Virus Treatment

Abstract

The present disclosure provides molecules for use in compositions, medicaments, and methods for treating or preventing RSV infection, its symptoms, and associated pathologies, and in some cases, infectious diseases caused or resulting from viral pathogens that do not bind, or do not primarily bind, to sialic acid-containing receptors during the pathogenesis. In particular, the present disclosure is based on the discovery that molecules having affinity for (or the ability to bind to) sialic acid (particularly sialoglycoconjugates) on cell surfaces (including sialic acid-containing glycoproteins and cell surface sialic acid receptors) find utility in treating and / or preventing symptoms, infections, diseases, and / or conditions associated with respiratory syncytial virus (RSV).

Description

[Technical Field]

[0001] The present invention provides molecules for use in compositions, medicaments and methods for the treatment or prevention of RSV infection, its symptoms and associated pathologies, and in some cases, infectious diseases caused or resulting from viral pathogens that do not bind, or do not primarily bind, to sialic acid-containing receptors during the pathogenesis. [Background technology]

[0002] Respiratory syncytial virus (RSV) is a syncytial virus that causes respiratory tract infection. It causes disease in humans and animals and is a major cause of lower respiratory tract infection, with children and the elderly being particularly at risk. Infection with RSV (including the form often referred to as human RSV (HRSV)) induces protective immunity, but studies have shown that the response is not long-lasting (or persistent) and cases of multiple infections are not uncommon. Unlike many other respiratory pathogens, RSV does not bind to sialoglycoconjugates for cell entry and infection. 3 However, due to the ubiquitous and predominant nature of sialic acid as the terminal glycan of respiratory epithelial cell surface glycoconjugates, it would be advantageous to utilize sialic acid-binding molecules as the basis for treatments against pathogens such as RSV. Summary of the Invention

[0003] The present disclosure is based on the discovery that molecules having affinity for (or the ability to bind to) sialic acid (particularly sialoglycoconjugates) present on cell surfaces (including sialic acid-containing glycoproteins and cell surface sialic acid receptors) find utility in the treatment and / or prevention of symptoms, infections, diseases and / or conditions associated with respiratory syncytial virus (RSV). The present disclosure provides sialic acid binding molecules for use in the treatment and / or prevention of symptoms, infections, diseases and / or conditions associated with respiratory syncytial virus (RSV). Further provided is the use of a sialic acid binding molecule in the manufacture of a medicament for use in the treatment and / or prevention of symptoms, infections, diseases and / or conditions associated with RSV. The present disclosure also provides a method for treating or preventing a RSV symptom, disease, infection or condition, comprising administering to a subject in need thereof a therapeutically effective amount of a sialic acid binding molecule. The present disclosure also provides sialic acid binding molecules, as well as agents and methods comprising sialic acid binding molecules, for use in methods of treating or preventing symptoms, diseases and / or conditions associated with RSV infection.

[0004] Throughout this specification, the terms "comprise," "comprising," and / or "comprises" are used to refer to aspects and embodiments of the invention that "comprise" particular features. It will be appreciated that these terms may also encompass aspects and / or embodiments that "consist essentially of" or "consist of" the relevant feature. RSV is a medium-sized (120-200 nm) enveloped virus containing a lipoprotein coat and a linear, negative-sense RNA genome. The genome encodes the F, G, and SH lipoproteins. The F (fusion) and G (attachment) lipoproteins (or glycoproteins) target the host cell membrane and control the initial stage of infection. These are also highly conserved among RSV isolates. In particular, the G protein targets ciliated cells in the respiratory tract, and the F protein fuses the virion membrane with the target cell membrane. Based on viral reactivity with monoclonal antibodies against the attachment (G) and fusion (F) glycoproteins, RSV is classified into two antigenic subgroups, A and B. As used herein, the term "RSV" includes all strains, forms, and antigenic variants of RSV, including all forms, strains, and variants of human (H) or animal RSV. Because RSV is a pathogen that does not bind or associate with sialic acid during pathogenesis, it does not bind to host cell sialic acid (or cell surface sialoglycoconjugates). Thus, the discovery that sialic acid-binding molecules (such as, for example, CBM40 molecules (and others defined below)) have utility in treating viral pathogens that do not utilize cell surface sialic acid (sialoglycoconjugates) during pathogenesis (or pathogens in which binding to sialic acid is not the primary means by which the pathogen binds to, colonizes, or enters / infects cells) is completely unexpected.

[0005] With regard to prior art disclosures in which sialic acid-binding molecules have been used as agents capable of blocking the binding of pathogens to cell surface sialic acid / sialoglycoconjugates, the usefulness of sialic acid-binding molecules is due to the fact that both sialic acid-binding molecules and pathogens bind to sialic acid, which is not the case with RSV. Because RSV does not bind to sialic acid, those skilled in the art would not recognize that sialic acid-binding molecules could be used to block RSV entry into cells. Without wishing to be bound by theory, it is suggested that when bound to cell surface sialic acid / sialoglycoconjugates, sialic acid-binding molecules prevent the G and / or F RSV proteins from reaching their targets on host cells. This in turn prevents RSV from colonizing and infecting cells. Furthermore, data presented below (see in particular the data presented in Example 6) indicate that the surface of the RSV virus contains glycoproteins that terminate in sialic acid, suggesting that sialic acid-binding molecules can bind to these glycoproteins (via the sialic acid element), further inhibiting the binding between RSV and host cells.

[0006] Although it may have been determined that sialic acid binding proteins can be used to stimulate or modulate the immune system, and that a stimulated or modulated immune response can also affect the pathology of many different pathogens (including pathogens that do not bind, or do not primarily bind, sialic acid during the course of pathogenesis), one skilled in the art would still not have discovered that molecules with affinity for sialic acid (e.g., CBMs such as CBM40-type molecules) can be used to physically block, impede, or neutralize RSV infection. However, the finding that sialic acid-binding molecules can be used to neutralize or prevent RSV infection extends the present disclosure to providing sialic acid-binding molecules for use in neutralizing or preventing RSV infection. Such uses may be utilized in in vitro or in vivo methods. The present disclosure further provides the use of a sialic acid binding molecule for the manufacture of a medicament for neutralizing or preventing RSV infection.

[0007] The present disclosure also relates to a method of neutralizing or preventing a RSV infection, comprising administering to a subject in need thereof a therapeutically effective amount of a sialic acid binding molecule. Alternatively, the present disclosure provides methods that can be used to render cells non-permissive to RSV (the term "non-permissive" refers to cells that resist viral attachment or colonization and / or subsequent infection and viral replication). Such methods (which may be in vitro or in vivo) may include contacting or incubating cells that are susceptible or vulnerable to RSV infection with a sialic acid-binding molecule described herein. The contacting or incubating step may include contacting or incubating the cells with the sialic acid-binding molecule before contacting them with RSV. Additionally or alternatively, contacting or incubating the cells with the sialic acid-binding molecule may be extended so that the sialic acid-binding molecule is in contact with the cells at the same time the cells are in contact with RSV. Without wishing to be bound by theory, it has been surprisingly found that during the incubation period, the sialic acid-binding molecules bind to sialic acid-containing cell surface receptors, and RSV itself does not bind to cell surface sialic acid moieties (sialoglycoconjugates), but that the binding between the sialic acid-binding molecules and the cell surface sialic acid-containing receptors / sialoglycoconjugates inhibits or prevents RSV from binding to cells, which in turn prevents RSV from infecting cells and intracellular RSV replication.

[0008] The findings reported in this disclosure have important implications for the formulation and administration of molecules with binding affinity for sialic acid and the subsequent use of these formulations in the treatment and / or prevention of RSV infection. For example, the discovery that sialic acid-binding molecules (e.g., CBM and / or CBM40-type molecules) can be used to prevent (or neutralize) RSV infection allows the use of sialic acid-binding molecules as formulations suitable for mucosal administration. The sialic acid-binding molecule-containing formulation for mucosal administration is (i) preventing RSV from binding to a host cell; and / or (ii) inhibit or neutralize RSV It may also be used for As specified, the terms "block" or "neutralize" refer to the blocking or neutralizing effect of sialic acid-binding molecules that bind to sialic acid-containing receptors / sialoglycoconjugates on host cells and (surprisingly) have been found to block RSV binding to (and subsequent infection of) the cells. Accordingly, the present disclosure provides compositions for mucosal administration comprising sialic acid binding molecules for use in the treatment and / or prevention of diseases and / or conditions associated with RSV.

[0009] It should be noted that the term "mucosal administration" includes compositions formulated for administration to any mucosal surface, including, for example, respiratory surfaces and the like. The term also includes compositions formulated for administration by inhalation. Compositions suitable for (or formulated for) mucosal administration may include compositions intended to be administered intranasally. Thus, compositions for mucosal administration may be formulated with excipients, diluents and / or buffers suitable for use in any type of mucosal administration. Compositions for mucosal (e.g., intranasal) administration may comprise solutions of the sialic acid-binding molecules to be administered and / or particles for aerosol dispersion or dispersion in drinking water (including the same). If dispersed, such compositions should desirably have a particle size in the range of 10-200 μm, for example, to enable retention in the nasal cavity; this can be achieved, if necessary, by using powders of appropriate particle size or by selecting an appropriate valve. Other suitable compositions include coarse powders having a particle size in the range of 20-500 μm for administration by rapid inhalation through the nostrils from a container held close to the nose, and nasal drops containing 0.2-5% w / v of the active compound in an aqueous or oily solution or suspension. Compositions for mucosal administration may also be provided in the form of a liquid spray.

[0010] In one embodiment, the present disclosure may not relate to the treatment and / or prevention of RSV by stimulating or modulating an immune response. The present disclosure may further provide sialic acid binding molecules for prophylactic use. In particular, the sialic acid molecules described herein may be used prophylactically to prevent RSV infection. Methods of prevention or preventing RSV infection can include administering a composition of the present disclosure to a subject in need thereof. As noted, compositions of the present disclosure can include compositions formulated for mucosal (e.g., intranasal) administration. As used herein and in any method of preventing RSV infection, "subject in need thereof" may be any subject that is susceptible to, susceptible to infection, or at risk of developing RSV infection.The subject may be a newborn, infant, or child.The subject may be an elderly person.The subject may be an immunocompromised subject.The subject may have one or more underlying or chronic health problems, particularly problems that affect the airway and / or breathing.For example, the subject may suffer from asthma.

[0011] Throughout this specification, we use the term "sialic acid-binding molecule," which encompasses any useful sialic acid-binding molecule. Useful sialic acid-binding molecules may take any form and / or belong to any class of molecules or compounds (e.g., they may be proteins, peptides, carbohydrates, antibodies, and the like). The term "sialic acid" encompasses all forms of N- or O-substituted neuraminic acid, including all synthetic, natural, and / or modified forms thereof. Sialic acid may also be found as a component of cell surface molecules, glycoproteins, and glycolipids. In most cases, sialic acid is present at the terminus (terminal region) of carbohydrate chains and / or proteins attached to cell membranes. For example, some cells of the human upper respiratory tract contain α-2,6-linked sialic acid receptors, while other cells of the upper and lower respiratory tract contain α-2,3-linked sialic acid receptors. The sialic acid family includes numerous (approximately 50) derivatives that may result from acetylation, glycolylation, lactonization, and methylation at C4, C5, C7, C8, and C9. All such derivatives are intended to be encompassed by the term "sialic acid."

[0012] Furthermore, sialic acid is found in an α(2,3) or α(2,6) linkage with Gal and GalNAc, or an α(2,8) or α(2,9) linkage with another sialic acid. Thus, it is important to understand that while the term "sialic acid" is used throughout this specification, it encompasses all derivatives, analogs, or variants thereof (whether naturally occurring or synthetically formed), as well as monomers, dimers, trimers, oligomers, polymers, or concatamers comprising the same. Thus, the sialic acid binding molecules of the present disclosure (and for use as described herein) include moieties that exhibit affinity for sialic acid, including all of the forms of sialic acid described above and any form of sialic acid present on the surface of cells, e.g., mammalian cells (possibly as part of a cell surface receptor). These various forms of sialic acid are sometimes collectively referred to as "sialic acid moieties." Because the sialic acid-binding molecules of the present disclosure exhibit affinity for sialic acid, they may bind / link and / or associate with one or more sialic acid moieties. Thus, the term "sialic acid-binding molecule" may further encompass any fragment of the entire sialic acid-binding molecule that retains the ability to bind or otherwise link or associate with a sialic acid moiety.

[0013] A sialic acid-binding molecule for use may comprise a single sialic acid-binding molecule (e.g., a monomeric or monovalent molecule) or two or more sialic acid-binding molecules, e.g., a polymeric or multivalent molecule, which may be the same or different. Sialic acid-binding molecules for use may comprise, consist essentially of, or consist of one or more sialic acid-binding molecules known as "carbohydrate-binding modules" (CBMs). CBMs suitable for use exhibit affinity for sialic acid. CBMs are classified into families, and CBMs classified as members of Family 40 CBMs (CBM40) may be useful. Family 40 CBMs comprise molecules of approximately 200 residues and are often found at the N-terminus of GH33 sialidases. They may also be found inserted into the β-propeller of GH33 sialidases. The present disclosure may also include the use of molecules, e.g., larger molecules, that include a sialic acid-binding element. As noted, the sialic acid-binding element (i.e., the sialic acid-binding molecule) itself may comprise (consist of, or consist essentially of) a CBM, e.g., CBM40. By way of (non-limiting) example, a molecule (e.g., a sialic acid-binding molecule) of the present disclosure may not only exhibit the ability to bind sialic acid, but may also have one or more other functions. For example, the molecule may have enzymatic activity. For example, a useful molecule may include a CBM (as described herein) and exhibit some sialidase activity.

