Viral therapy

Abstract

The present disclosure provides molecules in compositions, drugs and methods for treating or preventing RSV infection, its symptoms and related pathologies, and potential infections caused or contributed to by viral pathogens that do not bind or do not primarily bind to sialic acid-containing receptors during the course of disease. Specifically, the present disclosure is based on the discovery that molecules with affinity (or binding ability) for sialic acid (particularly sialic acid glycoconjugates) on the cell surface (these include sialic acid-containing glycoproteins and cell surface sialic acid receptors) can be used for the treatment and / or prevention of 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 treating or preventing RSV infection, its symptoms and associated pathologies, and latent infections caused or contributed to by viral pathogens that do not bind or do not primarily bind to sialic acid-containing receptors during disease progression. Background Art

[0002] Respiratory syncytial virus (RSV) is a syncytial virus that causes respiratory tract infections. It causes disease in humans and animals and is a major cause of lower respiratory tract infections, with the young and elderly being particularly at risk. RSV infection, including the form commonly known as human RSV (HRSV), does induce protective immunity, but studies have shown that this response is not prolonged (or sustained), and multiple infections are not uncommon.

[0003] Unlike many other respiratory pathogens, RSV does not bind to sialic acid glycoconjugates to enter cells and infect 3 However, since sialic acid is ubiquitous and dominant as the terminal glycan of glycoconjugates on the surface of respiratory epithelial cells, it would be advantageous if sialic acid-binding molecules could be used as the basis for treatment of pathogens such as RSV. Summary of the invention

[0004] The present disclosure is based on the discovery that molecules having affinity (or binding ability) for sialic acid (particularly sialic acid glycoconjugates) on the surface of cells (including molecules of glycoproteins containing sialic acid and cell surface sialic acid receptors) can be used for the treatment and / or prevention of symptoms, infections, diseases and / or disorders associated with respiratory syncytial virus (RSV).

[0005] The present disclosure provides sialic acid binding molecules for use in treating and / or preventing symptoms, infections, diseases and / or disorders associated with respiratory syncytial virus (RSV).

[0006] Further provided is the use of a sialic acid binding molecule in the preparation of a medicament for treating and / or preventing symptoms, infections, diseases and / or disorders associated with RSV.

[0007] The present disclosure also provides a method of treating or preventing RSV symptoms, diseases, infections or disorders, the method comprising administering to a subject in need thereof a therapeutically effective amount of a sialic acid binding molecule.

[0008] The present disclosure also provides sialic acid binding molecules, as well as medicaments and methods comprising the sialic acid binding molecules for use in methods of treating or preventing symptoms, diseases and / or disorders associated with RSV infection.

[0009] Throughout the specification, the terms "comprise", "comprising" and / or "comprises" are used to indicate aspects and embodiments of the invention that "comprise" one or more specified features. It should be understood that this / these terms may also include aspects and / or embodiments that "consist essentially of" or "consist of" the relevant one or more features.

[0010] RSV is a medium-sized (120–200 nm) enveloped virus that contains a lipoprotein coat and a linear negative sense RNA genome. The genome encodes F, G and SH lipoproteins. The F (fusion) and G (attachment) lipoproteins (or glycoproteins) target the host cell membrane and control the initial stages of infection. They are also highly conserved among RSV isolates. Specifically, the G protein targets ciliated cells in the respiratory tract, and the F protein causes the fusion of the virion membrane with the target cell membrane. RSV is divided into two antigenic subgroups, A and B, based on the reactivity of the virus with monoclonal antibodies against the attachment (G) and fusion (F) glycoproteins. The term "RSV" as used herein includes all strains, forms and antigenic variants of RSV - including all forms, strains and variants of human (H) or animal RSV.

[0011] RSV is not a pathogen that binds or associates with sialic acid during the course of disease; therefore, RSV does not bind host cell sialic acid (or cell surface sialic acid glycoconjugates). Therefore, the following discovery is completely unexpected: sialic acid binding molecules (e.g., CBM40 molecules (and other molecules defined below)) have utility in treating viral pathogens that do not utilize cell surface sialic acid (sialic acid glycoconjugates) during the course of disease (or pathogens for which sialic acid binding is not the primary means of pathogen binding, colonization, or entry / infection of cells).

[0012] The prior art disclosure that sialic acid binding molecules have been used as agents capable of blocking pathogens from binding to cell surface sialic acid / sialic acid glycoconjugates, the utility of sialic acid binding molecules is based on the fact that sialic acid binding molecules and pathogens bind sialic acid; and RSV is not the case. Because RSV does not bind sialic acid, the skilled person would not think that sialic acid binding molecules can be used to block RSV from entering cells.

[0013] Without wishing to be bound by theory, it is suggested that when combined with cell surface sialic acid / sialic acid glycoconjugates, sialic acid binding molecules prevent G and / or F RSV proteins from approaching their targets on host cells. This in turn prevents RSV from being planted and infected cells. In addition, the data provided below (particularly referring to the data provided in Example 6) show that RSV viral surface comprises a glycoprotein terminated with sialic acid; Therefore, it is shown that sialic acid binding molecules can be combined with these glycoproteins (by sialic acid components), and further suppress the combination between RSV and host cells.

[0014] Although it may have been determined that sialic acid binding proteins can be used to trigger or modulate the immune system, and that the immune responses triggered or modulated may affect the pathology of the entire host of different pathogens (including pathogens that do not bind or do not primarily bind sialic acid during the pathogenesis process), technicians have not yet discovered that molecules with affinity for sialic acid (such as CBMs, such as CBM40-type molecules) can be used to physically block, prevent or neutralize RSV infection.

[0015] However, the discovery that sialic acid binding molecules can still be used to neutralize or block RSV infection extends the disclosure to provide sialic acid binding molecules for neutralizing or blocking RSV infection. Such uses can be applied to in vitro or in vivo methods.

[0016] The present disclosure further provides the use of sialic acid binding molecules in the preparation of a medicament for neutralizing or blocking RSV infection.

[0017] The present disclosure also relates to methods of neutralizing or blocking RSV infection, the methods comprising administering to a subject in need thereof a therapeutically effective amount of a sialic acid binding molecule.

[0018] Alternatively, the disclosure provides methods that can be used to make cells impermissible to RSV (the term "impermissible" refers to cells that resist virus attachment or planting and / or subsequent infection and viral replication). Such methods (the method can be an in vitro or in vivo method) can include contacting or incubating cells that are sensitive to RSV infection or susceptible to RSV infection with sialic acid binding molecules as described herein. The step of contacting or incubating can include contacting or incubating cells with sialic acid binding molecules before contacting RSV. Additionally or alternatively, the step of contacting or incubating cells with sialic acid binding molecules or incubation can be extended so that the sialic acid binding molecules contact cells while cells are in contact with RSV.

[0019] Without wishing to be bound by theory, during incubation, cell surface receptors containing sialic acid will be bound by sialic acid binding molecules, and although RSV itself does not bind to sialic acid moieties (sialic acid glycoconjugates) on the cell surface, it is surprising that the binding between sialic acid binding molecules and receptors / sialic acid glycoconjugates containing cell surface sialic acid inhibits or prevents RSV cell binding. This in turn can prevent RSV cell infection and intracellular RSV replication.

[0020] The findings reported in this disclosure are of great significance for the preparation and administration of molecules with sialic acid binding affinity and the subsequent use of these preparations in treating and / or preventing 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 preparations suitable for mucosal administration.

[0021] Formulations containing sialic acid binding molecules for mucosal administration can be used to

[0022] (i) preventing RSV from binding to host cells; and / or

[0023] (ii) Blocking or neutralizing RSV.

[0024] As stated above, the terms "blocking" or "neutralizing" refer to the blocking or neutralizing effect of sialic acid binding molecules that bind to receptor / sialic acid glycoconjugates containing host cell sialic acid and have now (surprisingly) been found to block RSV cell binding (and subsequent infection).

[0025] Thus, the present disclosure provides compositions for mucosal administration comprising sialic acid binding molecules for treating and / or preventing diseases and / or disorders associated with RSV.

[0026] It should be noted that the term "mucosal administration" includes compositions that have been formulated for administration to any mucosal surface (including, for example, respiratory surfaces, etc.). The term also encompasses compositions formulated for administration by inhalation. Compositions suitable (or formulated) for mucosal administration may include compositions intended for intranasal administration.

[0027] Thus, compositions for mucosal administration may be formulated with excipients, diluents and / or buffers suitable for any type of mucosal administration.

[0028] Compositions for mucosal (e.g., intranasal) administration may include solutions of sialic acid binding molecules to be administered and / or particles (including sialic acid binding molecules) for aerosol dispersion or distribution in drinking water. When distributed, such compositions should ideally have a particle size in the range of 10 to 200 microns to be retained in, for example, the nasal cavity; this can be achieved by appropriately using a powder of a suitable particle size or selecting a suitable valve. Other suitable compositions include coarse powders with a particle size of 20 to 500 microns for administration by rapid inhalation through the nasal passages from a container close to the nose, and nasal drops containing 0.2 to 5% w / v of the active compound in an aqueous or oily solution or suspension. Compositions for mucosal administration can be provided in the form of a liquid spray.

[0029] In one embodiment, the present disclosure may not be directed to treating and / or preventing RSV by eliciting or modulating an immune response.

[0030] The present disclosure may further provide sialic acid binding molecules for prophylactic use. Specifically, the sialic acid molecules described herein may be used prophylactically to prevent RSV infection.

