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Title:
TREATMENT OF VIRAL INFECTION
Document Type and Number:
WIPO Patent Application WO/2021/176017
Kind Code:
A1
Abstract:
Disclosed are molecules useful in the treatment or prevention of viral infections in humans and animals and/or the treatment or prevention of the associated symptoms, diseases and/or conditions. The disclosure provides molecules which comprise sugar (carbohydrate/polysaccharide/sialic acid/glycan)-binding protein(s) which are useful in the treatment or prevention of Coronavirus infections and/or diseases, symptoms and/or conditions caused or contributed to by the same.

Inventors:
TAYLOR GARRY (GB)
CONNARIS HELEN (GB)
POTTER JANE (GB)
ROGERS GRAEME (GB)
AITKEN ANGUS (GB)
TORTAJADA ANTONI (GB)
THOMSON DOUGLAS (GB)
YANG LEI (GB)
Application Number:
PCT/EP2021/055515
Publication Date:
September 10, 2021
Filing Date:
March 04, 2021
Export Citation:
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Assignee:
PNEUMAGEN LTD (GB)
International Classes:
A61K38/16; A61P11/00; A61P31/14
Attorney, Agent or Firm:
MARKS & CLERK LLP (GB)
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Claims:
Claims

1. A glycan binding molecule for use in the treatment or prevention of a Coronavirus infection and/or a disease or condition caused or contributed by a Coronavirus.

2. The Glycan binding molecule of claim 1 for use of claim 1 , wherein the disease or condition caused or contributed by a Coronavirus is COVID-19 or SARS or MERS.

3. The glycan binding molecule of claims 1 or 2, for use of claims 1 or 2, wherein the treatment of a Coronavirus infection and/or a disease or condition caused or contributed by a Coronavirus comprises the treatment of one or more of the symptoms associated with the Coronavirus infection, disease or condition.

4. The glycan binding molecule of claim 3, for use of claim 3, wherein the symptom(s) is/are a continuous cough and/or a fever and/or a change/loss in/of taste/smell.

5. The glycan binding molecule of claims 1-4, for use of claims 1-4, wherein the glycan binding molecule comprises a carbohydrate binding module (CBM).

6. The glycan binding molecule of claim 5, for the use of claim 5, wherein the CBM is selected from the group consisting of:

(i) A Family 40 CBM;

(ii) A Family 32 CBM;

7. The glycan binding molecule of claim 5 or 6, for the use of claim 5 or 6, wherein the CBM is selected from the group consisting of:

(i) a Clostridium perfringens CBM32 (CpCBM32);

(ii) a Streptococcus pneumoniae CBM40 (SpCBM40);

(iii) a Vibrio cholerae CBM40 ( l/cCBM40) ;

(iv) a Vibrio cholerae Nan FI sialidase CBM;

(v) a Vibrio cholerae Nan FI sialidase CBM sialic acid binding fragment thereof.

(vi) a Streptococcus pneumoniae nanA sialidase CBM; and

(vii) a Streptococcus pneumoniae nanA sialidase CBM sialic acid binding fragment thereof.

8. The glycan acid binding molecule of any preceding claim, for the use of any preceding claim, wherein the Coronavirus is SARS-CoV-2.

9. The glycan acid binding molecule of any preceding claim, for use of any preceding claim wherein the Coronavirus is a SARS-CoV-2 variant.

10. The glycan acid binding molecule of claim 9, for use of claim 9, wherein the SARS- CoV-2 variant comprises a mutation within the spike protein, the mutation being an amino acid change relative to the amino acid sequence of the spike protein of the Wuhan-Hu-1 isolate with accession codes : QHD43416.1/YP_009724390.1.

11 . The glycan acid binding molecule of claims 9 or 10, for use of claims 9 or 10, wherein the variant comprises one or more of the following spike protein mutations:

(i) HV69-70 deletion; and/or

(ii) N501Y; and/or

(iii) E484K.

12. The glycan acid binding molecule of claims 9, 10 or 11 , for use of claims 9, 10 or 11 , wherein the SARS-CoV-2 variant is the B.1 .1.7 variant and/or the B.1525 variant and/or the B.1.351 variant.

13. A CBM for use in the treatment or prevention of a Coronavirus infection a disease or condition caused or contributed by a Coronavirus and/or a symptom of a Coronavirus infection/disease.

14. A CBM32 for use in the treatment or prevention of:

(i) a Coronavirus infection, a disease or condition caused or contributed by a Coronavirus and/or a symptom of a Coronavirus infection/disease; or

(ii) COVID-19, a disease or condition caused or contributed by SARS-CoV-2 and/or a symptom of COVID-19.

15. The CBM32 of claim 14, for use of claim 14, wherein the CBM32 comprises the amino acid sequence of SEQ ID NO: 1 :

AIIETAIPQSEMTASATSEEGQDPASSAIDGNTNTMWHTKWNGSDALPQSLSVNLGSSRKVSSIAITP RTSGNNGFITKYEIHAINNGVETLVAEGTWEENNLVKTVTFDSPIDAEEIKITAIQGVGGFASIAELN VYE or a glycan/carbohydrate binding fragment thereof.

16. A CBM40 for use in the treatment or prevention of: (i) a Coronavirus infection, a disease or condition caused or contributed by a Coronavirus or

(ii) COVID-19, a disease or condition caused or contributed by SARS-CoV-2 and/or a symptom of COVID-19.

17. The CBM40 of claim 16, for use of claim 16, wherein the CBM40 comprises the amino acid sequence of SEQ ID NO: 4:

ALFDYNATGDTEFDSPAKQGWMQDNTNNGSGVLTNADGMPAWLVQGIGGRAQWTYSLSTNQHAQASSFGWRMTTE MKVLSGGMITNYYANGTQRVLPIISLDSSGNLVVEFEGQTGRTVLATGTAATEYHKFELVFLPGSNPSASFYFDG KLIRDNIQPTASKQNMIVWGNGSSNTDGVAAYRDIKFEIQGD or SEQ ID NO: 6

VIEKEDVETNASNGQRVDLSSELDKLKKLENATVHMEFKPDAKAPAFYNLFSVSSATKKDEYFTMAVYNNTATLE

GRGSDGKQFYNNYNDAPLKVKPGQWNSVTFTVEKPTAELPKGRVRLYVNGVLSRTSLRSGNFIKDMPDVTHVQIG

ATKRANNTVWGSNLQIRNLTVYNRALTPEEVQKRS or a glycan/sialic acid binding fragment of either.

18. A modified CBM for use in the treatment or prevention of:

(i) a Coronavirus infection a disease or condition caused or contributed by a Coronavirus and/or a symptom of a Coronavirus infection/disease; or

(ii) COVID-19, a disease or condition caused or contributed by SARS-CoV-2 and/or a symptom of COVID-19.

19. The modified CBM of claim 18, for use of claim 18, wherein the modified CBM comprises a wild type CBM sequence which has been modified to include one or more mutations.

20. The modified CBM of claim 18 or 19, wherein the modified CBM comprises the sequence of SEQ ID NO: 16:

GAMVIEKEDVETNASNGQRVDLSSELDKLKKLENATVHMEFKPDPKAPAFYNLFSVSSATKKDEYFTM AVYNNTATLEGRGSDGKQFYNNYNDAPLKVKPGQWNSVTFTVEKPTAELPKGRARLYVNGGLSRTSLR SGNFIKDMPDVTHVQIGATKRANNTVWGSNLQIRNLTVYNRALTPEEVQKRSGGGSGVIEKEDVETNA SNGQRVDLSSELDKLKKLENATVHMEFKPDPKAPAFYNLFSVSSATKKDEYFTMAVYNNTATLEGRGS DGKQFYNNYNDAPLKVKPGQWNSVTFTVEKPTAELPKGRARLYVNGGLSRTSLRSGNFIKDMPDVTHV QIGATKRANNTVWGSNLQIRNLTVYNRALTPEEVQKRSGGSLGVPDFESDWFDVSSNSLYTLSHGLQR SPRRWVE FARSSSPSTWNIVMPSYFNDGGHKGSGAQVEVGSLNIKLGTGAAVWGTGYFGGIDNSATT RFATGYYRVRAWI or a glycan acid binding fragment thereof.

21 . A glycan acid binding molecule for use in the treatment or prevention of:

(i) a Coronavirus infection a disease or condition caused or contributed by a

Coronavirus and/or a symptom of a Coronavirus infection/disease; or

(ii) COVID-19, a disease or condition caused or contributed by SARS-CoV-2 and/or a symptom of COVID-19; wherein the glycan binding molecule is selected from the group consisting of:

(i) Cp2CBM32TD (comprising, consisting essentially of or consisting of: 2 CBMs (CBM32s) from Clostridium perfringens fused to a trimerisation domain);

(ii) Sp2CBM40TD (comprising, consisting essentially of or consisting of: 2 CBMs (CBM40s) from Streptococcus pneumoniae fused to a trimerisation domain;

(iii) f/c2CBM40TD (comprising, consisting essentially of or consisting of: 2 CBMs (CBM40s) from Vibrio cholerae fused to a trimerisation domain; and

(iv) f/c4CBM (comprising, consisting essentially of or consisting of: 4 CBMs (CBM40s) from Vibrio cholerae).

Description:
TREATMENT OF VIRAL INFECTION FIELD OF THE INVENTION

The disclosure provides molecules for use in compositions, medicaments and methods for the treatment or prevention of viral diseases and/or conditions.

BACKGROUND

Viral pathogens can cause an array of diseases and/or conditions in humans and while vaccines and antiviral therapies are available for use against some pathogens, for others there are no useful therapeutic options. In some cases, the treatment of a viral infection is limited to patient isolation (to minimise the risk of further spread) and treatment of the symptoms. In some cases a viral pathogen, in particular a respiratory viral pathogen, can lead to secondary complications such as shortness of breath, pneumonia, bronchitis and/or bronchiolitis. The young, old, immunocompromised, pregnant and individuals with underlying health conditions are often most at risk from viral pathogens.

The pathology of any given viral pathogen will vary, but those causing respiratory illness will most often be transmitted by aerosolised droplets generated by, for example, coughs and sneezes and fluids/secretions from/to mucus membranes (including the respiratory airways, the lungs, nose, mouth and eyes).

Occasionally, a viral outbreak concerns a species and/or strain of pathogen that is new to science. There is often no effective treatment for these infections and outbreaks must be contained and controlled to prevent uncontrolled spread.

Severe acute respiratory syndrome (SARS) is a viral respiratory disease of zoonotic origin. This disease is caused by the SARS Coronavirus and the symptoms may include a fever and/or ‘flu’-like illness with muscle pain, a cough, sore throat and other nonspecific symptoms (including, for example, lethargy/malaise). Infection with the SARS Coronavirus may also result in a shortness of breath, viral pneumonia and/or secondary bacterial pneumonia.

Vaccines are often difficult to develop, taking a considerable time to prepare and test. Also, antiviral drugs are variably effective and, in many cases, the virus infection can resolve before the drug takes effect.

Accordingly, there is a need for new and effective treatments to combat viral infections, in particular viral respiratory infections. SUMMARY

Disclosed herein are molecules which may be used in the treatment or prevention of viral infections in humans and animals and/or the treatment or prevention of the associated diseases and/or conditions.

The described molecules may find particular application in the treatment or prevention of viral respiratory pathogens, including for example pathogens belonging to the Family Coronavi dae.

It should be noted that the terms “comprise”, “comprising” and/or “comprises” is/are used to denote that the various aspects and embodiments of this disclosure “comprise” a particular feature or features. It should be understood that this/these terms may also encompass aspects and/or embodiments which “consist essentially of or “consist of the relevant feature or features.

The Coronavihdae are a group of enveloped, positive-sense, single-stranded RNA viruses. The Coronavihdae contain the genus Coronavirus. The name “ Coronavihdae " or “ Coronavirud’ is derived from the distinctive shape of the virus which contains a number of crown-like projections ("peplomers" or "spikes"). Coronaviruses cause respiratory tract infections in humans and outbreaks of deadly pneumonia worldwide.

The Coronaviruses are large enveloped, positive strand RNA viruses that have been classified into 4 genera: Alpha-, Beta-, Gamma-, and Deltacoronavirus. They have the largest genome across all RNA viruses (27 - 32kb), packed inside a helical capsid surrounded by an envelope. There are at least 3 structural proteins associated with the viral envelope: the membrane protein (M), the envelope protein (E) and the spike (glyco)protein (S). Some Coronavirus encode a hemagglutinin-esterase protein (HE).