[0014] Useful sialic acid-binding molecules may be fusion proteins comprising an enzymatic portion and a sialic acid-binding portion, where the sialic acid-binding portion comprises a CBM as described herein. In such cases, the enzymatic portion may be fused to the sialic acid-binding portion. As specified, the enzymatic portion of any useful fusion protein may comprise (or may have or exhibit) sialidase activity. In one embodiment, a sialic acid binding protein or CBM for various uses described herein may not be provided as or included as part of a molecule (e.g., a fusion protein) with enzymatic (e.g., sialidase) activity. Additionally or alternatively, the sialic acid binding molecule may (i) not bind to heparin or heparin sulfate, and / or (ii) not include a GAG-binding domain of a protein that binds to heparin or heparin sulfate moieties. Accordingly, the present disclosure provides a CBM or CBM40 for use in the treatment and / or prevention of diseases and / or conditions associated with respiratory syncytial virus (RSV).

[0015] Further provided is the use of CBM or CBM40 in the manufacture of a medicament for use in the treatment and / or prevention of diseases and / or conditions associated with RSV. The present disclosure also provides a method of treating or preventing a RSV infection, comprising administering to a subject in need thereof a therapeutically effective amount of CBM or CBM40. The present disclosure also provides sialic acid binding molecules and agents comprising CBM or CBM40 for use in methods of treating or preventing diseases and / or conditions associated with RSV infection and methods. The present disclosure also provides a CBM or CBM40 for use in neutralizing or preventing RSV infection. The present disclosure further provides the use of CBM and / or CBM40 for the manufacture of a medicament for neutralizing or preventing RSV infection. The present disclosure relates to methods of neutralizing or preventing RSV infection, comprising administering a therapeutically effective amount of CBM and / or CBM40 to a subject in need thereof.

[0016] Alternatively, the present disclosure provides methods that can be used to render cells non-permissive to RSV (the term "non-permissive" refers to cells that resist viral attachment or colonization and / or subsequent infection and viral replication). Such methods (which may be in vitro or in vivo methods) may include contacting or incubating cells that are susceptible or vulnerable to RSV infection with a CBM and / or CBM40 of the present disclosure. CBM and / or CBM40-containing formulations for mucosal administration include: (i) preventing RSV from binding to a host cell; and / or (ii) inhibit or neutralize RSV It may also be used for The present disclosure provides compositions for mucosal administration comprising CBM and / or CBM40 for use in treating and / or preventing diseases and / or conditions associated with RSV. As specified, compositions for mucosal administration may be formulated with excipients, diluents and / or buffers suitable for use in any type of mucosal administration. The present disclosure may further provide a CBM or CBM40 for prophylactic use. In particular, the CBM or CBM40 described herein may be used prophylactically to prevent RSV infection.

[0017] As noted, compositions of the present disclosure may include compositions formulated for mucosal (eg, intranasal) administration. Exemplary carbohydrate binding modules (CBMs) for use may include the sialic acid binding domain of Vibrio cholerae NanH sialidase (VcCBM:CBM40) and / or the corresponding (or identical) domain of Streptococcus pneumoniae NanA sialidase (SpCBM: also CBM40). Of course, similar or identical sialic acid binding modules present in other microorganisms are also intended to be encompassed within the scope of the term "CBM."

[0018] An exemplary Vibrio cholerae NanH sialidase amino acid sequence has been deposited under accession number A5F7A4 and is reproduced below as SEQ ID NO: 1 (781 amino acids). MRFKNVKKTA LMLAMFGMAT SSNAALFDYN ATGDTEFDSP AKQGWMQDNT NNGSGVLTNA DGMPAWLVQG IGGRAQWTYS LSTNQHAQAS SFGWRMTTEM KVLSGGMITN YYANGTQRVL PIISLDSSGN LVVEFEGQTG RTVLATGTAA TEYHKFELVF LPGSNPSASF YFDGKLIRDN IQPTASKQNM IVWGNGSSNT DGVAAYRDIK FEIQGDVIFR GPDRIPSIVA SSVTPGVVTA FAEKRVGGGD PGALSNTNDI ITRTSRDGGI TWDTELNLTE QINVSDEFDF SDPRPIYDPS SNTVLVSYAR WPTDAAQNGD RIKPWMPNGI FYSVYDVASG NWQAPIDVTD QVKERSFQIA GWGGSELYRR NTSLNSQQDW QSNAKIRIVD GAANQIQVAD GSRKYVVTLS IDESGGLVAN LNGVSAPIIL QSEHAKVHSF HDYELQYSAL NHTTTLFVDG QQITTWAGEV SQENNIQFGN ADAQIDGRLH VQKIVLTQQG HNLVEFDAFY LAQQTPEVEK DLEKLGWTKI KTGNTMSLYG NASVNPGPGH GITLTRQQNI SGSQNGRLIY PAIVLDRFFL NVMSIYSDDG GSNWQTGSTL PIPFRWKSSS ILETLEPSEA DMVELQNGDL LLTARLDFNQ IVNGVNYSPR QQFLSKDGGI TWSLLEANNA NVFSNISTGT VDASITRFEQ SDGSHFLLFT NPQGNPAGTN GRQNLGLWFS FDEVTWKGP IQLVNGASAY SDIYQLDSEN AIVIVETDNS NMRILRMPIT LLKQKLTLSQ N The CBM region of SEQ ID NO: 1 is amino acid residues 25 to 216, and this sequence may be SEQ ID NO:2.

[0019] An exemplary Streptococcus pneumoniae NanA sialidase amino acid sequence has been deposited under accession number P62575 and is reproduced below as SEQ ID NO: 3 (1035 amino acids). MSYFRNRDID IERNSMNRSV QERKCRYSIR KLSVGAVSMI VGAVVFGTSP VLAQEGASEQ PLANETQLSG ESSTLTDTEK SQPSSETELS GNKQEQERKD KQEEKIPRDY YARDLENVET VIEKEDVETN ASNGQRVDLS SELDKLKKLE NATVHMEFKP DAKAPAFYNL FSVSSATKKD EYFTMAVYNN TATLEGRGSD GKQFYNNYND APLKVKPGQW NSVTFTVEKP TAELPKGRVR LYVNGVLSRT SLRSGNFIKD MPDVTHVQIG ATKRANNTVW GSNLQIRNLT VYNRALTPEE VQKRSQLFKR SDLEKKLPEG AALTEKTDIF ESGRNGKPNK DGIKSYRIPA LLKTDKGTLI AGADERRLHS SDWGDIGMVI RRSEDNGKTW GDRVTITNLR DNPKASDPSI GSPVNIDMVL VQDPETKRIF SIYDMFPEGK GIFGMSSQKE EAYKKIDGKT YQILYREGEK GAYTIRENGT VYTPDGKATD YRVVVDPVKP AYSDKGDLYK GNQLLGNIYF TTNKTSPFRI AKDSYLWMSY SDDDGKTWSA PQDITPMVKA DWMKFLGVGP GTGIVLRNGP HKGRILIPVY TTNNVSHLNG SQSSRIIYSD DHGKTWHAGE AVNDNRQVDG QKIHSSTMNN RRAQNTESTV VQLNNGDVKL FMRGLTGDLQ VATSKDGGVT WEKDIKRYPQ VKDVYVQMSA IHTMHEGKEY IILSNAGGPK RENGMVHLAR VEENGELTWL KHNPIQKGEF AYNSLQELGN GEYGILYEHT EKGQNAYTLS FRKFNWDFLS KDLISPTEAK VKRTREMGKG VIGLEFDSEV LVNKAPTLQL ANGKTARFMT QYDTKTLLFT VDSEDMGQKV TGLAEGAIES MHNLPVSVAG TKLSNGMNGS EAAVHEVPEY TGPLGTSGEE PAPTVEKPEY TGPLGTSGEE PAPTVEKPEY TGPLGTAGEE AAPTVEKPEF TGGVNGTEPA VHEIAEYKGS DSLVTLTTKE DYTYKAPLAQ QALPETGNKE SDLLASLGLT AFFLGLFTLG KKREQ The CBM region of SEQ ID NO:3 is amino acid residues 121 to 305, and this sequence may be SEQ ID NO:4.

[0020] Thus, a CBM for use as a sialic acid binding molecule in various aspects and embodiments of the present disclosure may comprise a protein or peptide having the sequence of SEQ ID NO: 1, 2, 3 or 4, or a sequence fragment derived therefrom, which encodes a molecule capable of binding sialic acid (in other words, the sialic acid binding molecule-encoding portion of a fragment of SEQ ID NO: 1, 2, 3 or 4). Useful sialic acid-binding molecules may comprise a protein portion encoded by the sialic acid-binding domain of the nanH gene (encoding a sialidase) of V. cholerae (as represented by SEQ ID NO: 1) or a corresponding or matching gene present in another organism (e.g., the corresponding / matching nanA sialidase gene of S. pneumoniae: see SEQ ID NO: 3). Sialic acid-binding molecules for use may comprise from about residue 1, 5, 10, 15, 25, or 30 (i.e., 1-30 or any amino acid residue therebetween) to about residue 150, 175, 200, 210, 216, 220-781 (up to any residue from 150 to 781, including any residue therebetween) of the V. cholerae sialidase molecules of SEQ ID NO: 1 and 2. For example, a sialic acid-binding molecule for use may comprise a peptide having a sequence corresponding to residue 25 to about residue 216 of SEQ ID NO: 1 above.

[0021] Additional suitable sialic acid-binding molecules may include proteins or peptides having the sequence of SEQ ID NO: 3 or 4, or sialic acid-binding fragments thereof. For example, useful sialic acid-binding molecules may include protein portions encoded by the sialic acid-binding domain of the Streptococcus pneumoniae nanA gene (which encodes a sialidase). For example, sialic acid-binding molecules for use may include from about residues 80, 90, 100, 110, 120, 121-130 (i.e., any residue between about residues 80 and 130, including any residue therebetween) to about residues 250, 275, 300, 305, 310, 320-1035 (i.e., any residue between about residues 250 and 1035, including any residue therebetween) of the S. pneumoniae sialidase molecules of SEQ ID NO: 3 and 4. For example, sialic acid-binding molecules for use may include peptides having a sequence corresponding to residues 121 to about residue 305 of SEQ ID NO: 3, above.

[0022] A sialic acid-binding molecule for use may comprise one or more CBMs. For example, a suitable sialic acid-binding molecule may comprise a single CBM, e.g., a single VcCBM or a single SpCBM. Alternatively, a sialic acid-binding molecule for use may comprise a number or a plurality (i.e., two or more) CBMs. A sialic acid-binding molecule comprising multiple CBMs may also be referred to as a "multivalent sialic acid-binding molecule" or "multivalent CBM." A multivalent CBM may comprise, for example, two or more (e.g., 3, 4, 5, or 6) VcCBMs or two or more SpCBMs. A multivalent CBM may comprise a mixture of different CBMs, e.g., one or more VcCBMs and one or more SpCBMs. The sialic acid-binding molecule for use may further comprise an oligomerization domain. A suitable oligomerization domain may exhibit the ability to self-associate to form a multimeric structure, e.g., a trimer. The oligomerization domain for use may comprise any molecule having the above-mentioned oligomerization properties, or any functional fragment thereof. For example, one or more (e.g., two) sialic acid-binding molecules (e.g., CBMs) may be conjugated, linked, or fused to an oligomerization domain, and the resulting sialic acid-binding molecule::oligomerization domain "fusion" may then be used (together with one or more other such "fusions") as a molecule for regulating cell growth and / or activity and / or for treating or preventing any of the diseases and / or conditions disclosed herein.

[0023] A suitable oligomerization domain may be derived, for example, from Pseudomonas aeruginosa pseudoaminidase. The amino acid sequence of an exemplary Pseudomonas aeruginosa pseudoaminidase sequence has been deposited under accession number PAO579 and is reproduced below as SEQ ID NO: 5 (438 amino acids). MNTYFDIPHR LVGKALYESY YDHFGQMDIL SDGSLYLIYR RATEHVGGSD GRVVFSKLEG GIWSAPTIVA QAGGQDFRDV AGGTMPSGRI VAASTVYETG EVKVYVSDDS GVTWVHKFTL ARGGADYNFA HGKSFQVGAR YVIPLYAATG VNYELKWLES SDGGETWGEG STIYSGNTPY NETSYLPVGD GVILAVARVG SGAGGALRQF ISLDDGGTWT DQGNVTAQNG DSTDILVAPS LSYIYSEGGT PHVVLLYTNR TTHFCYYRTI LLAKAVAGSS GWTERVPVYS APAASGYTSQ VVLGGRRILG NLFRETSSTT SGAYQFEVYL GGVPDFESDW FSVSSNSLYT LSHGLQRSPR RVVVEFARSS SPSTWNIVMP SYFNDGGHKG SGAQVEVGSL NIRLGTGAAV WGTGYFGGID NSATTRFATG YYRVRAWI The oligomerization domain of SEQ ID NO:5 is amino acid residues 333 to 438, and this sequence may be SEQ ID NO:6.