[0031] Methods of preventing or protecting against RSV infection may comprise administering a composition of the present disclosure to a subject in need thereof.As described above, the compositions of the present disclosure may comprise compositions formulated for mucosal (eg, intranasal) administration.

[0032] As used herein and in any method for preventing RSV infection, a "subject in need thereof" may be any subject susceptible to, susceptible to, 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 affecting the respiratory tract and / or breathing. For example, the subject may suffer from asthma.

[0033] Throughout the specification, we use the term "sialic acid binding molecule", which includes any useful sialic acid binding molecule. Useful sialic acid binding molecules can take any form and / or belong to any type of molecule or compound (for example, they can be proteins, peptides, carbohydrates, antibodies, etc.), and the term "sialic acid" includes all forms of N- or O-substituted neuraminic acid, and includes all its synthetic, naturally occurring and / or modified forms. Sialic acid can be found as a component of cell surface molecules, glycoproteins and glycolipids. Sialic acid is most commonly found at the end (terminal region) of the sugar chain connected to the cell membrane and / or protein. For example, some cells of the upper respiratory tract of the human body contain α-2,6-connected sialic acid receptors, while other cells of the upper and lower respiratory tracts contain α-2,3-connected sialic acid receptors. The sialic acid family includes many (about 50) derivatives, which may be due to acetylation, glycosylation, lactonization and methylation at C4, C5, C7, C8 and C9. The term "sialic acid" includes all these derivatives.

[0034] In addition, sialic acid is found to be α(2,3) or α(2,6) linked to Gal and GalNAc, or α(2,8) or α(2,9) linked to another sialic acid. Therefore, it is important to understand that although the term "sialic acid" is used throughout the specification, it encompasses all derivatives, analogs or variants thereof (naturally occurring or synthetically produced) as well as monomers, dimers, trimers, oligomers, polymers or concatemers comprising the same.

[0035] Therefore, the sialic acid binding molecules of the present disclosure (and used as described herein) include moieties that exhibit affinity for sialic acid, including all forms of sialic acid described above and any form of sialic acid present on the surface of a cell (e.g., a mammalian cell) (perhaps as part of a cell surface receptor). These various forms of sialic acid may be collectively referred to as "sialic acid moieties".

[0036] The sialic acid binding molecules of the present disclosure exhibit affinity for sialic acid, and therefore they can bind / couple and / or associate with one or more sialic acid moieties. Therefore, the term "sialic acid binding molecule" may further include any fragment of a complete sialic acid binding molecule that retains the ability to bind or couple or associate with a sialic acid moiety.

[0037] The sialic acid binding molecules used may comprise a single sialic acid binding molecule (eg, a monomeric or monovalent molecule), or alternatively comprise two or more sialic acid binding molecules (the two or more molecules may be the same or different) - such as a polymeric or multivalent molecule.

[0038] The sialic acid binding molecules used may comprise, consist essentially of, or consist of one or more sialic acid binding molecules referred to as "carbohydrate binding modules" (CBMs). Suitable CBMs for use show affinity for sialic acid. CBMs are divided into multiple families, and CBMs classified as members of family 40 CBMs (CBM40) may be useful. Family 40 CBMs contain molecules of approximately 200 residues, typically found at the N-terminus of GH33 sialidases. They can also be found inserted into the β-propeller of GH33 sialidases.

[0039] The present disclosure may include the use of molecules comprising sialic acid binding components, such as larger molecules. As described above, the sialic acid binding component (i.e., sialic acid binding molecule) itself may include a CBM, such as CBM40 (consisting of or substantially consisting of it). As a (non-limiting) example, the molecules of the present disclosure (e.g., sialic acid binding molecules) can not only show the ability to bind sialic acid, but can also have one or more other functions. For example, molecules can have enzymatic activity. For example, useful molecules can include a CBM (as described herein) and show some sialidase activity.

[0040] Useful sialic acid binding molecules can be fusion proteins comprising an enzyme portion and a sialic acid binding portion, wherein the sialic acid binding portion comprises a CBM as described herein. In this case, the enzyme portion can be fused to the sialic acid binding portion. As described above, the enzymatic portion of any useful fusion protein can comprise (or have or display) sialidase activity.

[0041] In one embodiment, the sialic acid binding proteins or CBMs of various uses described herein may not be part of or included in a molecule (e.g., a fusion protein) having enzyme (e.g., sialidase) activity. Additionally or alternatively, the sialic acid binding molecule may not (i) bind heparin or heparin sulfate and / or (ii) contain a GAG-binding domain of a protein that binds a heparin or heparin sulfate moiety.

[0042] Thus, the present disclosure provides a CBM or CBM40 for use in treating and / or preventing diseases and / or conditions associated with respiratory syncytial virus (RSV).

[0043] Further provided is the use of CBM or CBM40 in the preparation of a medicament for treating and / or preventing diseases and / or conditions associated with RSV.

[0044] The present disclosure also provides a method of treating or preventing RSV infection, comprising administering a therapeutically effective amount of CBM or CBM40 to a subject in need thereof.

[0045] The present disclosure also provides sialic acid binding molecules and medicaments and methods comprising CBM or CBM40 for use in methods of treating or preventing diseases and / or conditions associated with RSV infection.

[0046] The present disclosure also provides CBM or CBM40 for neutralizing or blocking RSV infection.

[0047] The present disclosure further provides the use of CBM and / or CBM40 in the preparation of a medicament for neutralizing or blocking RSV infection.

[0048] The present disclosure relates to methods of neutralizing or blocking RSV infection comprising administering a therapeutically effective amount of CBM and / or CBM40 to a subject in need thereof.

[0049] Alternatively, the 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 can be in vitro or in vivo methods) can include contacting or incubating cells that are sensitive or susceptible to RSV infection with a CBM and / or CBM40 of the disclosure.

[0050] Formulations containing CBM and / or CBM40 for mucosal administration can be used to

[0051] (i) preventing RSV from binding to host cells; and / or

[0052] (ii) Blocking or neutralizing RSV.

[0053] The present disclosure provides compositions for mucosal administration, comprising CBM and / or CBM40 for treating and / or preventing diseases and / or conditions associated with RSV. As described above, compositions for mucosal administration can be formulated with excipients, diluents and / or buffers suitable for any type of mucosal administration.

[0054] The present disclosure may further provide CBM or CBM40 for prophylactic use. Specifically, to prevent RSV infection, the CBM or CBM40 described herein may be used prophylactically.

[0055] As noted above, the compositions of the present disclosure may include compositions formulated for mucosal (eg, intranasal) administration.

[0056] Exemplary carbohydrate binding modules (CBMs) used may include the sialic acid binding domain of Vibrio cholerae NanH sialidase (VcCBM: CBM40) and / or the equivalent (or homologous) domain from Streptococcus pneumoniae NanA sialidase (SpCBM: also CBM40). Of course, similar or homologous sialic acid binding modules present in other organisms will be included within the scope of the term "CBM".

[0057] An exemplary V. cholerae NanH sialidase amino acid sequence is deposited under accession number A5F7A4 and is reproduced below as SEQ ID NO: 1 (781 amino acids).

[0058]

[0059] The CBM region of SEQ ID NO: 1 is from amino acid residues 25 to 216—this sequence may be SEQ ID NO:2.

[0060] An exemplary S. 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).

[0061]

[0062] The CBM region of SEQ ID NO:3 is located at amino acid residues 121 to 305—this sequence may be SEQ ID NO:4.

[0063] Therefore, the CBM used as a sialic acid-binding molecule in various aspects and embodiments of the present disclosure may comprise a protein or peptide having a sequence of SEQ ID NO: 1, 2, 3 or 4, or a sequence fragment derived therefrom, which encodes a molecule having the ability to bind to sialic acid (in other words, a sialic acid-binding molecule encoding a portion of a fragment of SEQ ID NO: 1, 2, 3 or 4).

[0064] Useful sialic acid binding molecules may comprise a portion of a protein encoded by the sialic acid binding domain of the nanH gene (encoding sialidase) of Vibrio cholerae (provided by SEQ ID NO: 1) or an equivalent or homologous gene present in another organism (e.g., the equivalent / homologous nanA sialidase gene of Streptococcus pneumoniae: see SEQ ID NO: 3).

[0065] The sialic acid binding molecule used may comprise about residue 1, 5, 10, 15, 25 or 30 (i.e., from 1-30 or any amino acid residue therebetween) to about residue 150, 175, 200, 210, 216, 220-781 (to any residue between 150-781, including any residue therebetween) of the Vibrio cholerae sialidase molecule of SEQ ID NOs: 1 and 2. For example, the sialic acid binding molecule used may comprise a peptide having a sequence corresponding to residue 25 to about residue 216 of SEQ ID NO: 1 above.

[0066] Another suitable sialic acid binding molecule may comprise a protein or peptide having a sequence of SEQ ID NO: 3 or 4 or a sialic acid binding fragment thereof. For example, a useful sialic acid binding molecule may comprise a portion of a protein encoded by the sialic acid binding domain of the Streptococcus pneumoniae nanA gene (encoding sialidase). For example, the sialic acid binding molecule used may comprise about residues 80, 90, 100, 110, 120, 121 to 130 (i.e., any residue from about residues 80 to 130, including any residue therebetween) to about residues 250, 275, 300, 305, 310, 320-1035 (i.e., to any residue from about 250-1035, including any residue therebetween) of the Streptococcus pneumoniae sialidase molecule of SEQ ID NO: 3 and 4. For example, the sialic acid binding molecule used may comprise a peptide having a sequence corresponding to residue 121 to about residue 305 of SEQ ID NO: 3 above.