The S glycoprotein forms large protrusions on the viral surface (forming the “corona” or “crown”) and it is involved in viral entry to the host cell. The S protein has a large ectodomain divided into the following domains/regions: S1 (receptor binding domain); S2 (membrane fusion domain); a transmembrane anchor; and a short intracellular tail. The S1 domain is divided into 2 major domains: (i) N-terminal domain (S1 -NTD) - responsible for binding sugar; and (ii) the C-terminal domain (S1 -CTD) - responsible for recognizing protein receptors ACE2, APN, and DPP4. As used herein, the term “Coronavirus" embraces any virus classed as belonging to the Family Coronaviridiae and embraces the SARS Coronavirus ; the MERS Coronavirus ; and SARS- CoV-2 Coronavirus.

The term “ Coronavirus " also embraces all SARS-CoV-2 variants. For example, the term "Coronavirus” embraces variants B.1.1.7, B.1525, B.1.351 and the variant referred to as P1. These variants may be characterised by mutations (for example amino acid additions, substitutions and/or deletions) within the spike protein. For example, the term “Coronavirus” may embrace any variant with one or more of the following mutations within the spike protein:

(i) HV69-70 deletion; and/or

(ii) N501Y; and/or

(iii) E484K.

In all cases, a variant spike protein sequence may contain one or more mutations relative to a reference sequence. A suitable reference sequence may be the Wuhan-Hu-1 strain, S- proteins sequences from which are available under accession codes QHD43416.1 /YP_009724390.1 .

SARS-CoV-2 is classified as belonging to the same genus as both SARS and MERS (, Betacoronavirus ); it is in the same sub-genus grouping as SARS, sharing around 80% nucleotide identity across the whole genome. Both SARS and SARS-CoV-2 use the glycosylated ACE2 protein expressed on the host cell surface for cell entry. Glycosylation of ACE2 residue 90 has been shown to significantly inhibit the SARS virus. The SARS S protein has a predicted 21 glycosylation sites, with at least 18 of these residues being conserved in the SARS-CoV-2 sequence.

Without wishing to be bound by any particular theory, antibodies which neutralise Coronavirus (for example, the MERS Coronavirus) may target the receptor-binding domain (RBD) of the spike glycoprotein and block its binding to the cellular receptor dipeptidyl peptidase 4 (DPP4). Other (anti-S protein N-terminal domain (NTD)) antibodies have been shown to bind to the N- terminal domain (NTD) of the spike glycoprotein and inhibit host cell entry with high potency. Thus, by targeting the NTD part of the S-protein, it may be possible to inhibit host cell entry in a way which is not dependent on the RBD (S1-CTD) site; that inhibition may affect the conformational state of the S glycoprotein. It is also suggested that, like certain types of influenza (for example, influenza type C), Coronavirus can bind host cell sialic acid containing receptors - in particular sialoglycans, including, for example, cell surface components containing 9-O-acetylated sialic. Moreover (and again, without wishing to be bound by theory), Coronaviruses possess a glycoprotein profile that might be recognised by a variety of CBMs including those classified as belonging to CBM families 32, 40, 47, 67 and 70.

Thus, proteins with an affinity for certain carbohydrates (polysaccharides or glycans), including, for example, sialic acid (e.g. any of the CBM(s) as described herein) have the potential to disrupt the infectivity of Coronavirus, including SARS-CoV-2, by targeting glycans on the host and on the virus.

Disclosed are a number of molecules which comprise (consist of, or consist essentially of) sugar (carbohydrate/polysaccharide/glycan)-binding protein(s). Proteins of this type exhibit a particular affinity for glycans and/or sialic acid and are useful in the treatment or prevention of Coronavirus infections and/or diseases and/or conditions caused or contributed to by the same.

The phrase “disease or condition caused or contributed to by a Coronavirus’’ may include those diseases or conditions referred to as SARS and MERS and/or any other respiratory disease and/or condition associated with a Coronavirus infection. Additionally, the phrase “disease or condition caused or contributed to by a Coronavirus’’ includes the acute respiratory disease referred to as COVID-19 and which is caused by, or which is associated with, a SARS- CoV-2 infection.

As used herein, the term treatment may embrace a reduction in one or more of the symptoms associated with a disease/condition caused or contributed to by a Coronavirus infection. Such symptoms may include, for example, a (continuous) cough, a fever, change/loss in/of taste/smell. Accordingly, any of the glycan binding molecules described herein may be used in compositions and or methods for the treatment of one or more of the symptoms of a Coronavirus infection. By way of example, any one of the glycan binding molecules of this invention may be used to reduce the continuous cough that might develop as a consequence of a Coronavirus infection.

In view of the above, this disclosure provides a glycan binding molecule for use in the treatment or prevention of a Coronavirus infection and/or a disease or condition caused or contributed by a Coronavirus.

Also disclosed is a method of treating or preventing a Coronavirus infection and/or a disease or condition caused or contributed by a Coronavirus, said method comprising administering a subject in need thereof a glycan binding molecule. Provided herein is the use of a glycan binding molecule in the manufacture of a medicament for the treatment or prevention of prevention of a Coronavirus infection and/or a disease or condition caused or contributed by a Coronavirus.

This disclosure also provides a sialic acid binding molecule for use in the treatment or prevention of a Coronavirus infection and/or a disease or condition caused or contributed by a Coronavirus.

Also disclosed is a method of treating or preventing a Coronavirus infection and/or a disease or condition caused or contributed by a Coronavirus, said method comprising administering a subject in need thereof a sialic acid binding molecule.

Provided herein is the use of a sialic acid binding molecule in the manufacture of a medicament for the treatment or prevention of prevention of a Coronavirus infection and/or a disease or condition caused or contributed by a Coronavirus.

A particularly useful molecule is the carbohydrate-binding module (CBM). Thus, a carbohydrate, glycan and/or sialic acid binding molecule for the uses, methods and medicaments described herein may be, or may comprise, a CBM.

Accordingly, the disclosure further provides:

(i) A CBM for use in the treatment or prevention of a Coronavirus infection and/or a disease or condition caused or contributed by a Coronavirus·,

(ii) A method of treating or preventing a Coronavirus infection and/or a disease or condition caused or contributed by a Coronavirus, said method comprising administering a subject in need thereof a CBM.

(iii) Use of a CBM in the manufacture of a medicament for the treatment or prevention of prevention of a Coronavirus infection and/or a disease or condition caused or contributed by a Coronavirus.

Based on the above, this disclosure provides a CBM for use in the treatment or prevention of a COVID-19 infection and/or a disease or condition caused or contributed by SARS-CoV-2.

The disclosure also provides a method of treating or preventing a COVID-19 infection and/or a disease or condition caused or contributed to by SARS-CoV-2, said method comprising administering a subject in need, thereof a CBM. Described herein is the use of a CBM in the manufacture of a medicament for the treatment or prevention of prevention of a COVID-19 infection and/or a disease or condition caused or contributed by SARS-CoV-2.

Examples of useful CBMs are provided below, but for completeness it should be understood that the term “CBM” includes, for example, CBMs classified as belonging to CBM families 32, 40, 47, 67 and 70. One of skill will appreciate that there is a vast array of different CBMs and members of the abovementioned families can be derived from many different bacterial species Further information regarding CBMs, in particular the family 32, 40, 47, 67 and 70 CBMs, can be found at the public CAZY database (available at: http://www.cazv.org)· Some particularly useful CBMs may be derived from bacterial species within the Genera: Streptococcus, Vibrio and Clostridium. It should also be understood (and as is set out in more detail below) the disclosure embraces the use of molecules which comprise one or more CBMs and modified forms of any of the CBMs described herein. A modified CBM is a CBM which includes one or more mutated residues relative to the wild-type CBM sequence.

The CBMs for the various uses, medicaments and methods described herein are all types of carbohydrate, glycan and/or sialic acid binding molecule.

The term “sialic acid” embraces all forms of N- or O-substituted neuraminic acid and includes all synthetic, naturally occurring and/or modified forms thereof. Sialic acids may be found as components of cell surface molecules, glycoproteins and glycolipids. Most often, sialic acids are present at the end (terminal regions) of sugar chains connected to cell membranes and/or proteins. For example, some cells of the human upper respiratory tract comprise a-2,6-linked sialic acid receptors and other cells of the upper and lower respiratory tracts comprise a-2,3- linked sialic acid receptors. The sialic acid family encompasses a number (approximately 50) of derivatives that may result from acetylation, glycolylation, lactonisation and methylation at C4, C5, C7, C8 and C9. All such derivatives are to be embraced by the term “sialic acid”. Sialic acids may be found linked a(2,3) or a(2,6) to Gal and GalNAc or a(2,8) or a(2,9) to another sialic acid. Accordingly, it is important to understand that while the term “sialic acid” is used throughout this specification, it encompasses all anomers, derivatives, analogues or variants (either naturally occurring or synthetically generated) thereof as well as all monomers, dimers, trimers, oligomers, polymers or concatamers comprising the same.

Accordingly, a sialic acid binding molecule for the various methods, uses and medicaments of this disclosure may comprise a moiety with affinity for sialic acid in any of its various forms as described above. Indeed, a CBM for use, or a CBM for use in a medicament or method described herein, may exhibit an affinity for sialic acid in any of its various forms as described above and/or may bind/couple to and/or associate with sialic acid molecules as may be present in or on, a Coronavirus, a (mammalian) cell surface and/or a (mammalian) cell surface receptor.

Useful CBMs may take any form and/or belong to any class or type of CBM. CBMs from any one or more of the following CBM families, may be of use.

(i) Family 32 CBMs (CBM32);

(ii) Family 40 CBMs (CBM40);

(iii) Family 47 CBMs (CBM47);

(iv) Family 67 CBMs (CBM67); and

(v) Family 70 CBMs (CBM70).

For example, the disclosure provides a CBM32 for use in the treatment or prevention of:

(i) a Coronavirus infection and/or a disease or condition caused or contributed by a Coronavirus·, or

(ii) a COVID-19 infection and/or a disease or condition caused or contributed by SARS-CoV-2.

Also disclosed is a method of treating or preventing:

(i) a Coronavirus infection and/or a disease or condition caused or contributed by a Coronavirus·, or

(ii) a COVID-19 infection and/or a disease or condition caused or contributed by SARS-CoV-2; said method comprising administering a subject in need thereof one or more Family 32 CBM32.

Additionally disclosed, is the use of a CBM32 in the manufacture of a medicament for the treatment or prevention of a Coronavirus infection and/or a disease or condition caused or contributed by a Coronavirus.

This disclosure also provides a CBM40, or a CBM47, or a CBM67 or a CBM70 for use in the treatment or prevention of:

(i) a Coronavirus infection and/or a disease or condition caused or contributed by a Coronavirus·, or

(ii) a COVID-19 infection and/or a disease or condition caused or contributed by SARS-CoV-2. The disclosure further relates to a method of treating or preventing:

(i) a Coronavirus infection and/or a disease or condition caused or contributed by a Coronavirus·, or

(ii) a COVID-19 infection and/or a disease or condition caused or contributed by SARS-CoV-2; said method comprising administering a subject in need thereof one or more Family 40 CBM, a Family 47 CBM, a Family 67 CBM or a Family 70 CBM.

Additionally, the disclosure provides the use of a CBM40, or a CBM47, or a CBM67 or a CBM70 in the manufacture of a medicament for the treatment or prevention of prevention of a Coronavirus infection and/or a disease or condition caused or contributed by a Coronavirus.

It should be noted that the use of any one of the disclosed CBM32s, CBM40s, CBM47s, CBM67s or CBM70s, may be combined with any other carbohydrate binding protein, glycan binding protein, sialic acid binding molecule and/or CBM. Indeed, the use of a CBM32, a CBM40, a CBM47, a CBM67 or a CBM70 may be combined with the use of any other one of a CBM32, a CBM40, a CBM47, a CBM 67 or a CBM70.

Further detail concerning each CBM family is provided below - all of these CBMs (their sub- types, variants, orthologues etc.) are for use in the treatment and/or prevention of Coronavirus infections and/or Coronavirus associated diseases and/or conditions described herein.

CBM32

A useful CBM32 (i.e. a CBM32 for the various methods, medicaments and uses described herein) may be derived from any suitable source. For example, CBM32s for use may be obtained from microorganisms, including, for example, bacteria of the genera Cellvibrio, Yersinia, Micromonospora, Streptococcus, Bifidobacteria and Clostridium. For example, useful CBM32s may be obtained or derived from, for example, Cellvibrio mixtus, Yersinia enterolitica, Clostridium perfringens, Clostridium thermocellum, Streptococcus pneumoniae, Bifidobacterium longum and Micromonospora viridifaciens. Further details concerning the source, structure and function of the CBM32 family can be found within the Carbohydrate Active Enzymes database (freely available on the internet at: http://www.cazv.org/CBM32.html)·

An exemplary CBM32 sequence is provided by SEQ ID NO: 1 below: SEQ ID NO: 1

AIIETAIPQSEMTASATSEEGQDPASSAIDGNTNTMWHTKWNGSDALPQSLSVNLGS SRKVSSIAITP

RTSGNNGFITKYEIHAINNGVETLVAEGTWEENNLVKTVTFDSPIDAEEIKITAIQG VGGFASIAELN

VYE

Accordingly, a CBM for use may comprise, consist essentially or consist of a CBM having the sequence of SEQ ID NO: 1 or a carbohydrate binding portion thereof. A carbohydrate binding fragment of SEQ ID NO: 1 may comprise anywhere between about 5, 6, 7, 8, 9 or 10 (consecutive or contiguous) amino acids to about 138 (consecutive or contiguous) amino acids from SEQ ID NO: 1. Suitable fragments may comprise about 11, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 105, about 110, about 115, about 120, about 125, about 130 or about 135 (consecutive or contiguous) amino acids from SEQ ID NO: 1.

