tissues, N- linked glycans were isolated from HBEs (a representative upper respiratory cell line) and analyzed using MALDI-MS.
  • the predominant expression of a2-6 in HBEs was confirmed by pre-treating the sample with Sialidase S (a2-3 specific) and Sialidase A (cleaves and SA).
  • the predominant expression of glycans with long branch topology is supported by TOF-TOF fragmentation analysis of representative mass peaks (highlighted in cyan).
  • HT29 human colonic epithelial cells
  • This cell line was chosen because the current H5N1 viruses have been shown to infect gut cells.
  • Sialidase A and S pre-treatment controls showed predominant expression of a2-3 glycans (highlighted in red) in the HT-29 cells.
  • H5N1 HA human adaptation of the H5N1 HA would involve HA mutations that would enable high affinity binding to the diverse glycans expressed in the human upper respiratory tissues (e.g., umbrella glycans).
  • FIG. 11 Data mining platform. Shown in (A) are the main 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 (B), shows the key software modules that enable the user to apply the data mining process to the glycan array data.
  • FIG. 12 Features used in data mining analysis. This figure shows the features defined herein as representative motifs that illustrate the different types of pairs, triplets and quadruplets abstracted from the glycans on the glycan microarray. The rationale behind choosing these features is based on the binding of di-tetra saccharides to the glycan binding site of HA. The final dataset comprise features from the glycans as well as the binding signals for each of the HAs screened on the array.
  • the rule induction classification method was utilized. One of the main advantages of this method is that it generates IF-THEN rules which can be interpreted more easily when compared to the other statistical or mathematical methods.
  • the two main objectives of the classification were: (1) identifying features present on a set of high affinity glycan ligands, which enhance binding, and (2) identifying features that are in the low affinity glycan ligands that are not favorable for binding.
  • FIG. 13 Classifiers used in data mining analysis. This figure presents a table of classifier ids and rules.
  • FIG. 14 Conformational map and solvent accessibility ofNeu5Aca2-3Gal 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- 6GaI where the c ⁇ -conformation (encircled region 3) is observed in all the HA- ⁇ 2-6 sialylated glycan co-crystal structures.
  • Panel C shows difference between solvent accessible surface area (SASA) of Neu5Ac ⁇ 2-3 and ⁇ 2-6 sialylated oligosaccharides in the respective HA-glycan co- crystal structures.
  • SASA solvent accessible surface area
  • 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 Neu5Ac ⁇ 2-3Gal) or C1-O-C6-C5 (for Neu5Ac ⁇ 2-6Gal); ⁇ : O-C6-C5-H5 (for Neu5 Ac ⁇ 2-6Gal) linkages.
  • the ⁇ , ⁇ maps were obtained from GlycoMaps DB
  • FIG. 15 Residues involved in binding of Hl, H3 and H5 HA to a2-3/6 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 H1, 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 ⁇ 2-3/6 sialylated glycans in Hl, H3 and H5 HA. Certain key residues involved in binding to ⁇ 2-3 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 a.2-6 sialylated glycan (cone topology). Stereo view of surface rendered VietO4_H5 glycan binding site with Neu5 Ac ⁇ 2-6Gal 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 c ⁇ -conformation but can bind without any contact with Lysl93 in the extended conformation, albeit less optimally.
  • Figure 17 Production of WT Hl, H3 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/1203/2004), run on a 4-12% SDS-polyacrylamide gel and blotted onto nitrocellulose membranes.
  • HlNl HA was probed using goat anti-Influenza A antibody and anti-goat IgG- HRP.
  • H3N2 was probed 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.
  • FIG. 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. Without wishing to be bound by any particular theory, we note that this finding suggests that goblet cells predominantly express O-linked glycans while ciliated epithelial cells predominantly express N- linked glycans.
  • 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 respectively in ciliated and goblet cells on the apical side of the tracheal epithelium.
  • FIG. 19 Dose response binding of recombinant Hl, HS WT HA to upper and lower respiratory tissue sections. HA binding is shown in green against propidium iodide staining ⁇ red). The apical side of tracheal tissue predominantly expresses ⁇ 2-6 glycans with long branch topology. The alveolar tissue on the other hand predominantly expresses a2-3 glycans. Hl HA binds significantly to the apical surface of the trachea and its binding reduces gradually with dilution from 40 to 10 ⁇ g/ml. Hl HA also shows some weak binding to the alveolar tissue only at the highest concentration.
  • H3 HA shows significant binding to both tracheal and alveolar tissue sections at 40 and 20 ⁇ g/ml. However, at a concentration of 10 ⁇ g/ml, H3 HA shows binding primarily to the apical side of the tracheal tissue and little or no binding to the alveolar tissue. Together, these tissue binding data highlight the importance of high affinity binding to the apical side of tracheal tissue.
  • FIG. 20 Direct binding dose response of Hl, H3 and H5 WT HA. Shows from top to bottom are the binding signals (normalized to the saturation level of around 800000) respectively for wild type Hl, H3, and H5 HA at various concentrations.
  • the legend for the glycans is shown as an inset, where LN corresponds to GaIb 104GIcNAc and 3'SLN and 6'SLN, respectively, correspond to ⁇ 2-3 and ⁇ 2-6 linked sialic acid at the LN.
  • the characteristic binding pattern of the Hl and H3 HAs which are adapted to infect humans, is their binding at saturating levels to the long ⁇ 2-6 (6'SLN-LN) glycans over a range of dilution from 40 ⁇ g/ml down to 5 ⁇ g/ml. While Hl HA is highly specific for binding to the long ⁇ 2-6 sialylated glycans, H3 HA also binds to short ⁇ 2-6 sialylated glycans (6'SLN) with high affinity and to a long ⁇ 2-3 with lower affinity relative to ⁇ 2-6. This direct binding dose response of Hl and H3 HA is consistent with the tissue binding pattern.
  • Hl and H3 HA show the high affinity binding of Hl and H3 HA to long ⁇ 2-6 silalylated glycans with their extensive binding to the apical side of tracheal tissues (which expresses ⁇ 2-6 sialylated glycans with long branch topology).
  • This correlation provides valuable insights into the upper respiratory tissue tropism of human- adapted Hl and H3 Has.
  • the H5 HA shows the opposite glycan binding trend, binding with high affinity to ⁇ 2-3 (saturating signals from 40 ⁇ g/ml down to 2.5 ⁇ g/ml) as compared with its relatively low affinity for ⁇ 2-6 sialylated glycans (significant signals seen only at 20-40 ⁇ g/ml).
  • a necessary condition for human adaptation of an HA polypeptide is to gain the ability to bind to long ⁇ 2-6 sialylated glycans (e.g., umbrella topology glycans), which are predominantly expressed in the human upper airway, with high affinity.
  • FIG. 21 Direct binding of SNA-I and competitive inhibition of HA binding by SNA- 1.
  • Top panel shows from top to bottom the binding signals (normalized to the saturation level of around 800000) respectively for wild type SNA-I at various concentrations.
  • the legend for the glycans is shown as an inset, where LN corresponds to GaIb 1-4GIcNAc and 3'SLN and 6'SLN, respectively, correspond to ⁇ 2-3 and ⁇ 2-6 linked sialic acid at the LN.
  • Bottom panel shows immunofluorescence microsopy analysis of competition assay with SNA-I and SC 18 HA polypeptides. Green channel (top left) shows SNA-I detection. Red channel (top right) shows SC 18 HA detection. The merged image is at the bottom.
  • HA Sequence Element 1 is a sequence element corresponding approximately to residues 97-185 (where residue positions are assigned using H3 HA as reference) of many HA proteins found in natural influenza isolates. This sequence element has the basic structure:
  • Xi is approximately 30-45 amino acids long; X 2 is approximately 5-20 amino acids long; X 3 is approximately 25-30 amino acids long; and X 4 is approximately 2 amino acids long.
  • Xi is about 35-45, or about 35-43, or about 35, 36, 37, 38, 38, 40, 41, 42, or 43 amino acids long.
  • X 2 is about 9-15, or about 9-14, or about 9, 10, 11, 12, 13, or 14 amino acids long.
  • X 3 is about 26-28, or about 26, 27, or 28 amino acids long.
  • X 4 has the sequence (G/A) QJV).
  • X 4 has the sequence GI; in some embodiments, X 4 has the sequence GV; in some embodiments, X 4 has the sequence AI; in some embodiments, X 4 has the sequence AV.
  • HA Sequence Element 1 comprises a disulfide bond. In some embodiments, this disulfide bond bridges residues corresponding to positions 97 and 139 (based on the canonical H3 numbering system utilized herein).
  • Xi is about 43 amino acids long, and/or X 2 is about 13 amino acids long, and/or X 3 is about 26 amino acids long.
  • HA Sequence Element 1 has the structure:
  • HA Sequence Element 1 has the structure:
  • XiA is approximately 27-42, or approximately 32-42, or approximately 32- 40, or approximately 32, 33, 34, 35, 36, 37, 38, 39, or 40 amino acids long,
  • X 3A is approximately 23-28, or approximately 24-26, or approximately 24, 25, or 26 amino acids long, and X 2 and X 4 are as above.
  • HA Sequence Element 1 includes the sequence:
  • Xi is about 39 amino acids long, and/or X 2 is about 13 amino acids long, and/or X 3 is about 26 amino acids long.
  • HA Sequence Element 1 has the structure:
  • XiA is approximately 27-42, or approximately 32-42, or approximately 32- 40, or approximately 23-38, or approximately 28-38, or approximately 28-36, or approximately 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 amino acids long, and X 2 -X 4 are as above.
  • HA Sequence Element 1 has the structure:
  • Xi A is approximately 27-42, or approximately 32-42, or approximately 32- 40, or approximately 32, 33, 34, 35, 36, 37, 38, 39, or 40 amino acids long,
  • X 3 A is approximately 23-28, or approximately 24-26, or approximately 24, 25, or 26 amino acids long, and X 2 and X 4 are as above.
  • HA Sequence Element 1 includes the sequence:
  • Xi is about 42 amino acids long, and/or X 2 is about 13 amino acids long, and/or X3 is about 26 amino acids long.
  • HA Sequence Element 1 has the structure:
  • XiA is approximately 27-42, or approximately 32-42, or approximately 32- 40, or approximately 23-38, or approximately 28-38, or approximately 28-36, or approximately 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 amino acids long, and X 2 -X 4 are as.
  • HA Sequence Element 1 has the structure:
  • XiA is approximately 27-42, or approximately 32-42, or approximately 32- 40, or approximately 32, 33, 34, 35, 36, 37, 38, 39, or 40 amino acids long, and
  • X 3 A is approximately 23-28, or approximately 24-26, or approximately 24, 25, or 26 amino acids long, and X 2 and X 4 are as above.
  • HA Sequence Element 1 is extended (i.e., at a position corresponding to residues 186-193) by the sequence: N D A A E X X (KfR)
  • HA Sequence Element 1 includes the sequence:
  • YEELKHLXSXXNHFEK typically within X 1 , and especially beginning about residue 6 of Xi (as illustrated, for example, in Figures 1, 2, and 5).
  • HA Sequence Element 2 is a sequence element corresponding approximately to residues 324-340 (again using a numbering system based on H3 HA) of many HA proteins found in natural influenza isolates. This sequence element has the basic structure:
  • HA Sequence Element 2 has the sequence: P XiG A I A G F I E, wherein:
  • Xi is approximately 4-14 amino acids long, or about 8-12 amino acids long, or about 12, 11, 10, 9 or 8 amino acids long.
  • this sequence element provides the HAO cleavage site, allowing production of HAl and HA2.
  • HA Sequence Element 2 has the structure:
  • XiA is approximately 3 amino acids long; in some embodiments, X 1 A is G (L/I) F.
  • HA Sequence Element 2 has the structure:
  • XiA is approximately 3 amino acids long; in some embodiments, X 1 A is G (L/I) F.
  • HA Sequence Element 2 has the structure:
  • XiA is approximately 3 amino acids long; in some embodiments, X 1 A is G (L/I) F.
  • 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.
  • binding typically refers to a non-covalent association between or among agents. In many embodiments herein, binding is addressed with respect to particular glycans (e.g., umbrella topology glycans or cone topology glycans). It will be appreciated by those of ordinary skill in the art that such binding may be assessed in any of a variety of contexts. In some embodiments, binding is assessed with respect to free glycans. In some embodiments, binding is assessed with respect to glycans attached (e.g., covalently linked to) a carrier. In some such embodiments, the carrier is a polypeptide.
  • binding is assessed with respect to glycans attached to an HA receptor. In such embodiments, reference may be made to receptor binding or to glycan binding.
  • binding agent In general, the term "binding agent" is used herein to refer to any entity that binds to glycans ⁇ e.g., to umbrella-topology glycans) as described herein. Binding agents may be of any chemical type.
  • binding agents are polypeptides (including, e.g., antibodies or antibody fragments); in some such embodiments, binding agents are HA polypeptides; in other embodiments, binding agents are polypeptides whose amino acid sequence does not include an HA characteristic sequence (i.e., "Non-HA polypeptides). In some embodiments, binding agents are small molecules. In some embodiments, binding agents are nucleic acids. In some embodiments, binding agents are aptamers. In some embodiments, binding agents are polymers; in some embodiments, binding agents are non-polymeric. In some embodiments, binding agents are carbohydrates. In some embodiments, binding agents are lectins.
  • binding agents as described herein bind to sialylated glycans having an umbrella-like topology. In certain embodiments, binding agents bind to umbrella- topology glycans with high affinity and/or specificity. In some embodiments, binding agents show a binding preference for umbrella-topology glycans as compared with cone-topology glycans. In some embodiments, binding agents compete with hemagglutinin for binding to glycans on hemagglutinin receptors. In some embodiments, binding agents compete with hemagglutinin for binding to umbrella-topology glycans. In some embodiments, a binding agent provided herein is an umbrella topology blocking agent. In some embodiments, a binding agent provided herein is an umbrella topology specific blocking agent. In some embodiments, binding agents bind to umbrella topology glycan mimics.
  • 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 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.
  • 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 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.
  • the term "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 1-5), 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.
  • Degree of separation removed As used herein, 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 1-3 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 1, 2, and 4 and include, for example, those described herein with regard to H3 -specific embodiments of HA Sequence Elements.
  • H 5 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 1, 2, and 5 and include, for example, those described herein with regard to H5 -specific embodiments of HA Sequence Elements.
  • 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.
  • NCBI National Center for Biotechnology Information
  • HA polypeptides generally, and/or of particular HA polypeptides (e.g., Hl, H2, H3, H4, H5, H6, H7, H8, H9, HlO, HI l, Hl 2, 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, Trpl53, Hisl83, and Leu/Ilel94.
  • 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.
  • Linkage Specific Blocking Agent refers to an agent which binds to an HA receptor having an ⁇ 2-6 sialylated glycan.
  • an LSBA selectively binds to an HA receptor having an ⁇ 2-6 sialylated glycan with at least about 40, 50, or 75% of the affinity of that for an HA receptor having an ⁇ 2-3 sialylated glycan.
  • an LSBA selectively binds to an HA receptor having an ⁇ 2-6 sialylated glycan with at least about 2, 4, 5, or 10 times greater affinity than that for an HA receptor having an ⁇ 2-3 sialylated glycan.
  • an LSBA has an affinity for an ⁇ 2-6 sialylated glycan that is at least 50, 100, 150, or 200 % of its affinity for an ⁇ 2-3 sialylated glycan.
  • an LSBA may compete with hemagglutinin for binding to an HA receptor.