[0067] The sialic acid binding molecule used may comprise one or more CBMs. For example, a suitable sialic acid binding molecule may comprise a single CBM, such as a single VcCBM or a single SpCBM. Alternatively, the sialic acid binding molecule used may comprise a plurality or more (i.e., two or more) CBMs. Sialic acid binding molecules comprising multiple CBMs may be referred to as "multivalent sialic acid binding molecules" or "multivalent CBMs". Multivalent CBMs may, for example, include two or more (e.g., three, four, five or six) VcCBMs or two or more SpCBMs. Multivalent CBMs may comprise a mixture of different CBMs, such as a mixture of one or more VcCBMs and one or more SpCBMs.

[0068] The sialic acid binding molecules used may further include an oligomerization domain. Suitable oligomerization domains may exhibit the ability to self-associate to form a multimeric structure such as a trimer. The oligomerization domains used may include any molecule or any functional fragment thereof having the above-mentioned oligomeric properties. For example, one or more (e.g., two) sialic acid binding molecules (e.g., CBMs) may be combined, coupled or fused to an oligomerization domain - the resulting sialic acid binding molecule:: oligomerization domain "fusion" (with one or more other such "fusions") may then be used as a molecule for regulating cell growth and / or activity and / or for treating or preventing any disease and / or disorder disclosed herein.

[0069] Suitable oligomerization domains may be derived from, for example, Pseudomonas aeruginosa pseudaminidase. An exemplary Pseudomonas aeruginosa pseudaminidase sequence amino acid sequence has been deposited under accession number PAO579 and is reproduced below as SEQ ID NO: 5 (438 amino acids).

[0070]

[0071] The oligomerization domain of SEQ ID NO:5 is amino acid residues 333 to 438—this sequence may be SEQ ID NO:6.

[0072] Thus, the oligomerization domain used may comprise about residues 250, 275, 300, 310, 320, 333, 340 to 350 (i.e., about residue 250 to about residue 350, including about any residue therebetween) to about residue 400, 410, 420, 430, or 438 (i.e., about any residue from about residue 400 to about residue 438, including about any residue therebetween) of the Pseudomonas aeruginosa pseudoaminase trimerization domain (PaTD) provided in SEQ ID NO: 5. For example, a useful sialic acid binding molecule may utilize an oligomerization domain comprising residues 333 to 438 of SEQ ID NO: 6.

[0073] The sialic acid binding molecules used may comprise one or more Figure 1 CBM-based molecules as shown. For example, a suitable sialic acid binding molecule may comprise (consist essentially of or consist of) two or more VcCBMs, optionally fused, bound or conjugated to an oligomerization domain (e.g., PaTD or an oligomerization fragment thereof). The sialic acid binding molecule may comprise, consist of or consist essentially of two fused (or bound) VcCBMs, which in turn are fused to an oligomerization domain (e.g., see Figure 1 The molecule shown is Vc2CBMTD).

[0074] The other sialic acid binding domains used may comprise two or more SpCBMs optionally fused, bound or conjugated to an oligomerization domain (e.g., PaTD or an oligomeric fragment thereof). The sialic acid binding molecule used may comprise, consist of or consist essentially of two fused (or bound) SpCBMs, which in turn are fused to an oligomerization domain (e.g., see Figure 1 The molecule shown is Sp2CBMTD).

[0075] The terms "sialic acid binding molecule", "CBM" and / or "CBM40" may include modified forms of any molecule described herein. In addition, the term "modified" includes molecules that contain one or more mutations relative to a reference sequence.

[0076] A "reference sequence" can be any wild-type CBM sequence. For example, a reference sequence can comprise, consist essentially of, or consist of a wild-type Family 40 CBM sequence, such as a wild-type CBM sequence from Vibrio cholerae NanH sialidase or Streptococcus pneumoniae NanA sialidase (it should be understood that similar or homologous CBMs present in other organisms (including CBM40) should also be included within the scope of the term "CBM" and / or as CBM reference sequences). Reference sequences from which useful sialic acid binding molecules can be derived (including useful multivalent CBMs described herein) can include any of the specific sequences described herein (e.g., SEQ ID NOs: 1, 2, 3, 4, and 5).

[0077] For example, the modified CBM sequence used may be derived from a particular or specific wild-type CBM. Useful modified CBM sequences may comprise a wild-type CBM sequence that includes one or more mutations.

[0078] One or more mutations may be functional. For example, these mutations may change the overall primary sequence of the CBM used, but may not (substantially) change the properties of the CBM-thus, although the sequence of the modified CBM may be different from the wild-type sequence from which it is derived, the overall function of the modified CBM is (substantially) the same as that of the wild-type CBM. Alternatively, one or more mutations may individually (and / or independently) or collectively (e.g., synergistically) regulate (improve or prevent / inhibit) one or more physiological, biological, immunological and pharmacological characteristics of a wild-type CBM (e.g., a wild-type CBM from which the modified CBM is derived). In particular, one or more mutations may:

[0079] (i) altering the immunogenicity (or antigenicity) of the CBM; and / or

[0080] (ii) altering (e.g. improving) the efficacy (of a CBM or any multimeric molecule comprising a modified CBM); and / or

[0081] (iii) they can modulate (e.g. improve) the thermal stability of the CBM; and / or

[0082] (iv) they can modulate (e.g. improve) the solubility of the CBM; and / or

[0083] (v) They may modulate (eg improve) the in vivo half-life of the molecule.

[0084] "Mutation" can include any change to a wild-type CBM molecule. For example, the term "mutation" can include, for example:

[0085] (i) one or more amino acid substitutions (wherein one or more wild-type amino acids are exchanged or changed by another (different) amino acid, the term "substitution" including conservative amino acid substitutions); and / or

[0086] (ii) one or more amino acid deletions (in which one or more wild-type amino acid residues are removed); and / or

[0087] (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

[0088] (iv) one or more amino acid / sequence inversions (where typically two or more consecutive amino acids in the primary sequence are inverted); and / or

[0089] (v) one or more amino acid / sequence repeats (wherein one amino acid or a portion (e.g. a stretch of 5-10 amino acids) of the primary amino acid sequence is repeated).

[0090] Thus, useful modified CBMs (ie, CBMs for the medical uses and methods described herein) may comprise one or more mutations described herein.

[0091] As a non-limiting example, the following representative can be used to prepare a single unit (referred to as " HEX " unit) of hexamer sialic acid binding molecules, for use in various embodiments described herein (for example, for treating or preventing RSV infection in the method medium). In each case, the HEX unit includes two modified CBMs (expressed as CBM1 and CBM2), and the specific mutations introduced into each CBM are identified in brackets. It should be noted that the "----" symbol represents an amino acid linker (one CBM is connected to another or CBM is connected to an oligomerization domain). In this way, the hexamer sialic acid binding molecules can be composed of several (for example 3) HEX units. In each case, the oligomerization domain (expressed as " TD ") puts the unit together as a trimer. Although any given hexamer can include the same copy of the above-mentioned (and under the title HEX1, HEX2, HEX3, HEX4, HEX5, HEX6 and HEX17) unit, it will be appreciated by the technician that other selections can also be utilized. For example, a HEX unit may consist of two CBMs, each CBM having a different mutation (the mutation being selected from one or more of the selections detailed herein).

[0092] (i)HEX1

[0093] CBM1(L170T V239A V246G I286A Y292E)-----CBM2(L170T V239A V246G I286AY292E)-----TD(S342D L348D R403K)

[0094] (ii)HEX2

[0095] CBM1(v239A V246G I286A Y292E)----CBM2(V239A V24 6G I286A Y292E)----TD(S342D R403K)

[0096] (iii)HEX3

[0097] CBM1(V239A V246G I286A)-----CBM2(V239A V246G I286A)-----TD(S342DR403K)

[0098] (iv)HEX4

[0099] CBM1(V239A V246G)-----CBM2(V239A V246G)-----TD(S342D)

[0100] (v)HEX5

[0101] CBM1(v239A V246G)-----CBM2(V239A V246G)-----TD(R403K)

[0102] (vi)HEX6

[0103] CBM1(V239A V246G)-----CBM2(V239A V246G)-----TD(S342D R403K)

[0104] (vii)HEX17

[0105] CBM1(V239A V246G A162P)-----CBM2(V239A V246G A162P)-----TD(S342DR403K)

[0106] It should be noted that HEX6 and HEX17 are identical except for the additional A162P mutation. This proline mutation (substituting wild-type alanine at residue 162) has been shown to improve thermal stability (single CBM Tm 3-4°C). More information on the use of proline mutations can be obtained from Fu 2009, "Increasing protein stability by improving beta-turns" (DOI 10.1002 / prot.22509), which describes the general approach. The proline mutation did not affect (increase or decrease) the predicted immunogenicity of the CBM molecule, nor was it located near other mutations, the N-terminus or C-terminus, or the ligand binding site. Unexpectedly, in addition to a modest improvement in thermal stability, it was noted that the A162P mutation produced hexameric CBMs (i.e., molecules comprising 3 HEX17 units) that exhibited significant improvements in in vivo experiments - particularly compared to those same experiments conducted using hexameric molecules comprising 3 Hex6 units. For example, the modified molecules, in particular those containing three HEX17 units, showed modulation of proinflammatory cytokines, including, for example, IL-8. In fact, the modulation (in particular, inhibition) of IL-8 production was improved in the molecules containing three HEX17 units compared to the other modified molecules tested.