CBM32 or a protein comprising, consisting essentially or consisting of SEQ ID NO: 1 may bind, for example, galactose, A/-acetylgalactosamine (GalNAc), /V-acetylglucosamine (GlcNAc) and/or lactose. Accordingly, any fragment for use may also bind galactose, N- acetylgalactosamine (GalNAc), A/-acetylglucosamine (GlcNAc) and/or lactose. One of skill will appreciate that the binding affinity of any given CBM32 molecule may depend on the precise CBM32 subtype; by way of further examples, some CBM32s have shown affinity for a variety of ligands (examples include type II blood group H-trisaccharide (Fuca1-2Gal pi-4GlcNAc), /V-acetyl-D-lactosamine (LacNAc), galactose, lacto-/V-biose, disaccharide GlcNAc-a-1 ,4-Gal (which may be referred to as an A/-acetylglucosamine linked alpha 1 ,4 to galactose), and/or GlcNAc). It should also be noted that multiple CBM32 subtypes may be derived from a single organism; these different CBM subtypes may exhibit the same, similar or different binding specificities. For example, Clostridium perfringens contains two sialidases NanJ and NanH; NanJ contains one galactose-specific CBM32; NanH contains four putative CBM32s with different binding selectivity - for example, the CBM32 encoded by NanH binds GlcNAc. As used herein, the term CBM32 embraces all CBM32 variants, derivatives and sub-types.

SEQ ID NO: 1 is derived from the sequence deposited in the UniProt database under ID No: A0A2X2YJF2. This sequence is reproduced as SEQ ID NO: 2 below (SEQ ID NO: 1 appears as residues 42-180 - shown in bold in the sequence below).

SEQ ID NO: 2

MKSKKIIATL VASLVISNMG GYLVKANPNV NHKAVIIEDR QAIIETAIPQ SEMTASATSE EGQDPASSAI DGNTNTMWHT KWNGSDALPQ SLSVNLGSSR KVSSIAITPR TSGNNGFITK YEIHAINNGV ETLVAEGTWE ENNLVKTVTF DSPIDAEEIK ITAIQGVGGF ASIAELNVYE

IKGEVDEIAN YGNLKITKEE ERLNITRDLE KFSSLDEGTI VTRFNMNDTS IQSLIGLSDG NKANNYFSLY VSGGKVGYEL RRQEGNGDFN VHHSADVTFN KGINTLALKI EKGVGAKIFL NGSLVKTVSD PNIKFLNAIN LNSGFIGKTD RANGYNEYLF RGNIDFMNIY DKPVSDNYLL RKTGETRAPS EDSLLPDDVY KTQPVELFYP GYLESRGYRI PALETTKKGT VLASIDVRNN GDHDAPNNNI DVGIRRKEVN GEWEEGKVIL DYPGKSAAID TSLMSATIEE NGIEKERIFL IVTHFPEGYG FPNTEGGSGY KEIDGKYYFI LKDAQNNEYT VREDGIVYNS EGNETDYVMK NDKTLIQNGE EVGNALLSNS PLKAVGTAHI EMIYSDDDGN TWSEPEDLNP GLKKEWMKFF GTAPGKGIQI KNGEHKGRLV FPIYYTNQNN FQSSAVIYSD DFGETWKLGE SPIDTASVSS ETVSSGTQLT ECQWEMPNG QLKLFMRNTG SYTRIATSFD GGATWHDEVP EDTSLREPYC QLSVINYSGK INGKDAIIFS NPDASSRVNG SVKVGLINEN GTYENGQPRY EFDWIYNKTV KPGSFAYSCL TELPDGNLGL FYEGEGAGRM AYTEFDLNYL KFNASEDSPA ATVQSIESLD EDLIYNAGDE VSIKVNFNQL VSLIGDRKIT LDIGGVDVPL NMVNYEGKSS AIFKGTIPEG INPGNYEIK LKENNALELNT VYNKVSTLNG LDNTGINVQI GELKTTVGNS TIKVNEEVQV GSAFEAILGI KGLNGDTEVY SAEYLFEYNA EAFKLNEITS FSDSLFVKSK EVEPGKVRIL VASLGNEIEK DSELVKVNLT PKISSELEVL GLTTALVGAG DGNTHDLELS SKEVKINEEA SGEIWNPVQ NFEIPEINKK NVKLTWNAPI TTEGLEGYVI YKDGKKLSEV PAESTEFW S KLNRHTIYNF KVAAKYSNGE LSAKESKTIR TAR

SEQ ID NOS 1 and 2 are derived from Clostridium perfringens. A CBM for use in the various aspects of this disclosure may comprise one, two, three, four or more CBM32s. A CBM for use may comprise one, two, three, four or more proteins comprising SEQ ID NO: 1 or a carbohydrate binding fragment thereof. One of skill will appreciate that useful CBM32s may comprise sequences which exhibit some degree (for example, 99%, 95%, 90%, 85%, 80%, 75%, 70%, 65% or 60%) of sequence identity or homology with the CBM32 sequences of SEQ ID NOS: 1 and 2. All such variant or divergent sequences are to be embraced within the scope of this disclosure and by the term “CBM32”. Identical and/or homologous CBM32 sequences may have carbohydrate binding function.

CBM40

A useful CBM40 (i.e. a CBM40 for the various methods, medicaments and uses described herein) may be derived from any suitable source. For example, CBM40s for use may be obtained from microorganisms, including, for example, bacteria of the genera Clostridium, Enterococcus, Staphylococcus, Streptococcus and Vibrio. For example, useful CBM40s may be obtained or derived from, for example Clostridium perfringens, Streptococcus pneumoniae and Vibrio cholerae. Further details concerning the source, structure and function of the CBM40 family can be found within the Carbohydrate Active Enzymes database (freely available on the internet at: http://www.cazy.org/CBM40.html).

The Family 40 CBMs embrace molecules of approximately 200 residues and are often found at the N-terminus of GH33 sialidases. They may also be found inserted in the b-propeller of GH33 sialidases. At least the CBM40 of Vibrio cholerae binds the alpha-anomer of sialic acid and, for example, a(2,3)-, a(2,6)-, and a(2,8)-linked sialosides.

Exemplary CBM40s for use may comprise the sialic acid binding domain of Vibrio cholerae NanH sialidase (t cCBM: a CBM40) and/or the equivalent (or homologous) domain from Streptococcus pneumoniae NanA sialidase (SpCBM: also a CBM40). Of course, similar or homologous sialic acid binding modules present in other organisms are to be encompassed within the scope of the terms “CBM” and “CBM40”.

An exemplary Vibrio cholerae NanH sialidase amino acid sequence is deposited under accession number A5F7A4 and is reproduced below as SEQ ID NO: 3 (781 amino acids).

SEQ ID NO: 3 MRFKNVKKTA LMLAMFGMAT SSNAALFDYN ATGDTEFDSP AKQGWMQDNT NNGSGVLTNA DGMPAWLVQG IGGRAQWTYS LSTNQHAQAS SFGWRMTTEM KVLSGGMITN YYANGTQRVL PIISLDSSGN LWEFEGQTG RTVLATGTAA TEYHKFELVF LPGSNPSASF YFDGKLIRDN IQPTASKQNM IVWGNGSSNT DGVAAYRDIK

FEIQGDVIFR GPDRIPSIVA SSVTPGW TA FAEKRVGGGD PGALSNTNDI ITRTSRDGGI TWDTELNLTE QINVSDEFDF SDPRPIYDPS SNTVLVSYAR WPTDAAQNGD RIKPWMPNGI FYSVYDVASG NWQAPIDVTD QVKERSFQIA GWGGSELYRR NTSLNSQQDW QSNAKIRIVD GAANQIQVAD GSRKYWTLS 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 FDEGVTWKGP IQLVNGASAY SDIYQLDSEN AIVIVETDNS NMRILRMPIT LLKQKLTLSQ N

The CBM region of SEQ ID NO: 3 is from amino acid residue 25 to 216 (sequence shown in bold) - this sequence may be SEQ ID NO: 4.

An exemplary Streptococcus pneumoniae NanA sialidase amino acid sequence has been deposited under accession number P62575 and is reproduced below as SEQ ID NO: 5 (1035 amino acids). SEQ ID NO: 5

MSYFRNRDID IERNSMNRSV QERKCRYSIR KLSVGAVSMI VGAW FGTSP VLAQEGASEQ PLANETQLSG ESSTLTDTEK SQPSSETELS GNKQEQERKD KQEEKIPRDY YARDLEVET 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 YRVW DPVKP 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: 5 is from amino acid residue 121 to 305 (sequence shown in bold) - this sequence may be SEQ ID NO: 6.

CBMs for use in the various aspects and embodiments of this disclosure may comprise a protein or peptide having the sequence of SEQ ID NO: 3, 4, 5 or 6 or a carbohydrate binding fragment of any of these. For example, a useful molecule (namely a molecule for the uses, methods and medicaments described herein) may comprise a proteinaceous moiety encoded by the sialic acid binding domain of the nanH gene (encoding sialidase) of V. cholerae (as provided by SEQ ID NO: 3) or an equivalent or homologous gene present in another organism (for example the equivalent/homologous nanA sialidase gene of S. pneumoniae·, see SEQ ID NO: 5).

A molecule for the uses, methods and medicaments of this disclosure may comprise from about residue 1 , 5, 10, 15, 25 or 30 (i.e. from 1-30 or from any amino acid residue there between) to about residue 150, 175, 200, 210, 216, 220-781 (to any residue from 150 to 781 including any residue therebetween) of the V. cholerae sialidase molecule of SEQ ID NOS: 3 and 4.

For example, a use, method or medicament of this disclosure may comprise a peptide having a sequence corresponding to residue 25 to about residue 216 of SEQ ID NO: 3 above. A carbohydrate binding fragment of SEQ ID NOS: 3 may comprise anywhere between about 5, 6, 7, 8, 9 or 10 (consecutive or contiguous) amino acids to about 191 (consecutive or contiguous) amino acids from SEQ ID NO: 3. Suitable fragments may comprise about 11, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 105, about 110, about 115, about 120, about 125, about 130 or about 135, about 140, about 150, about 155, about 160, about 165, about 170, about 180, about 185, about 186, about 187, about 188, about 189 or about 190 (consecutive or contiguous) amino acids from SEQ ID NO: 3.

A molecule for a use, method or medicament of this disclosure may comprise from about residue 1, 5, 10, 15, 25 or 30 (i.e. from 1-30 or from any amino acid residue there between) to about residue 150, 175, 200, 210, 216, 220-781 (to any residue from 150 to 781 including any residue therebetween) of the V. cholerae sialidase molecule of SEQ ID NOS: 3 and 4. 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: 3 above.

A further suitable molecule may comprise a protein or peptide having the sequence of SEQ ID NO: 5 or 6 or a carbohydrate binding fragment thereof. For example, a useful sialic acid binding molecule may comprise a proteinaceous moiety encoded by the sialic acid binding domain of the Streptococcus pneumoniae nanA gene (encoding sialidase).

A sialic acid binding molecule for use may comprise from about residue 80, 90, 100, 110, 120, 121 to 130 (i.e. from any of about residues 80 to 130 including any residue therebetween) to about residue 250, 275, 300, 305, 310, 320-1035 (i.e. to any residue from about 250-1035 including to about any residue therebetween) of the S. pneumoniae sialidase molecule of SEQ ID NOS: 5 and 6.

For example, a sialic acid binding molecule for use may comprise a peptide having a sequence corresponding to residue 121 to about residue 305 of SEQ ID NO: 5 above.

A carbohydrate binding fragment may comprise anywhere between about 5, 6, 7, 8, 9 or 10 (consecutive or contiguous) amino acids to about 185 (consecutive or contiguous) amino acids from SEQ ID NO: 5. Suitable fragments may comprise about 11, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 105, about 110, about 115, about 120, about 125, about 130, about 135, about 140, about 145, about 150, about 155, about 160, about 165, about 170, about 175, 180, 181 , 182, 183 or 184 (consecutive or contiguous) amino acids from SEQ ID NO: 5.