  • an LSBA may selectively inhibit the binding of an influenza virus particle (e.g., human or avian influenza virus) to an HA receptor based on the linkage characteristics (e.g., ⁇ 2-6 sialylated glycan or ⁇ 2-3 sialylated glycan).
  • an LSBA is a polypeptide.
  • an LSBA polypeptide has an amino acid sequence that is substantially identical or substantially homologous to that of a naturally-occurring polypeptide.
  • an LSBA polypeptide is an HA polypeptide.
  • an LSBA polypeptide is a naturally-occurring HA polypeptide, or a fragment thereof.
  • an LSBA polypeptide has an amino acid sequence that is not related to that of an HA polypeptide.
  • an LSBA polypeptide is an antibody or fragment thereof.
  • an LSBA polypeptide is a lectin (e.g., SNA-I).
  • an LSBA is not a polypeptide.
  • an LSBA is a small molecule.
  • an LSBA is a nucleic acid. [0066] Long 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 refers to an entity o
  • 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 99% pure.
  • 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) from other potential binding partners (e.g., an avian HA receptor).
  • a particular ligand e.g., an HA polypeptide
  • 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
  • Substantial homology is used herein to refer to a comparison between amino acid or nucleic acid sequences. As will be appreciated by those of ordinaryy skill in the art, two sequences are generally considered to be “substantially homologous” if they contain homologous residues in corresponding positions. Homologous residues may be identical residues. Alternatively, homologous residues may be non-identical residues will appropriately similar structural and/or functional characteristics. For example, as is well known by those of ordinary skill in the art, certain amino acids are typically classified as
  • amino acid or nucleic acid sequences may be compared using any of a variety of algorithms, including those available in commercial computer programs such as BLASTN for nucleotide sequences and BLASTP, gapped BLAST, and PSI-BLAST for amino acid sequences.
  • Exemplary such programs are described in Altschul, et al., Basic local alignment search tool, J. MoI. Biol, 215(3): 403-410, 1990; Altschul, et al., Methods in Enzymology; Altschul, et al., "Gapped BLAST and PSI-BLAST: a new generation of protein database search programs", Nucleic Acids Res.
  • two sequences are considered to be substantially homologous if at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of their corresponding residues are homologous over a relevant stretch of residues.
  • the relevant stretch is a complete sequence.
  • the relevant stretch is at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500 or more residues.
  • Substantial identitiy is used herein to refer to a comparison between amino acid or nucleic acid sequences. As will be appreciated by those of ordinary skill in the art, two sequences are generally considered to be “substantially identical” if they contain identical residues in corresponding positions. As is well known in this art, amino acid or nucleic acid sequences may be compared using any of a variety of algorithms, including those available in commercial computer programs such as BLASTN for nucleotide sequences and BLASTP, gapped BLAST, and PSI-BLAST for amino acid sequences. Exemplary such programs are described in Altschul, et al., Basic local alignment search tool, J. MoI.
  • two sequences are considered to be substantially identical if at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of their corresponding residues are identical over a relevant stretch of residues.
  • the relevant stretch is a complete sequence.
  • the relevant stretch is at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500 or more residues.
  • 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.
  • umbrella topology is typically adopted only by ⁇ 2-6 sialylated glycans, and is typical of long (e.g., greater than tetrasaccharide) oligosaccharides.
  • umbrella-tocology glycans are glycans exhibiting a three-dimensional structure substantially similar to the structure presented in Figure 6 (right panel).
  • umbrella- topology glycans are glycans which contact HA polypeptides via the amino acid residues shown in Figure 6 (right panel).
  • umbrella-topology glycans are glycans which are able to contact and/or specifically bind to the amino acid binding pocket shown in Figure 6 (right panel).
  • glycan structural topology is classified based on parameter ⁇ defined as angle between C 2 of Sia, Ci of Gal, and Ci of GIcNAc.
  • Values of ⁇ ⁇ 100° represent cone-like topology adopted by ⁇ 2-3 and short ⁇ 2-6 glycans.
  • Values of ⁇ > 110° represent umbrella-like topology, such as topology adopted by long ⁇ 2-6 glycans ( Figure 6).
  • 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 can adopt an umbrella topology.
  • the long ⁇ 2-6 motifs presented in Figure 9 includes Neu5 Ac ⁇ 2-6 linked at the non-reducing end to a long chain (e.g. , at least a trisaccharide) found as a part of biological N-linked glycans, 0-linked glycans, and glyco lipids.
  • the boxed inset shows examples of the umbrella-topology long ⁇ 2-6 glycan moieties that are found as a part of biological glycans that bind to high affinity with HA.
  • umbrella-topology glycans (e.g., at a site) comprise a greater proportion of long (e.g.
  • umbrella-topology glycans comprise about 2- fold, about 3-fold, about 4-fold, about 5-fold, about 10-fold, about 20-fold, about 50-fold, or greater than about 50-fold more long ⁇ 2-6 oligosaccharide branches than short ⁇ 2-6 (e.g. single lactosamine) branches.
  • the unique characteristic of HA interactions with umbrella-topology glycans and/or glycan decoys is the HA contact with a glycan comprising sialic acid (SA) and/or SA analogs at the non-reducing end.
  • SA sialic acid
  • chain length of the oligosaccharide is at least a trisaccharide (excluding the SA or SA analog).
  • a combination of the numbered residues shown in the right-hand panel of Figure 6 is involved in contacts with umbrella-like topology.
  • umbrella topology glycans are oligosaccharides of the following form:
  • Neu5 Ac ⁇ 2-6 is typically (but not essentially) at the non-reducing end;
  • (i) is a hexose (frequently Gal or GIc) or hexosamine (GIcNAc or GaINAc) in ⁇ or ⁇ configuration (frequently ⁇ - for N- and O-linked extension and ⁇ - in the case of GalNAc ⁇ - that is O-linked to glycoprotein);
  • non-sugar moieties such as sulfate, phosphate, guanidium, amine, N- acetyl, etc. can be attached to non-reducing positions (typically 6 position) of Sugl (e.g., to improve contacts with HA);
  • hexose frequently Gal or GIc
  • GIcNAc hexosamine
  • GaINAc hexosamine
  • sugars such as Fuc
  • non-sugar moieties such as sulfate, phosphate, guanidium, amine, N-acetyl, etc.
  • sugars such as Fuc
  • non-sugar moieties such as sulfate, phosphate, guanidium, amine, N-acetyl, etc.
  • Linkage between any two sugars in the oligosaccharide apart from Neu5 Ac ⁇ 2-6 linkage can be 1-2, 1-3, 1-4, and/or 1-6 (typically 1-3 or 1-4); and/or
  • Umbrella topology blocking agent refers to an agent which binds to an HA receptor having an umbrella topology glycan.
  • a UTBA binds to an HA receptor having an umbrella topology glycan foundin human upper airways.
  • a UBTA can bind to either an umbrella topology glycan and/or to a cone topology glycan.
  • a UTBA selectively binds to an umbrella topology glycan with 50, 100, 150, or 200% of its affinity for a cone topology glycan.
  • a UTBA selectively binds to an umbrella topology glycan with 50-150% of its affinity for a cone topology glycan. In some embodiments, and in some embodiments a UTBA binds to an umbrella topology glycan with about the same affinity as for a cone topology glycan. For example, in some embodiments, a UTBA binds an umbrella topology glycan (e.g., 6'SLN-LN) with about 50-200%, 50-150%, or about the same affinity to which it binds a cone topology glycan (e.g., 3'SLN-LN).
  • an umbrella topology glycan e.g., 6'SLN-LN
  • a cone topology glycan e.g., 3'SLN-LN
  • a UTBA selectively inhibits the binding of an influenza virus particle (e.g., a human or avian influenza virus) to the HA receptor based on the glycan topology of the receptor (e.g., umbrella or cone).
  • a UTBA is a polypeptide.
  • a UTBA polypeptide has an amino acid sequence that is substantially identical or substantially homologous to to that of a naturally-occurring polypeptide.
  • a UTBA polypeptide is an HA polypeptide.
  • a UTBA polypeptide is a naturally- occurring HA polypeptide, or a fragment thereof.
  • a UTBA polypeptide has an amino acid sequence that is not related to that of an HA polypeptide.
  • a UTBA polypeptide is an antibody or fragment thereof.
  • a UTBA polypeptide is a lectin (e.g., SNA-I).
  • a UTBA is not a polypeptide.
  • a UTBA is a small molecule.
  • a UTBA is a nucleic acid.
  • Umbrella topology glycan mimic An "umbrella topology glycan mimic" is an agent, other than an umbrella topology glycan, that binds to binding agents as described herein.
  • umbrella topology glycan mimics are agents that bind to HA polypeptides.
  • umbrella topology glycan mimics are agents that interact with HA polypeptide residues selected from the group consisting of residues 136, 137, 145, 153, 155, 156, 159, 186, 187, 189, 190, 192, 193, 194, 196, 222, 225, 226, 228 and combinations thereof.
  • umbrella topology glycan mimics are agents that interact with HA polypeptide residues selected from the group consisting of residues. In some such embodiments, umbrella topology glycan mimics are agents that interact with HA polypeptide residues selected from the group consisting of residues 156, 159, 189, 192, 193, 196, and combinations thereof. In some such embodiments, umbrella topology glycan mimics are agents that interact with HA polypeptide residues selected from the group consisting of residues 186, 187, 189, 190, and combinations thereof.
  • umbrella topology glycan mimics are agents that interact with HA polypeptide residues selected from the group consisting of residues 137, 145, 190, 226, 228, and combinations thereof. In some such embodiments, umbrella topology glycan mimics are agents that interact with HA polypeptide residues selected from the group consisting of residues 190, 222, 225, 226, and combinations thereof. In some such embodiments, umbrella topology glycan mimics are agents that interact with HA polypeptide residues selected from the group consisting of residues 136, 153, 155, 194, and combinations thereof.
  • umbrella topology glycan mimics are agents that interact with HA polypeptide residues selected from the group consisting of residues 190 and 226. In some such embodiments, umbrella topology glycan mimics are agents that interact with HA polypeptide residues selected from the group consisting of residues 222, 225, and 226. In some such embodiments, umbrella topology glycan mimics are agents that interact with HA polypeptide residues selected from the group consisting of residues 190, 192, 193, and 225. In some such embodiments, umbrella topology glycan mimics are agents that interact with HA polypeptide residues selected from the group consisting of residues 186, 193, and 222. Note that amino acid positions stated above are based on H3 HA numbering. In certain embodiments, an HA topology glycan mimic is an agent that competes with umbrella topology glycans for interaction with an HA polypeptide.
  • Umbrella topology specific blocking agent As used herein, the term "umbrella topology specific blocking agent” refers to an agent which binds to an HA receptor having an umbrella topology glycan found in human upper airways.
  • a UTSBA selectively binds an umbrella topology glycan HA.
  • a UTSBA binds an umbrella topology glycan (e.g., 6'SLN-LN) with about at least 2, 4, 5, or 10 times greater affinity than it binds to a cone topology glycan (e.g., 3'SLN-LN).
  • the affinity of a UTSBA for an umbrella topology glycan is greater than 1 nM.
  • the affinity of a UTSBA for a cone topology glycan is less is at least within 2 to 3 orders of magnitude of the binding affinity of umbrella topology glycans to human adapted HAs such as SC 18, Mos99, Tx91, etc. and ⁇ 2-6 binding plant lectins such as SNA-I.
  • the binding affinity of UTSBA as measured by the dose-dependent direct binding assay would typically be at least 1 nM.
  • the affinity of a UTSBA for a cone topology glycan is at most 1 to 3 orders of magnitude less than the binding affinity of cone topology glycans to avian HAs such as Viet0405, AvI 8, etc.
  • a UTSBA selectively inhibits binding of an influenza virus particle (e.g., a human or avian influenza virus) to the HA receptor (e.g., an Hl, H2 or H3 or a human-adapted H5, H7 or H9) based on glycan topology (e.g., umbrella or cone).
  • a UTSBA is a polypeptide.
  • a UTSBA polypeptide has an amino acid sequence that is that is substantially identical or substantially homologous to that of a naturally-occurring polypeptide.
  • a UTSBA polypeptide is an HA polypeptide.
  • a UTSBA polypeptide is a naturally-occurring HA polypeptide, or a fragment thereof. In some embodiments, a UTSBA polypeptide has an amino acid sequence that is not related to that of an HA polypeptide. In some embodiments, a UTSBA polypeptide is an antibody or fragment thereof. In some embodiments, a UTSBA polypeptide is a lectin (e.g., SNA-I). In some embodiments, a UTSBA is not a polypeptide. In some embodiments, a UTSBA is a small molecule. In some embodiments, a UTSBA is a nucleic acid.
  • 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 polypeptide (e.g., HA polypeptide) of interest and a "parent" polypeptide to which its sequence is being compared.
  • a polypeptide of interest is considered to be a "variant" of a parent polypeptide if the 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. In some embodiments, a variant has 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 substituted residue as compared with a parent. Often, 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. Moreover, 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 polypeptide is one found in nature.
  • a parent HA polypeptide may be one found in a natural (e.g., wilde type) 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 generally refers to a normal form of a protein or nucleic acid, as is found in nature.
  • 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.
  • the present invention provides binding agents (e.g., HA polypeptides, LSBAs, UTBAs, UTSBAs, etc.) that bind to umbrella topology glycans.
  • binding agents e.g., HA polypeptides, LSBAs, UTBAs, UTSBAs, etc.
  • the present invention provides binding agents that bind to umbrella topology glycans found on HA receptors of a particular target species.
  • the present invention provides binding agents that bind to umbrella topology glycans found on human HA receptors, e.g., HA receptors found on human epithelial cells, and particularly binding agents that bind to umbrella topology glycans found on human HA receptors in the upper respiratory tract.
  • the present invention provides binding agents that bind to HA receptors found on cells in the human upper respiratory tract, and in particular provides binding agents that binds to such receptors (and/or to their glycans, particularly to their umbrella glycans) with a designated affinity and/or specificity.
  • the present invention encompasses the recognition that gaining an ability to bind umbrella topology glycans (e.g., long a2-6 sialylated glycans), and particularly an ability to bind with high affinity, may confer upon an HA polypeptide variant the ability to infect humans (where its parent HA polypeptide cannot).
  • umbrella topology glycans e.g., long a2-6 sialylated glycans
  • HA polypeptide e.g., long a2-6 sialylated glycans
  • the present inventors propose that binding to umbrella topology glycans may be paramount, and in particular that loss of binding to other glycan types may not be required.
  • the present invention further provides various reagents and methods associated with inventive binding agents (e.g., HA polypeptides, UTBAs, UTSBAs, etc.) including, for example, systems for identifying them, strategies for preparing them, antibodies that bind to them, and various diagnostic and therapeutic methods relating to them. Further description of certain embodiments of these aspects, and others, of the present invention, is presented below.
  • inventive binding agents e.g., HA polypeptides, UTBAs, UTSBAs, etc.
  • Influenza viruses are RNA viruses which are characterized by a lipid membrane envelope containing two glycoproteins, hemagglutinin (HA) and neuraminidase (NA), embedded in the membrane of the virus particular.
  • HA hemagglutinin
  • NA neuraminidase
  • HA exists in the membrane as a homotrimer of one of 16 subtypes, termed H1-H16. Only three of these subtypes (Hl, H2, and H3) have thus far become adapted for human infection.
  • HAs that have adapted to infect humans
  • HlNl (1918) and H3N2 (1967-68) influenza subtypes are their ability to preferentially bind to ⁇ 2-6 sialylated glycans in comparison with their avian progenitors that preferentially bind to ⁇ 2-3 sialylated glycans
  • Sialylated glycans Skehel & Wiley, Annu Rev Biochem, 69:531, 2000; Rogers, & Paulson, Virology, 127:361, 1983; Rogers et al, Nature, 304:76, 1983; Sauter et al, Biochemistry, 31 :9609, 1992; Connor et al., Virology, 205:17, 1994; Tumpey et al, Science, 310:77, 2005).