[0107] Relative to the amino acid sequence of Sp2CBMTD (also known as "SpOrig"), the amino acid sequences of HEX6 and HEX 17 molecules are:

[0108]

[0109]

[0110] Thus, various aspects and embodiments of the present disclosure (uses, sialic acid binding molecules for use, methods and medicaments) may utilize, consist of, or consist essentially of a sialic acid binding molecule selected from the group consisting of:

[0111] (i) one or more VcCBMs;

[0112] (ii) one or more SpCBMs;

[0113] (iii) one or more modified CBMs;

[0114] (iv) HEX17 molecules; and

[0115] (iii) Multivalent CBMs.

[0116] Thus, the present disclosure provides:

[0117] Sp2CBM;

[0118] HEX17;

[0119] Vc2CBM; and / or

[0120] Vc4CBM;

[0121] For the treatment and / or prevention of RSV infection.

[0122] Further provided is the use of Vc2CBM or Vc4CBM in preparing a medicament for treating and / or preventing RSV infection.

[0123] The present disclosure also provides use of HEX17 in preparing a medicament for treating and / or preventing RSV infection.

[0124] The present disclosure also relates to a method for treating or preventing RSV, the method comprising the steps of administering to a subject in need thereof a therapeutically effective amount of

[0125] HEX17;

[0126] Sp2CBM;

[0127] Vc2CBM; and / or

[0128] Vc4CBM.

[0129] 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, bound or conjugated together. An exemplary Vc2CBM sequence may comprise, consist essentially of, or consist of the following sequence:

[0130]

[0131] Vc4CBM comprises, consists essentially of, or consists of four Vibrio cholerae NanH sialidase CBM units linked, bound or conjugated together. An exemplary Vc4CBM sequence may comprise, consists essentially of, or consists of the following sequence:

[0132]

[0133] Sp2CBM comprises, consists essentially of, or consists of two S. pneumoniae NanA sialidase units linked, bound or conjugated together. An exemplary Sp2CBM sequence may comprise, consists essentially of, or consists of two copies of the following sequence:

[0134]

[0135] Two copies of the above sequence can be connected by any one of the peptide linker sequences described herein. For example, the Sp2CBM sequence can comprise the following sequence, or be essentially composed of it or be composed of it:

[0136]

[0137] Wherein [xxxxx], [xxxxxxxxxx] and [xxxxxxxxxxxxxxx] - represent the choice of linker peptide sequence, as described below. The two CBM sequences will be connected by one of the 5, 10 or 15 amino acid linker sequences described herein.

[0138] Based on the family 40 sialic acid binding domain (CBM) of the nanH gene encoding the sialidase of Vibrio cholerae, Vc2CBM and Vc4CBM can be described as tandemly repeated multivalent proteins. Based on the family 40 sialic acid binding domain (CBM) of the nanA gene encoding the sialidase of Streptococcus pneumoniae, Sp2CBM can be described as a tandemly repeated multivalent protein.

[0139] The disclosed molecules (for the uses and methods described herein) can be produced using PCR-based cloning techniques and suitable methods for producing multivalent molecules of this type, such as those described 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). For example, multivalent CBM molecules, including HEX17, Vc2CBM, Vc4CBM, and Sp2CBM, etc., can be prepared as constructs comprising multiple CBMs connected by amino acid / peptide linkers. Each CBM (e.g., VcCBM) can be connected to each other by, for example, a peptide comprising 5, 10, or 15 amino acids. For example, any one or more of the following peptides can be used to connect two or more CBMs to produce a multivalent CBM:

[0140] (i) 5 amino acid linker: ALXGS

[0141] LqCy

[0142] GGX

[0143] GGALG

[0144] GGGGS

[0145] (ii) 10 amino acid linker: ALXGSGGGSG

[0146] LQALGGGGSL

[0147] (iii) 15 amino acid linker: ALXGSGGGSGGGGSG

[0148] wherein "X" is any amino acid.

[0149] An exemplary Vc4CBM may take the following form:

[0150]

[0151] This schematic diagram is hereinafter referred to as Formula 1.

[0152] Thus, the Vc4CBM molecule may conform to Formula 1 as described above, wherein the peptide linkers A, B and / or C are selected from the linker options represented as (i), (ii) and / or (iii) above. It should be noted that the term "VcCBM40" includes not only the complete family 40CBM derived from Vibrio cholerae (NanH sialidase), but also the sialic acid binding fragments derived therefrom. In fact, each VcCBM unit shown in Formula 1 can be selected from the group consisting of:

[0153] (i) Vibrio cholerae NanH sialidase CBM; and

[0154] (ii) the Vibrio cholerae NanH sialidase CBM sialic acid binding fragment thereof.

[0155] Therefore, each VcCBM unit of the molecule represented by Formula 1 may be the same or different.

[0156] Furthermore, it should be noted that the various uses, methods and medicaments described herein can utilize one or more sialic acid binding molecules described herein. For example, two or more different sialic acid binding molecules can be administered to a subject together, simultaneously or separately.

[0157] The present disclosure can provide compositions for various purposes, medicines and methods as described herein. In this way, any sialic acid binding molecule as described herein can be formulated for use. For example, a sialic acid binding molecule (or multiple molecules) can be formulated into a treatment or pharmaceutical composition. Various compositions can include one or more sialic acid binding molecules as described herein, and any given treatment may require (together, simultaneously or separately) the use of one or more of these compositions.

[0158] Pharmaceutical compositions according to the invention, in particular those formulations for mucosal or intranasal administration, may be prepared conventionally comprising substances commonly used in medicine and described, for example, in Remington's The Sciences and Practice of Pharmacy, 22nd edition (Pharmaceutical Press 2012) and / or Handbook of Pharmaceutical Excipients, 7th edition (edited by Rowe et al., Pharmaceutical Press, 2012) - the entire contents of all these documents and references are incorporated herein by reference.

[0159] Any suitable amount of sialic acid binding molecules (e.g., any CBM-type molecules described herein, including CBM40) can be used. For example, whether the composition comprising the sialic acid binding molecule (e.g., CBM, e.g., Sp2CBM or Vc2CBMTD) is administered intravenously or mucosally (e.g., intranasally), the dose of the sialic acid binding molecule can comprise any amount between about 0.1 μg and about 1000 μg. For example, a dosage 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 of sialic acid binding molecules can be used. These amounts can be provided in an excipient, diluent or buffer of any suitable volume. For example, the amount of sialic acid binding molecules can be provided in any amount of an excipient, diluent or buffer between about 1 μl to about 5 ml. For example, the desired amount of sialic acid binding molecule can 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 0.1-1 mg (sialic acid binding protein) per ml (excipient, diluent or buffer) may be most useful.

[0160] Detailed description

[0161] The present invention will now be described in detail with reference to the following drawings:

[0162] Figure 1 : Structural units of the multivalent CBM forms and their affinity for sialic acid. a, VcCBM, residues 25–216 of Vibrio cholerae sialidase (PDB: 1w0p) with α-2,3-sialyllactose drawn as a sphere. b, SpCBM, residues 121–305 of Streptococcus pneumoniae NanA sialidase (PDB: 4c1w) with α-2,3-sialyllactose. c, TD, trimerization domain, residues 333–438, of Pseudomonas aeruginosa pseudosialidase (PDB: 2w38) in rainbow colors; the other two monomers are in monochrome. d, Multivalent forms: molecular weight, valence, and binding affinity for α2,3-sialyllactose as determined by surface plasmon resonance (SPR) at 25 °C (K for VcCBM, Vc2CBM, and Vc3CBM has been reported previouslyD value 7 ). The tandem repeat CBM and the oligomeric CBM fused to TD are connected via a 5-amino linker.

[0163] Figure 2. The preventive effect of mCBM40 on RSV-infected mammalian cell lines. 3 HEp2:NPro cells were incubated with different amounts (0.1–100 μg / well) of Sp2CBMTD or Vc2CBMTD for 1 hour before 400 μg / well of 1 μg / well of 1 μg / well of 1 μg / well of 2 ...

[0164] Figure 3. Effect of mCBM40 in RSV-infected mammalian cell lines. HEp2:NPro cells were incubated with different amounts (0.1–100 μg / well) of Sp2CBMTD or Vc2CBMTD and RSV (1.5 x 10 3 PFU) were incubated together. Shown are the absorbance readings of infected cells obtained at 24h, 48h, 72h and 90h after infection for cells treated with Sp2CBMTD (ad) and Vc2CBMTD (eh) compared to untreated infected cells. The bar represents the average absorbance change ± SD of six replicates. All values ​​are expressed as mean ± SD, and the statistical results are expressed as: *p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001.

[0165] Figure 4. Therapeutic effect of mCBM40 in RSV-infected mammalian cell lines. HEp2:NPro cells were incubated with RSV (1.5x10 cells / well) before adding different amounts (0.1–100 μg / well) of Sp2CBMTD or Vc2CBMTD at 24h, 48h, or 72h post-infection. 3 PFU) incubation. Shown are the absorbance readings of infected cells obtained 90 h after infection for cells treated with Sp2CBMTD (ac) and Vc2CBMTD (df) compared to untreated infected cells. The bars represent the average absorbance changes ± SD of six replicates. All values ​​are expressed as mean ± SD, and the statistical results are expressed as: *p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001.