[0024] Thus, an oligomerization domain for use may comprise from about residues 250, 275, 300, 310, 320, 333, 340-350 (i.e., from about residue 250 to about residue 350, including about any residue therebetween) to about residue 400, 410, 420, 430, or 438 (i.e., from about residue 400 to about any residue therebetween, including about any residue therebetween) of the P. aeruginosa pseudoaminidase trimerization domain (PaTD) set forth by SEQ ID NO: 5. For example, a useful sialic acid-binding molecule may utilize an oligomerization domain comprising residues 333-438 of SEQ ID NO: 6. Sialic acid-binding molecules for use may comprise one or more CBM-based molecules shown in Figure 1. For example, a suitable sialic acid-binding molecule may comprise (consist essentially of, or consist of) two or more VcCBMs, optionally fused, linked, or attached to an oligomerization domain (such as PaTD or an oligomerization fragment thereof). A sialic acid-binding molecule may comprise, consist, or consist essentially of two fused (or linked) VcCBMs, which are further fused to an oligomerization domain (see, e.g., the molecule Vc2CBMTD shown in Figure 1).

[0025] Other sialic acid binding domains for use may comprise two or more SpCBMs, optionally fused, linked, or attached to an oligomerization domain (such as PaTD or an oligomerization fragment thereof). A sialic acid binding molecule for use may comprise, consist of, or consist essentially of two fused (or linked) SpCBMs, which are further fused to an oligomerization domain (see, e.g., the molecule Sp2CBMTD shown in Figure 1). The terms "sialic acid binding molecule," "CBM," and / or "CBM40" may include modified forms of any of the molecules described herein. Additionally, the term "modified" encompasses molecules that contain one or more mutations relative to a reference sequence. A "reference sequence" may be any wild-type CBM sequence. For example, a reference sequence may comprise, consist essentially of, or consist of a wild-type Family 40 CBM sequence, such as the wild-type CBM sequence of Vibrio cholerae NanH sialidase or Streptococcus pneumoniae NanA sialidase (it should be recognized that similar or identical CBMs present in other microorganisms, including CBM40, are encompassed within the terms "CBM" and / or CBM reference sequence). Reference sequences from which useful sialic acid-binding molecules (including useful multivalent CBMs as described herein) may be derived may include any of the specific sequences described herein (e.g., SEQ ID NOs: 1, 2, 3, 4, and 5).

[0026] For example, a modified CBM sequence for use may be derived from a given or particular wild-type CBM. Useful modified CBM sequences may include wild-type CBM sequences containing one or more mutations. One or more mutations may be functional. For example, a mutation may alter the overall primary sequence of the CBM for use, but may not (substantially) alter the properties of the CBM; thus, the sequence of the modified CBM may differ from the wild-type sequence from which it is derived, but the overall function of the modified CBM will (substantially) correspond to that of the wild-type CBM. Alternatively, one or more mutations may individually (and / or independently) or collectively (e.g., synergistically) modulate (enhance or suppress / inhibit) one or more physiological, biological, immunological, and / or pharmacological properties of the wild-type CBM (e.g., the wild-type CBM from which the modified CBM is derived). In particular, one or more mutations may be: (i) may alter the immunogenicity (or antigenicity) of the CBM; and / or (ii) the potency (of the CBM or any multimeric molecule comprising the modified CBM) may be altered (e.g., improved); and / or (iii) they may modulate (e.g., improve) the thermal stability of the CBM; and / or (iv) they may modulate (e.g., enhance) the solubility of the CBM; and / or (v) They may modulate (e.g., enhance) the in vivo half-life of the molecule.

[0027] A "mutation" may include any alteration to a wild-type CBM molecule. For example, the term "mutation" may include, for example: (i) one or more amino acid substitutions (wherein one or more wild-type amino acids are replaced or changed with another (different) amino acid, the term "substitution" shall include conservative amino acid substitutions); and / or (ii) one or more amino acid deletions (wherein one or more wild-type amino acid residues are removed); and / or (iii) one or more amino acid additions / insertions (wherein additional amino acid residues are added to the wild-type (or reference) primary sequence); and / or (iv) one or more amino acid / sequence inversions (typically where two or more consecutive amino acids in a primary sequence are inverted; and / or (v) one or more amino acid / sequence duplications (wherein an amino acid or a portion thereof (e.g., a stretch of 5-10 amino acids) of the primary amino acid sequence is repeated);

[0028] Thus, a useful modified CBM (ie, a CBM for use in the medical uses and methods described herein) may contain one or more of the mutations described herein. As non-limiting examples, the following represent individual units (referred to as "HEX" units) that may be used to create hexameric sialic acid-binding molecules for use in various embodiments described herein (e.g., for use in methods of treating or preventing RSV infection, etc.). In each case, the HEX unit includes two modified CBMs (designated CBM1 and CBM2), with the specific mutations introduced into each CBM identified in parentheses. Note that the "----" symbol indicates an amino acid linker (connecting one CBM to another CBM, or a CBM to an oligomerization domain). Thus, a hexameric sialic acid-binding molecule may be composed of several (e.g., three) HEX units. In each case, an oligomerization domain (designated "TD") connects the units together as trimers. While any given hexamer may contain identical copies of the units described above (and below under the headings HEX1, HEX2, HEX3, HEX4, HEX5, HEX6, and HEX17), one of skill in the art will appreciate that further options are available. For example, a HEX unit may be composed of two CBMs, each with a different mutation (one or more of which are selected from the options detailed herein).

[0029] (i) HEX1 CBM1 (L170T V239A V246G I286A Y292E)----CBM2 (L170T V239A V246G I286A Y292E)----TD (S342D L348D R403K) (ii) HEX2 CBM1 (V239A V246G I286A Y292E)----CBM2 (V239A V246G I286A Y292E)----TD (S342D R403K) (iii) HEX3 CBM1 (V239A V246G I286A)----CBM2 (V239A V246G I286A)------TD (S342D R403K) (iv) HEX4 CBM1 (V239A V246G)----CBM2 (V239A V246G)----TD (S342D) (v) HEX5 CBM1 (V239A V246G)----CBM2 (V239A V246G)----TD (R403K) (vi) HEX6 CBM1 (V239A V246G)----CBM2 (V239A V246G)----TD (S342D R403K) (vii) HEX17 CBM1 (V239A V246G A162P)----CBM2 (V239A V246G A162P)------TD (S342D R403K)

[0030] Note that HEX6 and HEX17 are identical except for an additional A162P mutation. This proline mutation (substitution of wild-type alanine at residue 162) was shown to improve thermal stability (single CBM Tm by 3–4°C). Further information on the use of proline mutations can be derived from Fu 2009, 'Increasing protein stability by improving beta-turns' (DOI 10.1002 / prot.22509), which describes the general approach. The proline mutation does not affect (increase or decrease) the predicted immunogenicity of the CBM molecule, and it is not located near other mutations, the N- or C-termini, or the ligand-binding site. Rather unexpectedly, beyond a moderate improvement in thermal stability, the A162P mutation was confirmed to result in a hexameric CBM (i.e., a molecule containing 3 × HEX17 units) that exhibited a significant improvement in in vivo experiments, particularly compared to the same experiments performed using a hexameric molecule containing 3 × HEX6 units. For example, modified molecules (especially molecules containing 3xHEX17 units) exhibit modulation of pro-inflammatory cytokines, including IL-8, and indeed the regulatory effect (especially the inhibitory effect) on IL-8 production by molecules containing 3xHEX17 units was improved over the other modified molecules tested.

[0031] The amino acid sequences of the HEX6 and HEX17 molecules compared to the Sp2CBMTD (also known as "SpOrig") amino acid sequence are as follows: SpOrig GAMVIEKEDVETNASNGQRVDLSSELDKLKKLENATVHMEFKPDAKAPAFYNLFSVSSAT HEX6 GAMVIEKEDVETNASNGQRVDLSSELDKLKKLENATVHMEFKPDAKAPAFYNLFSVSSAT Hex17 GAMVIEKEDVETNASNGQRVDLSSELDKLKKLENATVHMEFKPDPKAPAFYNLFSVSSAT SpOrig KKDEYFTMAVYNNTATLEGRGSDGKQFYNNYNDAPLKVKPGQWNSVTFTVEKPTAELPKG HEX6 KKDEYFTMAVYNNTATLEGRGSDGKQFYNNYNDAPLKVKPGQWNSVTFTVEKPTAELPKG Hex17 KKDEYFTMAVYNNTATLEGRGSDGKQFYNNYNDAPLKVKPGQWNSVTFTVEKPTAELPKG SpOrig RVRLYVNGVLSRTSLRSGNFIKDMPDVTHVQIGATKRANNTVWGSNLQIRNLTVYNRALT HEX6 RARLYVNGGLSRTSLRSGNFIKDMPDVTHVQIGATKRANNTVWGSNLQIRNLTVYNRALT Hex17 RARLYVNGGLSRTSLRSGNFIKDMPDVTHVQIGATKRANNTVWGSNLQIRNLTVYNRALT SpOrig PEEVQKRSGGGSGGVIEKEDVETNASNGQRVDLSSELDKLKKLENATVHMEFKPDAKAPAF HEX6 PEEVQKRSGGGSGGVIEKEDVETNASNGQRVDLSSELDKLKKLENATVHMEFKPDAKAPAF Hex17 PEEVQKRSGGGSGVIEKEDVETNASNGQRVDLSSELDKLKKLENATVHMEFKPDPKAPAF SpOrig YNLFSVSSATKKDEYFTMAVYNNTATLEGRGSDGKQFYNNYNDAPLKVKPGQWNSVTFTV HEX6 YNLFSVSSATKKDEYFTMAVYNNTATLEGRGSDGKQFYNNYNDAPLKVKPGQWNSVTFTV Hex17 YNLFSVSSATKKDEYFTMAVYNNTATLEGRGSDGKQFYNNYNDAPLKVKPGQWNSVTFTV SpOrig EKPTAELPKGRVRLYVNGVLSRTSLRSGNFIKDMPDVTHVQIGATKRANNTTVWGSNLQIR HEX6 EKPTAELPKGRARLYVNGGLSRTSLRSGNFIKDMPDVTHVQIGATKRANNTVWGSNLQIR Hex17 EKPTAELPKGRARLYVNGGLSRTSLRSGNFIKDMPDVTHVQIGATKRANNTVWGSNLQIR SpOrig NLTVYNRALTPEEVQKRSGGALGVPDFESDWFSVSSNSLYTLSHGLQRSPRVVVEFARS HEX6 NLTVYNRALTPEEVQKRSGGSLGVPDFESDWFDVSSNSLYTLSHGLQRSPRRVVVEFARS Hex17 NLTVYNRALTPEEVQKRSGGSLGVPDFESDWFDVSSNSLYTLSHGLQRSPRRVVVEFARS SpOrig SSPSTWNIVMPSYFNDGGHKGSGAQVEVGSLNIRLGTGAAVWGTGYFGGIDNSATTRFAT HEX6 SSPSTWNIVMPSYFNDGGHKGSGAQVEVGSLNIKLGTGAAVWGTGYFGGIDNSATTRFAT Hex17 SSPSTWNIVMPSYFNDGGHKGSGAQVEVGSLNIKLGTGAAVWGTGYFGGIDNSATTRFAT SpOrig GYYRVRAWI HEX6 GYYRVRAWI Hex17 GYYRVRAWI

[0032] Accordingly, various aspects and embodiments of the disclosure (uses, sialic acid binding molecules for use, methods and agents) include: (i) one or more VcCBMs; (ii) one or more SpCBMs; (iii) one or more modified CBMs; (iv) a HEX17 molecule; and (iii) Multivalent CBM A sialic acid binding molecule may be utilized that comprises, consists of, or consists essentially of a sialic acid binding molecule selected from the group consisting of:

[0033] Thus, the present disclosure provides a method for the treatment and / or prevention of RSV infection. Sp2CBM; HEX17; Vc2CBM; and / or Vc4CBM; to provide. Further provided is the use of Vc2CBM or Vc4CBM in the manufacture of a medicament for use in the treatment and / or prevention of RSV infection. The present disclosure also provides the use of HEX17 in the manufacture of a medicament for use in the treatment and / or prevention of RSV infection.