  • the present invention encompasses the recognition that ability to infect human hosts correlates less with binding to glycans of a particular linkage, and more with binding to glycans of a particular topology.
  • the present invention demonstrates that HAs that mediate infection of humans bind to umbrella topology glycans, often showing preference for umbrella topology glycans over cone topology glycans (even though cone- topology glycans may be ⁇ 2-6 sialylated glycans).
  • the crystal structures of H5 (A/duck/Singapore/3/97) alone or bound to an ⁇ 2-3 or an ⁇ 2-6 sialylated oligosaccharide identifies certain amino acids that interact directly with bound glycans, and also amino acids that are one or more degree of separation removed (Stevens et al, Proc Natl Acad Sci USA 98:11181, 2001).
  • conformation of these residues is different in bound versus unbound states.
  • Glul90, Lysl93 and Gln226 all participate in direct-binding interactions and have different conformations in the bound versus the unbound state.
  • the conformation of Asnl86, which is proximal to Glul90, is also significantly different in the bound versus the unbound state.
  • the present invention encompasses the finding that binding to umbrella topology glycans correlates with ability to mediate infection of particular hosts, including for example, humans.
  • the present invention provides binding agents (e.g., HA polypeptides, LSBAs, UTBAs, UTSBAs, etc.) that bind to umbrella glycans (and/or to umbrella topology glycan mimics).
  • inventive binding agents bind to umbrella glycans (and/or to umbrella topology glycan mimics) with high affinity.
  • inventive binding agents bind to a plurality of different umbrella topology glycans, often with high affinity and/or specificity.
  • inventive binding agents bind to umbrella topology glycans (e.g., long ⁇ 2-6 silaylated glycans such as, for example, Neu5Ac ⁇ 2-6Gal ⁇ l-4GlcNAc ⁇ l- 3Gal ⁇ l -4GIcNAc-) with high affinity.
  • inventive binding agents bind to umbrella topology glycans with an affinity comparable to that observed for a wild type HA that mediates infection of a humans (e.g., HlNl HA or H3N2 HA).
  • inventive binding agents bind to umbrella glycans with an affinity that is at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of that observed under comparable conditions for a wild type HA that mediates infection of humans. In some embodiments, inventive binding agents bind to umbrella glycans with an affinity that is greater than that observed under comparable conditions for a wild type HA that mediates infection of humans.
  • binding affinity of inventive binding agents is assessed over a range of concentrations. Such a strategy provides significantly more information, particularly in multivalent binding assays, than do single-concentration analyses. In some embodiments, for example, binding affinities of inventive binding agents are assessed over concentrations ranging over at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or more fold.
  • inventive binding agents show high affinity if they show a saturating signal in a multivalent glycan array binding assay such as those described herein. In some embodiments, inventive binding agents show high affinity if they show a signal above about 400000 or more (e.g., above about 500000, 600000, 700000, 800000, etc) in such studies. In some embodiments, binding agents as described herein show saturating binding to umbrella glycans over a concentration range of at least 2 fold, 3 fold, 4 fold, 5 fold or more, and in some embodiments over a concentration range as large as 10 fold or more.
  • inventive binding agents bind to umbrella topology glycans (and/or to umbrella topology glycan mimics) more strongly than they bind to cone topology glycans.
  • inventive binding agents show a relative affinity for umbrella glycans vs cone glycans that is about 10, 9, 8, 7, 6, 5, 4, 3, or 2.
  • inventive binding agents bind to ⁇ 2-6 sialylated glycans; in some embodiments, inventive binding agents bind preferentially to ⁇ 2-6 sialylated glycans. In certain embodiments, inventive binding agents bind to a plurality of different ⁇ 2-6 sialylated glycans. In some embodiments, inventive binding agents are not able to bind to ⁇ 2-3 sialylated glycans, and in other embodiments inventive binding agents are able to bind to ⁇ 2-3 sialylated glycans.
  • inventive binding agents bind to receptors found on human upper respiratory epithelial cells. In certain embodiments, inventive binding agents bind to HA receptors in the bronchus and/or trachea. In some embodiments, inventive binding agents are not able to bind receptors in the deep lung, and in other embodiments, inventive binding agents are able to bind receptors in the deep lung.
  • inventive binding agents bind to at least about 10%, 15%,
  • glycans found on HA receptors in human upper respiratory tract tissues e.g., epithelial cells.
  • inventive binding agents bind to one or more of the glycans illustrated in Figure 9. In some embodiments, inventive binding agents bind to multiple glycans illustrated in Figure 9. In some embodiments, inventive binding agents bind with high affinity and/or specificity to glycans illustrated in Figure 9. In some embodiments, inventive binding agents bind to glycans illustrated in Figure 9 preferentially as compared with their binding to glycans illustrated in Figure 8. In some embodiments, inventive binding agents bind to an oligosaccharide of the following form:
  • Neu5 Ac ⁇ 2-6 is always or almost always at the non-reducing end
  • Sugl is a hexose (frequently Gal or GIc) or hexosamine (GIcNAc or GaINAc) in ⁇ or ⁇ configuration (frequently ⁇ - for N- and O-linked extension and ⁇ - in the case of GalNAc ⁇ - that is O-linked to glycoprotein); b. no sugars other than Neu5Ac ⁇ 2-6 should be attached to any of the non-reducing positions of Sugl (except when Sugl is GalNAc ⁇ - that is O-linked to the glycoprotein); and/or c. non- sugar moieties such as sulfate, phosphate, guanidium, amine, N-acetyl, etc. can be attached to non-reducing positions (typically 6 position) of Sugl to improve contacts with HA;
  • Sug2 and/or Sug3 a. hexose (frequently Gal or GIc) or hexosamine (GIcNAc or GaINAc) in ⁇ or ⁇ configuration (frequently ⁇ ); and/or b. sugars (such as Fuc) or non-sugar moieties such as sulfate, phosphate, guanidium, amine, N-acetyl, etc. can be attached to non-reducing positions of Sug2, Sug3, and/or Sug4;
  • Linkage between any two sugars in the oligosaccharide apart from Neu5Ac ⁇ 2-6 linkage can be 1-2, 1-3, 1-4, and/or 1-6 (typically 1-3 or 1-4); and/or
  • Neu5Ac ⁇ 2-6 is linked GalNAc ⁇ that is O-linked to the glycoprotein and additional sugars are linked to the non-reducing end of GalNAc ⁇ for example i. Neu5Ac ⁇ 2-6(Neu5Ac ⁇ 2-3Gal ⁇ l-3)GalNAc ⁇ - ii. Neu5Ac ⁇ 2-6(Gal ⁇ l-3)GalNAc ⁇ -
  • the present invention provides binding agents with designated binding specificity, and also provides binding agents with designated binding characteristics with respect to umbrella glycans.
  • inventive binding agents are HA polypeptides.
  • the present invention provides isolated HA polypeptides with designated binding specificity, and also provides engineered HA polypeptides with designated binding characteristics with respect to umbrella glycans.
  • provided HA polypeptides with designated binding characteristics are Hl polypeptides.
  • inventive HA polypeptides with designated binding characteristics are H2 polypeptides.
  • inventive HA polypeptides with designated binding characteristics are H3 polypeptides.
  • inventive HA polypeptides with designated binding characteristics are H4 polypeptides.
  • inventive HA polypeptides with designated binding characteristics are H5 polypeptides.
  • inventive HA polypeptides with designated binding characteristics are H6 polypeptides.
  • inventive HA polypeptides with designated binding characteristics are H7 polypeptides.
  • inventive HA polypeptides with designated binding characteristics are H8 polypeptides. In some embodiments, inventive HA polypeptides with designated binding characteristics are H9 polypeptides. In some embodiments, inventive HA polypeptides with designated binding characteristics are HlO polypeptides. In some embodiments, inventive HA polypeptides with designated binding characteristics are HI l polypeptides. In some embodiments, inventive HA polypeptides with designated binding characteristics are H12 polypeptides. In some embodiments, inventive HA polypeptides with designated binding characteristics are Hl 3 polypeptides. In some embodiments, inventive HA polypeptides with designated binding characteristics are H14 polypeptides. In some embodiments, inventive HA polypeptides with designated binding characteristics are H15 polypeptides. In some embodiments, inventive HA polypeptides with designated binding characteristics are Hl 6 polypeptides.
  • inventive HA polypeptides with designated binding characteristics are not Hl polypeptides, are not H2 polypeptides, and/or are not H3 polypeptides.
  • inventive HA polypeptides do not include the Hl protein from any of the strains: A/South Carolina/1/1918; A/Puerto Rico/8/1934; A/Taiwan/1/1986; A/Texas/36/1991; A/Beijing/262/1995; A/Johannesburg/92/1996; A/New Caledonia/20/1999; A/Solomon Islands/3/2006.
  • inventive HA polypeptides are not the H2 protein from any of the strains of the Asian flu epidemic of 1957-58). In some embodiments, inventive HA polypeptides do not include the H2 protein from any of the strains: A/Japan/305+/1957; A/Singapore/1/1957; A/Taiwan/1/1964; A/Taiwan/1/1967.
  • inventive HA polypeptides do not include the H3 protein from any of the strains: A/Aichi/2/1968; A/Phillipines/2/1982; A/Mississippi/1/1985; A/Leningrad/360/1986; A/Sichuan/2/1987; A/Shanghai/I 1/1987; A/Beijing/353/1989; A/Shandong/9/1993; A/Johannesburg/33/1994; A/Nanchang/813/1995; A/Sydney/5/1997; A/Moscow/10/1999; A/Panama/2007/1999; A/Wyoming/3/2003; A/Oklahoma/323/2003; A/California/7/2004; A/Wisconsin/65/2005.
  • a provided HA polypeptide is a variant of a parent HA polypeptide in that its amino acid sequence is identical to that of the parent HA but for a small number of particular sequence alterations.
  • the parent HA is an HA polypeptide found in a natural isolate of an influenza virus (e.g., a wild type HA polypeptide).
  • inventive HA polypeptide variants have different glycan binding characteristics than their corresponding parent HA polypeptides.
  • inventive HA variant polypeptides have greater affinity and/or specificity for umbrella glycans (e.g., as compared with for cone glycans) than do their cognate parent HA polypeptides.
  • such HA polypeptide variants are engineered variants.
  • HA polypeptide variants with altered glycan binding characteristics have sequence alternations in residues within or affecting the glycan binding site.
  • such substitutions are of amino acids that interact directly with bound glycan; in other embodiments, such substitutions are of amino acids that are one degree of separation removed from those that interact with bound glycan, in that the one degree of separation removed-amino acids either (1) interact with the direct-binding amino acids; (2) otherwise affect the ability of the direct-binding amino acids to interact with glycan, but do not interact directly with glycan themselves; or (3) otherwise affect the ability of the direct-binding amino acids to interact with glycan, and also interact directly with glycan themselves.
  • inventive HA polypeptide variants contain substitutions of one or more direct-binding amino acids, one or more first degree of separation-amino acids, one or more second degree of separation-amino acids, or any combination of these. In some embodiments, inventive HA polypeptide variants may contain substitutions of one or more amino acids with even higher degrees of separation. [00112] In some embodiments, HA polypeptide variants with altered glycan binding characteristics have sequence alterations in residues that make contact with sugars beyond Neu5Ac and Gal (see, for example, Figure 7).
  • HA polypeptide variants have at least one amino acid substitution, as compared with a wild type parent HA. In certain embodiments, inventive HA polypeptide variants have at least two, three, four, five or more amino acid substitutions as compared with a cognate wild type parent HA; in some embodiments inventive HA polypeptide variants have two, three, or four amino acid substitutions. In some embodiments, all such amino acid substitutions are located within the glycan binding site.
  • HA polypeptide variants have sequence substitutions at positions corresponding to one or more of residues 137, 145, 156, 159, 186, 187, 189, 190, 192, 193, 196, 222, 225, 226, and 228.
  • HA polypeptide variants have sequence substitutions at positions corresponding to one or more of residues 156, 159, 189, 192, 193, and 196; and/or at positions corresponding to one or more of residues 186, 187, 189, and 190; and/or at positions corresponding to one or more of residues 190, 222, 225, and 226; and/or at positions corresponding to one or more of residues 137, 145, 190, 226 and 228. In some embodiments, HA polypeptide variants have sequence substitutions at positions corresponding to one or more of residues 190, 225, 226, and 228.
  • HA polypeptide variants and particularly H5 polypeptide variants, have one or more amino acid substitutions relative to a wild type parent HA (e.g., H5) at residues selected from the group consisting of residues 98, 136, 138, 153, 155, 159, 183, 186, 187, 190, 193, 194, 195, 222, 225, 226, 227, and 228.
  • a wild type parent HA e.g., H5
  • residues selected from the group consisting of residues 98, 136, 138, 153, 155, 159, 183, 186, 187, 190, 193, 194, 195, 222, 225, 226, 227, and 228.
  • HA polypeptide variants and particularly H5 polypeptide variants, have one or more amino acid substitutions relative to a wild type parent HA at residues selected from amino acids located in the region of the receptor that directly binds to the glycan, including but not limited to residues 98, 136, 153, 155, 183, 190, 193, 194, 222, 225, 226, 227, and 228.
  • an HA polypeptide variant and particularly an H5 polypeptide variant, has one or more amino acid substitutions relative to a wild type parent HA at residues selected from amino acids located adjacent to the region of the receptor that directly binds the glycan, including but not limited to residues 98, 138, 186, 187, 195, and 228.
  • an inventive HA polypeptide variant, and particularly an H5 polypeptide variant has one or more amino acid substitutions relative to a wild type parent HA at residues selected from the group consisting of residues 138, 186, 187, 190, 193, 222, 225, 226, 227 and 228.
  • an inventive HA polypeptide variant, and particularly an H5 polypeptide variant has one or more amino acid substitutions relative to a wild type parent HA at residues selected from amino acids located in the region of the receptor that directly binds to the glycan, including but not limited to residues 190, 193, 222, 225, 226, 227, and 228.
  • an inventive HA polypeptide variant, and particularly an H5 polypeptide variant has one or more amino acid substitutions relative to a wild type parent HA at residues selected from amino acids located adjacent to the region of the receptor that directly binds the glycan, including but not limited to residues 138, 186, 187, and 228.
  • an HA polypeptide variant, and particularly an H5 polypeptide variant has one or more amino acid substitutions relative to a wild type parent HA at residues selected from the group consisting of residues 98, 136, 153, 155, 183, 194, and 195.
  • an HA polypeptide variant, and particularly an H5 polypeptide variant has one or more amino acid substitutions relative to a wild type parent HA at residues selected from amino acids located in the region of the receptor that directly binds to the glycan, including but not limited to residues 98, 136, 153, 155, 183, and 194.
  • an inventive HA polypeptide variant, and particularly an H5 polypeptide variant has one or more amino acid substitutions relative to a wild type parent HA at residues selected from amino acids located adjacent to the region of the receptor that directly binds the glycan, including but not limited to residues 98 and 195.
  • an HA polypeptide variant, and particularly an H5 polypeptide variant has one or more amino acid substitutions relative to a wild type parent HA at residues selected from amino acids that are one degree of separation removed from those that interact with bound glycan, in that the one degree of separation removed-amino acids either (1) interact with the direct-binding amino acids; (2) otherwise affect the ability of the direct-binding amino acids to interact with glycan, but do not interact directly with glycan themselves; or (3) otherwise affect the ability of the direct-binding amino acids to interact with glycan, and also interact directly with glycan themselves, including but not limited to residues 98, 138, 186, 187, 195, and 228.