SEQ ID NOS 3 and 4 are derived from Vibrio cholerae and SEQ ID NOS 5 and 6 are derived from Streptococcus pneumoniae. A CBM for use in the various aspects of this disclosure may comprise one, two, three, four or more CBM40s. A CBM for use may comprise one, two, three, four or more proteins comprising SEQ ID NO: 3, 4, 5 or 6 or a carbohydrate binding fragment any of these. One of skill will appreciate that useful CBM40s may comprise sequences which exhibit some degree (for example, 99%, 95%, 90%, 85%, 80%, 75%, 70%, 65% or 60%) of sequence identity or homology with the CBM40 sequences of SEQ ID NOS: 3, 4, 5 and/or 6. All such variant or divergent sequences are to be embraced within the scope of this disclosure and by the term ‘‘CBM40”. Identical and/or homologous CBM40 sequences may have carbohydrate/sialic acid binding function.

CBM47

A useful CBM47 (i.e. a CBM47 for the various methods, medicaments and uses described herein) may be obtained from microorganisms, including, for example, bacteria of the genera Acinetobacter, Bathymodiolus, Campylobacter, Planctomycetes, Streptococcus and Streptomyces. For example, useful CBM47s may be obtained or derived from, for example, Streptococcus mitis or Streptococcus pneumoniae. Further details concerning potential sources and the structure and function of the CBM47 family can be found within the Carbohydrate Active Enzymes database (freely available on the internet at: http://www.cazv.orq/CBM47.html).

An exemplary CBM47 sequence is provided by SEQ ID NO: 7 below:

SEQ ID NO: 7

TPDKFNDGNLNIAYAKPTTQSSVDYNGDPNRAVDGNRNGNFNSGSVTHTRADNPSWW EVDLKKMDKVG LVKIYNRTDAETQRLSNFDVILYDNNRNEVAKKHVTSTNLSGESVSLDFKEKGARYIKVK LLTSGVPLSL

AEVEVFRES

Accordingly, a CBM for use may comprise, consist essentially or consist of a CBM having the sequence of SEQ ID NO: 7 or a carbohydrate binding portion thereof. A carbohydrate binding fragment of SEQ ID NO: 7 may comprise anywhere between about 5, 6, 7, 8, 9 or 10 (consecutive or contiguous) amino acids to about 144 (consecutive or contiguous) amino acids from SEQ ID NO: 7. Suitable fragments may comprise about 11 , about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 105, about 110, about 115, about 120, about 125, about 130 or about 135, about 140 or about 143 (consecutive or contiguous) amino acids from SEQ ID NO: 7.

CBM47 or a protein comprising, consisting essentially or consisting of SEQ ID NO: 7 may bind L-fucose, fucosyllactose, H-trisaccharide and/or Lewis y antigen. Accordingly, any fragment for use may also bind L-fucose, fucosyllactose, H-trisaccharide and/or Lewis y antigen.

SEQ ID NO: 7 is derived from the sequence deposited in the UniProt database under ID No: A0A1Q2T229. This sequence is reproduced as SEQ ID NO: 8 below (SEQ ID NO: 7 appears as residues 601-745 - shown in bold in the sequence below):

SEQ ID NO: 8

MNKEKIKRKL ITILFVCIGM LCFGLLAGVK ADNRVQMRTT INNESPLLLS PLYGNDNGNG LWWGNTLKGA WEAIPEDVKP YAAIELHPAK VCKPTSCIPR DTKELREWYV KMLEEAQSLN IPVFLVIMSA GERNTVPPEW LDEQFQKYSV LKGVLNIENY WIYNNQLAPH SAKYLEVCAK YGAHFIWHDH EKWFWETIMN DPTFFEASQK YHKNLVLATK NTPIRDDAGT DSIVSGFWLS GLCDNWGSST DTWKWWEKHY TNTFETGRAR DMRSYASEPE SMIAMEMMNV YTGGGTVYNF ECAAYTFMTN DVPTPAFTKG IIPFFRHAIQ NPAPSKEEW NRTKAVFWNG EGRISSLNGF YQGLYSNDET MPLYNNGRYH ILPVIHEKID KEKISSIFPN AKILTKNSEE LSSKVNYLNS LYPKLYEGDG YAQRVGNSWY IYNSNANINK NQQVMLPMYT NNTKSLSLDL TPHTYAW KE NPNNLHILLN NYRTDKTAMW ALSGNFDASK SWKKEELELA NWISKNYSIN PVDNDFRTTT LTLKGHTGHK PQINISGDKN HYTYTENWDE NTHVYTITVN HNGMVEMSIN TEGTGPVSFP TPDKFNDGNL NIAYAKPTTQ SSVDYNGDPN RAVDGNRNGN FNSGSVTHTR ADNPSWWEVD LKKMDKVGLV KIYNRTDAET QRLSNFDVIL YDNNRNEVAK KHVNNLSGES VSLDFKEKGA RYIKVKLLTS GVPLSLAEVE VFRESDGKQS EEDIDKITED KW STNKVAT QSSTNYEGVA ALAVDGNKDG DYGHHSVTHT KEDSPSWWEI DLAQTEELEK LIIYNRTDAE IQRLSNFDII IYDSNDYEVF TQHIDSLESN NLSIDLKGLK GKKVRISLRN AGIPLSLAEV EVYTYK

SEQ ID NOS 7 and 8 are derived from Streptococcus pneumoniae. A CBM for use in the various aspects of this disclosure may comprise one, two, three, four or more CBM47s. A CBM for use may comprise one, two, three, four or more proteins comprising SEQ ID NO: 8 or a carbohydrate binding fragment thereof.

One of skill will appreciate that useful CBM47s may comprise sequences which exhibit some degree (for example, 99%, 95%, 90%, 85%, 80%, 75%, 70%, 65% or 60%) of sequence identity or homology with the CBM47 Sequences of SEQ ID NOS: 7 and 8 All such variant or divergent sequences are to be embraced within the scope of this disclosure and by the term "CBM47”. Identical and/or homologous CBM47 sequences may have carbohydrate binding function.

CBM67

A useful CBM67 (i.e. a CBM67 for the various methods, medicaments and uses described herein) may be derived from any suitable source. For example, CBM67s for use may be obtained from microorganisms, including, for example, bacteria of the genera Bacillus, Paenibacillus, Planctomycetes and Streptomyces. For example, a useful CBM67 may be obtained or derived from, for example, Streptomyces avermitilis. Further details concerning potential sources and the structure and function of the CBM67 family can be found within the

Carbohydrate Active Enzymes database (freely available on the internet at: http://www.cazy.org/CBM67.html).

An exemplary CBM67 sequence is provided by SEQ ID NO: 9 below:

SEQ ID NO: 9

APSLEGSSWIWFPEGEPANSAPAATRWFRRTVDLPDDITGATLAISADNVYAVSVDG AEVARTDLEAD

NEGWRRPAVIDVLDHVHSGNNTLAVSASNASVGPAGWICVLVLTTASGEKKIFSDAS WKSTDHEPADG

WREPDFDDSGWPAAKVAAAWGAGPWGRVA

Accordingly, a CBM for use may comprise, consist essentially or consist of a CBM having the sequence of SEQ ID NO: 9 or a carbohydrate binding portion thereof.

A carbohydrate binding fragment of SEQ ID NO: 9 may comprise anywhere between about 5, 6, 7, 8, 9 or 10 (consecutive or contiguous) amino acids to about 164 (consecutive or contiguous) amino acids from SEQ ID NO: 9. Suitable fragments may comprise about 11, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 105, about 110, about 115, about 120, about 125, about 130, about 135 about 140, about 145, about 150, about 155, about 160 or about 163 (consecutive or contiguous) amino acids from SEQ ID NO: 9.

CBM67 or a protein comprising, consisting essentially or consisting of SEQ ID NO: 9 may bind L-rhamnose. Accordingly, any fragment for use may also bind L-rhamnose. SEQ ID NO: 9 is derived from the sequence deposited in the UniProt database under ID No: Q82PP4. This sequence is reproduced as SEQ ID NO: 10 below (SEQ ID NO: 9 appears as residues 132- 296 - shown in bold in the sequence below):

SEQ ID NO: 10

MSALRVTSPS VEYVQRPLGL DAAHPRLSWP MASAAPGRRQ SAYQVRVASS AAGLSHPDVW DSGKW SDDS VLVPYAGPPL KPRTRYFWSV RVWDADGGAS EWSAPSWWET GLMGASQWSA KWISAPAPLT EAPSLEGSSW IWFPEGEPAN SAPAATRWFR RTVDLPDDIT GATLAISADN VYAVSVDGAE VARTDLEADN EGWRRPAVID VLDHVHSGNN TLAVSASNAS VGPAGWICVL VLTTASGEKK IFSDASWKST DHEPADGWRE PDFDDSGWPA AKVAAAWGAG PWGRVAPVAS AANQLRHEFR LPHKKVSRAR LYATALGLYE AHLNGRRVGR DQLAPGWTDY RKRVQYQTYD VTSSVRPGAN ALAAYVAPGW YAGNVGMFGP HQYGERPALL AQLEVEYADG TSERITSGPD WRAASGPIVS ADLLSGETYD ARKETAGWTS PGFDDRAWLA VRGADNDVPE QIVAQVDGPV RIAKELPARK VTEPKPGVFV LDLGQNMVGS VRLRVSGDAG TTVRLRHAEV LNPDGTIYTA NLRSAAATDT YTLKGQGEET YEPRFTFHGF RYVEVTGFPG KPSTTSVTGR VMHTSAPFTF EFETNVPMLN KLHSNITWGQ RGNFLSVPTD TPARDERLGW TGDINVFAPT AAYTMESARF LTKWLVDLRD AQTSDGAFTD VAPAVGNLGN GVAGWGDAGV TVPWALYQAY GDRQVLADAL PSVHAWLRYL EKHSDGLLRP ADGYGDWLNV SDETPKDVIA TAYFAHSADL AARMATELGK DAAPYTDLFT RIRKAFQTAY VASDGKVKGD TQSAYVLTLS MNLVPDALRK AAADRLVALI EAKDWHLSTG FLGTPRLLPV LTDTGHTDVA YRLLHQRTFP SWGYPIDKGS TTMWERWDSI QPDGGFQTPE MNSFNHYAYG SVGEWMYANI AGIAPGRAGY RQW IRPRPG GEVTSARATF ASLHGPVSTR WQQRSGGFVL TCSVPPNTTA EVWIPADHPD RVQHTHGTFV RAEDGCAVFE VGSGSHRFTV

SEQ ID NOS 9 and 10 are derived from Streptomyces avermitilis.

A CBM for use in the various aspects of this disclosure may comprise one, two, three, four or more CBM67s. A CBM for use may comprise one, two, three, four or more proteins comprising SEQ ID NO: 9 or a carbohydrate binding fragment thereof.

One of skill will appreciate that useful CBM67s may comprise sequences which exhibit some degree (for example 99%, 95%, 90%, 85%, 80%, 75%, 70%, 65% or 60%) of sequence identity or homology with the CBM67 Sequences of SEQ ID NOS: 9 and 10. All such variant or divergent sequences are to be embraced within the scope of this disclosure and by the term "CBM67”. Identical and/or homologous CBM67 sequences may have carbohydrate binding function.

CBM70

A useful CBM70 (i.e. a CBM70 for the various methods, medicaments and uses described herein) may be derived from any suitable source. For example, CBM70s for use may be obtained from microorganisms, including, for example, bacteria of the genera Bacillus, Paenibacillus, Planctomycetes and Streptococcus. For example, a useful CBM70s may be obtained or derived from, for example, Streptomyces pneumoniae. Further details concerning potential sources and the structure and function of the CBM70 family can be found within the Carbohydrate Active Enzymes database (freely available on the internet at: http://www.cazy.org/CBM70.html)

An exemplary CBM70 sequence is provided by SEQ ID NO: 11 below:

SEQ ID NO: 11

NLVENGDFGQTEDGSSPWTGSKAQGWSAWVDQKNSADASTRVIEAKDGAITISSHEK LRAALHRMVPI

EAKKKYKLRFKIKTDNKIGIAKVRIIEESGKDKRLWNSATTSGTKDWQTIEADYSPT LDVDKIKLELF

YETGTGTVSFKDIELVEVADQLS

A carbohydrate binding fragment of SEQ ID NO: 11 may comprise anywhere between about 5, 6, 7, 8, 9 or 10 (consecutive or contiguous) amino acids to about 158 (consecutive or contiguous) amino acids from SEQ ID NO: 11. Suitable fragments may comprise about 11, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 105, about 110, about 115, about 120, about 125, about 130, about 135, about 140, about 145, about 150, about 155 or about 157 (consecutive or contiguous) amino acids from SEQ ID NO: 11. CBM70 or a protein comprising, consisting essentially or consisting of SEQ ID NO: 11 may bind hyaluronan. Accordingly, any fragment for use may also bind hyaluronan.