[0034] The present disclosure also provides a method of treating or preventing RSV, comprising administering to a subject in need thereof a therapeutically effective amount of HEX17; Sp2CBM; Vc2CBM; and / or Vc4CBM The present invention relates to a method comprising administering For the avoidance of doubt, HEX17 is a multivalent modified CBM as described above. The Vc2CBM comprises, consists essentially of, or consists of two Vibrio cholerae NanH sialidase CBM units linked, linked, or attached together. Exemplary Vc2CBM sequences may comprise, consist essentially of, or consist of the following: GAMALFDYNATGDTEFDSPAKQGWMQDNTNNGSGVLTNADGMPAWLVQGIGGRAQWTYSLSTNQHAQASSFGWRMTTEMKVLSGGMITNYYANGTQRVLPIISLDSGNLVVEFEGQTGRTVLATGTAATEYHKFELVFLPGSNPSASFYFDGKLIRDNIQPTASKQNMIVWGNGSSNTDGVAAYRDIKFEIQGDA LNGSMALFDYNATGDTEFDSPAKQGWMQDNTNNGSGVLTNADGMPAWLVQGIGGRAQWTYSLSTNQHAQASSFGWRMTTEMKVLSGGMITNYYANGTQRVLPIISLDSGNLVVEFEGQTGRTVLATGTAATEYHKFELVFLPGSNPSASFYFDGKLIRDNIQPTASKQNMIVWGNGSSNTDGVAAYRDIKFEIQGD

[0035] Vc4CBM comprises, consists essentially of, or consists of four Vibrio cholerae NanH sialidase CBM units linked, linked, or bound together. An exemplary Vc4CBM sequence may comprise, consist essentially of, or consist of the following sequence: GAMALFDYNATGDTEFDSPAKQGWMQDNTNNGSGVLTNADGMPAWLVQGIGGRAQWTYSLSTNQHAQASSFGWRMTTEMKVLSGGMITNYYANGTQRVLPIISLDSGNLVVEFEGQTGRTVLATGTAATEYHKFELVFLPGSNPSASFYFDGKLIRDNIQPTASKQNMIVWGNGSSNTDGVAAYRDIKFEIQGDAL NGSMALFDYNATGDTEFDSPAKQGWMQDNTNNGSGVLTNADGMPAWLVQGIGGRAQWTYSLSTNQHAQASSFGWRMTTEMKVLSGGMITNYYANGTQRVLPIISLDSGNLVVEFEGQTGRTVLATGTAATEYHKFELVFLPGSNPSASFYFDGKLIRDNIQPTASKQNMIVWGNGSSNTDGVAAYRDIKFEIQGDL QALGMALFDYNATGDTEFDSPAKQGWMQDNTNNGSGVLTNADGMPAWLVQGIGGRAQWTYSLSTNQHAQASSFGWRMTTEMKVLSGGMITNYYANGTQRVLPIISLDSGNLVVEFEGQTGRTVLATGTAATEYHKFELVFLPGSNPSASFYFDGKLIRDNIQPTASKQNMIVWGNGSSNTDGVAAYRDIKFEIQGD GGNSGMALFDYNATGDTEFDSPAKQGWMQDNTNNGSGVLTNADGMPAWLVQGIGGRAQWTYSLSTNQHAQASSFGWRMTTEMKVLSGGMITNYYANGTQRVLPIISLDSGNLVVEFEGQTGRTVLATGTAATEYHKFELVFLPGSNPSASFYFDGKLIRDNIQPTASKQNMIVWGNGSSNTDGVAAYRDIKFEIQGD

[0036] Sp2CBM comprises, consists essentially of, or consists of two Streptococcus pneumoniae NanA sialidase units linked, linked, or bound together. An exemplary Sp2CBM sequence may comprise, consist essentially of, or consist of two copies of the following sequence: GSMVIEKEDVETNASNGQRVDLSSELDKLKKLENATVHMEFKPDAKAPAFYNLFSVSSATKKDEYFTMAVYNNTATLEGRGSDGKQFYNNYNDAPLKVKPGQWNSVTFTVEKPTAELPKGRVRLYVNGVLSRTSLRSGNFIKDMPDVTHVQIGATKRANNTVWGSNLQIRNLTVYNRALTPEEVQKRS The two copies of the above sequence may be linked via any one of the peptide linker sequences described herein. For example, the Sp2CBM sequence may comprise, consist essentially of, or consist of: GSMVIEKEDVETNASNGQRVDLSSELDKLKKLENATVHMEFKPDAKAPAFYNLFSVSSATKKDEYFTMAVYNNTATLEGRGSDGKQFYNNYNDAPLKVKPGQWNSVTFTVEKPTAELPKGRVRLYVNGVLSRTSLRSGNFIKDMPDVTHVQIGATKRANNTVWGSNLQIRNLTVYNRALTPEEVQKRS[ xxxxx][xxxxxxxxxx][xxxxxxxxxxxxxxx] GSMVIEKEDVETNASNGQRVDLSSELDKLKKLENATVHMEFKPDAKAPAFYNLFSVSSATKKDEYFTMAVYNNTATLEGRGSDGKQFYNNYNDAPLKVKPGQWNSVTFTVEKPTAELPKGRVRLYVNGVLSRTSLRSGNFIKDMPDVTHVQIGATKRANNTVWGSNLQIRNLTVYNRALTPEEVQKRS where [xxxxx], [xxxxxxxxxx] and [xxxxxxxxxxxxxxx] represent linker peptide sequence options as outlined below. Both CBM sequences will be linked by one of the 5, 10 or 15 amino acid linker sequences described herein.

[0037] Vc2CBM and Vc4CBM are sometimes referred to as tandem repeat multivalent proteins based on the family 40 sialic acid binding domain (CBM) of the nanH gene, which encodes the sialidase of V. cholerae. Sp2CBM is sometimes referred to as a tandem repeat multivalent protein based on the family 40 sialic acid binding domain (CBM) of the nanA gene, which encodes the sialidase of S. pneumoniae. Molecules of the present disclosure (for the uses and methods described herein) may be formed using PCR-based cloning techniques; suitable methods for forming this type of multivalent molecule are described, for example, in Connaris et al., 2009 (Enhancing the Receptor Affinity of the Sialic Acid-Binding Domain of Vibrio cholerae Sialidase through Multivalency; J. Biol. Chem; Vol. 284(11); pp 7339-7351). Multivalent CBM molecules, including, for example, HEX17, Vc2CBM, Vc4CBM, and Sp2CBM, may be generated as constructs containing multiple CBMs linked by amino acid / peptide linkers. Each CBM (e.g., VcCBM) may be linked to another by a peptide containing, for example, 5, 10, or 15 amino acids. By way of example, any one or more of the following peptides may be used to link two or more CBMs to generate a multivalent CBM: (i) 5 amino acid linker: ALXGS LQALG GGXSG GGALG GGGGS (ii) 10 amino acid linker: ALXGSGGGSG LQALGGGGSL (iii) 15 amino acid linker: ALXGSGGGSGGGGSG where "X" is any amino acid.

[0038] An example Vc4CBM may take the following form: [ka]

[0039] This schematic will be referred to hereafter as General Formula 1. Thus, a Vc4CBM molecule may conform to general formula 1 above, where the peptide linkers A, B, and / or C are selected from the linker options presented above as (i), (ii), and / or (iii). It should be noted that the term "VcCBM40" includes not only the complete family 40 CBM from Vibrio cholerae (NanH sialidase), but also sialic acid-binding fragments thereof. Indeed, each of the VcCBM units shown in general formula 1 may be: (i) Vibrio cholerae NanH sialidase CBM; and (ii) its Vibrio cholerae NanH sialidase CBM sialic acid-binding fragment may be selected from the group consisting of:

[0040] Thus, each of the VcCBM units of the molecule represented by general formula 1 may be the same or different. Furthermore, it should be noted that the various uses, methods, and medicaments described herein may utilize one or more of the sialic acid-binding molecules described herein, for example, two or more different sialic acid-binding molecules may be administered to a subject together, simultaneously, or separately. The present disclosure can provide compositions for use in the various uses, medicaments, and methods described herein. Accordingly, any sialic acid-binding molecule described herein can be formulated for use. For example, a sialic acid-binding molecule can be formulated as a therapeutic or pharmaceutical composition. Various compositions can include one or more sialic acid-binding molecules described herein, and any given treatment may require the administration (together, simultaneously, or separately) of one or more of these compositions.

[0041] Pharmaceutical compositions according to the invention, particularly formulations thereof for mucosal or intranasal administration, contain substances customarily used in medicine and may be conventionally prepared, for example, as described in Remington's The Sciences and Practice of Pharmacy, 22nd Edition (Pharmaceutical Press 2012) and / or Handbook of Pharmaceutical Excipients, 7th edition (compiled by Rowe et al, Pharmaceutical Press, 2012), the entire contents of all of which literature and references are incorporated by reference.

[0042] Any suitable amount of sialic acid-binding molecule (e.g., any CBM-type molecule described herein, including CBM40) may be used. For example, whether a composition comprising a sialic acid-binding molecule (e.g., a CBM such as Sp2CBM or Vc2CBMTD) is administered intravenously or mucosally (e.g., intranasally), the dose of sialic acid-binding molecule may comprise anywhere from about 0.1 μg to about 1000 μg. For example, doses of sialic acid-binding molecules of about (e.g., + / - 0.5 μg) 0.1 μg, 0.5 μg, 1 μg, 5 μg, 10 μg, 11 μg, 12 μg, 13 μg, 14 μg, 15 μg, 20 μg, 30 μg, 40 μg, 50 μg, 100 μg, 200 μg, 300 μg, 400 μg, 500 μg, 600 μg, 700 μg, 800 μg, 900 μg, or 950 μg may be used. These amounts may be provided in any suitable volume of excipient, diluent, or buffer. For example, the amount of sialic acid-binding molecule may be provided in anywhere from about 1 μl to about 5 ml of excipient, diluent, or buffer. For example, the required amount of sialic acid binding molecule may be combined (or formulated) with about 5 μl, 10 μl, 15 μl, 20 μl, 25 μl, 30 μl, 35 μl, 40 μl, 45 μl, 50 μl, 55 μl, 60 μl, 65 μl, 70 μl, 75 μl, 80 μl, 85 μl, 90 μl, 95 μl, 100 μl, 200 μl, 300 μl, 400 μl, 500 μl, 600 μl, 700 μl, 800 μl, 900 μl, 1 ml, 2 ml, 3 ml, or 4 ml. A concentration of 0.1 to 1 mg (sialic acid binding protein) per ml (excipient, diluent, or buffer) may be most useful. The present invention will now be described in detail with reference to the following figures, which show: [Brief explanation of the drawings]