  • an HA polypeptide variant and particularly an H5 polypeptide variant, has one or more amino acid substitutions relative to a wild type parent HA at residues selected from amino acids that are one degree of separation removed from those that interact with bound glycan, in that the one degree of separation removed-amino acids either (1) interact with the direct-binding amino acids; (2) otherwise affect the ability of the direct-binding amino acids to interact with glycan, but do not interact directly with glycan themselves; or (3) otherwise affect the ability of the direct-binding amino acids to interact with glycan, and also interact directly with glycan themselves, including but not limited to residues 138, 186, 187, and 228.
  • an HA polypeptide variant, and particularly an H5 polypeptide variant has one or more amino acid substitutions relative to a wild type parent HA at residues selected from amino acids that are one degree of separation removed from those that interact with bound glycan, in that the one degree of separation removed-amino acids either (1) interact with the direct-binding amino acids; (2) otherwise affect the ability of the direct-binding amino acids to interact with glycan, but do not interact directly with glycan themselves; or (3) otherwise affect the ability of the direct-binding amino acids to interact with glycan, and also interact directly with glycan themselves, including but not limited to residues 98 and 195.
  • an HA polypeptide variant, and particularly an H5 polypeptide variant has an amino acid substitution relative to a wild type parent HA at residue 159.
  • an HA polypeptide variant, and particularly an H5 polypeptide variant has one or more amino acid substitutions relative to a wild type parent HA at residues selected from 190, 193, 225, and 226. In some embodiments, an HA polypeptide variant, and particularly an H5 polypeptide variant, has one or more amino acid substitutions relative to a wild type parent HA at residues selected from 190, 193, 226, and 228.
  • an inventive HA polypeptide variant, and particularly an H5 variant has one or more of the following amino acid substitutions: Serl37Ala, Lysl56Glu,
  • an inventive HA polypeptide variant, and particularly an H5 variant has one or more of the following sets of amino acid substitutions:
  • Lysl56Glu Alal89Lys, Lysl93Asn, Gln226Leu, Gly228Ser;
  • Lysl56Glu Alal89Lys, Lysl93Asn, Gly225Asp;
  • the HA polypeptide has at least one further substitution as compared with a wild type HA, such that affinity and/or specificity of the variant for umbrella glycans is increased.
  • inventive HA polypeptides (including HA polypeptide variants) have sequences that include D 190, D225, L226, and/or S228. In some embodiments, inventive HA polypeptides have sequences that include D 190 and D225; in some embodiments, inventive HA polypeptides have sequences that include L226 and S228. [00127] In some embodiments, inventive HA polypeptide variants have an open binding site as compared with a parent HA, and particularly with a parent wild type HAs.
  • the present invention further provides characteristic portions (which may or may not be binding agents) of inventive HA polypeptides and nucleic acids that encode them.
  • a characteristic portion is one that contains a continuous stretch of amino acids, or a collection of continuous stretches of amino acids, that together are characteristic of the HA polypeptide. Each such continuous stretch generally will contain at least two amino acids.
  • 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 H5 HA polypeptide.
  • a characteristic portion is one that, in addition to the sequence identity specified above, shares at least one functional characteristic with the relevant intact HA polypeptide.
  • inventive characteristic portions of HA polypeptides share glycan binding characteristics with the relevant full-length HA polypeptides.
  • binding agents provided in accordance with the present invention are polypeptides whose amino acid sequence does not include a characteristic HA sequence. Such polypeptides are referred to herein as "Non-HA polypeptides".
  • a Non-HA polypeptide has an amino acid sequence selected in advance ⁇ e.g., via rational design, including for example, introduction of strategic amino acid alterations [additions, deletions, and/or substitutions] as compared with a reference sequence).
  • a Non-HA polypeptide has an amino acid sequence that is determined stochastically and, for example, identified on the basis of the desirable binding characteristics defined herein.
  • binding agents provided in accordance with the present invention are antibodies (e.g., that bind to umbrella topology glycans and/or to umbrella topology glycan mimics).
  • Antibodies suitable for the invention include antibodies or fragments of antibodies that bind immunospecifically to any umbrella topology glycan epitope.
  • the term "antibodies" is intended to include immunoglobulins and fragments thereof which are specifically reactive to the designated protein or peptide, or fragments thereof.
  • Suitable antibodies include, but are not limited to, human antibodies, primatized antibodies, chimeric antibodies, bi-specif ⁇ c antibodies, humanized antibodies, conjugated antibodies (i.e., antibodies conjugated or fused to other proteins, radiolabels, cytotoxins), Small Modular ImmunoPharmaceuticals ("SMIPsTM ), single chain antibodies, cameloid antibodies, and antibody fragments.
  • SMIPsTM Small Modular ImmunoPharmaceuticals
  • single chain antibodies cameloid antibodies
  • antibody fragments also includes intact monoclonal antibodies, polyclonal antibodies, single domain antibodies (e.g., shark single domain antibodies (e.g., IgNAR or fragments thereof)), multispecif ⁇ c antibodies (e.g. bi-specif ⁇ c antibodies) formed from at least two intact antibodies, and antibody fragments so long as they exhibit the desired biological activity.
  • Antibody polyepeptides for use herein may be of any type (e.g., IgA, IgD, IgE, IgG, IgM).
  • an "antibody fragment” includes a portion of an intact antibody, such as, for example, the antigen-binding or variable region of an antibody.
  • antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; triabodies; tetrabodies; linear antibodies; single-chain antibody molecules; and multi specific antibodies formed from antibody fragments.
  • antibody fragment also includes any synthetic or genetically engineered protein that acts like an antibody by binding to a specific antigen to form a complex.
  • antibody fragments include isolated fragments, "Fv” fragments, consisting of the variable regions of the heavy and light chains, recombinant single chain polypeptide molecules in which light and heavy chain variable regions are connected by a peptide linker ("ScFv proteins”), and minimal recognition units consisting of the amino acid residues that mimic the hypervariable region.
  • Fv fragments
  • ScFv proteins peptide linker
  • Antibodies can be generated using methods well known in the art. For example, protocols for antibody production are described by Harlow and Lane, Antibodies: A Laboratory Manual, (1988). Typically, antibodies can be generated in mouse, rat, guinea pig, hamster, camel, llama, shark, or other appropriate host. Alternatively, antibodies may be made in chickens, producing IgY molecules (Schade et al, (1996) ALTEX 13(5):80-85). In some embodiments, antibodies suitable for the present invention are subhuman primate antibodies. For example, general techniques for raising therapeutically useful antibodies in baboons may be found, for example, in Goldenberg et al., international patent publication No.
  • monoclonal antibodies may be prepared using hybridoma methods (Milstein and Cuello, (1983) Nature 305(5934):537-40.). In some embodiments, monoclonal antibodies may also be made by recombinant methods (U.S. Pat. No. 4,166,452, 1979).
  • antibodies suitable for the invention may include humanized or human antibodies.
  • Humanized forms of non-human antibodies are chimeric Igs, Ig chains or fragments (such as Fv, Fab, Fab', F(ab')2 or other antigen-binding subsequences of Abs) that contain minimal sequence derived from non-human Ig.
  • a humanized antibody has one or more amino acid residues introduced from a non-human source. These non-human amino acid residues are often referred to as "import" residues, which are typically taken from an "import” variable domain.
  • Humanization is accomplished by substituting rodent complementarity determining regions (CDRs) or CDR sequences for the corresponding sequences of a human antibody (Riechmann et al, Nature 332(6162):323-7, 1988; Verhoeyen et al, Science. 239(4847): 1534-6, 1988.).
  • CDRs rodent complementarity determining regions
  • Such "humanized” antibodies are chimeric Abs (U.S. Pat. No. 4,816,567, 1989), wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species.
  • humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent Abs.
  • Humanized antibodies include human Igs (recipient antibody) in which residues from a CDR of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit, having the desired specificity, affinity and capacity. In some instances, corresponding non-human residues replace Fv framework residues of the human Ig. Humanized antibodies may comprise residues that are found neither in the recipient antibody nor in the imported CDR or framework sequences. In general, the humanized antibody comprises substantially all of at least one, and typically two, variable domains, in which most if not all of the CDR regions correspond to those of a non-human Ig and most if not all of the FR regions are those of a human Ig consensus sequence.
  • the humanized antibody optimally also comprises at least a portion of an Ig constant region (Fc), typically that of a human Ig (Riechmann et al, Nature 332(6162):323-7, 1988; Verhoeyen et al, Science. 239(4847): 1534-6, 1988.).
  • Fc Ig constant region
  • Human antibodies can also be produced using various techniques, including phage display libraries (Hoogenboom et al, Mol Immunol. (1991) 28(9):1027-37; Marks et al, J MoI Biol (1991) 222(3):581-97) and the preparation of human monoclonal antibodies (Reisfeld and Sell, 1985, Cancer Surv. 4(l):271-90). Similarly, introducing human Ig genes into transgenic animals in which the endogenous Ig genes have been partially or completely inactivated can be exploited to synthesize human antibodies.
  • binding agents provided in accordance with the present invention are lectins.
  • Lectins are sugar-binding proteins which may bind to a soluble carbohydrate or to a carbohydrate moiety which is a part of a glycoconjugate (e.g., a glycopeptide or glyco lipid).
  • Lectins typically agglutinate certain animal cells and/or precipitate glycoconjugates by recognizing a particular sugar moiety.
  • SNA-I is a lectin that has a high affinity for ⁇ 2-6 sialic acids.
  • polyporus squamosus lectins have high affinity for binding sialylated glycoconjugates containing Neu5Ac ⁇ 2,6Gal ⁇ l,4Glc/GlcNAc trisaccharide sequences of asparagine-linked glycoproteins.
  • Non- limiting exemplary lectins that may act as binding agents include SNA-I, SNA-I ', PSLIa, PSLIb, and polypeptides derived therefrom.
  • binding agents provided in accordance with the present invention are aptamers.
  • Aptamers are macromolecules composed of nucleic acid (e.g., RNA, DNA) that bind tightly to a specific molecular target (e.g., an umbrella topology glycan).
  • a particular aptamer may be described by a linear nucleotide sequence and is typically about 15-60 nucleotides in length. Without wishing to be bound by any theory, it is contemplated that the chain of nucleotides in an aptamer form intramolecular interactions that fold the molecule into a complex three-dimensional shape, and this three-dimensional shape allows the aptamer to bind tightly to the surface of its target molecule.
  • aptamers may be obtained for a wide array of molecular targets, including proteins and small molecules.
  • aptamers have very high affinities for their targets (e.g., affinities in the picomolar to low nanomolar range for proteins).
  • Aptamers are chemically stable and can be boiled or frozen without loss of activity. Because they are synthetic molecules, they are amenable to a variety of modifications, which can optimize their function for particular applications. For example, aptamers can be modified to dramatically reduce their sensitivity to degradation by enzymes in the blood for use in in vivo applications. In addition, aptamers can be modified to alter their biodistribution or plasma residence time.
  • aptamers that can bind umbrella topology glycans (and/or to umbrella topology glycan mimics) can be achieved through methods known in the art.
  • aptamers can be selected using the SELEX (Systematic Evolution of Ligands by Exponential Enrichment) method (Tuerk, C, and Gold, L., Science 249:505-510 (1990)).
  • SELEX Systematic Evolution of Ligands by Exponential Enrichment
  • a large library of nucleic acid molecules e.g., 10 15 different molecules
  • the target molecule e.g., an umbrella topology glycan of umbrella topology glycan epitope.
  • the target molecule is allowed to incubate with the library of nucleotide sequences for a period of time.
  • Several methods known in the art, can then be used to physically isolate the aptamer target molecules from the unbound molecules in the mixture, which can be discarded.
  • the aptamers with the highest affinity for the target molecule can then be purified away from the target molecule and amplified enzymatically to produce a new library of molecules that is substantially enriched for aptamers that can bind the target molecule.
  • the enriched library can then be used to initiate a new cycle of selection, partitioning, and amplification.
  • the library is reduced to a small number of aptamers that bind tightly to the target molecule.
  • Individual molecules in the mixture can then be isolated, their nucleotide sequences determined, and their properties with respect to binding affinity and specificity measured and compared. Isolated aptamers can then be further refined to eliminate any nucleotides that do not contribute to target binding and/or aptamer structure, thereby producing aptamers truncated to their core binding domain.
  • S ee Jayasena, S. D. Clin. Chem. 45:1628-1650 (1999) for review of aptamer technology; the entire teachings of which are incorporated herein by reference).
  • Inventive polypeptides ⁇ e.g., HA polypeptides and/or Non-HA polypeptides), and/or characteristic portions thereof, or nucleic acids encoding them, may be produced by any available means.
  • Inventive polypeptides may be produced, for example, by utilizing a host cell system engineered to express an inventive polypeptide-encoding nucleic acid.
  • polypeptides or characteristic portions
  • polypeptides or characteristic portions
  • baculovirus such as egg, baculovirus, plant, yeast, Madin-Darby Canine Kidney cells (MDCK), or Vera (African green monkey kidney) cells.
  • MDCK Madin-Darby Canine Kidney cells
  • Vera African green monkey kidney cells.
  • polypeptides (or characteristic portions) can be expressed in cells using recombinant techniques, such as through the use of an expression vector (Sambrook et ah, Molecular Cloning: A Laboratory Manual, CSHL Press,
  • inventive polypeptides can be produced by synthetic means.
  • inventive polypeptides may be produced in the context of intact virus, whether otherwise wild type, attenuated, killed, etc.
  • inventive polypeptides ⁇ e.g., HA polypeptides), or characteristic portions thereof, may also be produced in the context of virus like particles.
  • HA polypeptides can be isolated and/or purified from influenza virus.
  • virus may be grown in eggs, such as embryonated hen eggs, in which case the harvested material is typically allantoic fluid.
  • influenza virus may be derived from any method using tissue culture to grow the virus.
  • Suitable cell substrates for growing the virus include, for example, dog kidney cells such as MDCK or cells from a clone of MDCK, MDCK- like cells, monkey kidney cells such as AGMK cells including Vero cells, cultured epithelial cells as continuous cell lines, 293T cells, BK-21 cells, CV-I cells, or any other mammalian cell type suitable for the production of influenza virus for vaccine purposes, readily available from commercial sources (e.g., ATCC, Rockville, Md.). Suitable cell substrates also include human cells such as MRC-5 cells. Suitable cell substrates are not limited to cell lines; for example primary cells such as chicken embryo fibroblasts are also included.
  • polypeptides and particularly variant HA polypeptides as described herein, may be generated, identified, isolated, and/or produced by culturing cells or organisms that produce the polypeptide (whether alone or as part of a complex, including as part of a virus particle or virus), under conditions that allow ready screening and/or selection of polypeptides capable of binding to umbrella-topology glycans.
  • polypeptides e.g., HA variant polypeptides
  • a collection of polypeptides results from evolution in nature.
  • a collection of polypeptides results from engineering.
  • such a collection of polypeptides results from a combination of engineering and natural evolution.
  • HA interacts with the surface of cells by binding to a glycoprotein receptor. Binding of HA to HA receptors is predominantly mediated by N-linked glycans on the HA receptors. Specifically, 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.