SEQ ID NO: 11 is derived from the sequence deposited in the UniProt database under ID No: Q54873. This sequence is reproduced as SEQ ID NO: 12 below (SEQ ID NO: 11 appears as residues 54-212 - shown in bold in the sequence below):

SEQ ID NO: 12

MQTKTKKLIV SLSSLVLSGF LLNHYMTIGA EETTTNTIQQ SQKEVQYQQR DTKNLVENGD F6QTEDGSSP WTGSKAQGWS AWVDQKNSAD ASTRVIEAKD GAITISSHEK LRAALHRMVP IEAKKKYKLR FKIKTDNKIG IAKVRIIEES GKDKRLWNSA TTSGTKDWQT IEADYSPTLD VDKIKLELFY ETGTGTVSFK DIELVEVADQ LSEDSQTDKQ LEEKIDLPIG KKHVFSLADY

TYKVENPDVA SVKNGILEPL KEGTTNVIVS KDGKEVKKIP LKILASVKDA YTDRLDDWNG IIAGNQYYDS KNEQMAKLNQ ELEGKVADSL SSISSQADRT YLWEKFSNYK TSANLTATYR KLEEMAKQVT NPSSRYYQDE TW RTVRDSM EWMHKHVYNS EKSIVGNWWD YEIGTPRAIN NTLSLMKEYF SDEEIKKYTD VIEKFVPDPE HFRKTTDNPF KALGGNLVDM GRVKVIAGLL RKDDQEISST IRSIEQVFKL VDQGEGFYQD GSYIDHTNVA YTGAYGNVLI DGLSQLLPVI QKTKNPIDKD KMQTMYHWID KSFAPLLVNG ELMDMSRGRS ISRANSEGHV AAVEVLRGIH RIADMSEGET KQCLQSLVKT IVQSDSYYDV FKNLKTYKDI SLMQSLLSDA GVASVPRPSY LSAFNKMDKT AMYNAEKGFG FGLSLFSSRT LNYEHMNKEN KRGWYTSDGM FYLYNGDLSH YSDGYWPTVN PYKMPGTTET DAKRADSDTG KVLPSAFVGT SKLDDANATA TMDFTNWNQT LTAHKSWFML KDKIAFLGSN IQNTSTDTAA TTIDQRKLES GNPYKVYVND KEASLTEQEK DYPETQSVFL ESFDSKKNIG YFFFKKSSIS MSKALQKGAW KDINEGQSDK EVENEFLTIS QAHKQNRDSY GYMLIPNVDR ATFNQMIKEL ESSLIENNET LQSVYDAKQG VWGIVKYDDS VSTISNQFQV LKRGVYTIRK EGDEYKIAYY NPETQESAPD QEVFKKLEQA AQPQVQNSKE KEKSEEEKNH SDQKNLPQTG EGQSILASLG FLLLGAFYLF RRGKNN

SEQ ID NOS 11 and 12 are derived from Streptococcus pneumoniae.

A CBM for use in the various aspects of this disclosure may comprise one, two, three, four or more CBM70s.

A CBM for use may comprise one, two, three, four or more proteins comprising SEQ ID NO: 11 or a carbohydrate binding fragment thereof. One of skill will appreciate that useful CBM70s may comprise sequences which exhibit some degree (for example 99%, 95%, 90%, 85%, 80%, 75%, 70%, 65% or 60%) of sequence identity or homology with the CBM70 Sequences of SEQ ID NOS: 11 and 12. All such variant or divergent sequences are to be embraced within the scope of this disclosure and by the term "CBM70”. Identical and/or homologous CBM70 sequences may have carbohydrate binding function.

The various molecules described herein (including the various carbohydrate, sialic acid and/or glycan binding proteins/molecules) which are all for the various uses, medicaments and methods described herein, may further comprise an oligomerisation domain.

Suitable oligomerisation domains may exhibit an ability to self-associate to form multimeric structures, for example trimers. An oligomerisation domain for use may comprise any molecule with oligomerisation properties or any functional fragment thereof. For example, one or more (for example, two) sialic acid/glycan binding molecules (for example, CBMs) may be bound, coupled or fused to an oligomerisation domain - the resulting sialic acid/glycan binding molecule::oligomerisation domain "fusion” may then be used (with one or more other such "fusions”) as a molecule for treating or preventing a Coronavirus infection and/or a disease or condition associated therewith.

Suitable oligomerisation 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 PA0579 and is reproduced below as SEQ ID NO: 13 (438 amino acids).

SEQ ID NO: 13

MNTYFDIPHR LVGKALYESY YDHFGQMDIL SDGSLYLIYR RATEHVGGSD GRW FSKLEG GIWSAPTIVA QAGGQDFRDV AGGTMPSGRI VAASTVYETG EVKVYVSDDS GVTWVHKFTL ARGGADYNFA HGKSFQVGAR YVIPLYAATG VNYELKWLES SDGGETWGEG STIYSGNTPY NETSYLPVGD GVILAVARVG SGAGGALRQF ISLDDGGTWT DQGNVTAQNG DSTDILVAPS LSYIYSEGGT PHW LLYTNR TTHFCYYRTI LLAKAVAGSS GWTERVPVYS APAASGYTSQ W LGGRRILG NLFRETSSTT SGAYQFEVYL GGVPDFESDW FSVSSNSLYT LSHGLQRSPR RVWEFARSS SPSTWNIVMP SYFNDGGHKG SGAQVEVGSL NIRLGTGAAV WGTGYFGGID NSATTRFATG YYRVRAWI The oligomerisation domain of SEQ ID NO: 13 is from amino acid residue 333 to 438 - this sequence may be SEQ ID NO: 14 (sequence shown in bold).

Thus, an oligomerisation domain for use may comprise from about residue 250, 275, 300, 310, 320, 333, 340 to 350 (i.e. from about residue 250 to about residue 350 including from about any residue therebetween) to about residue 400, 410, 420, 430 or 438 (i.e. to about any residue from about residue 400 residue 438 including to about any residue therebetween) of the P. aeruginosa pseudaminidase trimerisation domain (PaTD) provided by SEQ ID NO: 5. For example, a useful sialic acid/glycan binding molecule may exploit an oligomerisation domain comprising residues 333 to 438 of SEQ ID NO: 13.

Sialic acid/glycan binding molecules for any of the methods, uses and/or medicaments described herein may include modified forms of any of the CBMs described herein.

The term “modified” embraces molecules which 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, e.g. the wild type CBM sequences from Vibrio cholerae NanH sialidase or Streptococcus pneumoniae NanA sialidase (it should be appreciated that similar or homologous CBMs (including CBM40s) present in other organisms are to be encompassed within the scope of the term “CBM” and/or as CBM reference sequences). A reference sequence may comprise (or consist of, or consist essentially of) any of SEQ ID NOS: 1-12 above.

Accordingly, a modified CBM sequence may be derived from a specific or particular wild type CBM sequence.

A modified CBM sequence may comprise a wild type CBM sequence which has been modified to include one or more mutations.

The one or more mutations may be functional - that is to say they may individually (and/or independently) or collectively (for example, synergistically) modulate (alter, improve or suppress/inhibit) one or more of the physiological, biological immunological and/or pharmacological properties characteristic of a wild type CBM (for example the wild type CBM from which the modified CBM is derived). In particular, the one or more mutations may:

(i) alter the immunogenicity (or antigenicity) of the CBM; and/or (ii) alter (for example improve) the efficacy (of the CBM or of any multimeric molecule comprising a modified CBM)’ and/or

(iii) they may modulate (for example improve) the thermostability of the CBM; and/or

(iv) they may modulate (for example improve) the solubility of the CBM; and/or

(v) they may modulate the binding affinity of the CBM for its target (for example a glycan, sialic acid, galactose, lactose, polygalacturonic acid, LacNAc, fucose, L- rhamnose; hyaluronan); and/or

(vi) they may modulate (for example improve) the in vivo half-life of the molecule.

A “mutation” may include any alteration to a wild-type CBM molecule. For example, the term "mutation” may embrace, for example:

(i) one or more amino acid substitution(s) (where one or more of the wild type amino acid(s) is/are swapped or changed for another (different) amino acid - the term “substitutions” would include conservative amino acid substitutions); and/or

(ii) one or more amino acid deletion(s) (where one or more of the wild type amino acid residue(s) are removed); and/or

(iii) one or more amino acid addition(s)/insertion(s) (where additional amino acid residue(s) are added to a wild type (or reference) primary sequence); and/or

(iv) one or more amino acid/sequence inversions (usually where two or more consecutive amino acids in a primary sequence are reversed; and/or

(v) one or more amino acid/sequence duplications (where an amino acid or a part of the primary amino acid sequence (for example a stretch of 5-10 amino acids) is repeated).

Accordingly, a modified CBM according to this disclosure may comprise one or more of the mutations described herein.

An exemplary wild type CBM (in other words a reference sequence from which a useful modified CBM may be derived) is the Streptococcus pneumoniae NanA sialidase, the amino acid sequence for which has been deposited under accession number P62575 and is reproduced above as SEQ ID NO: 5 (1035 amino acids). As also noted above, the CBM region of SEQ ID NO: 5 is from amino acid residue 121 to 305 - this sequence is designated SEQ ID NO: 6.

Thus, this disclosure provides carbohydrate binding molecules with an affinity for sialic acid, which molecules comprise modified forms of SEQ ID NO: 6 (and/or SEQ ID NO: 5). A modified form of SEQ ID NO: 6 may comprise one or more mutated residues - the mutations being, for example, amino acid substitutions, additions/insertions, duplications, deletions and/or inversions made relative to the sequence of SEQ ID NO: 6.

An exemplary Vibrio choierae NanH sialidase amino acid sequence is deposited under accession umber A5F7A4 and is reproduced above as SEQ ID NO: 7 (781 amino acids). The CBM region of SEQ ID NO: 7 is from amino acid residue 25 to 216 - this is SEQ ID NO: 8.

Thus, this disclosure provides sialic acid/glycan binding molecules which comprise modified forms of SEQ ID NO: 8 (and/or SEQ ID NO: 7). A modified form of SEQ ID NO: 8 may comprise one or more mutated residues - the mutations being, for example, amino acid substitutions, additions/insertions, duplications, deletions and/or inversions made relative to the sequence of SEQ ID NO: 8.

Thus, a useful modified CBM (i.e. a CBM for use in the medical uses and methods described herein) may comprise one or more of the mutations described herein.

By way of non-limiting example, the following represent individual units (referred to as “HEX” units) which may be used to make modified sialic acid/glycan binding molecules for the various uses, methods and medicaments described herein (for example, for use in methods of treating or preventing Coronavirus infections).

(i) HEX1

CBMX1 (L170T V239A V246G I286A Y292E) - CBMX2 (L170T V239A V246G I286A

Y292E) - TD (S342D L348D R403K)

(ii) HEX2

CBMX1 (V239A V246G I286A Y292E)— -CBMX2 (V239A V246G I286A Y292E)— -TD (S342D R403K)

(iii) HEX3

CBMX1 (V239A V246G I286A) - CBMX2 (V239A V246G I286A) - TD (S342D R403K)

(iv) HEX4

CBMX1 (V239A V246G) - CBMX2 (V239A V246G) - TD (S342D)

(v) HEX5 CBMX1 (V239A V246G) - CBMX2 (V239A V246G) - TD (R403K)

(vi) HEX6

CBMX1 (V239A V246G) - CBMX2 (V239A V246G) - TD (S342D R403K)

(vii) HEX17

CBMX1 (V239A V246G A162P) - CBMX2 (V239A V246G A162P) - TD (S342D R403K)

A suitable HEX unit may comprise two modified CBMs (denoted CBMX1 and CBMX2 above) with the specific mutations introduced to each CBM being identified in parenthesis. In one teaching the units CBMX1 and CBMX2 may comprise (consist of, or consist essentially of) any type of CBM (for example any member of the CBM40, any CBM32, any CBM47, any CBM67 and/or any CBM70 groups/classes). It should be noted that a symbol indicates an amino acid linker (linking one modified CBM to another modified CBM or a modified CBM to an oligomerisation domain).

In each case, the oligomerisation domain (denoted “TD”) present in each HEX unit conjugates the units together as a trimer. While any given hexamer may comprise three identical copies of one of the units described above, one of skill will appreciate that further options are available. For example, a HEX unit may be made up of two CBMs, each having different mutations (the mutations being one or more selected from the options detailed herein).