[0043] [Figure 1]Figure 1 shows the building blocks of multivalent CBM types and their affinities for sialic acid. a) VcCBM, residues 25–216 of V. cholerae sialidase (PDB: 1w0p) with α-2,3-sialyllactose depicted as a sphere. b) SpCBM, residues 121–305 of S. pneumoniae NanA sialidase with α-2,3-sialyllactose (PDB: 4c1w). c) TD, trimerization domain, residues 333–438 of iridescent P. aeruginosa pseudoaminidase (PDB: 2w38); the other two monomers in monochromatic form. d) Multivalent types: their molecular weights, valencies, and binding affinities for α2,3-sialyllactose determined by surface plasmon resonance (SPR) at 25 °C (KD values ​​for VcCBM, Vc2CBM, and Vc3CBM have been previously reported). The tandem repeat CBM and the oligomeric CBM fused to the TD are linked by a 5-amino linker. [Figure 2-1] This graph shows the preventive effect of mCBM40 on RSV-infected mammalian cell lines. Hep2:NPro cells were incubated with various amounts (0.1–100 μg / well) of either Sp2CBMTD or Vc2CBMTD for 1 hour before the addition of RSV (1.5 × 10 PFU per well). Absorbance readings of infected cells for Sp2CBMTD (a–d) and Vc2CBMTD (e–h)-treated cells compared to untreated infected cells were measured at 24, 48, 72, and 90 h postinfection. Bars indicate the mean absorbance change ± SD from six replicates. All values ​​are presented as mean ± SD, with statistical results indicated as follows: *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001. [Figure 2-2]This graph shows the preventive effect of mCBM40 on RSV-infected mammalian cell lines. Hep2:NPro cells were incubated with various amounts (0.1–100 μg / well) of either Sp2CBMTD or Vc2CBMTD for 1 hour before the addition of RSV (1.5 × 10 PFU per well). Absorbance readings of infected cells for Sp2CBMTD (a–d) and Vc2CBMTD (e–h)-treated cells compared to untreated infected cells were measured at 24, 48, 72, and 90 h postinfection. Bars indicate the mean absorbance change ± SD from six replicates. All values ​​are presented as mean ± SD, with statistical results indicated as follows: *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001. [Figure 2-3] This graph shows the preventive effect of mCBM40 on RSV-infected mammalian cell lines. Hep2:NPro cells were incubated with various amounts (0.1–100 μg / well) of either Sp2CBMTD or Vc2CBMTD for 1 hour before the addition of RSV (1.5 × 10 PFU per well). Absorbance readings of infected cells for Sp2CBMTD (a–d) and Vc2CBMTD (e–h)-treated cells compared to untreated infected cells were measured at 24, 48, 72, and 90 h postinfection. Bars indicate the mean absorbance change ± SD from six replicates. All values ​​are presented as mean ± SD, with statistical results indicated as follows: *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001. [Figure 2-4]This graph shows the preventive effect of mCBM40 on RSV-infected mammalian cell lines. Hep2:NPro cells were incubated with various amounts (0.1–100 μg / well) of either Sp2CBMTD or Vc2CBMTD for 1 hour before the addition of RSV (1.5 × 10 PFU per well). Absorbance readings of infected cells for Sp2CBMTD (a–d) and Vc2CBMTD (e–h)-treated cells compared to untreated infected cells were measured at 24, 48, 72, and 90 h postinfection. Bars indicate the mean absorbance change ± SD from six replicates. All values ​​are presented as mean ± SD, with statistical results indicated as follows: *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001. [Figure 3-1] This graph shows the effect of mCBM40 on RSV-infected mammalian cell lines. Hep2:NPro cells were incubated with various amounts (0.1–100 μg / well) of either Sp2CBMTD or Vc2CBMTD with RSV (1.5 × 10 PFU per well) before adding it to the cells. Absorbance readings of infected cells for Sp2CBMTD (a–d) and Vc2CBMTD (e–h)-treated cells compared to untreated infected cells were measured at 24 h, 48 h, 72 h, and 90 h postinfection. Bars indicate the mean absorbance change ± SD from six replicates. All values ​​are presented as mean ± SD, with statistical results indicated as follows: *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001. [Figure 3-2]This graph shows the effect of mCBM40 on RSV-infected mammalian cell lines. Hep2:NPro cells were incubated with various amounts (0.1–100 μg / well) of either Sp2CBMTD or Vc2CBMTD with RSV (1.5 × 10 PFU per well) before adding it to the cells. Absorbance readings of infected cells for Sp2CBMTD (a–d) and Vc2CBMTD (e–h)-treated cells compared to untreated infected cells were measured at 24 h, 48 h, 72 h, and 90 h postinfection. Bars indicate the mean absorbance change ± SD from six replicates. All values ​​are presented as mean ± SD, with statistical results indicated as follows: *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001. [Figure 3-3] This graph shows the effect of mCBM40 on RSV-infected mammalian cell lines. Hep2:NPro cells were incubated with various amounts (0.1–100 μg / well) of either Sp2CBMTD or Vc2CBMTD with RSV (1.5 × 10 PFU per well) before adding it to the cells. Absorbance readings of infected cells for Sp2CBMTD (a–d) and Vc2CBMTD (e–h)-treated cells compared to untreated infected cells were measured at 24 h, 48 h, 72 h, and 90 h postinfection. Bars indicate the mean absorbance change ± SD from six replicates. All values ​​are presented as mean ± SD, with statistical results indicated as follows: *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001. [Figure 3-4]This graph shows the effect of mCBM40 on RSV-infected mammalian cell lines. Hep2:NPro cells were incubated with various amounts (0.1–100 μg / well) of either Sp2CBMTD or Vc2CBMTD with RSV (1.5 × 10 PFU per well) before adding it to the cells. Absorbance readings of infected cells for Sp2CBMTD (a–d) and Vc2CBMTD (e–h)-treated cells compared to untreated infected cells were measured at 24 h, 48 h, 72 h, and 90 h postinfection. Bars indicate the mean absorbance change ± SD from six replicates. All values ​​are presented as mean ± SD, with statistical results indicated as follows: *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001. [Figure 4-1] This graph shows the therapeutic effect of mCBM40 in RSV-infected mammalian cell lines. Hep2:NPro cells were incubated with RSV (1.5 x 103 PFU per well) followed by the addition of various amounts (0.1-100 μg / well) of either Sp2CBMTD or Vc2CBMTD at 24, 48, or 72 h postinfection. Absorbance readings of infected cells for Sp2CBMTD (a-c) and Vc2CBMTD (d-f)-treated cells compared to untreated infected cells measured at 90 h postinfection are shown. Bars indicate the mean absorbance change ± SD from six replicates. All values ​​are presented as mean ± SD with statistical results indicated as follows: *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001. [Figure 4-2]This graph shows the therapeutic effect of mCBM40 in RSV-infected mammalian cell lines. Hep2:NPro cells were incubated with RSV (1.5 x 103 PFU per well) followed by the addition of various amounts (0.1-100 μg / well) of either Sp2CBMTD or Vc2CBMTD at 24, 48, or 72 h postinfection. Absorbance readings of infected cells for Sp2CBMTD (a-c) and Vc2CBMTD (d-f)-treated cells compared to untreated infected cells measured at 90 h postinfection are shown. Bars indicate the mean absorbance change ± SD from six replicates. All values ​​are presented as mean ± SD with statistical results indicated as follows: *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001. [Figure 4-3] This graph shows the therapeutic effect of mCBM40 in RSV-infected mammalian cell lines. Hep2:NPro cells were incubated with RSV (1.5 x 103 PFU per well) followed by the addition of various amounts (0.1-100 μg / well) of either Sp2CBMTD or Vc2CBMTD at 24, 48, or 72 h postinfection. Absorbance readings of infected cells for Sp2CBMTD (a-c) and Vc2CBMTD (d-f)-treated cells compared to untreated infected cells measured at 90 h postinfection are shown. Bars indicate the mean absorbance change ± SD from six replicates. All values ​​are presented as mean ± SD with statistical results indicated as follows: *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001. [Figure 4-4]This graph shows the therapeutic effect of mCBM40 in RSV-infected mammalian cell lines. Hep2:NPro cells were incubated with RSV (1.5 x 103 PFU per well) followed by the addition of various amounts (0.1-100 μg / well) of either Sp2CBMTD or Vc2CBMTD at 24, 48, or 72 h postinfection. Absorbance readings of infected cells for Sp2CBMTD (a-c) and Vc2CBMTD (d-f)-treated cells compared to untreated infected cells measured at 90 h postinfection are shown. Bars indicate the mean absorbance change ± SD from six replicates. All values ​​are presented as mean ± SD with statistical results indicated as follows: *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001. [Figure 5] Figure 1 shows ProPred predictions of antigenic peptides. A. SpCBM sequence. B. PaTD sequence. Predicted binders are colored blue, with the first residue of each binding region shown in red. Antigenic peptides predicted by Nordic Biopharma (green bars) and Proimmune (purple bars) are shown below the sequences. [Figure 6] Expression studies of wild-type and mutant domains. Lane 1, M12 standard; lane 2, WT SpCBM; lanes 3-11, Im15-Im23; lanes 12-15, Im24-Im27; lane 16, WT PaTD. A) Whole cell extract, B) Soluble extract. [Figure 7] FIG. 1 shows the positions of peptides 167 to 181 in the SpCBM structure. [Figure 8] FIG. 1 shows the expression and Ni-NTA pulldown of variants Im28 to Im34. [Figure 9]Figure 1 shows the locations of HEX17 mutations on the hexameric structure. The quaternary structure of HEX17 was modeled by combining the crystal structures of the individual SpCBMs (pdb code 4c1x) and PaTDs (pdb code 2w38) to create a hexamer (i.e., six copies of SpCBM and three copies of PaTD per molecule). The location of the bound ligand (α2,3-sialyllactose) is shown as a stick (orange). The locations of the mutations are also indicated: blue, site of the A162P mutation; light blue, sites of the other two CBM mutations; magenta, site of the TD mutation. [Figure 10] 1 is a graph showing IL-8 stimulation. A549 cells were stimulated by adding 10 μg of a biological agent (Hex17). Cell supernatants were collected at 24 or 48 h, and IL-8 content was determined by ELISA. Statistical significance between control and treated cells was determined using one-way analysis of variance with Tukey's multiple comparison test. [Figure 11A] Figure 1 shows multiplex analysis of inflammatory mediators. A549 cells were stimulated by adding 10 μg of biologics (Sp2CBMTD (also known as SpOrig), HEX6, or HEX17). Cell supernatants were collected at 6 h, 24, or 48 h and analyzed for inflammatory mediators using a human Cytokine 12-plex assay. Statistical significance between control and / or WT hexamer and hexamer variants was determined using one-way ANOVA (Tukey's multiple comparison test). [Figure 11B] Figure 1 shows multiplex analysis of inflammatory mediators. A549 cells were stimulated by adding 10 μg of biologics (Sp2CBMTD (also known as SpOrig), HEX6, or HEX17). Cell supernatants were collected at 6 h, 24, or 48 h and analyzed for inflammatory mediators using a human Cytokine 12-plex assay. Statistical significance between control and / or WT hexamer and hexamer variants was determined using one-way ANOVA (Tukey's multiple comparison test). [Figure 11C]Figure 1 shows multiplex analysis of inflammatory mediators. A549 cells were stimulated by adding 10 μg of biologics (Sp2CBMTD (also known as SpOrig), HEX6, or HEX17). Cell supernatants were collected at 6 h, 24, or 48 h and analyzed for inflammatory mediators using a human Cytokine 12-plex assay. Statistical significance between control and / or WT hexamer and hexamer variants was determined using one-way ANOVA (Tukey's multiple comparison test). [Figure 11D] Figure 1 shows multiplex analysis of inflammatory mediators. A549 cells were stimulated by adding 10 μg of biologics (Sp2CBMTD (also known as SpOrig), HEX6, or HEX17). Cell supernatants were collected at 6 h, 24, or 48 h and analyzed for inflammatory mediators using a human Cytokine 12-plex assay. Statistical significance between control and / or WT hexamer and hexamer variants was determined using one-way ANOVA (Tukey's multiple comparison test). [Figure 11E] Figure 1 shows multiplex analysis of inflammatory mediators. A549 cells were stimulated by adding 10 μg of biologics (Sp2CBMTD (also known as SpOrig), HEX6, or HEX17). Cell supernatants were collected at 6 h, 24, or 48 h and analyzed for inflammatory mediators using a human Cytokine 12-plex assay. Statistical significance between control and / or WT hexamer and hexamer variants was determined using one-way ANOVA (Tukey's multiple comparison test). [Figure 11F] Figure 1 shows multiplex analysis of inflammatory mediators. A549 cells were stimulated by adding 10 μg of biologics (Sp2CBMTD (also known as SpOrig), HEX6, or HEX17). Cell supernatants were collected at 6 h, 24, or 48 h and analyzed for inflammatory mediators using a human Cytokine 12-plex assay. Statistical significance between control and / or WT hexamer and hexamer variants was determined using one-way ANOVA (Tukey's multiple comparison test). [Figure 12] Figure 1 shows the survival percentage of CBM-treated and untreated mice after lethal challenge with H. influenzae strain PR8. CBM2, CBM3, and CBM4 represent HEX17, HEX6, and WT (SpOrig), respectively. Animals treated with a single dose of CBM received 100 μg of CBM one day before lethal challenge with PR8, while animals treated repeatedly received 2 × 0.1 μg of CBM 3 and 1 days before PR8 challenge. [Figure 13] Figure 1 shows the clinical scores of CBM-treated and untreated mice during PR8 infection. CBM2, CBM3, and CBM4 represent HEX17, HEX6, and WT (SpOrig), respectively. An ascending clinical score of 1 to 5 indicates asymptomatic (1) to coma and death (5), respectively. [Figure 14] Graph showing the percentage of weight loss in CBM-treated and untreated mice during PR8 infection. CBM2, CBM3, and CBM4 represent Hex17, Hex6, and WT(SpOrig), respectively. [Figure 15] Graph showing anti-mCBM antibody analysis of lung homogenates and serum tissues from the PR8 challenge mouse study on day 21. Statistical significance between WT hexamer and hexamer variants was determined using one-way ANOVA (Tukey's multiple comparison test). [Figure 16A] 1 is a graph of ELISA results showing the interaction of Sp2CBMTD with RSV. [Figure 16B] 1 is a graph of ELISA results showing the interaction of Vc2CBMTD with RSV. [Figure 17A] 1 is an image of an assay showing the binding of GFP-Sp2CBMTD and Vc2CBMTD to RSV. PBS was used as a control assay. [Figure 17B] 1 is an image of an assay showing the binding of GFP-Sp2CBMTD and Vc2CBMTD to RSV. PBS was used as a control assay. [Figure 17C]1 is an image of an assay showing the binding of GFP-Sp2CBMTD and Vc2CBMTD to RSV. PBS was used as a control assay. [Figures 18A-18B] 1 is a graph of ELISA results showing the interaction between Sp2CBMTD and Sp2CBMTD-R274Q (0.1 μg and 10 ng) of RSV. [Figure 19A] 1 is a graph of ELISA results showing the interaction of Sp2CBMTD with neuraminidase-treated RSV. [Figure 19B] 1 is a graph of ELISA results showing the interaction of Vc2CBMTD with neuraminidase-treated RSV. [Figure 20A] 1 is a graph of data showing the interaction between 3'-SL treated Sp2CBMTD and RSV. [Figure 20B] 1 is a graph of data showing the interaction between 3'-SL treated Vc2CBMTD and RSV. [Figure 21A] 1 is a graph of data showing that protection of mammalian cells (Hep2:NPro cells) from RSV by CBM (Sp2CBMTD) is reduced after the cells are treated with neuraminidase. [Figure 21B] 1 is a graph of data showing that protection of mammalian cells (Hep2:NPro cells) from RSV by CBM (Vc2CBMTD) is reduced after the cells are treated with neuraminidase. [Figure 22](Panels A-F) Graphs showing the effect of mCBM40 on RSV-infected mammalian cell lines. Hep2:NPro cells were incubated with various amounts (0.1-100 μg / well) of either Vc4CBM or HEX17 with RSV (1.5 × 10 PFU per well) before adding to the cells. Absorbance readings of infected cells for Vc4CBM (A, C, and E) and HEX17 (B, D, and F)-treated cells compared to untreated infected cells measured at 24 h, 48 h, and 72 h postinfection are shown. Bars indicate the mean absorbance change ± SD from six replicates. All values ​​are shown as mean ± SD with statistical results indicated as follows: *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001. [Figure 23] Graphs of RSV-B viral genome copy number quantification of infected MucilAir inserts treated with CBM at 48 h post-infection compared to virus-only infected cells: A) Sp2CBMTD, B) Vc2CBMTD. DETAILED DESCRIPTION OF THE INVENTION

[0044] Methods and Results [Example]

[0045] Example 1 CBM40 proteins, Sp2CBMTD, Vc2CBMTD, and Vc4CBM (endotoxin-free) were purified using the method described in Connaris et al. (2014). 1 Hex17 was generated as described above. The mammalian cell line Hep2:NPro (a cell line engineered to constitutively overexpress the BVDV N-terminal protease fragment, Npro, which only responds to, but does not produce, IFN) was purchased from ATCC. 4 Cells were purchased from SF-DMEM. Cells were maintained in DMEM / 10% FBS (with 1% penicillin / streptomycin) in T75 flasks and incubated at 37°C and 5% CO. RSV-A2 strains were cultured at 1.5 x 10 in SF-DMEM.4 10 corresponding to PFU / ml -2 Dilutions of 1000 mg / mL were prepared. For infection studies, 100 μL of cells (2.5 × 10 ) in DMEM / 10% FCS were added to a 96-well plate (Nunc). 5 CBM40 protein (100 μL, 10-fold serial dilutions of a 10 mg / mL stock solution in SF-DMEM) was added to the wells of the 100-well plate, and RSV (100 μL, 1.5 × 10 cells per well) was added to the wells of the 100-well plate. 3 CBM40 and / or RSV were either added to cells prior to infection, mixed with RSV on ice for 1 hour before addition to the cells, or added to cells after RSV infection. In each case, CBM40 and / or RSV were left in place for 1 hour before removal. Cells were then covered with 300 μL of AVICEL, and plates were left to incubate at 37°C and 5% CO2 for 24, 48, 72, or 90 hours.