  • Some crystal structures of exemplary HA-glycan interactions have been identified and are presented in Table 1 : Table 1. Crystal Structures ofHA-Glycan Complexes
  • APR34 H1 23 (IRVX) A/Puerto Rico/8/34 (HlNl) Neu5 Ac ⁇ 3 Gal ⁇ 4GlcNAc
  • APR34 H1 26 (IRVZ) A/Puerto Rico/8/34 (HlNl) Neu5Ac ⁇ 6Gal ⁇ 4GlcNAc
  • ADS97_H5_23 (USN) A/Duck/Singapore/3/97 (H5N3) Neu5 Ac ⁇ 3 Gal ⁇ 3 GIcNAc
  • HA - ⁇ 2-6 sialylated glycan complexes were generated by superimposition of the CA trace of the HAl subunit of ADU63 H3 and ADS97 H5 and VietO4_H5 on ASI30 H1 26 and APR34 H1 26 (Hl).
  • H3N2 human A/Aichi/2/68
  • ⁇ 2-6 sialylated glycans are published (Eisen et ah, 1997, Virology, 232:19), their coordinates were not available in the Protein Data Bank.
  • 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 can affect the conformation of the receptor's HA-binding site, thus affecting 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 (according to the present invention), are characterized by glycans, including ⁇ 2-3 sialylated and/or ⁇ 2-6 sialylated glycans, which predominantly adopt cone topologies.
  • HA receptors having such cone-topology glycans may be referred to herein as CTH Ars.
  • human HA receptors in the bronchus and trachea of the upper respiratory tract are modified by ⁇ 2-6 sialylated glycans.
  • the ⁇ 2-6 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
  • UTH Ars umbrella-topology glycans
  • humans are not usually infected by viruses containing many wild type avian HAs (e.g., avian H5).
  • viruses containing many wild type avian HAs e.g., avian H5
  • 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.
  • GBP glycan-glycan binding protein
  • CFG Functional Glycomics
  • each array comprises 264 glycans with low (10 ⁇ M) and high (100 ⁇ M) concentrations, and six spots for each concentration (see http://www.functionalglycomics.org/static/consortium/resources/resourcecoreh5.shtml).
  • the arrays predominantly comprise synthetic glycans that capture the physiological diversity of N- and 0-linked glycans.
  • N-linked glycan mixtures derived from different mammalian glycoproteins are also represented on the array.
  • a glycan "array” refers to a set of one or more glycans, optionally immobilized on a solid support.
  • an "array” is a collection of glycans present as an organized arrangement or pattern at two or more locations that are physically separated in space.
  • a glycan array will have at least 4, 8, 16, 24, 48, 96 or several hundred or thousand discrete locations.
  • inventive glycan arrays may have any of a variety of formats.
  • Various different array formats applicable to biomolecules are known in the art. For example, a huge number of protein and/or nucleic acid arrays are well known. Those of ordinary skill in the art will immediately appreciate standard array formats appropriate for glycan arrays of the present invention.
  • inventive glycan arrays are present in "microarray" formats.
  • a microarray may typically have sample locations separated by a distance of 50-200 microns or less and immobilized sample in the nano to micromolar range or nano to picogram range.
  • Array formats known in the art include, for example, those in which each discrete sample location has a scale of, for example, ten microns.
  • inventive glycan arrays comprise a plurality of glycans spatially immobilized on a support.
  • the present invention provides glycan molecules arrayed on a support.
  • support refers to any material which is suitable to be used to array glycan molecules.
  • any of a wide variety of materials may be employed.
  • support materials which may be of use in the invention include hydrophobic membranes, for example, nitrocellulose,
  • PVDF nylon membranes.
  • nylon membranes are well known in the art and can be obtained from, for example, Bio-Rad, Hemel Hempstead, UK.
  • the support on which glycans are arrayed may comprise a metal oxide.
  • Suitable metal oxides include, but are not limited to, titanium oxide, tantalum oxide, and aluminum oxide. Examples of such materials may be obtained from Sigma-Aldrich
  • such a support is or comprises a metal oxide gel.
  • a metal oxide gel is considered to provide a large surface area within a given macroscopic area to aid immobilization of the carbohydrate-containing molecules.
  • Additional or alternative support materials which may be used in accordance with the present invention include gels, for example silica gels or aluminum oxide gels. Examples of such materials may be obtained from, for example, Merck KGaA, Darmstadt, Germany.
  • glycan arrays are immobilized on a support that can resist change in size or shape during normal use.
  • a support may be a glass slide coated with a component material suitable to be used to array glycans.
  • some composite materials can desirable provide solidity to a support.
  • inventive arrays are useful for the identification and/or characterization of different HA polypeptides and their binding characteristics.
  • inventive HA polypeptides are tested on such arrays to assess their ability to bind to umbrella topology glycans (e.g., to ⁇ 2-6 sialylated glycans, and particularly to long ⁇ 2-6 sialylated glycans arranged in an umbrella topology).
  • the present invention provides arrays of ⁇ 2-6 sialylated glycans, and optionally ⁇ 2-3 sialylated glycans, that can be used to characterize HA polypeptide binding capabilities and/or as a diagnostic to detect, for example, human-binding HA polypeptides.
  • inventive arrays contain glycans (e.g., ⁇ 2-6 sialylated glycans, and particularly long ⁇ 2-6 sialylated glycans) in an umbrella topology.
  • glycans e.g., ⁇ 2-6 sialylated glycans, and particularly long ⁇ 2-6 sialylated glycans
  • such arrays are useful for characterizing or detecting any HA polypeptides, including for example, those found in natural influenza isolates in addition to those designed and/or prepared by researchers.
  • such arrays include glycans representative of about 10%, 15%, 20%, 25%, 30% 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% 95%, or more of the glycans (e.g., the umbrella glycans, which will often be ⁇ 2-6 sialylated glycans, particularly long ⁇ 2-6 sialylated glycans) found on human HA receptors, and particularly on human upper respiratory tract HA receptors.
  • the umbrella glycans e.g., the umbrella glycans, which will often be ⁇ 2-6 sialylated glycans, particularly long ⁇ 2-6 sialylated glycans
  • inventive arrays include some or all of the glycan structures depicted in Figure 10 In some embodiments, arrays include at least about 10%, 15%, 20%, 25%, 30% 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% 95%, or more of these depicted glycans.
  • the present invention provides methods for identifying or characterizing HA proteins using glycan arrays.
  • such methods comprise steps of (1) providing a sample containing HA polypeptide, (2) contacting the sample with a glycan array comprising, and (3) detecting binding of HA polypeptide to one or more glycans on the array.
  • Suitable sources for samples containing HA polypeptides to be contacted with glycan arrays according to the present invention include, but are not limited to, pathological samples, such as blood, serum/plasma, peripheral blood mononuclear cells/peripheral blood lymphocytes (PBMC/PBL), sputum, urine, feces, throat swabs, dermal lesion swabs, cerebrospinal fluids, cervical smears, pus samples, food matrices, and tissues from various parts of the body such as brain, spleen, and liver.
  • PBMC/PBL peripheral blood mononuclear cells/peripheral blood lymphocytes
  • sputum sputum
  • urine feces
  • throat swabs sputum
  • dermal lesion swabs cerebrospinal fluids
  • cervical smears cerebrospinal fluids
  • pus samples food matrices
  • food matrices and tissues from various parts of the body
  • HA polypeptides can be detectably labeled (directly or indirectly) prior to or after being contacted with the array; binding can then be detected by detection of localized label.
  • scanning devices can be utilized to examine particular locations on an array.
  • binding to arrayed glycans can be measured using, for example, calorimetric, fluorescence, or radioactive detection systems, or other labeling methods, or other methods that do not require labeling.
  • fluorescent detection typically involves directly probing the array with a fluorescent molecule and monitoring fluorescent signals.
  • arrays can be probed with a molecule that is tagged (for example, with biotin) for indirect fluorescence detection (in this case, by testing for binding of fluorescently-labeled streptavidin).
  • fluorescence quenching methods can be utilized in which the arrayed glycans are fluorescently labeled and probed with a test molecule (which may or may not be labeled with a different fluorophore).
  • binding to the array acts to squelch the fluorescence emitted from the arrayed glycan, therefore binding is detected by loss of fluorescent emission.
  • arrayed glycans can be probed with a live tissue sample that has been grown in the presence of a radioactive substance, yielding a radioactively labeled probe. Binding in such embodiments can be detected by measuring radioactive emission.
  • Such methods are useful to determine the fact of binding and/or the extent of binding by HA polypeptides to inventive glycan arrays. In some embodiments of the invention, such methods can further be used to identify and/or characterize agents that interfere with or otherwise alter glycan-HA polypeptide interactions.
  • Methods described below may be of particular use in, for example, identifying whether a molecule thought to be capable of interacting with a carbohydrate can actually do so, or to identify whether a molecule unexpectedly has the capability of interacting with a carbohydrate.
  • inventive arrays for example, to detect a particular agent in a test sample.
  • methods may comprise steps of (1) contacting a glycan array with a test sample (e.g., with a sample thought to contain an HA polypeptide); and, (2) detecting the binding of any agent in the test sample to the array.
  • binding to inventive arrays may be utilized, for example, to determine kinetics of interaction between binding agent and glycan.
  • inventive methods for determining interaction kinetics may include steps of (1) contacting a glycan array with the molecule being tested; and, (2) measuring kinetics of interaction between the binding agent and arrayed glycan(s).
  • the kinetics of interaction of a binding agent with any of the glycans in an inventive array can be measured by real time changes in, for example, colorimetric or fluorescent signals, as detailed above. Such methods may be of particular use in, for example, determining whether a particular binding agent is able to interact with a specific carbohydrate with a higher degree of binding than does a different binding agent interacting with the same carbohydrate.
  • inventive HA polypeptides can be evaluated on glycan samples or sources not present in an array format per se.
  • inventive HA polypeptides can be bound to tissue samples and/or cell lines to assess their glycan binding characteristics.
  • Appropriate cell lines include, for example, any of a variety of mammalian cell lines, particularly those expressing HA receptors containing umbrella topology glycans (e.g., at least some of which may be ⁇ 2-6 sialylated glycans, and particularly long ⁇ 2-6 sialylated glycans).
  • utilized cell lines express individual glycans with umbrella topology.
  • utilized cell lines express a diversity of glycans.
  • cell lines are obtained from clinical isolates; in some they are maintained or manipulated to have a desired glycan distribution and/or prevalence.
  • tissue samples and/or cell lines express glycans characteristic of mammalian upper respiratory epithelial cells.
  • HA polypeptides can be identified and/or characterized by mining data from glycan binding studies, structural information (e.g., HA crystal structures), and/or protein structure prediction programs.
  • structural information e.g., HA crystal structures
  • protein structure prediction programs e.g., protein structure prediction programs.
  • FIG 11. The main steps involved in the particular data mining process utilized by the present inventors (and exemplified herein) are illustrated in Figure 11. These steps involved operations on three elements: data objects, features, and classifiers.
  • Data objects were the raw data that were stored in a database.
  • glycan array data the chemical description of glycan structures in terms of monosaccharides and linkages and their binding signals with different GBPs screened constituted the data objects.
  • Properties of the data objects were "features.” Rules or patterns obtained based on the features were chosen to describe a data object. “Classifiers” were the rules or patterns that were used to either cluster data objects into specific classes or determine relationships between or among features. The classifiers provided specific features that were satisfied by the glycans that bind with high affinity to a GBP. These rules were 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 utilized herein comprised software modules that interact with each other ( Figure 11) to perform the operations described above.
  • the feature extractor interfaces to the CFG database to extract features, and the object-based relational database used by CFG facilitates the flexible definition of features.
  • Table 5 Features extracted from the glycans on the glycan array.
  • Pairs Pair refers to a pair of monosaccharide, connected covalently by a linkage.
  • the pairs are classified into two categories, regular [B] and terminal [T] 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 Clusters In the case of surface triplets and quadruplets above, if the linkage information is ignored, we get a set of monosaccharide clusters, and their frequency of occurrence (composition) is tabulated. These features were chosen to analyze the importance of types of linkages between the monosaccharides.
  • 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.
  • a threshold that distinguished low affinity and high affinity binding was defined for each of the glycan array screening data sets.
  • the present invention provides nucleic acids which encode an
  • the invention provides nucleic acids which are complementary to nucleic acids which encode an HA polypeptide or a characteristic or biologically active portion of an HA polypeptide.
  • the invention provides nucleic acid molecules which hybridize to nucleic acids encoding an HA polypeptide or a characteristic or biologically active portion of an HA polypeptide.
  • nucleic acids can be used, for example, as primers or as probes. To give but a few examples, such nucleic acids can be used as primers in polymerase chain reaction
  • PCR as probes for hybridization (including in situ hybridization), and/or as primers for reverse transcription-PCR (RT-PCR).
  • nucleic acids can be DNA or RNA, and can be single stranded or double-stranded.
  • inventive nucleic acids may include one or more non-natural nucleotides; in other embodiments, inventive nucleic acids include only natural nucleotides.
  • inventive binding agent polypeptides e.g., HA polypeptides
  • HA polypeptides may be monoclonal or polyclonal and may be prepared by any of a variety of techniques known to those of ordinary skill in the art (e.g., see Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988).
  • antibodies can be produced by cell culture techniques, including the generation of monoclonal antibodies, or via transfection of antibody genes into suitable bacterial or mammalian cell hosts, in order to allow for the production of recombinant antibodies.
  • an "animal host” includes any animal model suitable for influenza research.
  • animal hosts suitable for the invention can be any mammalian hosts, including primates, ferrets, cats, dogs, cows, horses, rodents such as, mice, hamsters, rabbits, and rats.
  • an animal host used for the invention is a ferret.
  • an animal host is na ⁇ ve to viral exposure or infection prior to administration of an inventive binding agent (optionally in an inventive composition).
  • the animal host is inoculated with, infected with, or otherwise exposed to virus prior to or concurrent with administration of an inventive binding agent.
  • An animal host used in the practice of the present invention can be innoculated with, infected with, or otherwise exposed to virus by any method known in the art.
  • an animal host may be innoculated with, infected with, or exposed to virus intranasally.
  • a suitable animal host may have a similar distribution of umbrella vs. cone topology glycans and/or ⁇ 2-6 glycans vs. ⁇ 2-3 glycans to the distribution found in the human respiratory tract.
  • a ferret as an animal host may be more representative than a mouse when used as model of disease caused by influenza viruses in humans (Tumpey, et al. Science (2007) 315; 655-659).
  • the present invention encompasses the idea that ferrets may have a more similar distributution of glycans in the respiratory tract to those in the human respiratory tract than mouse does to human.
  • Na ⁇ ve and/or innoculated animals may be used for any of a variety of studies.
  • animal models may be used for virus transmission studies as in known in the art. It is comtemplated that the use of ferrets in virus transmission studies may serve as a reliable predictor for virus transmission in humans.
  • air transmission of viral influenza from innoculated animals (e.g., ferrets) to na ⁇ ve animals is known in the art (Tumpey, et al. Science (2007) 315; 655-659).
  • Virus transmission studies may be used to test inventive binding agent poylpeptides (e.g., HA polypeptides).
  • inventive binding agents may be administered to a suitable animal host before, during or after virus transmission studies in order to determine the efficacy of said binding agent in blocking virus binding and/or infectivity in the animal host.
  • virus transmission studies in order to determine the efficacy of said binding agent in blocking virus binding and/or infectivity in the animal host.
  • the present invention provides for pharmaceutical compositions including inventive binding agents (e.g., HA polypeptides, LSBAs, UTBAs, UTBSAs, etc.) and/or related entities.
  • inventive binding agents e.g., HA polypeptides, LSBAs, UTBAs, UTBSAs, etc.