[0166] Figure 5: ProPred predictions of antigenic peptides. A. SpCBM sequence. B. PaTD sequence. Predicted binders are in blue, and the first residue of each binding region is shown in red. Antigenic peptides predicted by Nordic Biopharma (green bars) and ProImmune (purple bars) are shown below the sequences.

[0167] Figure 6 : Expression test of wild type and mutant domains. Lane 1, M12 standard; Lane 2, WTSpCBM; Lanes 3-11, Im15-Im23; Lanes 12-15, Im24-Im27; Lane 16, WT PaTD. A) Whole cell extract, B) Soluble extract.

[0168] Figure 7 : Position of peptide 167-181 in the SpCBM structure.

[0169] Figure 8 : Expression and Ni-NTA pull-down of variants Im28 to Im34.

[0170] Fig. 9 : Sites of HEX17 mutations on the hexameric structure. The quaternary structure of HEX17 was simulated by assembling the crystal structures of individual SpCBM (pdb code 4c1x) and PaTD (pdb code 2w38) into a hexamer (i.e., 6 copies of SpCBM and 3 copies of PaTD per molecule). The position of the bound ligand (α2,3-sialyllactose) is shown in sticks (orange). The positions of the mutations are also shown: blue, site of A162P mutation; cyan, site of the other two CBM mutations; magenta, site of TD mutation.

[0171] Fig.10 : IL-8 stimulation. A549 cells were stimulated by adding 10 μg of biological agent (Hex17). Cell supernatants were collected at 24 hours or 48 hours, and the content of IL-8 was determined by ELISA. The statistical significance between control cells and treated cells was determined by one-way analysis of variance using Tukey's multiple comparison test.

[0172] Figure 11: Multiplex analysis of inflammatory mediators. A549 cells were stimulated by adding 10 μg of biological agents (Sp2CBMTD (also known as SpOrig), HEX6 or HEX17). Cell supernatants were collected at 6 hours, 24 hours or 48 hours and analyzed for inflammatory mediators using the 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).

[0173] Fig.12 : Percent survival of CBM-treated and untreated mice after lethal challenge with influenza strain PR8. CBM2, CBM3 and CBM4 represent HEX17, HEX6 and WT (SpOrig), respectively. Animals that received a single CBM dose were given 100 μg CBM one day before lethal challenge with PR8; animals that received repeated doses were given 2x0.1 μg CBM on days 3 and 1 before PR8 challenge.

[0174] Fig.13 : Clinical scores of CBM-treated and untreated mice during PR8 infection. CBM2, CBM3, and CBM4 represent HEX17, HEX6, and WT (SpOrig), respectively. Increasing clinical scores from 1 to 5 represent asymptomatic (1) to lethargy and death (5), respectively.

[0175] Fig.14 : Percentage of body weight loss in CBM-treated and untreated mice during PR8 infection. CBM2, CBM3, and CBM4 represent Hex17, Hex6, and WT (SpOrig), respectively.

[0176] Fig.15 : Anti-mCBM antibody analysis of lung homogenates and serum tissues from day 21 of the PR8-challenged mouse study. Statistical significance between the WT hexamer and the hexamer variants was determined using one-way ANOVA (Tukey's multiple comparison test).

[0177] Figure 16A and B: ELISA results showing the interaction of Sp2CBMTD and Vc2CBMTD with RSV.

[0178] Fig.17A -E: Assay showing GFP-Sp2CBMTD and Vc2CBMTD binding to RSV. PBS was used in the control assay.

[0179] Figures 18A and B: ELISA results showing the interaction between Sp2CBMTD and Sp2CBMTD-R274Q (0.1 μg and 10 ng) and RSV.

[0180] Fig.19A and B: ELISA results showing the interaction between Sp2CBMTD and Vc2CBMTD and neuraminidase-treated RSV.

[0181] Fig. 20A and B: data showing the interaction between 3'-SL-treated Sp2CBMTD / Vc2CBMTD and RSV.

[0182] Fig.21A and B: Data show that the protective effect of CBMs (Sp2CBMTD and Vc2CBMTD) on mammalian cells (HEp2:NPro cells) against RSV is reduced after treating the cells with neuraminidase.

[0183] Figure 22 (Panels AF): Effects of mCBM40 in RSV-infected mammalian cell lines. HEp2:NPro cells were incubated with different amounts (0.1–100 μg / well) of Vc4CBM or HEX17 with RSV (1.5 x 10 3 PFU) were incubated together. Shown are the absorbance readings of infected cells obtained at 24h, 48h and 72h after infection for cells treated with Vc4CBM (A, C and E) and HEX17 (B, D and F) compared to untreated infected cells. The bars represent the average absorbance change ± SD of six replicates. All values ​​are expressed as mean ± SD, and the statistical results are expressed as: *p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001.

[0184] Fig.23 A and B: Quantification of RSV-B viral genome copy number in CBM-treated infected MucilAir inserts 48 h post-infection compared to cells infected with virus alone. A) Sp2CBMTD, B) Vc2CBMTD.

[0185] Methods and Results

[0186] Example 1

[0187] As Connaris et al. (2014) 1 CBM40 protein, Sp2CBMTD, Vc2CBMTD and Vc4CBM (endotoxin-free) were prepared as described in . Hex17 was prepared as described above. The mammalian cell line HEp2:NPro (a cell line designed to constitutively overexpress the BVDV N-terminal protease fragment Npro, which only responds to IFN but does not produce IFN) was purchased from ATCC 4The cells were maintained in DMEM / 10% FBS (with 1% penicillin / streptomycin) in T75 flasks and cultured at 37°C and 5% CO. 2 RSV-A2 strain was prepared in SF-DMEM at 10 -2 dilution, equivalent to 1.5x10 4 PFU / ml.

[0188] For infection studies, 96-well plates (Nunc) were seeded with 100 μL of cells (2.5×10 5 cells / well) and incubate at 37°C and 5% CO 2 Incubate for 24 hours until cells reach 80-90% confluence. 3 CBM40 protein (100 μL, 10-fold serial dilutions of a 10 mg / ml stock in SF-DMEM) was added to the cells before 100 μL of 10 PFU, mixed with RSV on ice for 1 h before addition to the cells, or added to the cells after RSV infection. In each case, CBM40s and / or RSV were left for 1 h before removal. The cells were then covered with 300 μL of Avicel and the plates were incubated at 37°C and 5% CO 2 Incubate at 4 °C for 24, 48, 72 or 90 h.

[0189] To determine the level of RSV in cells after infection, AVICEL was removed and cells were fixed with 10% PFA in PBS for 10 minutes at room temperature, followed by immunostaining with mouse anti-RSV F primary antibody (1:200 dilution, Serotec) and goat anti-mouse HRPIgG antibody (1:2000, Santa Cruz). The presence of RSV was detected using the colorimetric substrate TMB (Sigma). Absorbance was measured at a wavelength of 450 nm (620 nm wavelength was used as a reference).

[0190] Statistical analysis. Group comparisons were performed using one-way ANOVA and Dunnett's multiple comparison test. All analyses were performed using GraphPad Prism 7 (GraphPad Software). Tests with p<0.05 were considered statistically significant. Unless otherwise indicated, all values ​​are expressed as mean ± SD, and statistical results are expressed as: *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001.

[0191] Summarize

[0192] The results are summarized in Figures 2, 3, 4 and 22.

[0193] 1. In the HEp2:NPro mammalian cell line, a single dose of mCBM40 administered before or simultaneously with RSV appears to reduce or inhibit RSV attachment to the cells.

[0194] 2. All mCBM40s (up to the highest concentration of 0.1–1 mg / ml) appeared to be significantly effective in reducing RSV infection when administered concurrently with RSV, compared to prophylactic administration when cells were tested 90 h post-infection.

[0195] 3. Administration of mCBM40 24h after RSV infection demonstrated a reduction in viral infection when given at the highest dose. The highest dose of Vc2CBMTD appeared to beneficially affect RSV levels in the HEp2:NPro mammalian cell line when given 24h, 48h and 72h after RSV administration.

[0196] 4. Among the mCBM40s tested, Vc2CBMTD appears to be the most effective against RSV infection in mammalian cell lines.

[0197] References

[0198] [1]Connaris H, Crocker, PR and Taylor, GL (2009). Enhancing the ReceptorAffinity of the Sialic Acid-binding Domain of Vibrio cholerae Sialidasethrough Multivalency. JBC 284:7339–7351.

[0199] [2]Connaris H, Govorkova EA, Ligertwood Y, Dutia BM, Yang L, Tauber S, Taylor MA, Alias

[0200] N,Hagan R,Nash AA,Webster RG,Taylor GL(2014).Prevention of influenza by targeting

[0201] host receptors using engineered proteins. Proc Natl Acad Sci USA111:6401–6406.

[0202] [3]Harris,J and Werling,D(2003).Binding and entry of respiratory syncytial virus into host cells and initiation of the innate immuneresponse.Cellular Microbiology 5:671–680.

[0203] [4] Hilton, L., Moganeradj, K., Zhang, G., Chen, YH, Randall, RE, McCauley, JW, and Goodbourn, S (2006). The NPro product of bovine viral diarrhea virus inhibits DNA binding by interferon regulatory factor 3and targets it for proteasomal degradation. J Virol 80(23), 11723-32.