[0046] To determine the level of RSV in infected cells, AVICEL was removed, and cells were fixed with 10% PFA in PBS for 10 minutes at room temperature and then immunostained with mouse anti-RSV F primary antibody (1:200 dilution, Serotec) followed by goat anti-mouse HRP IgG antibody (1:2000, Santa Cruz). The presence of RSV was detected using the chromogenic substrate TMB (Sigma). Absorbance was measured at 450 nm (620 nm used as a reference). Statistical Analysis. Group comparisons were performed using one-way analysis of variance and Dunnett's multiple comparison test. GraphPad Prism 7 (GraphPad Software) was used for all analyses. A test of p<0.05 was considered statistically significant. Unless otherwise stated, all values ​​are presented as mean ± SD with statistical results indicated as follows: * p<0.05, ** p<0.01, *** p<0.001, and **** p<0.0001. overview Figures 2, 3, 4 and 22 summarize the results. 1. A single dose of mCBM40 appeared to reduce or inhibit RSV adhesion to cells when given prior to or simultaneously with RSV in the Hep2:NPro mammalian cell line. 2. When given concomitantly with RSV, all mCBM40 concentrations up to 0.1-1 mg / ml appeared to have a significant effect on reducing RSV infection compared to prophylactic dosing when cells were tested up to 90 h post-infection. 3. Administration of mCBM40 24 h after RSV infection showed a reduction in viral infection when given at the highest dose. The highest dose of Vc2CBMTD appeared to favorably affect RSV levels in the Hep2:NPro mammalian cell line when given at 24, 48, and 72 h after RSV challenge. 4. Of the mCBM40s tested, Vc2CBMTD appeared to be the most effective against RSV infection in mammalian cell lines. References TIFF0007637507000002.tif101164

[0047] Example 2 Sp2CBMTD: immunogenicity region prediction Nordic Biopharma in silico screening In silico T cell epitope screening identified four prominent and two borderline immunogenic populations. Notable: Domain Residue Range Sequence SpCBM 245~254 GVLSRTSLRS PaTD 340~349 WFSVSSNSLY PatTD 351~359 LSHGLQRSP PatTD 398~406 GSLNIRLGT boundary: Domain Residue Range Sequence SpCBM 167~178 FYNLFSVSSATK SpCBM 239~251 VRLYVNGVLSRTS

[0048] Proimmune Human Donor T Cell Proliferation Assay The Proimmune study highlighted two highly antigenic and two moderately antigenic regions. Highly antigenic: Domain Residue Range Sequence SpCBM 236~250 KGRVRLYVNGVLSRT PaTD 392~406 GAQVEVGSLNIRLGT Moderately antigenic: Domain Residue Range Sequence SpCBM 167~181 FYNLFSVSSATKKDE PaTD 338~352 SDWFSVSSNSLYTLS

[0049] ProPred in silico analysis Further in silico tools, online ProPred server 4 The results from the ProPred server are shown in Figure 5. The relative locations of the Nordic Biopharma / Proimmune epitopes are also highlighted, showing reasonable agreement between the three methods. In addition to the epitopes listed above, ProPred strongly predicted one other immunogenic epitope in the SpCBM domain. Domain Residue Range Sequence SpCBM 286~294 IRNLTVYNR Mutations in individual CBM and TD domains To guide the design of mutations that would reduce immunogenicity, ProPred was used to test the effect of substituting every residue in these peptides with an alternative residue. Those that most significantly reduced the predicted number of allele binders were identified. Because the crystal structures of both the SpCBM and TD domains are known, these mutations were also modeled to reduce the likelihood of introducing mutations that would significantly disrupt protein structure.

[0050] Initially, nine single mutations were introduced into SpCBM and four single mutations into PaTD, which are listed below ("Im" stands for immunogenic mutant): TIFF0007637507000003.tif69155 NOTE: Im1–Im14 (not shown) were introduced by mutagenesis into a non-codon-optimized background before Proimmune data became available.

[0051] Synthesis of WT and mutant constructs Genes encoding WT SpCBM, WT PaTD, and variants Im15–Im27 were codon-optimized for E. coli expression and synthesized by GeneArt, which then cloned them in-house into the pHISTEV vector for expression as 6His-tagged proteins. Expression and biophysical characterization Initial expression studies were performed to assess solubility. Results indicate that all were expressed, but not all were soluble (Figure 6). Note: Solubility (or lack thereof) is not necessarily a predictor of usefulness. Those skilled in the art will understand that when manufacturing or producing proteins, certain processes require the use of insoluble material for ease of purification (from inclusion bodies and the like). Downstream protocols may then reshape the protein to adjust characteristics such as solubility.

[0052] The results of the expression studies show the following: Im16(L170A) is insoluble or very poorly soluble Im25 (TD, S345D) is insoluble Im15(Y168W) and Im17(L170T) have reduced solubility · Im18 (V173G) and Im22 (I286A) are slightly decreased. The rest show soluble expression. The 13 soluble proteins were expressed in E. coli and purified by immobilized metal affinity chromatography (IMAC) followed by TEV digestion to remove the 6His tag, then reverse IMAC and size exclusion chromatography (SEC). The 10 purified domains (WTSp, Im19, Im20, Im21, Im22, Im23, WTTD, Im24, Im26, and Im27) were further characterized by: (i) Thermofluor for measuring melting temperature (Tm) (ii) near-UV circular dichroism (CD) to compare the tertiary structure with WT (iii) Dynamic light scattering (DLS) to investigate the oligomeric state in solution (iv) Surface plasmon resonance (SPR) to measure binding affinity to sialyllactose (v) Measurement of IL-8 cytokine stimulation

[0053] A summary of the results is shown in Table 1. [Table 1] Table 1. Qualitative summary of biophysical characterization of WT domains and their variants. Color coding is from green to red (including green', orange, and yellow), where green indicates that the variant is closely similar to its WT counterpart for that particular feature, and light green (green') or yellow indicates an increasing degree of difference. Red or orange indicates significant differences. N / A: These characterizations were not performed due to insufficient protein solubility / purity. N / D: Not determined.

[0054] Sp peptides 167-181: Im15, Im16, Im17, and Im18 are all insoluble or poorly soluble (although as noted, this does not necessarily affect the utility of the proteins). They lie in the "moderately" antigenic region 167-181 (FYNLFSVSSATKKDE), a region that is apparently highly susceptible to change. Previous results indicate that M156F adjacent to L170 (and I286) increases Tm by approximately 4°C.

[0055] Therefore, it could be combined with L170T. M156F does not increase the predicted immunogenicity. M185I increases the Tm by 5°C, paralleling that of L170 (Figure 7). This mutation could also be included. Note that, like M156F, M185I does not increase predicted immunogenicity but slightly reduces the number of predicted allele binders. Sp peptides 236-250: Im19, Im20, and Im21 all behaved similarly to WT. These lie in the "highly" antigenic region 236-250 (KGRVRLYVNGVLSRT). Im19 (V239A) was chosen over the threonine mutation (Im20, V239T). While there is no difference in predicted immunogenicity, Im19 is more consistent with the WT Thermofluor Tm and near-UV spectrum. It will be combined with Im21 (V246G).

[0056] Sp peptides 286-294: Im22(I286A) is broadly similar to WT, while Im23(Y292E) appears to exhibit lower ligand affinity. This region, 286-294IRNLTVYNR, was not flagged by Proimmune but is strongly predicted to be immunogenic by ProPred. There are some indications that Im22 has a lower Tm than WT. This residue is adjacent to M156 and may therefore behave differently if M156F is included. TD peptides 338-352: Im24(S342D) and Im26(L348D) show similar characteristics to the WT trimerization domain, but suggest a somewhat reduced Tm for Im26. These lie in the "moderately" antigenic region 338-352SDWFSVSSNSLYTLS. The WT sequence is predicted to bind nine alleles, whereas Im24 predicts two alleles and the Im24 / Im26 double mutant predicts one allele. TD peptides 392-406: Im27 (R403K) is similar to the WT. This mutation is part of the "highly" antigenic region 392-406GAQVEVGSLNIRLGT. This mutation reduces the predicted allele count from 21 to 3.

[0057] Synthesis of Im28-34, a combination of multiple mutations The following mutations were introduced: i) M156F / L170T ii) M156F / L170T / M185I: In ProPred, the predicted alleles for this region are reduced from 31 in WT to 19 in this combination. iii) V239A / V246G: In ProPred, the alleles for this region are reduced from 44 to 3. iv) I286A / Y292E: In ProPred, the allele is reduced from 41 to 1. v) V239A / V246G / I286A / Y292E combines the preceding two. vi) M156F / L170T / M185I / V239A / V246G / I286A / Y292E combines all Sp mutations vii) TD: S342D / L348D / R403K: The predicted alleles are reduced from 9 to 1 for TD peptide 338-352, and from 21 to 3 for peptide TD peptide 392-406. This triple mutant combines all TD variants, all of which are surface-exposed and distal to the N-terminus of the TD, and would not be expected to interfere with hexameric SpCBM assembly.

[0058] The constructs are referred to as Im28-Im34. (SpCBM) variant mutation Im28 M156F / L170T Im29 M156F / L170T / M185I Im30 V239A / V246G Im31 I286A / Y292E Im32 V239A / V246G / I286A / Y292E Im33 M156F / L170T / M185I / V239A / V246G / I286A / Y292E (PaTD) variant mutation Im34 S342D / L348D / R403K

[0059] 2.5 Expression and biophysical characterization of Im28–Im34 Similar to the single mutations, the combination Im28–Im34 was synthesized by GeneArt and subcloned into pHISTEV for expression analysis. Nickel bead pulldowns on His-tagged soluble extracts were also performed (Figure 8). Hexameric type Design of hexameric constructs HEX1 to HEX17 Genes encoding the hexameric forms (termed HEX1 to HEX17) were synthesized by GeneArt. Sp2CBMTD TIFF0007637507000005.tif69160

[0060] To circumvent the problems associated with synthesizing repeats of tandem CBM copies, the hexameric form was synthesized in two parts: the first gene contained the first CBM, and the second part encompassed the second CBM plus TD. These could then be cloned simultaneously into pHISTEV to create the Sp2CBMTD construct, which formed a trimer upon expression. The first hexamer, HEX1, contained the mutations L170T / V239A / V246G / I286A / Y292E in the CBM and S342D / L348D / R403K in the TD. Solubility data for the individual domains indicated that HEX1 was unlikely to be soluble (again, not necessarily reflective of the utility of the molecule). A further construct, HEX3, was synthesized. HEX2 contained the same mutations as HEX3, but with the addition of Y292E.

[0061] HEX3 was synthesized and subcloned into the pHISTEV vector. Expression was insoluble under all conditions tested (varying temperature, IPTG concentration, cell density at induction, with or without heat shock). The CBM-only domain containing the same three mutations (V239A V246G I286A) was soluble. The double mutant (V239A V246G) behaved very similarly to WT. Therefore, additional variants (HEX4, HEX5, and HEX6) lacking I286A and containing either one or both TD mutations were designed and constructed by PCR / ligation. During work on HEX6, several other versions containing different combinations of HEX6 mutations were designed (numbered HEX7 to HEX16; uncharacterized). HEX17 contains the HEX6 mutation along with an additional A162P mutation. This proline mutation was shown to increase the single CBM Tm by 3-4°C. The proline mutation is not near any other mutations, the N- or C-termini, or the ligand binding site.