  • binding agent polypeptide(s) e.g., HA polypeptides
  • nucleic acids encoding such polypeptides e.g., HA polypeptides
  • characteristic or biologically active fragments of such polypeptides or nucleic acids e.g., antibodies that bind to and/or compete with such polypeptides or fragments
  • small molecules that interact with or compete with such polypeptides or with glycans that bind to them, etc.
  • inventive pharmaceutical compositions are administered to a subject suffering from or susceptible to an influenza infection.
  • a subject is considered to be suffering from an influenza infection in the subject is displaying one or more symptoms commonly associated with influenza infection.
  • the subject is known or believed to have been exposed to the influenza virus.
  • a subject is considered to be susceptible to an influenza infection if the subject is known or believed to have been exposed to the influenza virus.
  • a subject is known or believed to have been exposed to the influenza virus if the subject has been in contact with other individuals known or suspected to have been infected with the influenza virus and/or if the subject is or has been present in a location in which influzena infection is known or thought to be prevalent.
  • subjects suffering from or susceptible to influenza infection are tested for antibodies to inventive binding agents prior to, during, or after administration of inventive pharmaceutical compositions.
  • subjects having such antibodies are not administered pharmaceutical compositions comprising inventive binding agents.
  • an appropriate dose of pharmaceutical composition and/or binding agent is selected based on detection (or lack thereof) of such antibodies.
  • selection of a particular subject for treatment, particular binding agent or composition for administration, and/or particular dose or regimen for administration is memorialized, for example in a written, printed, or electronic storage form.
  • Inventive compositions may be admininstered prior to or after development of one or more symptoms of influenza infection.
  • the invention encompasses treatment of influenza infections by administration of compounds described herein.
  • treatment of influenza infections according to the present invention is accomplished by administration of a vaccine.
  • inventive binding agents e.g., HA polypeptides, LSBAs, UTBAs, UTBSAs, etc.
  • binding agents that bind to umbrella glycans (e.g., ⁇ 2-6 linked umbrella glycans such as, for example, long ⁇ 2-6 sialylated glycans).
  • inventive vaccines are formulated utilizing one or more strategies (see, for example, Enserink, Science, 309:996, 2005) intended to allow use of lower dose of H5 HA protein, and/or to achieve higher immunogenicity.
  • multivalency is improved (e.g., via use of dendrimers); in some embodiments, one or more adjuvants is utilized, etc.
  • vaccines are compositions comprising one or more of the following: (1) inactivated virus, (2) live attenuated influenza virus, for example, replication-defective virus, (3) inventive binding agent (e.g., HA polypeptides, LSBAs, UTBAs, UTBSAs, etc.)), (4) nucleic acid encoding binding agent polypeptide (e.g., HA polypeptide) or characteristic or biologically active portion thereof, (5) DNA vector that encodes inventive binding agent polypeptide (e.g., HA polypeptide) or characteristic or biologically active portion thereof , and/or (6) expression system, for example, cells expressing one or more influenza proteins to be used as antigens.
  • inventive binding agent e.g., HA polypeptides, LSBAs, UTBAs, UTBSAs, etc.
  • inventive binding agent polypeptide e.g., HA polypeptides, LSBAs, UTBAs, UTBSAs, etc.
  • inactivated flu vaccines comprise one of three types of antigen preparation: inactivated whole virus, sub-virions where purified virus particles are disrupted with detergents or other reagents to solubilize the lipid envelope ("split" vaccine) or purified HA polypeptide ("subunit” vaccine).
  • virus can be inactivated by treatment with formaldehyde, beta-propiolactone, ether, ether with detergent (such as Tween-80), cetyl trimethyl ammonium bromide (CTAB) and Triton NlOl, sodium deoxycholate and tri(n-butyl) phosphate.
  • Inactivation can occur after or prior to clarification of allantoic fluid (from virus produced in eggs); the virions are isolated and purified by centrifugation (Nicholson et al., eds., Textbook of Influenza, Blackwell Science, Maiden, MA, 1998).
  • SRD single radial immunodiffusion
  • the present invention also provides live, attenuated flu vaccines, and methods for attenuation are well known in the art. In certain embodiments, attenuation is achieved through the use of reverse genetics, such as site-directed mutagenesis.
  • influenza virus for use in vaccines is grown in eggs, for example, in embryonated hen eggs, in which case the harvested material is allantoic fluid.
  • influenza virus may be derived from any method using tissue culture to grow the virus.
  • Suitable cell substrates for growing the virus include, for example, dog kidney cells such as MDCK or cells from a clone of MDCK, MDCK- like cells, monkey kidney cells such as AGMK cells including Vero cells, cultured epithelial cells as continuous cell lines, 293T cells, BK-21 cells, CV-I cells, or any other mammalian cell type suitable for the production of influenza virus (including upper airway epithelial cells) for vaccine purposes, readily available from commercial sources (e.g., ATCC, Rockville, Md.).
  • Suitable cell substrates also include human cells such as MRC-5 cells. Suitable cell substrates are not limited to cell lines; for example primary cells such as chicken embryo fibroblasts are also included.
  • inventive vaccines further comprise one or more adjuvants.
  • adjuvants for example, aluminum salts (Baylor et al., Vaccine, 20:S18, 2002) and monophosphoryl lipid A (MPL; Ribi et al., (1986, Immunology and Immunopharmaco logy of bacterial endotoxins, Plenum Publ. Corp., NY, p407, 1986) can be used as adjuvants in human vaccines.
  • new compounds are currently being tested as adjuvants in human vaccines, such as MF59 (Chiron Corp., http://www.chiron.com/investors/pressreleases/2005/051028.html), CPG 7909 (Cooper et al., Vaccine, 22:3136, 2004), and saponins, such as QS21 (Grochikyan et al., Vaccine, 24:2275, 2006).
  • vacunasin vaccines such as poly[di(carboxylatophenoxy)phosphazene] (PCCP; Payne et al., Vaccine, 16:92, 1998), interferon- ⁇ (Cao et al., Vaccine, 10:238, 1992), block copolymer P 1205 (CRL1005; Katz et al, Vaccine,. 18:2177, 2000), interleukin-2 (IL-2; Mbwuike et al, Vaccine, 8:347, 1990), and polymethyl methacrylate (PMMA; Kreuter et al, J. Pharm. ScL, 70:367, 1981).
  • PCCP poly[di(carboxylatophenoxy)phosphazene]
  • interferon- ⁇ Cao et al., Vaccine, 10:238, 1992
  • block copolymer P 1205 CRL1005; Katz et al, Vaccine,. 18:2177, 2000
  • inventive compositions do not include adjuvants (e.g., provided compositions are essentially free of adjugants).
  • inventive compositions do not include an alum adjuvant (e.g., provided compositions are essentially free of alum).
  • the present invention provides other therapeutic compositions useful in the treatment of viral infections.
  • treatment is accomplished by administration of an agent that interferes with expression or activity of an HA polypeptide.
  • the present invention provides pharmaceutical compositions comprising antibodies or other agents related to provided polypeptides.
  • compositions containing antibodies recognize virus particles containing a particular HA polypeptide (e.g., an HA polypeptide that binds to umbrella glycans), nucleic acids (such as nucleic acid sequences complementary to HA sequences, which can be used for RNAi), glycans that compete for binding to HA receptors, small molecules or glycomometics that compete the glycan-HA polypeptide interaction, or any combination thereof.
  • collections of different agents, having diverse structures are utilized.
  • therapeutic compositions comprise one or more multivalent agents.
  • treatment comprises urgent administration shortly after exposure or suspicion of exposure.
  • a pharmaceutical composition will include a therapeutic agent in addition to one or more inactive agents such as a sterile, biocompatible carrier including, but not limited to, sterile water, saline, buffered saline, or dextrose solution.
  • a sterile, biocompatible carrier including, but not limited to, sterile water, saline, buffered saline, or dextrose solution.
  • the composition can contain any of a variety of additives, such as stabilizers, buffers, excipients (e.g., sugars, amino acids, etc), or preservatives.
  • an inventive pharmaceutical composition contains a binding agent (e.g., an HA polypeptide, LSBA, UTBA, UTSBA, etc.) that binds to umbrella topology glycans (and/or to umbrella topology glycan mimics).
  • a binding agent e.g., an HA polypeptide, LSBA, UTBA, UTSBA, etc.
  • the inventive composition is substantially free of related agents (e.g., of other HA polypeptides, etc.) that do not bind to umbrella-topology glycans.
  • the inventive pharmaceutical compositions contains not more than 50%, 40%, 30%, 20%, 10%, 5%, or 1% of an agent that binds to HA receptor glycans other than umbrella topology glycans.
  • a pharmaceutical composition will include a therapeutic agent that is encapsulated, trapped, or bound within a lipid vesicle, a bioavailable and/or biocompatible and/or biodegradable matrix, or other microparticle.
  • a provided pharmaceutical composition will include a binding agent (e.g., an HA polypeptide, LSBA, UTBA, UTSBA, etc.) that is not aggregated.
  • a binding agent e.g., an HA polypeptide, LSBA, UTBA, UTSBA, etc.
  • a provided pharmaceutical composition will include a binding agent (e.g., an HA polypeptide, LSBA, UTBA, UTSBA, etc.) that is not denatured.
  • a binding agent e.g., an HA polypeptide, LSBA, UTBA, UTSBA, etc.
  • a binding agent e.g., an HA polypeptide, LSBA, UTBA, UTSBA, etc.
  • a binding agent e.g., an HA polypeptide, LSBA, UTBA, UTSBA, etc.
  • a provided pharmaceutical composition will include a binding agent (e.g., an HA polypeptide, LSBA, UTBA, UTSBA, etc.) that is not inactive.
  • a binding agent e.g., an HA polypeptide, LSBA, UTBA, UTSBA, etc.
  • less than 1%, 2%, 5%, 10%, 20%, or 30%, by dry weight or number, of the UTSBA administered is inactive.
  • inventive pharmaceutical compositions are formulated to reduce immunogenicity of provided binding agents.
  • a provided binding agent is associated with (e.g., bound to) an agent, such as polyethylene glycol and/or carboxymethyl cellulose, that masks its immunogenicity.
  • a provided binding agent has additional glycosylation that reduces immunogenicity.
  • compositions of the present invention may be administered either alone or in combination with one or more other therapeutic agents including, but not limited to, vaccines and/or antibodies.
  • “in combination with” it is not intended to imply that the agents must be administered at the same time or formulated for delivery together, although these methods of delivery are within the scope of the invention.
  • each agent will be administered at a dose and on a time schedule determined for that agent.
  • the invention encompasses the delivery of the inventive pharmaceutical compositions in combination with agents that may improve their bioavailability, reduce or modify their metabolism, inhibit their excretion, or modify their distribution within the body.
  • the pharmaceutical compositions of the present invention can be used for treatment of any subject
  • inventive pharmaceutical compositions and/or binding agents are administered in combination with one or more of an anti-viral agent (e.g., Oseltamivir [tamiflu],
  • Zanamavir [Releza], etc.) and/or a sialydase are examples of Zanamavir [Releza], etc. and/or a sialydase.
  • compositions of the present invention can be administered by a variety of routes, including oral, intravenous, intramuscular, intra-arterial, subcutaneous, intraventricular, transdermal, interdermal, rectal, intravaginal, intraperitoneal, topical (as by powders, ointments, creams, or drops), mucosal, nasal, buccal, enteral, sublingual; by intratracheal instillation, bronchial instillation, and/or inhalation; and/or as an oral spray, nasal spray, and/or aerosol.
  • routes including oral, intravenous, intramuscular, intra-arterial, subcutaneous, intraventricular, transdermal, interdermal, rectal, intravaginal, intraperitoneal, topical (as by powders, ointments, creams, or drops), mucosal, nasal, buccal, enteral, sublingual; by intratracheal instillation, bronchial instillation, and/or inhalation; and/or as an oral spray,
  • oral or nasal spray or aerosol route (e.g., by inhalation) is most commonly used to deliver therapeutic agents directly to the lungs and respiratory system.
  • the invention encompasses the delivery of the inventive pharmaceutical composition by any appropriate route taking into consideration likely advances in the sciences of drug delivery.
  • preparations for inhaled or aerosol delivery comprise a plurality of particles. In some embodiments, such preparations have a mean particle size of 4, 5,
  • preparations for inhaled or aerosol delivery are formulated as a dry powder.
  • preparations for inhaled or aerosol delivery are formulated as a wet powder, for example through inclusion of a wetting agent, in some embodiments, the wetting agent is selected from the group consisting of water, saline, or other liquid of physiological pH.
  • inventive compositions are administered as drops to the nasal or buccal cavity.
  • a dose may comprise a plurality of drops (e.g., 1-100, 1-
  • inventive compositions are administered using a device that delivers a metered dosage of composition (e.g., of binding agent).
  • Suitable devices for use in delivering intradermal pharmaceutical compositions described herein include short needle devices such as those described in U.S. Pat. No. 4,886,499,
  • Intradermal compositions may also be administered by devices which limit the effective penetration length of a needle into the skin, such as those described in WO99/34850, incorporated herein by reference, and functional equivalents thereof.
  • jet injection devices which deliver liquid vaccines to the dermis via a liquid jet injector or via a needle which pierces the stratum corneum and produces a jet which reaches the dermis. Jet injection devices are described for example in U.S. Pat. No. 5,480,381, U.S. Pat. No. 5,599,302, U.S. Pat. No. 5,334,144, U.S. Pat. No. 5,993,412, U.S. Pat. No. 5,649,912, U.S. Pat. No.
  • compositions may be administered in any dose appropriate to achieve a desired outcome.
  • the desired outcome is reduction in intesity, severity, and/or frequency, and/or delay of onset of one or more symptoms of influenza infection.
  • inventive pharmacuetical compositions are formulated to administer a dose of binding agent effective to compete with influenza HA for binding to umbrella topology glycans.
  • binding by influenza HA is reduced after administration of one or more doses of an inventive composition as compared with its level absent such administration.
  • inventive pharmaceutical compositions are formulated to administer a dose of binding agent effective to saturate at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more HA binding sites (e.g., HA binding sites containing umbrella topology glycans) present in the subject (e.g., in the respiratory tract of the subject) receiving the composition.
  • HA binding sites e.g., HA binding sites containing umbrella topology glycans
  • inventive pharmaceutical compositions are formulated to deliver a unit dose of binding agent within the range of 0.0001 to 1000 nmg/kg.
  • inventive pharmaceutical compositions are administered in multiple doses.
  • inventive pharmaceutical compositions are administered in multiple doses/day.
  • inventive pharmaceutical compositions are administered according to a continuous dosing regimen, such that the subject does not undergo periods of less than therapeutic dosing interposed between periods of therapeutic dosing.
  • inventive pharmaceutical compositions are administerd according to an intermittent dosing regimen, such that the subject undergoes at least one period of less than therapeutic dosing interposed between two periods of therapeutic dosing.
  • the present invention provides kits for detecting binding agents (e.g., HA polypeptides, LSBAs, UTBAs, UTSBAs, etc), and particular for detecting binding agents with particular glycan binding characteristics (e.g., binding to umbrella glycans, to ⁇ 2-6 sialylated glycans, to long ⁇ 2-6 sialylated glycans, etc.) in pathological samples, including, but not limited to, blood, serum/plasma, peripheral blood mononuclear cells/peripheral blood lymphocytes (PBMC/PBL), sputum, urine, feces, throat swabs, dermal lesion swabs, cerebrospinal fluids, cervical smears, pus samples, food matrices, and tissues from various parts of the body such as brain, spleen, and liver.
  • binding agents e.g., HA polypeptides, LSBAs, UTBAs, UTSBAs, etc
  • the present invention also provides kits for detecting binding agents (e.g., HA polypeptides, LSBAs, UTBAs, UTSBAs, etc) of interest in environmental samples, including, but not limited to, soil, water, and flora. Other samples that have not been listed may also be applicable.