[0204] Example 2

[0205] Sp2CBMTD: prediction of immunogenic regions

[0206] Nordic Biopharma computer screening

[0207] In silico T-cell epitope screening identified four significant and two borderline immunogenic clusters:

[0208] Notable:

[0209]

[0210] Critical:

[0211] Domain residue range sequence

[0212] SpCBM 167 to 178 FYNLFSVSSATK

[0213] SpCBM 239 to 251 VRLYVNGVLSRTS

[0214] ProImmune Human Donor T Cell Proliferation Assay

[0215] The ProImmune study highlighted two regions of high antigenicity and two regions of moderate antigenicity:

[0216] High antigenicity:

[0217] Domain residue range sequence

[0218] SpCBM 236 to 250 KGRVRLYVNGVLSRT

[0219] PaTD 392 to 406 GAQVEVGSLNIRLGT

[0220] Moderate antigenicity:

[0221] Domain residue range sequence

[0222] SpCBM 167 to 181 FYNLFSVSSATKKDE

[0223] PaTD 338 to 352 SDWFSVSSNSLYTLS

[0224] ProPred computer analysis

[0225] Another computer tool was used, the online ProPred server 4 The output of the ProPred server is shown in Figure 5. The relative positions of the NordicBiopharma / ProImmune epitopes are also highlighted and show reasonable agreement between the three methods. In addition to the epitopes listed above, ProPred strongly predicted another immunogenic epitope in the SpCBM domain:

[0226] Domain residue range sequence

[0227] SpCBM 286 to 294 IRNLTVYNR

[0228] Mutations in individual CBM and TD domains

[0229] To guide the design of mutations that might reduce immunogenicity, ProPred was used to test the effect of changing each residue in these peptides to each alternative residue. Those changes that minimized the number of predicted allele binders were noted. Since 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 significantly disrupt protein structure.

[0230] Initially, nine single mutations in SpCBM and four single mutations in PaTD were introduced and are listed below ("Im" is an abbreviation for immunogenic mutants):

[0231] (SpCBM) variant mutation (PaTD) variant mutation

[0232] WTSp - WTTD -

[0233] Im15 Y168W Im24 S342D

[0234] Im16 L170A Im25 S345D

[0235] Im17 L170T Im26 L348D

[0236] Im18 V173G Im27 R403K

[0237] Im19 V239A

[0238] Im20 V239T

[0239] Im21 V246G

[0240] Im22 I286A

[0241] Im23 Y292E

[0242] Note: Im1 to Im14 (not shown) were introduced into a non-codon optimized background by mutagenesis prior to acquisition of ProImmune data.

[0243] Synthesis of WT and mutant constructs

[0244] The genes encoding WT SpCBM, WT PaTD, and Im15 to Im27 variants were codon-optimized for expression in E. coli and synthesized by GeneArt. The genes were then cloned in-house into the pHISTEV vector and expressed as 6His-tagged proteins.

[0245] Expression and biophysical characterization

[0246] Initial expression tests were performed to assess solubility. Results showed that all were expressed, but not all were soluble ( Figure 6 ). Note: Solubility (or lack thereof) is not necessarily a predictor of utility. The skilled artisan will appreciate that when preparing or producing a protein, some processes require the use of insoluble material because it is easily purified (from inclusion bodies, etc.). Downstream protocols may then redesign the protein to modulate characteristics such as solubility.

[0247] The results of the expression test showed that:

[0248] Im16 (L170A) is insoluble or very difficult to dissolve

[0249] ·Im25 (TD, S345D) is insoluble

[0250] · Reduced solubility of Im15 (Y168W) and Im17 (L170T)

[0251] Im18 (V173G) and Im22 (I286A) were slightly decreased.

[0252] • The rest showed soluble expression.

[0253] 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, followed by reversed-phase IMAC and size exclusion chromatography (SEC).

[0254] The ten purified domains (WTSp, Im19, Im20, Im21, Im22, Im23, WTTD, Im24, Im26 and Im27) were also characterized by:

[0255] (i) Melting temperature (Tm) was measured by Thermofluor

[0256] (ii) Comparison of tertiary structure with WT by near-ultraviolet circular dichroism (CD)

[0257] (iii) Examination of oligomeric states in solution by dynamic light scattering (DLS)

[0258] (iv) Measurement of binding affinity to sialyllactose by surface plasmon resonance (SPR)

[0259] (v) Measurement of IL-8 cytokine stimulation

[0260] The results are summarized in Table 1.

[0261]

[0262]

[0263] Table 1. Qualitative summary of biophysical characteristics of the WT domain and its variants. Color coding ranges from green to red (including green, orange and yellow), where green indicates that the variant is very similar to its WT counterpart in a particular feature, while light green (green) or yellow indicates increasing differences. Red or orange indicates significant differences. N / A: These characterizations were not performed due to poor solubility / purity of the protein. N / D: Not determined.

[0264] Sp peptide 167-181:

[0265] Im15, Im16, Im17, Im18 are all insoluble or poorly soluble (as mentioned above, this does not necessarily affect the utility of the protein). They are in the "medium" antigenic region 167-181 (FYNLFSVSSATKKDE). This region is apparently very sensitive to change.

[0266] Early results showed that M156F adjacent to L170 (and I286) increased Tm by approximately 4°C.

[0267] Therefore, it can be combined with L170T. M156F does not increase the predicted immunogenicity.

[0268] M185I increased Tm by 5°C and paralleled L170 ( Figure 7 ). This mutation could also be included. Note that like M156F, M185I does not increase predicted immunogenicity but slightly decreases the number of predicted allele binders.

[0269] Sp peptide 236-250:

[0270] Im19, Im20, Im21 all behave similarly to WT. These are located in the "highly" antigenic region 236-250 (KGRVRLYVNGVLSRT).

[0271] Im19 (V239A) was selected instead of the threonine mutation (Im20, V239T). There was no difference in the predicted immunogenicity, but Im19 was closer to the WT Thermofluor Tm and near-UV spectrum. It will be combined with Im21 (V246G).

[0272] Sp peptide 286-294:

[0273] Im22 (I286A) was largely similar to WT, while Im23 (Y292E) showed reduced ligand affinity. ProImmune did not label this region 286-294IRNLTVYNR, but ProPred strongly predicted that this region was immunogenic.

[0274] There is some indication that Im22 has a lower Tm than WT. This residue is adjacent to M156, so it is possible that its behavior would be different if M156F was included.

[0275] TD peptide 338-352:

[0276] Im24(S342D) and Im26(L348D) show similar features to the WT trimerization domain, but there is evidence of a reduced Tm in Im26. They are in the "medium" antigenic region 338-352SDWFSVSSNSLYTLS. The WT sequence is predicted to bind 9 alleles, while Im24 is predicted to bind 2 alleles and the Im24 / Im26 double mutant is predicted to bind 1 allele.

[0277] TD peptide 392-406:

[0278] Im27(R403K) is similar to WT. It is part of the "high" antigenic region 392-406GAQVEVGSLNIRLGT. After introducing this mutation, the predicted alleles were reduced from 21 to 3.

[0279] Synthesis of multiple mutation combinations Im28-34

[0280] The following mutations were introduced:

[0281] i)M156F / L170T

[0282] ii) M156F / L170T / M185I: In ProPred, the predicted alleles for this region were reduced from 31 in WT to 19 for this combination.

[0283] iii) V239A / V246G: In ProPred, the alleles in this region were reduced from 44 to 3.

[0284] iv) I286A / Y292E: In ProPred, the alleles were reduced from 41 to 1.

[0285] v) V239A / V246G / I286A / Y292E combines the first two double mutations.

[0286] vi) M156F / L170T / M185I / V239A / V246G / I286A / Y292E combines all Sp mutations

[0287] vii) TD: S342D / L348D / R403K: The predicted alleles for TD peptide 338-352 were reduced from 9 to 1, and the predicted alleles for TD peptide 392-406 were reduced from 21 to 3. This triple mutant combines all TD mutants. They are all surface exposed and located at the N-terminal end of TD, so it is not expected to interfere with SpCBM in a hexameric form.

[0288] These constructs were named Im28 to Im34:

[0289]

[0290]

[0291] 2.5 Expression and biophysical characterization of Im28-Im34

[0292] As with the individual mutations, combinations of Im28 to Im34 were synthesized by GeneArt and subcloned into pHISTEV for expression analysis. Nickel bead pull-downs were also performed on His-tagged soluble extracts ( Figure 8 ).

[0293] Hexameric form

[0294] Design of hexameric constructs HEX1 to HEX17

[0295] Genes encoding the hexameric forms (called HEX1 to HEX17) were synthesized by GeneArt:

[0296] Sp2CBMTD

[0297]

[0298] The hexameric form is synthesized in two parts to avoid problems associated with synthesizing repetitive sequences in tandem CBM copies. The first gene covers the first CBM and the second part contains the second CBM plus the TD. These can then be cloned simultaneously into pHISTEV to create the Sp2CBMTD construct that trimerizes when expressed.

[0299] The first hexamer, HEX1, contained the mutations L170T / V239A / V246G / I286A / Y292E in the CBM and S342D / L348D / R403K in the TD.

[0300] Solubility data for the individual domains suggested that HEX1 was unlikely to be soluble (again, not necessarily reflective of the utility of the molecule); an additional construct, HEX3, was synthesized. Note that HEX2 contains the same mutations as HEX3, but with the addition of Y292E.