It will be noted that HEX6 and HEX17 are identical except for the additional A162P mutation. This proline mutation (a substitution for the wild type alanine at residue 162) has been shown to improve thermostability (the single CBM Tm by 3-4 ° C). Further information regarding the use of proline mutations may 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, is not located near the other mutations, the N- or C-termini or the ligand binding site. Rather unexpectedly, beyond the modest improvement in thermostability, it was noted that the A162P mutation yields a molecule exhibiting a marked improvement in in vivo experiments - in particular in comparison to those same experiments conducted using a hexameric molecule comprising other (for example, HEX6) HEX units. For example, the modified molecules (in particular, a molecule comprising a HEX17 unit) exhibit modulation over pro-inflammatory cytokines, including, for example, IL-8. Indeed, the modulatory effect (specifically an inhibitory effect) on the production of IL-8 by a molecule comprising a HEX17 unit, was improved over other tested modified molecules. Relative to the amino acid sequences of Sp2CBMTD (aka “SpOrig”) the amino acid sequence of the HEX6 and HEX17 molecules is:

SpOrig GAMVIEKEDVETNASNGQRVDLSSELDKLKKLENATVHMEFKPDAKAPAFYNLFSVSSAT HEX6 GAMVIEKEDVETNASNGQRVDLSSELDKLKKLENATVHMEFKPDAKAPAFYNLFSVSSAT HEX17 GAMVIEKEDVETNASNGQRVDLSSELDKLKKLENATVHMEFKPDPKAPAFYNLFSVSSAT

SpOrig KKDEYFTMAVYNNTATLEGRGSDGKQFYNNYNDAPLKVKPGQWNSVTFTVEKPTAELPKG HEX6 KKDEYFTMAVYNNTATLEGRGSDGKQFYNNYNDAPLKVKPGQWNSVTFTVEKPTAELPKG HEX17 KKDEYFTMAVYNNTATLEGRGSDGKQFYNNYNDAPLKVKPGQWNSVTFTVEKPTAELPKG

SpOrig RVRLYVNGVLSRTSLRSGNFIKDMPDVTHVQIGATKRANNTVWGSNLQIRNLTVYNRALT HEX6 RARLYVNGGLSRTSLRSGNFIKDMPDVTHVQIGATKRANNTVWGSNLQIRNLTVYNRALT HEX17 RARLYVNGGLSRTSLRSGNFIKDMPDVTHVQIGATKRANNTVWGSNLQIRNLTVYNRALT

SpOrig PEEVQKRSGGGSGVIEKEDVETNASNGQRVDLSSELDKLKKLENATVHMEFKPDAKAPAF HEX6 PEEVQKRSGGGSGVIEKEDVETNASNGQRVDLSSELDKLKKLENATVHMEFKPDAKAPAF HEX17 PEEVQKRSGGGSGVIEKEDVETNASNGQRVDLSSELDKLKKLENATVHMEFKPDPKAPAF

SpOrig YNLFSVSSATKKDEYFTMAVYNNTATLEGRGSDGKQFYNNYNDAPLKVKPGQWNSVTFTV HEX6 YNLFSVSSATKKDEYFTMAVYNNTATLEGRGSDGKQFYNNYNDAPLKVKPGQWNSVTFTV HEX17 YNLFSVSSATKKDEYFTMAVYNNTATLEGRGSDGKQFYNNYNDAPLKVKPGQWNSVTFTV

SpOrig EKPTAELPKGRVRLYVNGVLSRTSLRSGNFIKDMPDVTHVQIGATKRANNTVWGSNLQIR HEX6 EKPTAELPKGRARLYVNGGLSRTSLRSGNFIKDMPDVTHVQIGATKRANNTVWGSNLQIR

HEX17 EKPTAELPKGRARLYVNGGLSRTSLRSGNFIKDMPDVTHVQIGATKRANNTVWGSNLQIR SpOrig NLTVYNRALTPEEVQKRSGGALGVPDFESDWFSVSSNSLYTLSHGLQRSPRRVW EFARS

HEX6 NLTVYNRALTPEEVQKRSGGSLGVPDFESDWFDVSSNSLYTLSHGLQRSPRRW VEFARS HEX17 NLTVYNRALTPEEVQKRSGGSLGVPDFESDWFDVSSNSLYTLSHGLQRSPRRW VEFARS

SpOrig SSPSTWNIVMPSYFNDGGHKGSGAQVEVGSLNIRLGTGAAVWGTGYFGGIDNSATTRFAT HEX6 SSPSTWNIVMPSYFNDGGHKGSGAQVEVGSLNIKLGTGAAVWGTGYFGGIDNSATTRFAT HEX17 SSPSTWNIVMPSYFNDGGHKGSGAQVEVGSLNIKLGTGAAVWGTGYFGGIDNSATTRFAT

SpOrig GYYRVRAWI HEX6 GYYRVRAWI HEX17 GYYRVRAWI

[SpOrig (SEQ ID NO: 15); HEX6 (SEQ ID NO: 16) and HEX17 (SEQ ID NO: 17)]

SpOrig may be modified to include the following mutation (R274Q). This mutation modulates the binding affinity of the CBM for sialic acid.

In view of the above, the disclosure provides a modified CBM, for use in the treatment or prevention of:

(i) a Coronavirus infection and/or a disease or condition caused or contributed by a Coronavirus·, or

(ii) a COVID-19 infection and/or a disease or condition caused or contributed by SARS-CoV-2.

Also disclosed is a method of treating or preventing:

(i) a Coronavirus infection and/or a disease or condition caused or contributed by a Coronavirus·, or

(ii) a COVID-19 infection and/or a disease or condition caused or contributed by SARS-CoV-2; said method comprising administering a subject in need thereof a modified CBM.

Additionally disclosed, is the use of a modified CBM in the manufacture of a medicament for the treatment or prevention of a Coronavirus infection and/or a disease or condition caused or contributed by a Coronavirus. Additionally, the disclosure provides HEX17, for use in the treatment or prevention of:

(i) a Coronavirus infection and/or a disease or condition caused or contributed by a Coronavirus·, or

(ii) a COVID-19 infection and/or a disease or condition caused or contributed by SARS-CoV-2.

Also disclosed is a method of treating or preventing:

(i) a Coronavirus infection and/or a disease or condition caused or contributed by a Coronavirus·, or

(ii) a COVID-19 infection and/or a disease or condition caused or contributed by SARS-CoV-2; said method comprising administering a subject in need thereof HEX17.

Additionally disclosed, is the use of HEX17 in the manufacture of a medicament for the treatment or prevention of a Coronavirus infection and/or a disease or condition caused or contributed by a Coronavirus.

A molecule for the uses, methods and medicaments described herein may comprise any one or more of the CBM molecules described herein. Molecules of this type may further comprise the abovementioned trimerisation domain allowing the formation of sialic acid/glycan binding molecules which are multivalent CBMs.

For example, a molecule for use may comprise a plurality or multiple (i.e. two, three, four or more) CBMs. Molecules which comprise a plurality of CBMs may be termed “multivalent glycan or sialic acid binding molecules” or “multivalent CBMs”.

Multivalent CBM molecules, may be prepared as constructs comprising multiple (identical or different) CBMs linked by amino acid/peptide linkers. Each CBM (for example, VcCBM, SpCBM or modified CBM) may be linked to another (for example, VcCBM, SpCBM or modified CBM) or to a trimerisation domain (TD) by, for example, peptides comprising 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 or a CBM to a trimerisation domain, to produce a multivalent CBM:

(i) 5 amino acid linkers: ALNGS LQALG

GGNSG

GGGSG

GGALG

(ii) 10 amino acid linkers: ALNGSGGGSG

LQALGGGGSL

(iii) 15 amino acid linkers: ALNGSGGGSGGGGSG

A multivalent CBM for the methods, uses and medicaments described herein may, for example, comprise two or more Family 32 CBMs, two or more Family 40 CBMs, two or more Family 47 CBMs, two or more Family 67 CBMs, two or more Family 70 CBMs. A multivalent CBM may comprise a mix of different CBM types, for example CBMs from different CBM families, or repeats of the same CBM.

Various examples of useful mono- and multivalent glycan/sialic acid binding molecules are described herein.

For example, the molecules presented in Figure 1 may be for use in the methods, uses and medicaments of this disclosure. A suitable molecule may comprise (consist essentially of, or consist of) one or more (for example, two, three, four or more) VcCBMs (that is a CBM40 derived from Vibrio cholerae ). The CBM (for example the VcCBM) may be fused, bound or conjugated to an oligomerisation domain (such as a PaTD or oligomerisation fragment thereof). A glycan/sialic acid binding molecule may comprise, consist or consist essentially of two fused (or bound) CBMs which are in turn fused to an oligomerisation domain (see, for example, molecule Vc2CBMTD shown in Figure 1).

Every one of the disclosed molecules alone or in combination with another disclosed molecule, may be for use in treating or preventing a Coronavirus infection or a disease or conditions associated therewith a method of treating a Coronavirus infection or a disease or conditions associated therewith or for use in the manufacture of a medicament for treating or preventing a Coronavirus infection or a disease or conditions associated therewith.

Any of the disclosed molecules (for the uses, medicaments and methods described herein) - especially the modified CBM molecules, may be generated using PCR-based cloning techniques and a suitable method for the generation of multivalent molecules of this type is described in, for example, 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 the likes of HEX17, f/c2CBM, f/c4CBM and Sp2CBM may be prepared as constructs comprising multiple CBMs linked by amino acid/peptide linkers - such as those described above.

In the context of this disclosure, molecules (carbohydrate binding molecules, sialic acid binding molecules, glycan binding proteins/molecules and/or CBMs) for use in treating or preventing Coronavirus infections, may comprise one or more CBMs selected from the group consisting of:

(i) one or more (for example, 2, 3, 4 or more) Family 32 CBMs;

(ii) one or more (for example, 2, 3, 4 or more) Family 40 CBMs;

(iii) one or more (for example, 2, 3, 4 or more) Family 47 CBMs;

(iv) one or more (for example, 2, 3, 4 or more) Family 67 CBMs;

(v) one or more (for example, 2, 3, 4 or more) Family 70 CBMs; and

(vi) a modified CBM (as described herein).

Additionally, molecules (carbohydrate binding molecules, sialic acid binding molecules, glycan binding molecules/proteins and/or CBMs) for use in treating or preventing Coronavirus infections, may comprise a CBM selected from the group consisting of:

(i) a Clostridium perfringens CBM32 (CpCBM32);

(ii) a Streptococcus pneumoniae CBM40 (SpCBM40);

(iii) a Vibrio cholerae CBM40 ( l/cCBM40) ;

(iv) a Streptococcus pneumoniae CBM47(SpCBM47);

(v) a Streptomyces avermitilis CBM67 (SaCBM67);

(vi) a Streptococcus pneumoniae CBM70 (SpCBM70);

(vii) a Vibrio cholerae Nan FI sialidase CBM;

(vii) a Vibrio cholerae Nan FI sialidase CBM sialic acid binding fragment thereof.

(xi) a Streptococcus pneumoniae nanA sialidase CBM; and

(x) a Streptococcus pneumoniae nanA sialidase CBM sialic acid binding fragment thereof.

A multivalent CBM for the various uses, methods and medicaments described herein may comprise:

(i) a 1/cCBM; or

(ii) two (or more) f/cCBM(s); or (iii) three or four (or more) t/cCBM(s); or

(iv) a SpCBM; or

(v) two (or more) SpCBM(s); or

(vi) three or four (or more) SpCBM(s); or

(vii) a CpCBM; or

(viiiO two (or more) CpCBM(s); or

(viii) three or four (or more) CpCBM(s); or

(ix) a SaCBM; or

(x) two (or more) SaCBM(s); or

(xi) three or four (or more) SaCBM(s).

A multivalent CBM for a use, method or medicament of this disclosure may comprise a mixture of different CBMs, for example, one or more CBM32s with one more other CBMs selected from the group consisting of:

(i) a Family 40 CBM

(ii) a Family 47 CBM

(iii) a Family 67 CBM

(iv) a Family 70 CBM

For example, a method, use or medicament described herein may exploit the combination of a CBM32 with a Family 40 CBM (a CBM40).

Alternatively, a multivalent CBM for the uses, methods and medicaments described herein may comprise a mixture of different CBMs, for example one or more CBM40s with one more other CBMs selected from the group consisting of:

(i) a Family 32 CBM

(ii) a Family 47 CBM

(iii) a Family 67 CBM

(iv) a Family 70 CBM

For example, a method, use or medicament described herein may exploit the combination of a CBM40 with a Family 32 CBM (a CBM32).