  • binding agents e.g., HA polypeptides, LSBAs, UTBAs, UTSBAs, etc
  • kits for detecting HA polypeptides as described herein whether or not such polypeptides are binding agents may include one or more agents that specifically detect binding agents (e.g., HA polypeptides, LSBAs, UTBAs, UTSBAs, etc) with particular glycan binding characteristics.
  • binding agents e.g., HA polypeptides, LSBAs, UTBAs, UTSBAs, etc
  • Such detecting agents may include, for example, antibodies that specifically recognize certain binding agents (e.g., binding agents that bind to umbrella glycans and/or to ⁇ 2-6 sialylated glycans and/or to long ⁇ 2-6 sialylated glycans), which can be used to specifically detect such binding agents by ELISA, immunofluorescence, and/or immunoblotting.
  • binding agents e.g., binding agents that bind to umbrella glycans and/or to ⁇ 2-6 sialylated glycans and/or to long ⁇ 2-6 sialylated glycans
  • Antibodies that bind to HA polypeptides can also be used in virus neutralization tests, in which a sample is treated with antibody specific to HA polypeptides of interest, and tested for its ability to infect cultured cells relative to untreated sample. If the virus in that sample contains such HA polypeptides, the antibody will neutralize the virus and prevent it from infecting the cultured cells. Alternatively or additionally, such antibodies can also be used in HA-inhibition tests, in which the HA protein is isolated from a given sample, treated with antibody specific to a particular HA polypeptide or set of HA polypeptides, and tested for its ability to agglutinate erythrocytes relative to untreated sample.
  • such agents may include nucleic acids that specifically bind to nucleotides that encode particular HA polypeptides and that can be used to specifically detect such HA polypeptides by RT-PCR or in situ hybridization
  • nucleic acids which have been isolated from a sample are amplified prior to detection.
  • diagnostic reagents can be detectably labeled.
  • the present invention also provides kits containing reagents according to the invention for the generation of influenza viruses and vaccines.
  • kits include, but are not limited to, expression plasmids containing HA nucleotides (or characteristic or biologically active portions) encoding HA polypeptides of interest (or characteristic or biologically active portions).
  • kits may contain expression plasmids that express HA polypeptides of interest (or characteristic or biologically active portions).
  • Expression plasmids containing no virus genes may also be included so that users are capable of incorporating HA nucleotides from any influenza virus of interest.
  • Mammalian cell lines may also be included with the kits, including but not limited to, Vera and MDCK cell lines.
  • diagnostic reagents can be detectably labeled.
  • kits for use in accordance with the present invention may include, a reference sample, instructions for processing samples, performing the test, instructions for interpreting the results, buffers and/or other reagents necessary for performing the test.
  • the kit can comprise a panel of antibodies.
  • glycan arrays as discussed above, may be utilized as diagnostics and/or kits.
  • inventive glycan arrays and/or kits are used to perform dose response studies to assess binding of HA polypeptides to umbrella glycans at multiple doses (e.g., as described herein). Such studies give particularly valuable insight into the binding characteristics of tested HA polypeptides, and are particularly useful to assess specific binding. Dose response binding studies of this type find many useful applications. To give but one example, they can be helpful in tracking the evolution of binding characteristics in a related series of HA polypeptide variants, whether the series is generated through natural evolution, intentional engineering, or a combination of the two.
  • inventive glycan arrays and/or kits are used to induce, identify, and/or select binding agents (e.g. , HA polypeptides, and/or HA polypeptide variants) having desired binding characteristics.
  • inventive glycan arrays and/or kits are used to exert evolutionary (e.g., screening and/or selection) pressure on a population of polypeptide binding agents (e.g., HA polypeptides).
  • kits for administration of inventive pharmaceutical compositions For example, in some embodiments, the invention provides a kit comprising at least one dose of a binding agent. In some embodiments, the invention provides a kit comprising an initial unit dose and a subsequent unit dose of a binding agent. In some such embodiments, the initial unit dose is greater than the subsequent unit dose or wherein the two doses are equal.
  • inventive kits (particularly those for administration of inventive pharmaceutical compositions) comprise at least one component of a delivery device, e.g., an inhaler. In some such embodiments, the invention provides a kit comprising at least one component of a delivery device, e.g., an inhaler and a dose of an of a binding agent.
  • provided kits comprise instructions for use.
  • Example 1 Framework for binding specificity of Hl, H3 and H5 HAs to ⁇ 2-3 and ⁇ 2-6 sialylated glycans
  • 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 ⁇ 2-6 sialylated glycans.
  • the Asp 190GIu mutation in the HA of the 1918 human pandemic virus reversed its specificity from ⁇ 2-6 to ⁇ 2-3 sialylated glycans (Stevens et al, J. MoI. Biol., 355:1143, 2006; Glaser et al, J. Virol, 79:11533, 2005).
  • the double mutation Glul90Asp and Gly225Asp on an avian Hl reversed its specificity from ⁇ 2-3 to ⁇ 2-6 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 al., Nature, 304:76, 1983).
  • 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 ⁇ 2-3 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 3).
  • the common feature in all these three classes is that the Neu5Ac ⁇ 2- 3GaI 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 Neu5 Ac and Gal monosaccharides ( Figure 15, Panels C,D).
  • APR34 a human Hl subtype, contains all the four amino acids Alal38, Glul90, Gln226 and Gly228 and binds to ⁇ 2- 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 3 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 3).
  • a basic amino acid at position 222 could interact with 4-0 sulfated GIcNAc in Neu5Ac ⁇ 2-3Gal ⁇ l -3GIcNAc motif or 6-0 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.
  • 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 supports the above notion wherein the ⁇ ⁇ -60 (referred to as cis conformation) causes the sugars beyond Neu5 Ac ⁇ 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 ASI30 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.
  • Asp 190 and Ser/Asnl93 are positioned to interact with additional monosaccharides GlcNAc ⁇ l-3Gal of the Neu5Ac ⁇ 2-6Gal ⁇ l-4GlcNAc ⁇ l-3Gal 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 3).
  • the Glul90Asp/Gly225Asp double mutant of the avian Hl HA reverses its binding to ⁇ 2-6 sialylated glycans (Table 3).
  • the Lys222Leu mutant of human ANY 18 Hl 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 ⁇ 2-6 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 ⁇ 2-6 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 GIu 190 Asp and Gly225 Asp mutations in H5 HA could potentially improve the contacts with ⁇ 2-6 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 Neu5Ac ⁇ 2-6Gal motif.
  • ADU63 H3 26, AAI68 H3, ADS97 H5 26 and VietO4_H5 provides insights into the H3-like binding of H5 HA to ⁇ 2-6 sialylated glycans.
  • the double mutant does not bind to any glycan structure since it loses the amino acid GIu 190 for binding ⁇ 2-3 sialylated glycans and has the steric interference from Lysl93 for binding to ⁇ 2-6 sialylated glycans.
  • the double mutant Gln226Leu/Gly228Ser binds to some of the ⁇ 2-3 sialylated glycans ( ⁇ 2-3 Type B classifier) but only to a single biantennary ⁇ 2-6 sialylated glycan ( ⁇ 2-6 Type A classifier).
  • a necessary condition for human adaptation of influenza A virus HAs is to gain the ability to bind to long ⁇ 2-6 (predominantly expressed in human upper airway) with high affinity.
  • ⁇ 2-6 predominantly expressed in human upper airway
  • 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 3).
  • the full length construct of HA has a TV-terminal signal peptide and a C-terminal transmembrane sequence.
  • a shortened construct of HA is used which allows the protein to be secreted.
  • This shortened soluble construct is created by replacing the HA' s TV-terminal signal peptide with a Gp67 signal peptide sequence and the C-terminal transmembrane region is replaced by a 'foldon' sequence followed by a tryptic cleavage site and a 6X-His tag (Stevens et al, J. MoI. Biol, 355:1143, 2006).
  • Both full length and the soluble form of HA were expressed in insect cells. Suspension cultures of Sf- 9 cells in Sf900 II SFM medium (Invitrogen) were infected with baculoviruses containing either full length or soluble form of HA. The cells were harvested 72-96 hours post infection.
  • Hemagglutinin (HA) from A/Vietnam/ 1203/2004 was a kind gift from Adolfo Garcia-Sastre.
  • This "wild type"(WT) HA was used as template to create two different mutant constructs, DSLS and DSDL, using QuikChange II XL Site-Directed Mutagenesis Kit (Stratagene) and QuikChange Multi Site -Directed Mutagenesis Kit (Stratagene).
  • the primers used for mutagenesis were designed using the web based program, PrimerX (http://bioinformatics.org/primerx/), and synthesized by Invitrogen.
  • the WT and mutant HA genes were sub-cloned into the entry vector pENTR-D-TOPO (Invitrogen) using TOPO ligation.
  • the entry vectors containing the WT and mutant genes were recombined with BaculoDirect linear DNA (Invitrogen) using Gateway cloning technology. DNA sequencing was performed at each sub-cloning step to confirm the accuracy of the sequences.
  • the recombinant baculovirus DNA produced was used to transfect Spodoptera frugiperda Sf-9 cells (Invitrogen) to yield primary stock of virus.
  • the full length HA was purified from the membrane fraction of the infected cells by a method modified from Wang et al. (2006) Vaccine, 24:2176. Briefly, the cells from the 150 ml culture were harvested by centrifugation and the cell pellet was extracted with 30 ml of 1% Tergitol NP-9 in buffer A (20 mM sodium phosphate, 1.0 mM EDTA, 0.01 % Tergitol-NP9, 5% glycerol, pH 5.93) at 4 0 C for 30 min. The extract was then subjected to centrifugation at 6,000 g for 15 min.
  • the supernatant was filtered using a 0.45 micron filter and loaded on Q/SP columns (GE healthcare, Piscataway, NJ) that were previously equilibrated with Buffer A. After loading, the columns were washed with 20 ml of Buffer A. Then, the anion exchange column Q was disconnected and the SP column was used for elution of protein using five 5 ml fractions of buffer B (20 mM sodium phosphate, 0.03 % Tergitol, 5% glycerol, pH 8.2) and two 5 ml fractions of buffer C (20 mM sodium phosphate, 150 mM NaCl, 0.03% Tergitol, 5% glycerol, pH 8.2). The fractions containing the protein of interest were pooled together and subjected to ultrafiltration using Amicon Ultra 100 K NMWL membrane filters (Millipore). The protein was concentrated and reconstituted in PBS.
  • Q/SP columns GE healthcare, Piscataway, NJ
  • the soluble form of HA was purified from the supernatant of the infected cells using the protocol described in Stevens et al. (2004). Briefly, the supernatant was concentrated and the soluble HA was recovered from the concentrated cell supernatant by performing affinity chromatography using Ni-NTA beads (Qiagen). Eluting fractions containing HA were pooled and dialyzed against 10 mM Tris-HCl, 50 mM NaCl; pH 8.0. Ion exchange chromatography was performed on the dialyzed samples using a Mono-Q HR 10/ 10 column (Pharmacia).
  • the fractions containing HA were pooled together and subjected to ultrafiltration using Amicon Ultra 100 K NMWL membrane filters (Millipore). The protein was concentrated and reconstituted in PBS. [00266] The presence of the protein in the samples was verified by performing western blot analysis with anti avian H5N1 HA antibody. Through dot-blot immunoassay (using WT H5 HA obtained from Protein Sciences Inc as the reference) the protein concentration of WT and the mutants were determined. In the various experiments that were performed the protein concentration of the H5 HA (WT and mutants) were typically found to be in 20-50 microgram/ml range. Based on the protein concentration for a given lot appropriate serial dilutions in the ranges of 1 : 10 - 1 : 100 were used (see Figure 17).
  • Example 3 Application of Data Mining Platform to investigate glycan binding specificity of HA
  • 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 3).
  • 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 ⁇ 2-3 and ⁇ 2-6 sialylated glycans, including glycans of different topologies, as discussed below.
  • HA polypeptides are H5 polypeptides.
  • inventive H5 polypeptides show binding (e.g., high affinity and/or specificity binding) to umbrella glycans.
  • inventive H5 polypeptides are termed "broad spectrum human binding" (BSHB) H5 polypeptides.
  • BSHB broad spectrum human binding
  • H5 polypeptides bind to HA receptors found in human epithelial tissues, and particularly to human HA receptors characterized by ⁇ 2-6 sialylated glycans.
  • inventive BSHB H5 HA polypeptides bind to receptors found on human upper respiratory epithelial cells.
  • inventive BSHB H5 HA polypeptides bind to receptors found on human upper respiratory epithelial cells.
  • HA polypeptides bind to a plurality of different ⁇ 2-6 sialylated glycans.
  • BSHB H5 HA polypeptides bind to umbrella glycans.
  • inventive BSHB H5 HA polypeptides bind to HA receptors in the bronchus and/or trachea.
  • BSHB H5 HA polypeptides are not able to bind receptors in the deep lung, and in other embodiments, BSHB H5 HA polypeptides are able to bind receptors in the deep lung.
  • BSHB H5 HA polypeptides are not able to bind to ⁇ 2-3 sialylated glycans, and in other embodiments BSHB H5 HA polypeptides are able to bind to ⁇ 2-3 sialylated glycans.
  • inventive BSHB H5 HA polypeptides are variants of a parent
  • inventive BSHB H5 HA polypeptides have at least one amino acid substitution, as compared with wild type H5 HA, within or affecting the glycan binding site.
  • such substitutions are of amino acids that interact directly with bound glycan; in other embodiments, such substitutions are of amino acids that are one degree of separation removed from those that interact with bound glycan, in that the one degree of separation removed-amino acids either (1) interact with the direct-binding amino acids; (2) otherwise affect the ability of the direct-binding amino acids to interact with glycan, but do not interact directly with glycan themselves; or (3) otherwise affect the ability of the direct-binding amino acids to interact with glycan, and also interact directly with glycan themselves.
  • inventive BSHB H5 HA polypeptides contain substitutions of one or more direct-binding amino acids, one or more first degree of separation-amino acids, one or more second degree of separation-amino acids, or any combination of these. In some embodiments, inventive BSHB H5 HA polypeptides may contain substitutions of one or more amino acids with even higher degrees of separation. [00275] In certain embodiments, inventive BSHB H5 HA polypeptides have at least two, three, four, five or more amino acid substitutions as compared with wild type H5 HA; in some embodiments inventive BSHB H5 HA polypeptides have two, three, or four amino acid substitutions. In some embodiments, all such amino acid substitutions are located within the glycan binding site.
  • a BSHB H5 HA polypeptide has one or more amino acid substitutions relative to wild type H5 HA at residues selected from the group consisting of residues 98, 136, 138, 153, 155, 159, 183, 186, 187, 190, 193, 194, 195, 222, 225, 226, 227, and 228.
  • a BSHB H5 HA polypeptide has one or more amino acid substitutions relative to wild type H5 HA at residues selected from amino acids located in the region of the receptor that directly binds to the glycan, including but not limited to residues 98, 136, 153, 155, 183, 190, 193, 194, 222, 225, 226, 227, and 228.
  • a BSHB H5 HA polypeptide has one or more amino acid substitutions relative to wild type H5 HA at residues selected from amino acids located adjacent to the region of the receptor that directly binds the glycan, including but not limited to residues 98, 138, 186, 187, 195, and 228.
  • a BSHB H5 HA polypeptide has one or more amino acid substitutions relative to wild type H5 HA at residues selected from the group consisting of residues 138, 186, 187, 190, 193, 222, 225, 226, 227 and 228.