[0301] HEX3 was synthesized and subcloned into pHISTEV vector. Under all tested conditions (variable temperature, IPTG concentration, cell density during induction, with or without heat shock), expression was insoluble. Only the CBM domain containing the same three mutations (V239A V246G1286A) was soluble. The behavior of the double mutant (V239A V246G) was very similar to WT. Therefore, further variants (HEX4, HEX5 and HEX6) were designed and constructed by PCR / ligation, which did not include I286A and contained one or two TD mutations.

[0302] During the work on HEX6, a number of additional versions were designed containing different combinations of HEX6 mutations (numbered HEX7 to HEX16; not characterized).

[0303] HEX17 contains the HEX6 mutation with an additional A162P mutation. This proline mutation has been shown to increase the single CBM Tm by 3-4°C. The proline mutation is not close to other mutations, the N- or C-termini, or the ligand binding site.

[0304] Characterization of hexameric variants

[0305] The expression, purification and characterization results are shown in Table 2. Based on these results, HEX6 and HEX17 were proposed. The position of the HEX17 mutation on the hexamer is shown in Fig. 9 shown.

[0306] Table 2

[0307]

[0308]

[0309] Table 2. Qualitative summary of biophysical characterization of hexameric Sp2CBMTD variants. Color coding ranges from green to red, where green indicates that the variant is very similar to its WT counterpart in a particular feature, while light green (green) or yellow indicates increasing differences. Red or orange indicates significant differences. N / A: These characterizations were not performed due to poor solubility / purity of the protein. N / D: Not determined.

[0310] Example 3: Inflammatory mediators

[0311] Objective: To determine the innate immune response of human lung epithelial cells (A549) treated with mCBM by analyzing the levels of inflammatory mediators over time.

[0312] Administration of Sp2CBMTD to mammalian cells stimulates proinflammatory responses both in vitro and in vivo 1,2To determine whether this could still be observed with modified hexameric sialic acid binding molecules, mammalian A549 cells were stimulated by adding 10 μg of biologic (Sp2CBMTD (also known as SpOrig), HEX6 (i.e., a sialic acid binding molecule comprising 3xHEX6 units), or HEX17 (i.e., a sialic acid binding molecule comprising 3xHEX17 units), and cell culture media were collected at specific time points after administration. Concentrations of inflammatory mediators were measured by ELISA and multiplex assays.

[0313] Human IL-8 (a benchmark cytokine for research) responses were tested using the Human 1x Mouse CXCL1 / KC Quantikine ELISA Kit (R&D BioSystems). IL-8 concentration levels in stimulated A549 cells were as follows: Fig.10 It is evident that when A549 cells were stimulated with the modified hexamer HEX17, IL-8 levels were significantly lower than those in cells stimulated with Sp2CBMTD (also known as SpOrig).

[0314] Human Cytokine 12-plex Assay (Bio-PlexPro TM , Bio-Rad) tested the inflammatory mediator response. Figure 11 shows the analysis of 12 inflammatory mediators from the culture medium after A549 cells were stimulated with Sp2CBMTD (WT, also known as SpOrig), HEX6 and HEX17 (variants) at specific time points (6h, 24h, 48h). Before analysis, the samples were thawed and diluted 1:4 in PBS, and then human HS cytokine 12-reanalysis method (R&D Systems) was used. The data show that:

[0315] HEX17 affected the levels of almost all cytokines tested compared to SpOrig and HEX6. At 48h, significantly lower concentrations (pg / ml) were observed for the analytes IL-6, IL-8, GM-CSF, and IFN-γ compared to SpOrig and HEX6.

[0316] - HEX17 appears to result in increased levels of all cytokines tested compared to controls at 48h, with the exception of IL-5 and VEGF (to be confirmed).

[0317] HEX6 showed only reduced IL-6 stimulation compared to SpOrig at 48h.

[0318] Example 4: In vivo PR8 mouse data

[0319] The aim of this study was to evaluate the efficacy of Sp2CBMTD (SpOrig) and its variants in a mouse model of lethal influenza virus infection. Each candidate protein was also administered in the absence of influenza infection to assess whether they alone caused any morbidity or mortality.

[0320] Survival rate, clinical scores, and weight loss

[0321] The results showed that CBM2 (HEX17), CBM3 (HEX6) or CBM4 (WT, SpOrig) alone did not cause any significant morbidity or mortality. A single 100 μg dose of CBM2 (HEX17), CBM3 (HEX6) and CBM4 (WT, SpOrig) administered one day before a lethal challenge with PR8 influenza virus induced protection against PR8 infection, with HEX17 (100% survival) having the highest efficacy, followed by SpOrig and then HEX6 ( Fig.12 HEX17 also had lower clinical scores compared to SpOrig and HEX6 ( Fig.13 Compared with untreated infected mice, surviving mice from the single high-dose treatment group all lost weight at the peak of infection but recovered quickly ( Fig.14 ).

[0322] Anti-mCBM antibody analysis of lung homogenates and serum tissues from PR8-challenged mouse studies.

[0323] The aim of this study was to determine whether modified immunogenic epitopes of modified variants of Sp2CBMTD (SpOrig) would show reduced antibody levels in mice in a PR8 challenge study (it should be noted that the epitope was modified based on human MHC-II class binding information). To this end, mice surviving the PR8 challenge study were culled on day 21 and lungs and serum were collected and tested in an ELISA format for anti-mCBM antibodies - IgG, IgA, IgE and IgM - against the coating antigen SpOrig (1 μg / well). Fig.15 The data shown in the

[0324] The modified protein HEX17 did show a significant reduction in mouse lung IgM levels compared to SPORIG (p<0.05). Since only one surviving mouse received HEX6 treatment, only HEX17 and SPORIG data were statistically analyzed.

[0325] There was an indication of a slight trend toward decreased antibody levels (lung IgA and IgM) in mice treated with modified CBM compared to SpOrig.

[0326] Example 5

[0327] The purpose of the following experiment was to determine whether there is a direct interaction between CBM (Sp2CBMTD and Vc2CBMTD) and human RSV in a cell-free system. The ELISA experiment was designed as follows:

[0328] Experiment 1

[0329] At 4°C, 96-well plates were pre-coated with different concentrations of RSV (A2 strain), CBM, Sp2CBMTD and Vc2CBMTD and PBS (vector, negative control) overnight. The wells were blocked with blocking buffer (BB, PBS containing 1% w / v BSA). Different concentrations of CBMs were diluted in BB and added to the wells after removing the coated antigen and blotting the plate. The plate was incubated for 1 hour at room temperature. The wells were then washed with washing buffer (WB, PBS supplemented with 0.5% (v / v) Tween-20) and incubated for 1-2 hours at room temperature before adding the primary antibody (rabbit anti-SpCBM antibody or rabbit anti-VcCBM antibody, diluted 1:5000 in BB, Eurogentec). Goat anti-rabbit IgG-HRP (diluted 1:2500 in BB, Sigma) was used as a secondary antibody, and TMB (Sigma) was used as a substrate. Binding of CBM to RSV was determined by comparing the absorbance at 450 nm (620 nm as background reference) between treated and untreated control wells after reaction with TMB (substrate for HRP).

[0330] The results showed that both Sp2CBMTD and Vc2CBMTD directly interacted with RSV in a dose-dependent manner (see Figures 16A and B).

[0331] Experiment 2

[0332] 96-well plates were pre-coated with 10 μg GFP-Sp2CBMTD, GFP-Vc2CBMTD, GFP, GFP-Sp2CBMTD-R274Q (sialic acid binding mutant of Sp2CBMTD) or 32000PFU RSV and PBS (carrier, negative control) as coating antigens. Holes were incubated at room temperature for 1 hour with the associated proteins. Images of GFP fluorescence binding were captured using a microscope with a 10x objective (in the GFP channel on the EVOS FL cell imaging system).

[0333] The results showed that both GFP-Sp2CBMTD and GFP-Vc2CBMTD were able to bind directly to RSV. Negligible binding to RSV was observed with the sialic acid binding mutant GFP-Sp2CBMTD-R274Q (see Fig.17A -E).

[0334] Example 6

[0335] The purpose of the following experiments was to support the view that the interaction between CBM (Sp2CBMTD and Vc2CBMTD) and RSV is based on the binding of CBM to viral surface glycoproteins terminated with sialic acid. Therefore, three ELISA experiments were designed.

[0336] Experiment 1

[0337] Wild-type Sp2CBMTD and mutant Sp2CBMTD-R274Q (sialic acid binding mutant) were used in an ELISA format as described in Experiment 1 of Example 5. The results showed that wild-type Sp2CBMTD bound RSV with higher affinity than mutant Sp2CBMTD-R274Q at the same concentration (see Figures 18A and B).

[0338] Experiment 2

[0339] Neuraminidase (Nu) from Clostridium perfringens (Sigma) is a glycosidase that can cleave terminal sialic acid from sialylated sugar conjugates on the cell surface, and has a connection specificity for Gal, GlcNac, GalNAc, AcNeu, GlcNeu, oligosaccharides, glycolipids or glycoproteins linked to α-2,3-α-2,6-α-2,8-. To test whether the viral surface glycoproteins belong to this situation, RSV was treated with or without neuraminidase before adding CBM. An ELISA experiment similar to Experiment 1 in Example 5 was established. A 96-well plate was pre-coated with different concentrations of RSV, CBM (as a positive control) and PBS (as a negative control). For some wells coated with RSV, neuraminidase (30mU / well) was added for 1 hour, followed by CBM (100ng or 10ng). As described in Project 1, CBM binding was evaluated using anti-CBM antibodies first, followed by detection of anti-IgG-HRP.