A multivalent CBM for the uses, methods and medicaments described herein may comprise a mixture of different CBMs, for example one or more CBM47s with one more other CBMs selected from the group consisting of: (i) a Family 32 CBM

(ii) a Family 40 CBM

(iii) a Family 67 CBM

(iv) a Family 70 CBM

A multivalent CBM for the uses, methods and medicaments described herein may comprise a mixture of different CBMs, for example one or more CBM67s with one more other CBMs selected from the group consisting of:

(i) a Family 32 CBM

(ii) a Family 40 CBM

(iii) a Family 47 CBM

(iv) a Family 70 CBM

A multivalent CBM for the uses, methods and medicaments described herein may comprise a mixture of different CBMs, for example one or more CBM70s with one more other CBMs selected from the group consisting of:

(i) a Family 32 CBM

(ii) a Family 40 CBM

(iii) a Family 47 CBM

(iv) a Family 67 CBM

Multivalent CBMs for the various uses, methods and medicaments disclosed herein may include, for example, molecules selected from the group consisting of:

(i) Cp2CBM32TD (comprising, consisting essentially of or consisting of: 2 CBMs (CBM32s) from Clostridium perfringens fused to a trimerisation domain);

(ii) Sp2CBM40TD (comprising, consisting essentially of or consisting of: 2 CBMs (CBM40s) from Streptococcus pneumoniae fused to a trimerisation domain;

(iii) t/c2CBM40TD (comprising, consisting essentially of or consisting of: 2 CBMs (CBM40s) from Vibrio cholerae fused to a trimerisation domain; and

(iv) t/c4CBM (comprising, consisting essentially of or consisting of: 4 CBMs (CBM40s) from Vibrio cholerae)

(v) Sp2CBM47TD comprising, consisting essentially of or consisting of: 2 CBMs (CBM47s) from Streptococcus pneumoniae fused to a trimerisation domain);

(vi) Sa2CBM67TD comprising, consisting essentially of or consisting of: 2 CBMs (CBM67s) from Streptococcus avermitilis fused to a trimerisation domain); (vii) Sp2CBM70TD comprising, consisting essentially of or consisting of: 2 CBMs (CBM70s) from Streptococcus pneumoniae fused to a trimerisation domain).

It should be noted that this disclosure provides uses (compositions, methods and medicaments) which comprise not only any one or more of the CBMs disclosed herein, in isolated form (that is uses in which at least the CBM component (of the therapeutic molecule) comprises or consists essentially of one or more of the CBM sequence(s) described herein and/or functional fragments thereof), but also uses in which the CBM component of the therapeutic molecule is comprised within a larger molecule. By way of example, the various CBMs described herein may be provided and/or used in the form of large molecules comprising a CBM component. The CBM component (e.g. the sialic acid binding molecule) may itself comprise (consist of or consist essentially of), for example, any one of the CBMs described herein (including, for example CBM32, CBM40, CBM47, CBM67 and CBM70). By way of (non-limiting) example, molecules (e.g. the CBMs and/or glycan/sialic acid binding molecules) of this disclosure may not only exhibit an ability to bind a glycan (or a component thereof) or sialic acid, but may also have one or more other functions. For example, the molecules may have enzymatic activity. For example, a useful molecule may comprise a CBM (as described herein) and exhibit some sialidase activity.

A useful molecule may be a fusion protein comprising an enzymatic portion and a glycan/sialic acid binding portion - wherein the glycan/sialic acid binding portion comprises a CBM as described herein. In such cases, the enzymatic portion may be fused to the glycan/sialic acid binding portion. As stated, the enzymatic portion of any useful fusion protein may comprise (or have, or exhibit) sialidase activity.

In one embodiment, the sialic acid binding molecule, glycan binding molecule, or CBM for the various uses described herein, may not be provided as part of, or comprised within, a molecule (for example a fusion protein) with enzymatic (for example sialidase) activity. Additionally or alternatively, the sialic acid/glycan binding molecule may not (i) bind heparin or heparin sulfate and/or (ii) comprise the GAG-binding domain of a protein that binds heparin or heparin sulfate moieties. A construct comprising the sialic acid/glycan binding molecule, glycan binding molecule, or CBM, may not show or exhibit enzymatic (for example sialidase) activity.

This disclosure also provides compositions, in particular pharmaceutical compositions which may be exploited in the described uses, methods and medicaments. As such, any of the useful molecule(s) (for example, the CBMs (modified, multivalent or otherwise) or glycan binding molecules) described herein may be formulated for subsequent use. For convenience, it should be noted that the term "CBM” embraces, all monovalent, multivalent and modified CBM molecules described herein.

For example, a sialic acid binding molecule, a glycan binding molecule or CBM may be formulated as a therapeutic or pharmaceutical composition. The various compositions may comprise one or more of the sialic acid binding molecule(s)/glycan binding molecule(s)/CBM(s) as described herein and one or more pharmaceutically acceptable excipients. For example, the pharmaceutical preparations comprising the molecules described herein may be mixed with stabilisers, wetting agents, emulsifiers, salts (for use in influencing osmotic pressure), buffers and/or other substances that do not react deleteriously with the active compounds.

Any given therapeutic use or method of treatment may require the administration (together, concurrently or separately) of one or more of these compositions, which compositions may comprise one or more different CBMs.

Pharmaceutical compositions according to the present disclosure may be prepared for oral, mucosal, intranasal or parenteral (intravenous) administration. Those formulations for mucosal or intranasal administration may be prepared conventionally, comprising substances that are customarily used in pharmaceuticals and as described in, for example, 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 content of all of these documents and references being incorporated by reference.

Liquid dosage forms for oral and/or intranasal administration may include emulsions, solutions, suspensions, syrups, and elixirs. In addition to the compound or composition, the liquid dosage forms may contain inert diluents commonly used in the art, such as water or other solvents, solubilising agents and emulsifiers.

Any suitable amount of a sialic acid binding molecule, glycan binding molecule or CBM may be used. For example, whether a composition comprising a sialic acid binding molecule, glycan binding molecule or CBM is to be administered intravenously or mucosally (for example, intranasally) the dose of sialic acid binding molecule/glycan binding molecule/CBM may comprise anywhere between about 0.1 pg and about 6000 pg. For example, a dose of about (for example +/- 0.5 pg) 0.1 pg, 0.5 pg, 1 pg, 5 pg, 10 pg, 11 pg, 12 pg, 13 pg, 14 pg, 15 pg, 20 pg, 30 pg, 40 pg, 50 pg, 100 pg, 200 pg, 300 pg, 400 pg, 500 pg, 600 pg, 700 pg, 800 pg, 900 pg, 950 pg 10OOpg, 1500pg, 2000pg, 2500pg, 3000pg, 3500pg, 4000pg, 4500pg, 5000pg, 5500pg, or 6000pg of the sialic acid binding molecule/glycan binding molecule/CBM 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/glycan binding molecule/CBM may be provided in anywhere between about 1 pi to about 0.5 ml of excipient, diluent or buffer. For example, the required amount of sialic acid binding molecule/glycan binding molecule or CBM may be combined (or formulated) with about 5 pi, 10 pi, 15 pi, 20 pi, 25 pi, 30 pi, 35 pi, 40 pi, 45 pi, 50 pi, 55 pi, 60 pi, 65 pi, 70 pi, 75 pi, 80 pi, 85 pi, 90 pi, 95 pi, 100 pi, 140pl 200 pi, 280pl, 300 pi, 400 pi, 500 pi, 560pl, 600 pi, 700 pi, 800 pi, 900 pi, or 1 ml, Concentrations of 0.1-15mg (for example 0.5 mg, 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 11 mg, 12 mg, 13 mg or 14 mg) (sialic acid/glycan binding protein) per ml (excipient, diluent or buffer) may be most useful. A concentration of 10mg/ml (excipient, diluent or buffer) may be very useful.

A composition of this disclosure (for example, a composition comprising any of the sialic acid binding molecules, glycan binding molecules or CBMs disclosed herein) may be administered (prophylactically) to a subject at regular and/or predetermined times. For example, a composition described herein may be administered at regular and/or predetermined times both before, during and after the subject enters or encounters a scenario during which they might be vulnerable and/or susceptible to a Coronavirus infection.

A composition of this disclosure may be administered every day and/or every few days.

A composition of this disclosure may be administered multiple times throughout any given day.

A composition described herein may be administered over a period of weeks or months or years. The precise administration regimen will depend on the subject, the health of that subject and the period of time that subject is deemed to be at risk of or vulnerable to, a Coronavirus infection.

The present disclosure will now be described with reference to the following figures which show:

Figure 1 : Building blocks of the multivalent CBM forms and their affinities for sialic acid a, t/cCBM, residues 25-216 of the V. cholerae sialidase (PDB:1w0p) with a-2,3-sialyllactose drawn as spheres b, SpCBM, residues 121 -305 of S. pneumoniae NanA sialidase with a-2,3- sialyllactose (PDB:4c1w). c, TD, the trimerisation domain, residues 333-438, of the P. aeruginosa pseudaminidase (PDB:2w38) in rainbow colours; the other two monomers in single colours d, Multivalent forms: their molecular weights, valencies and binding affinities fora2,3-sialyllactose as determined by surface plasmon resonance (SPR) at 25°C (KD values for f/cCBM, f/c2CBM and f/c3CBM had been reported previously (Connaris et al, 2009)). Tandem repeat CBMs, and oligomeric CBMs fused to TD are linked by a 5-amino linker (details in Connaris, H. et al., (2014). PNAS 111 :6401 -6406).

Figure 2: Graph showing the results of the condition 1 assay. There is an observable anti-viral effect with all CBM compounds especially when tested at 3mg/mL.

Figure 3: Plaque assay showing the results of the condition 1 assay; again, there is an anti viral effect shown for all 3 CBMs especially when used at the higher concentration.

Figure 4: Graph showing the results of the condition 2 assay. In this case, cells were exposed to CBM before SARS-CoV-2 infection; thus, the condition 2 assay represents a prophylaxis model. An anti-viral effect is shown for at least CBM2 & CBM3

Figure 5: Graph showing the results of the condition 3 assay. In this case, infected cells (with SARS-CoV-2) were treated with the various CBMs. An anti-viral effect is shown for all 3 CBMs - particularly CBM3.

Figure 6: Plaque assay showing the results of the condition 3 assay. Again, this shows the anti-viral effect of all 3 CBMs - particularly CBM3.

Figure 7: the mean and SEM of the total clinical observations for group 2 (Control) and 3 (Neumifil) from 0 DPC until day of cull (7 DPC). Figure 7 also shows the mean (with SEM) percentage weight change for the same period (right y-axis).

Figure 8: Detection of Neumifil (HEX17) binding to SARS-CoV-2 Spike S1 variants. The dotted lines represent 4PL curve fits of the data. Inset: EC50 values for each variant.

Figure 9: Detection of Neumifil (HEX17) binding to recombinant human ACE2. The dotted line represents a 4PL curve fitting of the data. Inset: EC50 value.

Methods

Plaque reduction Assay

Thaw vial of titrated SARS CoV-2 on ice. Prepare 2 working stocks of SARS-CoV-2, diluting the virus in serum free (SF) DMEM to 500 pfu/mL (working stock for condition 1 ) and 250 pfu/mL (working stock for conditions 2 and 3). Store the prepared stocks on ice.

For test agent preparation, thaw an aliquot of each CBM (100 mI_ per vial at 10 mg/ml_) on ice and transfer contents into a new sterile 1.5 ml. Eppendorf tube an centrifuge at 13,000 rpm for 5 min to pellet any precipitates that may have formed. Transfer the supernatant into new sterile 1.5 ml. Eppendorf tubes.

From the CBM supernatants, prepare 2 working stock concentrations (3 mg/ml_ and 1 mg/ml_, from a master stock of 10 mg/ml_) of the 3 CBMs to be tested, diluting the CBMs in a 50:50 mix of serum-free DMEM:PBS (see Table 1)

The final concentration of CBMs will vary between conditions. Condition 1 : final CBM concentrations 1.5 mg/ml_ and 0.5 mg/ml_, and for conditions 2 and 3, final CBM concentrations 3 mg/ml_ and 1 mg/mL.