  • a BSHB H5 HA polypeptide has one or more amino acid substitutions relative to wild type H5 HA at residues selected from amino acids located in the region of the receptor that directly binds to the glycan, including but not limited to residues 190, 193, 222, 225, 226, 227, and 228.
  • a BSHB H5 HA polypeptide has one or more amino acid substitutions relative to wild type H5 HA at residues selected from amino acids located adjacent to the region of the receptor that directly binds the glycan, including but not limited to residues 138, 186, 187, and
  • a BSHB H5 HA polypeptide has one or more amino acid substitutions relative to wild type H5 HA at residues selected from the group consisting of residues 98, 136, 153, 155, 183, 194, and 195.
  • a BSHB H5 HA polypeptide has one or more amino acid substitutions relative to wild type H5 HA at residues selected from amino acids located in the region of the receptor that directly binds to the glycan, including but not limited to residues 98, 136, 153, 155, 183, and 194.
  • a BSHB H5 HA polypeptide has one or more amino acid substitutions relative to wild type H5 HA at residues selected from amino acids located adjacent to the region of the receptor that directly binds the glycan, including but not limited to residues 98 and 195.
  • a BSHB H5 HA polypeptide has one or more amino acid substitutions relative to wild type H5 HA at residues selected from amino acids that are one degree of separation removed from those that interact with bound glycan, in that the one degree of separation removed-amino acids either (1) interact with the direct-binding amino acids; (2) otherwise affect the ability of the direct-binding amino acids to interact with glycan, but do not interact directly with glycan themselves; or (3) otherwise affect the ability of the direct-binding amino acids to interact with glycan, and also interact directly with glycan themselves, including but not limited to residues 98, 138, 186, 187, 195, and 228.
  • a BSHB H5 HA polypeptide has one or more amino acid substitutions relative to wild type H5 HA at residues selected from amino acids that are one degree of separation removed from those that interact with bound glycan, in that the one degree of separation removed-amino acids either (1) interact with the direct-binding amino acids; (2) otherwise affect the ability of the direct-binding amino acids to interact with glycan, but do not interact directly with glycan themselves; or (3) otherwise affect the ability of the direct-binding amino acids to interact with glycan, and also interact directly with glycan themselves, including but not limited to residues 138, 186, 187, and 228.
  • a BSHB H5 HA polypeptide has one or more amino acid substitutions relative to wild type H5 HA at residues selected from amino acids that are one degree of separation removed from those that interact with bound glycan, in that the one degree of separation removed-amino acids either (1) interact with the direct-binding amino acids; (2) otherwise affect the ability of the direct-binding amino acids to interact with glycan, but do not interact directly with glycan themselves; or (3) otherwise affect the ability of the direct-binding amino acids to interact with glycan, and also interact directly with glycan themselves, including but not limited to residues 98 and 195.
  • a BSHB H5 HA polypeptide has an amino acid substitution relative to wild type H5 HA at residue 159.
  • a BSHB H5 HA polypeptide has one or more amino acid substitutions relative to wild type H5 HA at residues selected from 190, 193, 225, and 226. In some embodiments, a BSHB H5 HA polypeptide has one or more amino acid substitutions relative to wild type H5 HA at residues selected from 190, 193, 226, and 228. In some embodiments, an inventive HA polypeptide variant, and particularly an H5 variant has one or more of the following amino acid substitutions: Serl37Ala, Lysl56Glu, Asnl86Pro, Aspl87Ser,
  • Lysl93Asn Lysl93His, Lysl93Ser, Gly225Asp, Gln226Ile, Gln226Leu, Gln226Val,
  • an inventive HA polypeptide variant, and particularly an H5 variant has one or more of the following sets of amino acid substitutions:
  • Lysl56Glu Alal89Lys, Lysl93Asn, Gln226Leu, Gly228Ser;
  • Lysl56Glu Alal89Lys, Lysl93Asn, Gly225Asp;
  • the HA polypeptide has at least one further substitution as compared with a wild type HA, such that affinity and/or specificity of the variant for umbrella glycans is increased.
  • inventive BSHB H5 HA polypeptides have amino acid sequences characteristic of Hl HAs.
  • Hl-like BSHB amino acid sequences characteristic of Hl HAs.
  • H5 HA polypeptides have substitutions Glul90Asp, Lysl93Ser, Gly225Asp and Gln226Leu.
  • inventive BSHB H5 HA polypeptides have amino acid sequences characteristic of Hl HAs.
  • H5 HAs contain substitutions Glul90Asp, Lysl93Ser, Gln226Leu and Gly228Ser.
  • inventive BSHB H5 HA polypeptides have an open binding site as compared with wild type H5 HAs. In some embodiments, inventive BSHB H5 HA
  • polypeptides bind to the following ⁇ 2-6 sialylated glycans: (" O " ) , (--II— ) , (— Q— ) , combinations thereof.
  • inventive BSHB H5 HA polypeptides bind to glycans of the structure: ) and combinations thereof; and/or i cC- ⁇
  • inventive BSHB H5 HA polypeptides bind to
  • inventive BSHB H5 HA polypeptides bind to umbrella topology glycans. In some embodiments, inventive BSHB H5 HA polypeptides bind to at least some of the glycans (e.g., ⁇ 2-6 silaylated glycans) depicted in Figure 9. In some embodiments, inventive BSHB H5 HA polypeptides bind to multiple glycans depicted in Figure 9.
  • inventive BSHB H5 HA polypeptides bind to at least about 10%, 15%, 20%, 25%, 30% 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% 95% or more of the glycans found on HA receptors in human upper respiratory tract tissues (e.g., epithelial cells).
  • human upper respiratory tract tissues e.g., epithelial cells.
  • Example 5 Glycan diversity in the human upper respiratory tissues
  • MALDI-MS glycan profiling analyses showed a substantial diversity ( Figure 10) as well as predominant expression of ⁇ 2-6 sialylated glycans on the human upper airways.
  • fragmentation of representative mass peaks using MALDI TOF-TOF supports glycan topologies where longer oligosaccharide branches with multiple lactosamine repeats are extensively distributed as compared to short oligosaccharide branches ( Figure 10).
  • MALDI-MS analysis was performed on N-linked glycans from human colonic epithelial cells (HT29).
  • Sections were then incubated with FITC labeled Jacalin (specific for O-linked glycans), biotinylated Concanavalin A (Con A, specific for ⁇ - linked mannose residues, which are part of the core oligosaccharide structure that constitute TV- linked glycans), biotinylated Maackia amurensis lectin (MAL, specific for SA ⁇ 2,3-gal) and biotinylated Sambuccus nigra agglutinin (SNA, specific for SA ⁇ 2,6-gal) (Vector labs; 10 ⁇ g/ml in PBS with 0.5% Tween-20) for 3 hrs.
  • Jacalin specific for O-linked glycans
  • Con A specific for ⁇ - linked mannose residues, which are part of the core oligosaccharide structure that constitute TV- linked glycans
  • MAL specific for SA ⁇ 2,3-gal
  • SNA biotinylated
  • TBST Tris buffered saline with 1% Tween-20
  • Alexa fluor 546 streptavidin (2 ⁇ g/ml in PBS with 0.5% Tween-20) for 1 hr.
  • Slides were washed with TBST and viewed under a confocal microscope (Zeiss LSM510 laser scanning confocal microscopy). All incubations were performed at room temperature (RT).
  • 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 were analyzed by MALDI-TOF MS in positive and negative modes respectively with soft ionization conditions (accelerating voltage 22 kV, grid voltage 93%, guide wire 0.3% and extraction delay time of 150 ns). The peaks were calibrated as non-sodiated species.
  • the predominant expression of ⁇ 2-6 sialylated glycans was confirmed by pretreatment of samples using Sialidase A and S.
  • the isolated glycans were subsequently incubated with 0.1 U of Arthrobacter ureafaciens sialidase (Sialidase A, non-specific) or Streptococcus pneumoniae sialidase (Sialidase S, specific for ⁇ 2-3 sialylated glycans) in a final volume of 100 mL of 50 mM sodium phosphate, pH 6.0 at 37 0 C for 24 hrs.
  • the neutral and the acidic fractions were analyzed by MALDI-TOF MS in positive and negative modes respectively.
  • Example 6 Dose response binding of Hl and H3 HA to human lung tissues [00295]
  • the apical side of tracheal tissue predominantly expresses ⁇ 2-6 glycans with long branch topology.
  • the alveolar tissue on the other hand predominantly expresses ⁇ 2-3 glycans.
  • Hl HA binds significantly to the apical surface of the trachea and its binding reduces gradually with dilution from 40 to 10 ⁇ g/ml ( Figure 19).
  • Hl HA also shows some weak binding to the alveolar tissue only at the highest concentration.
  • H3 HA The binding pattern of H3 HA is different from that of Hl HA where in H3 HA shows significant binding to both tracheal and alveolar tissue sections at 40 and 20 ⁇ g/ml ( Figure 19). However, at a concentration of 10 ⁇ g/ml, the HA shows binding primarily to the apical side of the tracheal tissue and little to no binding to the alveolar tissue. Together, the tissue binding data point to 1) the high affinity binding of Hl and H3 HA to the apical side of the tracheal tissue and 2) while H3 HA shows affinity for ⁇ 2-3 (relatively lower than ⁇ 2-6) Hl HA is highly specific for ⁇ 2-6.
  • the complexes formed were diluted in 1%BS A-PBS to a final HA concentration of 40, 20 or 10 ⁇ g/ml. Tissue sections were then incubated with the HA- antibody complexes for 3 hours at RT. Sections were counterstained with propidium iodide (Invitrogen; 1 : 100 in TBST), washed extensively and then viewed under a confocal microscope (Zeiss LSM510 laser scanning confocal microscopy).
  • Example 7 Dose response direct binding of wild type HA polypeptides to glycans of different topology
  • the present invention encompasses the recognition that binding by HA polypeptides to glycans having a particular topology, herein termed "umbrella" topology, correlates with ability of the HA polypeptides to mediate infection of human hosts.
  • the present Example describes results of direct binding studies with different HA polypeptides that mediate infection of different hosts, and illustrates the correlation between human infection and umbrella glycan binding.
  • Direct binding assays typically utilize glycan arrays in which defined glycan structures (e.g., monovalent or multivalent) are presented on a support (e.g., glass slides or well plates), often using a polymer backbone.
  • glycan arrays in which defined glycan structures (e.g., monovalent or multivalent) are presented on a support (e.g., glass slides or well plates), often using a polymer backbone.
  • glycan arrays in which defined glycan structures (e.g., monovalent or multivalent) are presented on a support (e.g., glass slides or well plates), often using a polymer backbone.
  • a support e.g., glass slides or well plates
  • trimeric HA polypeptide is bound to the array and then is detected, for example using labeled (e.g., with FITC or horse radish peroxidase) primary and secondary antibodies.
  • trimeric HA is first complexed with primary and secondary antibodies (typically in a 4:2:1 HA:primary:secondary ratio), such that there are 12 glycan binding sites per pre-complexed HA, and is then contacted with the array. Binding assays are typically carried out over a range of HA concentrations, so that information is obtained regarding relative affinities for different glycans in the array.
  • Hl HA is their binding at saturating levels to the long ⁇ 2-6 (6'SLN-LN) over a range of dilution from 40 down to 5 ⁇ g/ml ( Figure 20). While Hl HA is highly specific for binding to the long ⁇ 2-6, H3 HA also binds to short ⁇ 2-6 (6'SLN) with high affinity and to a long ⁇ 2-3 with a lower affinity relative to ⁇ 2-6 ( Figure 20). The direct binding dose response of Hl and H3 HA is consistent with the tissue binding pattern.
  • Hl and H3 HA correlates with their extensive binding to apical side of the tracheal tissues which expresses ⁇ 2-6 glycans with long branch topology.
  • This correlation provides valuable insights into the upper respiratory tissue tropism of human adapted Hl and H3 HAs.
  • the tested H5 HA shows the opposite glycan binding trend wherein it binds with high affinity to ⁇ 2-3 (saturating signals from 40 down to 2.5 ⁇ g/ml) as compared to its relatively low affinity for ⁇ 2-6 (significant signals seen only at 20-40 ⁇ g/ml) (Figure 20).
  • Example 8 SNA-I is a binding agents that blocks HA polypeptide interaction with umbrella topology glycans
  • the present invention encompasses the recognition that binding by binding agents to glycans having a particular topology, herein termed "umbrella" topology, correlates with ability of the binding agent to compete with binding of HA polypeptides.
  • the present Example describes results of direct binding studies with different binding agents and competitive binding studies with HA polypeptides, that illustrates the competitive inhibition of HA binding to umbrella topology glycans by binding agents.
  • Direct binding assays typically utilize glycan arrays in which defined glycan structures (e.g., monovalent or multivalent) are presented on a support (e.g., glass slides or well plates), often using a polymer backbone.
  • glycan arrays in which defined glycan structures (e.g., monovalent or multivalent) are presented on a support (e.g., glass slides or well plates), often using a polymer backbone.
  • a binding agent is bound to the array and then is detected, for example using labeled (e.g., with FITC or horse radish peroxidase) primary and secondary antibodies.
  • a binding agent is first complexed with primary and secondary antibodies (typically in a 4:2:1 binding agent:primary:secondary ratio), such that there are 12 glycan binding sites per pre-complexed binding agent, and is then contacted with the array. Binding assays are typically carried out over a range of binding agent concentrations, so that information is obtained regarding relative affinities for different glycans in the array.
  • Example 9 Testing inventive binding agents in an animal host
  • animal hosts e.g., ferrets
  • inventive binding agents e.g., HA polypeptides
  • the present Example desrcibes a virus transmission assay that can be used in the presence or absence of inventive binding agents to determine viral transmission in a suitable animal model.
  • Animal hosts e.g., ferrets
  • Animal hosts can be housed in adjacent cages that prevent direct and indirect contact between animals. However, these housing conditions allow the spread of influenza virus through the air.
  • a first portion of the animals are innoculated via methods known in the art, e.g., intranasally, with an effective amount of virus ("innoculated animals"). Na ⁇ ve animals can then be introduced into cages adjacent to the innoculated animals one, two, three or more days later.
  • Animals used in the study can be killed at any time one, two, three or more days post- innoculation or transmission for analysis. Suitable analysis for virus transmission studies can include, but is not limited to determination of infectious virus titers (e.g., by nasal washes), observation of physical symptoms in the animals (e.g., lethargy, anorexia, rhinorrhea, sneezing, high fever, and/or death), immunohistochemical analysis of respiratory tissues, among others.
  • the virus transmission assay described above can also incorporate the treatement of the animal host with an inventiv binding agent described herein before, during or after innoculation or transmission of virus. Analytic methods described herein are then used to determine the efficacy of the binding agent(s) in blocking transimssion and/or infection of the animal host with the virus.

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Abstract

La présente invention porte sur un système permettant d'analyser des interactions entre des glycanes et des partenaires d'interaction qui se lient à ceux-ci. La présente invention porte également sur des polypeptides HA qui se lient à des glycanes à topologie en parapluie, ainsi que sur des réactifs et des procédés s'y rapportant.
EP09701014A 2008-01-03 2009-01-02 Polypeptides d'hémagglutinine, réactifs, et procédés s'y rapportant Withdrawn EP2238146A4 (fr)

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KUMARI KSHAMA ET AL: "Receptor binding specificity of recent human H3N2 influenza viruses", VIROLOGY JOURNAL, BIOMED CENTRAL, LONDON, GB, vol. 4, no. 1, 9 May 2007 (2007-05-09), page 42, XP021025487, ISSN: 1743-422X, DOI: 10.1186/1743-422X-4-42 *
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