[0340] The results showed that both Sp2CBMTD and Vc2CBMTD interacted with RSV in a dose-dependent manner in the absence of neuraminidase treatment, as previously observed (below and in Example 5 Experiment 1). The interaction was partially abolished after neuraminidase treatment of RSV in the presence of Sp2CBMTD, but was almost completely abolished when Vc2CBMTD was used as an interaction partner (see Fig.19A and B).

[0341] These data provide evidence that RSV surface glycoproteins are sialylated, suggesting that CBMs may have a direct-acting antiviral role in RSV infection.

[0342] Experiment 3:

[0343] In order to determine whether the competitive ligand can block the sialic acid binding site of CBM and interfere with the interaction of CBM with RSV, CBM (Sp2CBMTD and Vc2CBMTD, 100ng / well) was pre-incubated with different concentrations of α2,3 sialyllactose (3'-SL, 10mM, 100mM, Glycotech) at room temperature for 1 hour. The mixture was added to a 96-well plate coated with RSV (32000PFU / well) together with a control (PBS, CBM only). As in the ELISA experiment in Example 5, CBM was added to the wells. The number of repetitions in the study was n=3, and the raw data were analyzed by one-way ANOVA using GraphPad Prism. p value <0.05 was considered statistically significant (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001).

[0344] The results showed that the competitive sialoside 3'-SL could reduce the interaction between RSV and Sp2CBMTD and Vc2CBMTD in a dose-dependent manner (see Fig. 20A and B).

[0345] Example 7

[0346] An in vitro study was performed to demonstrate that the protective effect of CBMs (Sp2CBMTD and Vc2CBMTD) on mammalian HEp2:NPro cells against RSV was reduced after treatment of the cells with neuraminidase.

[0347] The experiment was designed as follows: adherent HEp2:NPro cells were cultured in 96-well plates with or without neuraminidase at 37°C, 5% CO 2 Incubate for 1 hour at 4 °C, then add CBM (100 μg / well) and incubate for another hour, and finally add RSV (3000 PFU / well) and incubate for 1 hour. (Sigma) medium and incubate at 37°C with 5% CO 2Incubate for 96 hours at 4 °C. The amount of RSV infected cells was assessed using mouse anti-RSV F antibody (1:200 dilution, MCA490, BioRad), followed by anti-mouse IgG-HRP secondary antibody (1:2000 dilution, Cell Signalling Technology), thereby determining RSV-infected cells. After reaction with TMB (substrate for HRP), the inhibitory effect on RSV infectivity was determined by comparing the absorbance at 450 nm (620 nm as background reference) at wells treated with neuraminidase and untreated control wells. Repeat samples n=4, and the raw data were analyzed by one-way ANOVA using GraphPad Prism. p value <0.05 was considered statistically significant (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001).

[0348] For the RSV-only control, the results did not show any difference in infectivity between neuraminidase-treated and untreated cells (first two bars in the graph). However, after neuraminidase treatment of the cells, the cytoprotective effect of both CBMs was reduced (fourth bar in the graph), but to a different extent than in cells initially treated with the CBM and subsequently with RSV, which was more pronounced with Vc2CBMTD. The data suggest that CBMs may also protect cells from RSV by targeting cell surface sialic acid (see Fig.21A and B).

[0349] Example 8

[0350] Utility of engineered carbohydrate-binding module proteins (Sp2CBMTD and Vc2CBMTD) in RSV-B infection using an in vitro 3D model of human airway epithelial cells.

[0351] The aim of this study was to evaluate the effects of mCBM40, Sp2CBMTD, and Vc2CBMTD on RSV replication in a 3D model of human respiratory epithelium. TM Carry on, MucilAir TM The 3D model of human respiratory epithelium is a fully differentiated and easy-to-use model composed of primary human epithelial cells freshly isolated from nasal, tracheal, or bronchial biopsies (Epithelix). For infection studies, RSV-B strains (10 6Genome copies / ml RSV, Geneva clinical strain, 2017) and applied to the apical side of pre-washed (with PBS) MucilAir inserts on day 0. For mCBM treatment (n=3), Sp2CBMTD and Vc2CBMTD (at 10 μg or 100 μg doses) were applied apically a) one hour before virus infection as a single dose or b) one day and one hour before virus infection as repeated doses. Both regimens were further exposed to mCBM at 3.5 hours post infection (pi). Untreated infected MucilAir (n=2) was used as a control. Apical washes (200 μL) were collected at 3.5 h pi and then at 24, 48 and 96 h pi.

[0352] The viral genome copy number was determined using the QIAampR viral RNA kit (Qiagen) using 20 μL of top wash extracted from viral RNA. The viral RNA was quantified using QuantiTect Probe RT-PCR (Qiagen) using 5 μL of viral RNA and two RSV-B specific primers. The data from the readings were corrected for the dilution factor and expressed as the genome copy number per milliliter. Statistical analysis was performed using Prism 6.0GraphPad software. Student's unpaired t-test was used to compare the two groups of data, with *p<0.05.

[0353] Summarize

[0354] Both Sp2CBMTD and Vc2CBMTD were shown to reduce apical viral loads in RSV-infected human respiratory epithelial cells. The extent of the reduction depended on the type of mCBM and the number of doses administered. Due to variability in the data, exposure of infected MucilAIrinserts to Sp2CBMTD did not show a significant effect on viral replication, but at the highest dose (100 μg) there was a significant reduction in viral replication after two doses at 48 h pi compared to virus-infected controls (see Fig.23 A) and a slight reduction was observed after three doses at 96 h pi. In both administration regimens, exposure to Vc2CBMTD (100 μg) was more effective in reducing RSV viral replication than using the 10 μg administration regimen. Although not statistically significant, a reduction of greater than 1 log ( Fig.23 B).

Claims

1. Use of a sialic acid binding molecule in the preparation of a medicament for treating and / or preventing symptoms, infections, diseases and / or conditions associated with respiratory syncytial virus (RSV), wherein the sialic acid binding molecule comprises one or more family 40 carbohydrate binding modules (CBM40).

2. The method of claim 1, wherein the treatment and / or prevention of symptoms, infections, diseases and / or conditions associated with respiratory syncytial virus (RSV) comprises neutralizing or blocking RSV infection.

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

4. The use according to claim 1 or 2, wherein the sialic acid binding molecule is provided as a composition for mucosal administration.

5. The use according to claim 1 or 2, wherein the sialic acid binding molecule is used prophylactically to prevent RSV infection.

6. The use according to claim 1 or 2, wherein the sialic acid binding molecule: (i) not showing sialidase activity; (ii) does not bind heparin or heparin sulfate; and / or (iii) does not contain a GAG binding domain of a protein that binds heparin or heparan sulfate.

7. The use according to claim 1 or 2, wherein the sialic acid binding molecule comprises: (i) the sialic acid binding domain of Vibrio cholerae NanH sialidase; and / or (ii) The sialic acid binding domain of Streptococcus pneumoniae NanA sialidase.

8. The use according to claim 7, wherein: The Vibrio cholerae NanH sialidase comprises the amino acid sequence of SEQ ID NO: 1 or 2; and / or The Streptococcus pneumoniae NanA sialidase comprises the amino acid sequence of SEQ ID NO: 3 or 4.

9. The use according to claim 1 or 2, wherein the sialic acid binding molecule is Sp2CBM, Sp2CBMTD, Vc4CBM, Vc2CBM or Vc2CBMTD.

10. The use according to claim 1 or 2, wherein the sialic acid binding molecule comprises the following structure: CBM1(V239A V246G A162P)-CBM2(V239A V246G A162P)-TD (S342D R403K) CBM1 and CBM 2 are derived from the CBM40 sequence, and TD is derived from the trimerization domain.

11. The use according to claim 1 or 2, wherein the sialic acid binding molecule comprises the following sequence: 。 12. The in vitro method according to claim 3, wherein the sialic acid binding molecule: (i) not showing sialidase activity; (ii) does not bind heparin or heparin sulfate; and / or (iii) does not contain a GAG binding domain of a protein that binds heparin or heparan sulfate.

13. The in vitro method according to claim 3 or 12, wherein the sialic acid binding molecule comprises: (i) the sialic acid binding domain of Vibrio cholerae NanH sialidase; and / or (ii) Sialic acid binding domain of S. pneumoniae NanA sialidase.

14. The in vitro method according to claim 13, wherein: The Vibrio cholerae NanH sialidase comprises the amino acid sequence of SEQ ID NO: 1 or 2; and / or The Streptococcus pneumoniae NanA sialidase comprises the amino acid sequence of SEQ ID NO: 3 or 4.

15. The in vitro method according to claim 3, wherein the sialic acid binding molecule is Sp2CBM, Sp2CBMTD, Vc4CBM, Vc2CBM or Vc2CBMTD.

16. The in vitro method of claim 3, wherein the sialic acid binding molecule comprises the following structure: CBM1(V239A V246G A162P)-CBM2(V239A V246G A162P)-TD (S342D R403K) CBM1 and CBM 2 are derived from the CBM40 sequence, and TD is derived from the trimerization domain.

17. The in vitro method of claim 3, wherein the sialic acid binding molecule comprises the following sequence: 。

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