[0062] Characterization of hexameric variants The expression, purification, and characterization results are shown in Table 2. Based on these results, HEX6 and HEX17 were carried forward. The location of the HEX17 mutation on the hexamer is shown in Figure 9. [Table 2] Table 2. Qualitative summary of biophysical characterization of hexameric Sp2CBMTD variants. Color coding is green to red, where green indicates that the variant is closely similar to its WT counterpart with respect to that particular feature, and light green ('green') or yellow indicates an increasing degree of difference. Red or orange indicates significant differences. N / A: These characterizations were not performed due to insufficient protein solubility / purity. N / D: Not determined.

[0063] Example 3 Inflammatory mediators. Objective: To evaluate the innate immune response of mCBM-treated human lung epithelial cells (A549) by analyzing the levels of inflammatory mediators over time. Administration of Sp2CBMTD to mammalian cells stimulated a pro-inflammatory response both in vitro and in vivo 1、2 To determine whether this was still observed with engineered hexameric sialic acid-binding molecules, mammalian A549 cells were stimulated by the addition of 10 μg of a biologic (Sp2CBMTD (also known as SpOrig), HEX6 (i.e., a sialic acid-binding molecule containing 3 × HEX6 units), or HEX17 (i.e., a sialic acid-binding molecule containing 3 × HEX17 units). Cell culture medium was collected at specific time points after administration, and the concentrations of inflammatory mediators were measured by both ELISA and multiplex assay.

[0064] Human IL-8 (reference cytokine for testing) response using the Human 1xMouse CXCL1 / KC Quantikine ELISA Kit (R&D BioSystems). IL-8 concentration levels from stimulated A549 cells are shown in Figure 10. It is clear that when A549 cells are stimulated with the modified hexamer HEX17, IL-8 levels are significantly lower than when compared to Sp2CBMTD (also known as SpOrig) stimulated cells. Inflammatory mediator responses using the Human Cytokine 12-plex assay (Bio-Plex Pro™, Bio-Rad). Figure 11 shows the analysis of 12 inflammatory mediators from culture medium at specific time points (6 h, 24 h, 48 h) after stimulation of A549 cells with Sp2CBMTD (WT, also known as SpOrig), HEX6, and HEX17 (variants). Prior to analysis, samples were thawed and diluted 1:4 in PBS before using the Human HS Cytokine-12 plex assay (R&D Systems). The data show the following: HEX17 affects the levels of almost all cytokines tested compared to SpOrig and HEX6, with significantly lower observed concentrations (pg / ml) of the analytes IL-6, IL-8, GM-CSF, and IFN-gamma at 48 h compared to SpOrig and HEX6. When compared to controls at 48 h, HEX17 appeared to increase the levels of all cytokines tested except for IL-5 and VEGF (yet to be confirmed). HEX6 only showed reduced IL-6 stimulation compared to SpOrig at 48 h.

[0065] Example 4 In vivo PR8 mouse data. The objective of the study was to evaluate the efficacy of Sp2CBMTD (SpOrig) and its variants in a mouse model of lethal influenza infection. Each candidate protein was also administered in the absence of influenza infection to assess whether it alone caused morbidity or mortality. Survival, clinical scores and weight loss. These results demonstrate that neither CBM2 (HEX17), CBM3 (HEX6), nor CBM4 (WT, SpOrig) alone caused any overt morbidity or mass mortality. Administration of a single 100 μg dose of either CBM2 (HEX17), CBM3 (HEX6), or CBM4 (WT, SpOrig) 1 day before lethal challenge with PR8 influenza virus protected against PR8 infection, with the greatest efficacy seen with HEX17 (100% survival), followed by SpOrig and then HEX6 (Figure 12). Clinical scores were also lower with HEX17 compared with SpOrig and HEX6 (Figure 13). All surviving mice in the single high-dose treatment group lost weight at peak infection but quickly recovered, in contrast to untreated infected mice (Figure 14).

[0066] Anti-mCBM antibody analysis of lung homogenates and serum tissues from the PR8 challenge mouse study. The purpose of this study was to determine whether the modified immunogenic epitopes of the engineered variants of Sp2CBMTD (SpOrig) could elicit reduced antibody levels in mice following PR8 challenge (it should be noted that the epitopes were modified based on human MHC class II binding information). To this end, mice surviving PR8 challenge were selected on day 21, and lungs and serum were collected and tested for anti-mCBM antibodies, IgG, IgA, IgE, and IgM, against the coated antigen SpOrig (1 μg / well) in an ELISA format. The data presented in Figure 15 demonstrated the following: The engineered protein HEX17 showed a significant (p<0.05) reduction in mouse lung IgM levels compared to SPORIG. Because only one mouse survived the HEX6 treatment, only the data from HEX17 and SpORIG were statistically analyzed. There is some indication of a slight trend towards lower antibody levels (lung IgA and IgM) in mice treated with modified CBM compared to SpOrig.

[0067] Example 5 The purpose of the following experiment was to determine whether there was a direct interaction between the CBMs (Sp2CBMTD and Vc2CBMTD) and human RSV in a cell-free system. The ELISA experiment was designed as follows. Experiment 1 A 96-well plate was precoated overnight at 4°C with various concentrations of RSV (strain type A2), CBM, Sp2CBMTD, and Vc2CBMTD, and PBS (vehicle, negative control). The wells were blocked with blocking buffer (BB, PBS containing 1% w / v BSA). Various concentrations of CBM were diluted in BB and then added to the wells after removing the coating antigen and blotting the plate. The plate was incubated for 1 h at room temperature. After washing the wells with wash buffer (WB, PBS containing 0.5% (v / v) Tween-20), primary antibodies (rabbit anti-SpCBM antibody or rabbit anti-VcCBM antibody, diluted 1:5000 in BB, Eurogentec) were added and incubated for 1–2 h at room temperature. Goat anti-rabbit IgG-HRP (diluted 1:2500 in BB, Sigma) was used as the secondary antibody, and TMB (Sigma) was used as the substrate. Binding of CBM to RSV was determined by comparing the absorbance at 450 nm (620 nm as background reference) of treated versus untreated control wells after reaction with TMB (a substrate for HRP). The results indicate that both Sp2CBMTD and Vc2CBMTD directly interact with RSV in a dose-dependent manner (see Figures 16A and B).

[0068] Experiment 2 A 96-well plate was precoated with 10 μg of either GFP-Sp2CBMTD, GFP-Vc2CBMTD, GFP, GFP-Sp2CBMTD-R274Q (a sialic acid-binding mutant of Sp2CBMTD), or 32,000 PFU RSV as a coating antigen and PBS (vehicle, negative control). The wells were incubated with the relevant proteins for 1 h at room temperature. Images of GFP fluorescence binding were recorded using a microscope equipped with a 10x objective (in the GFP channel of an EVOS FL Cellular Imaging System). These results demonstrate that both GFP-Sp2CBMTD and GFP-Vc2CBMTD can directly bind to RSV. Negligible binding to RSV was observed with the sialic acid-binding mutant GFP-Sp2CBMTD-R274Q (see Figures 17A-E).

[0069] Example 6 The purpose of the following experiments was to confirm the finding that the interaction between CBMs (Sp2CBMTD and Vc2CBMTD) and RSV is based on binding of the CBMs to viral surface glycoproteins terminated with sialic acid. Therefore, three ELISA experiments were designed. Experiment 1 Both wild-type Sp2CBMTD and mutant Sp2CBMTD-R274Q (sialic acid binding mutant) were used in the ELISA format described in Experiment 1 of Example 5. The results show that wild-type Sp2CBMTD binds to RSV with much higher affinity than mutant Sp2CBMTD-R274Q at the same concentration (see Figures 18A and B).

[0070] Experiment 2 Clostridium perfringens (Sigma) neuraminidase (Nu) is a glycosidase capable of cleaving terminal sialic acids from cell surface sialylated glycoconjugates and has binding specificity for α-2,3, α-2,6, and α-2,8-linked Gal, GlcNac, GalNAc, AcNeu, GlcNeu, oligosaccharides, glycolipids, or glycoproteins. To test whether this is true for viral surface glycoproteins, RSV was treated with and without neuraminidase prior to the addition of CBM. An ELISA experiment similar to Experiment 1 in Example 5 was designed. 96-well plates were precoated with various concentrations of RSV, CBM (as a positive control), and PBS (as a negative control). To some RSV-coated wells, neuraminidase (30 mU / well) was added for 1 h, followed by the addition of CBM (100 ng or 10 ng). CBM binding was assessed using anti-CBM antibody followed by detection anti-IgG·HRP as described in Plan 1. The results show that in the absence of neuraminidase treatment, both Sp2CBMTD and Vc2CBMTD interact with RSV in a dose-dependent manner, as previously observed (see below and Experiment 1 in Example 5). After neuraminidase treatment of RSV in the presence of Sp2CBMTD, the interaction is somewhat abolished, but is almost completely abolished when Vc2CBMTD is used as the interacting partner (see Figures 19A and B). The data provide evidence that RSV surface glycoproteins are sialylated, suggesting that CBM may play a direct antiviral role in RSV infection.

[0071] Test 3: To determine whether a competing ligand could block the sialic acid binding site of CBM and prevent its interaction with RSV, CBM (Sp2CBMTD and Vc2CBMTD, 100 ng / well) was pre-incubated with different concentrations of α2,3 sialyllactose (3'-SL, 10 mM, 100 mM, Glycotech) at room temperature for 1 h. This mixture, along with controls (PBS, CBM only), was added to a 96-well plate coated with RSV (32,000 PFU / well). CBM was added to the wells as in the ELISA experiment described in Example 5. The number of replicates in this study was n=3, and the raw data was analyzed by one-way ANOVA using GraphPad Prism. A p-value of <0.05 was considered statistically significant ( * p<0.05; ** p<0.01; *** p<0.001; **** p<0.0001). The results demonstrate that the competing sialoside, 3'-SL, can reduce the interaction between RSV and Sp2CBMTD and Vc2CBMTD in a dose-dependent manner (see Figures 20A and B).

[0072] Example 7 In vitro studies were undertaken to demonstrate that protection of mammalian Hep2:NPro cells from RSV by CBMs (Sp2CBMTD and Vc2CBMTD) was reduced after the cells were treated with neuraminidase. This experiment was designed to incubate Hep2:NPro cells adherent to 96-well plates with or without neuraminidase at 37°C and 5% CO2 for 1 h, followed by CBM (100 μg / well) for another 1 h, and finally RSV (3000 PFU / well) for 1 h. The plates were then covered with AVICEL® (Sigma) medium and left to incubate at 37°C and 5% CO2 for 96 h. RSV infection was determined by assessing the amount of RSV that infected the cells using a mouse anti-RSV F antibody (1:200 dilution, MCA490, BioRad) followed by an anti-mouse IgG·HRP secondary antibody (1:2000 dilution, Cell Signaling Technology). Inhibition of RSV infectivity was determined by comparing the absorbance at 450 nm (620 nm as background reference) of neuraminidase-treated wells versus untreated control wells after reaction with TMB (a substrate for HRP). n=4 replicates, raw data were analyzed by one-way ANOVA using GraphPad Prism. A p-value of <0.05 was considered statistically significant ( * p<0.05; ** p<0.01; *** p<0.001; **** p<0.0001).

[0073] Relative to the RSV-only control, the results show no difference in infectivity between neuraminidase-treated and untreated cells (first two bars on the graph). However, protection of cells from both CBMs after neuraminidase treatment is reduced (fourth bar on the graph), although not to the same extent as cells treated first with CBM and then with RSV, which is more evident with Vc2CBMTD. This data suggests that CBMs also protect cells from RSV by targeting cell surface sialic acid (see Figures 21A and B).

[0074] Example 8 Utility of engineered carbohydrate-binding module proteins (Sp2CBMTD and Vc2CBMTD) against RSV-B infection using an in vitro 3D model of human respiratory epithelium. The purpose of this study was to evaluate the effects of mCBM40, Sp2CBMTD, and Vc2CBMTD on RSV replication in a 3D model of the human respiratory epithelium. The assay was performed using MucilAir™ (Epithelix), a fully differentiated, ready-to-use 3D model of the human airway epithelium composed of primary human epithelial cells freshly isolated from nasal, tracheal, or bronchial biopsy samples. For infection studies, RSV-B strain (10 6 A total of 100 genome copies / ml of RSV (Geneva Clinical Strain, 2017) was applied to the apical side of pre-washed (with PBS) MucilAir inserts on day 0. For mCBM treatments (n=3), Sp2CBMTD and Vc2CBMTD (10 μg or 100 μg doses) were applied apically either a) as a single dose 1 h prior to viral infection or b) as repeated doses 1 day and 1 h prior to viral infection. Both treatment regimens included an additional exposure to mCBM at 3.5 h postinfection (pi). Untreated infected MucilAir (n=2) served as a control. Apical lavage fluid (200 μL) was collected at 3.5 h postinfection, followed by 24, 48, and 96 h postinfection.

[0075] Viral genome copy number was determined using 20 μL of apical lavage fluid for viral RNA extraction using the QIAamp® Viral RNA Kit (Qiagen). Viral RNA was quantified using QuantiTect Probe RT-PCR (Qiagen) using 5 μL of viral RNA and two RSV-B-specific primers. Data were corrected for dilution factors and presented as genome copies per ml. Statistical analysis was performed using Prism 6.0 GraphPad software. An unpaired Student's t-test was used to compare two sets of data; in this case, * p<0.05.