Table 1 * 50-50 mix of serum free DMEM:PBS

Condition 1 : virus and CBM mixed prior to adding to cells • Mix equal volumes of SARS CoV-2 and CBM and incubate on ice for 1 hour. For the positive control mix SARS-CoV-2 with SF diluent (50-50 mix of SF DMEM and PBS) in place of the CBM. For the negative control mix CBM 1 at 3 mg/ml_ with SF DMEM in place of SARS-CoV-2. o Tube 1 : 300 mI_ SARS-CoV-2 at 500 pfu/mL plus 300 mI_ CBM1 at 3 mg/mL o Tube 2: 300 mI_ SARS-CoV-2 at 500 pfu/mL plus 300 mI_ CBM2 at 3 mg/mL o Tube 3: 300 mI_ SARS-CoV-2 at 500 pfu/mL plus 300 mI_ CBM3 at 3 mg/mL o Tube 4: 300 mI_ SARS-CoV-2 at 500 pfu/mL plus 300 mI_ CBM1 at 1 mg/mL o Tube 5: 300 mI_ SARS-CoV-2 at 500 pfu/mL plus 300 mI_ CBM1 at 1 mg/mL o Tube 6: 300 mI_ SARS-CoV-2 at 500 pfu/mL plus 300 mI_ CBM1 at 1 mg/mL o Tube NC: negative control: 300 mI_ SF DMEM plus 300 mI_ CBM diluent o Tube PC: positive control, 300 mI_ SARS-CoV-2 at 500 pfu/mL plus 300 pL CBM diluent

• After 1 hr, remove serum free media from the cells and wash with sterile PBS (<0.5 mL/well)

• After the PBS wash, add 200 mI_ of the prepared inoculums to the appropriate wells, with tubes 1-3 (and controls) on plate 1 and tubes 4-6 (and controls) on plate 2.

• Place plates in a flat-bottomed, sealable container and transfer to incubator. Incubate at 37 °C 5% CO2 for 1 hr.

• After 1 hr, remove the inoculum and add 1 ml. overlay to each well.

• T ransfer plates to a flat-bottomed sealable container

• Incubate for 5 days at 37 °C 5% CO2.

Condition 2: cells exposed to CBM before virus infection

• Remove serum free DMEM and wash with Sterile PBS (<0.5 mL/well).

• After the PBS wash, add 200 pL CBM, with CBMs at 3 mg/mL on plate 3 and 1 mg/mL on plate 4. Incubate at 37 °C 5% CO2 for 1 hr.

• After 1 hr, remove CBM and wash with sterile PBS (<0.5 mL/well).

• After the PBS wash, add 200pL SARS-CoV-2 diluted to 250 pfu/mL to each well except the negative control wells. To the negative control wells, add 200 pL SF DMEM.

• Place plates in a flat-bottomed, sealable container and transfer to incubator. Incubate at 37 °C 5% CO2 for 1 hr.

• After 1 hr, remove the inoculum and add 1 mL overlay to each well.

• T ransfer plates to a flat-bottomed sealable container

• Incubate for 5 days at 37 °C 5% CO2. Condition 3: cells infected with SARS-CoV-2 and then treated with CBM.

• Remove serum free DMEM and wash with sterile PBS (0.5 mL/well)

• After the PBS wash, add 200 mI_ SARS-CoV-2 diluted to 250 pfu/mL to each well except the negative control. To the negative control, add 200 mI_ SF DMEM.

• Place plates in a flat-bottomed, sealable container and transfer to incubator. Incubate at 37 °C 5% CO2 for 1 hr.

• After 1 hr, remove the inoculum and add 200 mI_ CBM to each well, except the negative control (CBMs at 3 mg/ml_ on plate 5 and 1 mg/ml_ on plate 6). To the negative control, add 200 mI_ CBM diluent (SF DMEM-PBS).

• Place plates in a flat-bottomed sealable container and transfer to incubator. Incubate at 37 °C 5% CO2 for 1 hr.

• After 1 hr, remove the CBMs and add 1 ml. overlay to each well.

• T ransfer plates to a flat-bottomed sealable container

• Incubate for 4 days at 37 °C 5% C02.

Specific details:

CBMs:

• CBM1 : Vc2CBM40TD (trimeric (hexavalent) form based on V. cholerae CBM40) · CBM2: Neumifil (HEX17: trimeric (hexavalent) form based on S.pneumoniae CBM40)

• CBM3: Cp2CBM32TD (trimeric (hexavalentform based on C. perfringens CBM32) CBMs added at two concentrations (1 mg/ml_ & 3 mg/ml_), and (as stated) tested in three conditions:

• Condition 1 : SARS-CoV-2 and CBM mixed prior to adding to cells · Condition 2: Cells exposed to CBM before SARS-CoV-2 infection (prophylaxis model)

• Condition 3: Cells infected with SARS-CoV-2 and then CBM added (treatment model)

For making up of CBM: 3 mg/ml_: 450 mI_ stock + 1050 mI_ DMEM:PBS.

1 mg/ml_: 150 mI_ stock + 1350 mI_ DMEM:PBS

SARS-CoV-2

Vial labelled: SARS CoV2, England 2, P2 HCM/V/52, 05.03.20 Stock at 2.4x10 5 pfu/mL ® 100 mI_ stock + 900 mI_ media = 2.4x10 4 pfu/mL

® 200 mI_ above + 1800 mI_ media = 2.4x10 3 pfu/mL ® 1.5 mL above + 6 mL media = 480 pfu/mL <— Used for studies

VeroE6 cells

P15, split 1 :225/3/20 (obtained from PHE HCM group)

Reagent details

► DMEM Sigma D5796, Lot RNBH6732, Exp 06/2020

► PBS Gibco 10010-023, Lot 2098597, Exp 30/6/2021 >- Overlay (aliquots made, each sufficient for 2 plates):

• 7.5 mL 4% CMC PHE media, MR/19/1044, Prod date 04/DEC/19

• 7.5 mL 2% CMC PHE media, MR/19/1043, Prod date 04/DEC/19

• 15 mL 2xMEM Gibco 21935-028, Lot 2150451 , Exp 31/12/2020

• 800 pL FCS Gibco 10100-139, Lot 216386RP, Exp 05/2024

• 400 pL anti-anti Sigma A5955, Lot 035M4800V RESULTS

Condition 1 : Virus and CBM mixed prior to adding to cells

In this phase of testing, SARS-CoV-2 virus and the CBM compounds were incubated for 1 hour prior to addition to cells. Plate 1 were compounds tested at a working concentration of 3 mg/mL and plate 2 at 1 mg/mL.

Negative control wells were all intact and the positive control gave counts between 119-164 plaques per well. The results are shown in Figures 2 (graph) and 3 (plaque assay). The results show that there is an observable anti-viral effect with all CBM compounds.

3.2. Condition 2: Cells exposed to CBM before virus infection

In this phase of testing, VeroE6 cells were exposed to CBM compounds prior to addition of SARS-CoV- 2 virus. Plate 3 were compounds tested at a working concentration of 3 mg/mL and plate 4 at 1 mg/mL. Plaques were counted after staining.

As shown in Figure 4, there was an observable anti-viral effect with the CBM2 and CBM3 compounds when tested at a working concentration of 1 mg/mL.

3.3. Condition 3: Cells infected with SARS-CoV-2 and then treated with CBM

In this phase of testing, VeroE6 cells were infected with SARS-CoV-2 virus before addition of CBM compounds. Plate 5 were compounds tested at a working concentration of 3 mg/mL and plate 6 at 1 mg/mL. Plaques were counted after staining. Negative control wells were all intact and the positive control gave counts between 117-132 plaques per well. When plotted on a graph (see Figure 5), all 3 CBM compounds are shown to have an anti viral effect, particularly CBM3 which showed the greatest reduction. Results from both working concentrations of CBM1 and CBM2 were similar. The results of the plaque assay are also shown in Figure 6.

Conclusion

All CBMs showed some anti-viral activity against SARS-CoV-2 virus in vitro

CBM2 (Neumifil: Flex! 7) and CBM3 (CBM32-based) are very promising. The results demonstrated here provide the basis for urgent in vivo studies in a suitable animal model.

Example 2: testing CBM vs OC43

OC43 is a human coronavirus OC43-(HCoV-OC43); it is a Betacoronavirus and is associated with occurrences of a ‘common cold’ type illness. Its S protein binds to sugar- based receptor-determinants, specifically to 9- O-acetylated sialic acids (9- O-Ac-Sias) attached as terminal residues to glycan chains on glycoproteins and lipids.

A series of experiments were completed to determine whether or not CBMs could be used to treat or prevent OC43 infections.

In each experiment the following CBMs were used:

1) 1/C2CBM40TD (VC2)

2) Cp2CBM32TD (CBM32)

3) HEX17

Each CBM was used at 1 mg/ml and at 3 mg/ml.

Condition 1 : Prophylactic CBM treatment

Cells were treated with CBMs for 1 hour before incubation with hCoV-OC43 for 1 hour. The assay showed that treatment with any of the CBMs tested was able to prevent a subsequent hCoV-OC43 infection as compared to cells infected but not pre-treated with any CBM.

The effect was most pronounced with VC2 (at 3 mg/ml and at 1 mg/ml), FIEX17 (at 1 mg/ml) and CBM32 (at 3 mg/ml and 1 mg/ml).

Condition 2: Simultaneous CBM treatment

Cells treated with CBMs and hCoV-OC43 for 1 hour simultaneously. The assay showed that simultaneous treatment with any of the CBMs and at any concentration (1 mg/ml or 3 mg/ml) was able to reduce hCoV-OC43 infection as compared to cells infected but not treated with any CBM. Example 3

Animal (Hamster) studies testing Neumifil against SARS-CoV-2 Protocol/Study design

Table 2: detail study protocol Table 3: study design summary

Results

Table 4: clinical observations for group 2 (PBS) and 3 (Neumifil) following challenge with 5E+04 PFU SARS-CoV-2.

H = Healthy, A = Arched/Hunched, R = Ruffled fur, Lb = Laboured breathing, P = Pinched / Wasp waisted, D = Dehydrated not drinking Clinical observations were made twice-daily from 7 days prior to challenge onwards. No clinical symptoms were observed in any group prior to challenge. Note also that in the Neumifil treated group, the average number of symptoms (A, R, Lb, P and/or D) is generally lower than in the control group. Moreover there are fewer instances of Lb in the Neumifil treated group.

Additional results are presented in Figure 7. As compared to the control group(s), the Neumifil treated group (group 3) exhibited fewer total clinical observations

Example 4

Angiotensin-converting enzyme 2 (ACE2) plays a major role in SARS-CoV-2 recognition, binding, fusion and entry into host cells [1]. Glycans, including sialic acid, may also be important in this interaction. There are eight glycosylation sites within the ACE2 receptor, three of which (N90, N322 and N546) may play a critical role in the interaction with SARS-CoV-2 Spike. Glycans on the SARS-CoV-2 Spike may also modulate the conformation of the Spike’s receptor binding domain (RBD), which is responsible for ACE2 recognition and binding. Deletion of these glycans significantly reduces ACE2 binding.

Multiple new variants of SARS-CoV-2 have emerged and are circulating globally [1 ,2]. Of particular concern are the B.1 .1 .7 and B.1525 variants identified in the UK, the B.1 .351 South African variant and the P.1 Brazil variant. The B.1.1.7 UK (‘Kent’) virus is characterized by the HV69-70 deletion and N501Y in the Spike protein which increases transmissibility and may be associated with increased risk of death. The B.1.351 S. African variant shares some mutations with B.1 .1 .7 alongside additional differences, including the potential vaccine escape mutation E484K. The Brazil variant, P.1 is spreading to multiple countries and is associated with reinfections. P.1 also includes E484K, in addition to the N501Y mutation linked to increased infectivity of the UK variant. A further variant, B1525, has been identified in the UK and contains the important E484K mutation alongside the Kent B.1 .1 .7 mutations.

Aims: 1) To determine whether Neumifil interacts with SARS-CoV-2 Spike S1 protein and whether the affinity is affected by new variant mutations. 2) To determine whether Neumifil interacts with human ACE2.

Methods:

Table 1 summarizes the variant S1 Spike sequence information provided by the manufacturers. Each of the Spike and ACE2 proteins were recombinantly expressed in HEK293 cells.

Table 5. Spike S1 protein variants used in the study with corresponding accession codes and mutations. EC50 (half-maximal effective concentration) values were determined by ELISA. Spike or ACE2 proteins were immobilized overnight at 4°C on a high-binding ELISA plate at a concentration of 1 pg/mL. The wells were then incubated for 1.5 h with Neumifil (3-fold dilution series: 29160, 9720, 3240, 1080, 360, 120, 40, 0 ng/mL) in triplicate. Immunodetection of Neumifil binding was performed by incubation with rabbit anti-Neumifil (1 h), followed by HRP- labelled anti-rabbit IgG (1 h) and TMB substrate development. Binding curves were analysed using 4 parameter logistic (4 PL) curve fitting to determine the inflection point (EC50). Results:

As shown in Figure 8, Neumifil binds to the original Spike protein (consisting of the Wuhan- Hu-1 December 2019 isolate sequence) with an EC50 of 174 ng/mL. The binding profiles and EC50s for the variants indicate that the affinity is not significantly affected by the mutations present in the South African and UK (Kent) sequences. Figure 9 shows Neumifil binding to ACE2 with an EC50 of 235 ng/ml.

References

1. Understanding variants of SARS-CoV-2 (2021) The Lancet World Report 397(102), P462 2. McNally, A. (2021 ) BMJ 372, 504