[0076] overview Both Sp2CBMTD and Vc2CBMTD were shown to reduce apical viral load in RSV-infected human airway epithelial cells. The extent of reduction depended on the type of mCBM and the number of doses administered. Exposure of infected MucilAir inserts to Sp2CBMTD did not show a significant effect on viral replication due to data variability; however, a slight reduction was observed after two doses at 48 h postinfection with the highest dose (100 μg) (see Figure 23A) and after three doses at 96 h postinfection compared to virus-infected controls. Exposure to Vc2CBMTD (100 μg) was more effective at reducing RSV viral replication than using the 10 μg dosing regimen, with both regimens. Although not statistically significant, a greater than 1-log reduction in viral load was observed at 48 h postinfection (Figure 23B).

Claims

1. A composition for use in the treatment and / or prevention of respiratory syncytial virus (RSV) infection, the composition comprising a sialic acid binding molecule comprising one or more family 40 carbohydrate binding modules (CBM40).

2. A composition for use in neutralizing or preventing a respiratory syncytial virus (RSV) infection, the composition comprising a sialic acid binding molecule that contains one or more family 40 carbohydrate binding modules (CBM40).

3. An in vitro method for rendering a cell non-permissive to respiratory syncytial virus (RSV), comprising contacting or incubating a cell that is susceptible or vulnerable to RSV infection with a sialic acid binding molecule under conditions that allow binding between the sialic acid binding molecule and cell surface sialic acid / sialoglycoconjugates, wherein the sialic acid binding molecule comprises one or more family 40 carbohydrate binding modules (CBM40).

4. A composition for mucosal administration and for use in the treatment and / or prevention of respiratory syncytial virus (RSV) infection, the composition comprising a sialic acid binding molecule comprising one or more family 40 carbohydrate binding modules (CBM40).

5. A composition for prophylactic use in preventing RSV infection, the composition comprising a sialic acid binding molecule comprising one or more family 40 carbohydrate binding modules (CBM40).

6. The composition of any one of claims 1, 2, 4 and 5, wherein the sialic acid binding molecule does not exhibit sialidase activity.

7. The composition of any one of claims 1, 2, and 4 to 6, wherein the sialic acid binding molecule does not bind to heparin or heparin sulfate and / or does not contain a GAG binding domain of a protein that binds to heparin or heparin sulfate.

8. The composition of any one of claims 1, 2, and 4-7, wherein the sialic acid binding molecule comprises the sialic acid binding domain of Vibrio cholerae NanH sialidase and / or the sialic acid binding domain of Streptococcus pneumoniae NanA sialidase.

9. 9. The composition of claim 8, wherein the Vibrio cholerae NanH sialidase comprises the amino acid sequence of SEQ ID NO: 1 or 2.

10. 9. The composition of claim 8, wherein the Streptococcus pneumoniae NanA sialidase comprises the amino acid sequence of SEQ ID NO: 3 or 4.

11. The composition of any one of claims 1, 2, and 4 to 7, wherein the sialic acid binding molecule comprises an oligomerization domain of Pseudomonas aeruginosa pseudoaminidase, wherein the pseudoaminidase consists of the amino acid sequence of SEQ ID NO:5, or wherein the oligomerization domain consists of the amino acid sequence of SEQ ID NO:

6.

12. The composition of any one of claims 1, 2, and 4 to 11, wherein the sialic acid binding molecule is Sp2CBM, Sp2CBMTD, Vc4CBM, Vc2CBM, or Vc2CBMTD.

13. the sialic acid binding molecule comprises one or more modified family 40 carbohydrate binding modules (CBM40); The composition of any one of claims 1, 2, and 4 to 12, wherein the modification comprises one or more amino acid mutations.

14. one or more modified CBM40s contain one or more mutations relative to the reference sequence; The reference sequence is (i) wild-type family 40 CBM sequence; (ii) the wild-type CBM40 sequence of Vibrio cholerae; (iii) the NanH sialidase sequence of Vibrio cholerae; (iv) the wild-type CBM40 sequence of Streptococcus pneumoniae; (v) the NanA sialidase sequence of Streptococcus pneumoniae; (vi) the sequence of SEQ ID NO:1; (vii) the sequence of SEQ ID NO:2; (viii) the sequence of SEQ ID NO:3; and (ix) a sequence of SEQ ID NO: 4 The composition of claim 13, selected from the group consisting of:

15. The mutation, (i) one or more amino acid substitutions; (ii) removal of one or more amino acids; (iii) one or more amino acid additions / insertions; (iv) one or more amino acid / sequence inversions; and (v) one or more amino acid / sequence overlaps The composition of claim 14 selected from the group consisting of:

16. the sialic acid binding molecule comprises an engineered oligomerization domain; the oligomerization domain is an oligomerization domain of a Pseudomonas aeruginosa pseudoaminidase, said pseudoaminidase consisting of the amino acid sequence of SEQ ID NO:5, or said oligomerization domain consisting of the amino acid sequence of SEQ ID NO:6; The composition according to any one of claims 13 to 15, wherein the modification comprises one or more amino acid mutations.

17. The sialic acid binding molecule has the following structure: CBM1(V239A V246G A162P)--CBM2(V239A V246G A162P)--TD(S342D R403K) Including, CBM1 and CBM2 are each derived from the carbohydrate binding module (CBM) of Streptococcus pneumoniae NanA sialidase and contain the following mutations relative to SEQ ID NO:3: V239A, V246G, and A162P, respectively; TD is derived from the trimerization domain of Pseudomonas aeruginosa pseudoaminidase and comprises the mutations S342D and R403K relative to SEQ ID NO:5; The composition of any one of claims 1, 2 and 4 to 16, wherein -- represents an amino acid linker.

18. The sialic acid binding molecule comprising the following sequence: GAMVIEKEDVETNASNGQRVDLSSELDKLKKLENATVHMEFKPDPKAPAFYNLFSVSSATKKDEYFTMAVYNNTATLEGRGSDGKQFYNNYNDAPLKVKPGQWNSVTFTVEKPTAELPKGRA RLYVNGGLSRTSLRSGNFIKDMPDVTHVQIGATKRANNTVWGSNLQIRNLTVYNRALTPEEVQKRSGGGSGGVIEKEDVETNASNGQRVDLSSELDKLKKLENATVHMEFKPDPKAPAFYNLF SVSSATKKDEYFTMAVYNNTATLEGRGSDGKQFYNNYNDAPLKVKPGQWNSVTFTVEKPTAELPKGRARLYVNGGLSRTSLRSGNFIKDMPDVTHVQIGATKRANNTVWGSNLQIRNLTVYN RALTPEEVQKRSGGSLGVPDFESDWFDVSSNSLYTLSHGLQRSPRRVVVEFARSSSPSTWNIVMPSYFNDGGHKGSGAQVEVGSLNIKLGTGAAVWGTGYFGGIDNSATTRFATGYYRVRAWI The composition of any one of claims 1, 2 and 4 to 16, comprising:

19. The method of claim 3, wherein the sialic acid binding molecule does not exhibit sialidase activity.

20. 20. The method of claim 3 or 19, wherein the sialic acid binding molecule does not bind to heparin or heparin sulfate and / or does not contain a GAG binding domain of a protein that binds to heparin or heparin sulfate.

21. 21. The method of any one of claims 3, 19 and 20, wherein the sialic acid binding molecule comprises the sialic acid binding domain of Vibrio cholerae NanH sialidase and / or the sialic acid binding domain of Streptococcus pneumoniae NanA sialidase.

22. 22. The method of claim 21 , wherein the Vibrio cholerae NanH sialidase comprises the amino acid sequence of SEQ ID NO: 1 or 2.

23. 22. The method of claim 21 , wherein the Streptococcus pneumoniae NanA sialidase comprises the amino acid sequence of SEQ ID NO: 3 or 4.

24. The method of any one of claims 3, 19 and 20, wherein the sialic acid binding molecule comprises an oligomerization domain of Pseudomonas aeruginosa pseudoaminidase, the pseudoaminidase consisting of the amino acid sequence of SEQ ID NO:5, or the oligomerization domain consisting of the amino acid sequence of SEQ ID NO:

6.

25. 24. The method of any one of claims 3 and 19 to 23, wherein the sialic acid binding molecule is Sp2CBM, Sp2CBMTD, Vc4CBM, Vc2CBM or Vc2CBMTD.

26. the sialic acid binding molecule comprises one or more modified family 40 carbohydrate binding modules (CBM40); 26. The method of any one of claims 3 and 19 to 25, wherein the modification comprises one or more amino acid mutations.

27. one or more modified CBM40s contain one or more mutations relative to the reference sequence; The reference sequence is (i) wild-type family 40 CBM sequence; (ii) the wild-type CBM40 sequence of Vibrio cholerae; (iii) the NanH sialidase sequence of Vibrio cholerae; (iv) the wild-type CBM40 sequence of Streptococcus pneumoniae; (v) the NanA sialidase sequence of Streptococcus pneumoniae; (vi) the sequence of SEQ ID NO:1; (vii) the sequence of SEQ ID NO:2; (viii) the sequence of SEQ ID NO:3; and (ix) a sequence of SEQ ID NO: 4 27. The method of claim 26, selected from the group consisting of:

28. The mutation, (i) one or more amino acid substitutions; (ii) removal of one or more amino acids; (iii) one or more amino acid additions / insertions; (iv) one or more amino acid / sequence inversions; and (v) one or more amino acid / sequence overlaps 28. The method of claim 27, selected from the group consisting of:

29. the sialic acid binding molecule comprises an engineered oligomerization domain; the oligomerization domain is an oligomerization domain of a Pseudomonas aeruginosa pseudoaminidase, said pseudoaminidase consisting of the amino acid sequence of SEQ ID NO:5, or said oligomerization domain consisting of the amino acid sequence of SEQ ID NO:6; 29. The method of claim 27 or 28, wherein the modification comprises one or more amino acid mutations.

30. The sialic acid binding molecule has the following structure: CBM1(V239A V246G A162P)--CBM2(V239A V246G A162P)--TD(S342D R403K) Including, CBM1 and CBM2 are each derived from the carbohydrate binding module (CBM) of Streptococcus pneumoniae NanA sialidase and contain the following mutations relative to SEQ ID NO:3: V239A, V246G, and A162P, respectively; TD is derived from the trimerization domain of Pseudomonas aeruginosa pseudoaminidase and comprises the mutations S342D and R403K relative to SEQ ID NO:5; 30. The method of any one of claims 3 and 19 to 29, wherein -- represents an amino acid linker.

31. The sialic acid binding molecule, comprising the following sequence: GAMVIEKEDVETNASNGQRVDLSSELDKLKKLENATVHMEFKPDPKAPAFYNLFSVSSATKKDEYFTMAVYNNTATLEGRGSDGKQFYNNYNDAPLKVKPGQWNSVTFTVEKPTAELPKGRA RLYVNGGLSRTSLRSGNFIKDMPDVTHVQIGATKRANNTVWGSNLQIRNLTVYNRALTPEEVQKRSGGGSGGVIEKEDVETNASNGQRVDLSSELDKLKKLENATVHMEFKPDPKAPAFYNLF SVSSATKKDEYFTMAVYNNTATLEGRGSDGKQFYNNYNDAPLKVKPGQWNSVTFTVEKPTAELPKGRARLYVNGGLSRTSLRSGNFIKDMPDVTHVQIGATKRANNTVWGSNLQIRNLTVYN RALTPEEVQKRSGGSLGVPDFESDWFDVSSNSLYTLSHGLQRSPRRVVVEFARSSSPSTWNIVMPSYFNDGGHKGSGAQVEVGSLNIKLGTGAAVWGTGYFGGIDNSATTRFATGYYRVRAWI 30. The method of any one of claims 3 and 19 to 29, comprising:

32. 1. A composition for use in the treatment and / or prevention of a respiratory syncytial virus (RSV) infection comprising the following sequence: GAMVIEKEDVETNASNGQRVDLSSELDKLKKLENATVHMEFKPDPKAPAFYNLFSVSSATKKDEYFTMAVYNNTATLEGRGSDGKQFYNNYNDAPLKVKPGQWNSVTFTVEKPTAELPKGRA RLYVNGGLSRTSLRSGNFIKDMPDVTHVQIGATKRANNTVWGSNLQIRNLTVYNRALTPEEVQKRSGGGSGGVIEKEDVETNASNGQRVDLSSELDKLKKLENATVHMEFKPDPKAPAFYNLF SVSSATKKDEYFTMAVYNNTATLEGRGSDGKQFYNNYNDAPLKVKPGQWNSVTFTVEKPTAELPKGRARLYVNGGLSRTSLRSGNFIKDMPDVTHVQIGATKRANNTVWGSNLQIRNLTVYN RALTPEEVQKRSGGSLGVPDFESDWFDVSSNSLYTLSHGLQRSPRRVVVEFARSSSPSTWNIVMPSYFNDGGHKGSGAQVEVGSLNIKLGTGAAVWGTGYFGGIDNSATTRFATGYYRVRAWI The composition comprising a sialic acid binding molecule comprising:

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