BACTERIAL VAGINOSIS DIAGNOSTIC

FIELD OF THE INVENTION

The present invention relates to detecting cleavage activity of a sialidase enzyme and the use thereof in detecting bacterial vaginosis. In particular, the invention provides specifically-designed peptides and indicator molecules useful for detecting cleavage activity of a sialidase enzyme. Various other aspects of the invention include an enzyme detection device, kit, method and use for detecting or measuring the presence in a test sample of the activity of a sialidase enzyme capable of cleaving an indicator molecule of the invention.

BACKGROUND TO THE INVENTION

Sialidase enzymes (otherwise known as neuraminidases) are glycoside hydrolase enzymes split into two main classes that cleave exo or endo (poly-)sialic acids (EC 3.2.1.18 and EC 3.2.1.129 respectively). Sialic acids are N- or O-substituted derivatives of neuraminic acid (shown below):

Sialic acids are generally found in nature in glycoproteins and glycolipids (in particular gangliosides). Viral, bacterial and mammalian sialidases are all known in nature. Elevated levels of bacterial sialidase activity are known to act as a diagnostic marker for bacterial vaginosis (BV; Briselden et. al. , J. Clin. Microbiol., 1992, vol. 30, pages 663-666). The theory behind this is based on the bacterial imbalance that can occur in the vagina and the transition from a typical healthy environment high in lactobacilli to an anaerobic driven microbiome (Petrova et. al., Front. Physiol., 2015, 6:81). Certain anaerobes excrete sialidase enzyme and compromise the natural mucus barriers in the vagina. Nonetheless, the exact mechanism for BV is still to be fully established. Patients suffering from symptomatic BV can experience significant discomfort, vaginal discharge, an unpleasant odour and more generally a feeling of lack of control over their vaginal health (Bilardi et. al., Plos One, 2013, vol. 8, e74378). Treatment pathways typically involve topical or oral antibiotics. Topical prebiotic treatments (e.g.: VH essentials ®), and pH gel (e.g. balance active BV) are also available over the counter. BV can be intractable and patients can suffer a relapse after antibiotic treatment (Bilardi et. al., Plos One, 2013, vol. 8, e74378).

Some of the intractable cases are believed to be linked to the formation of biofilms in the vagina (Verstraelen & Swidsinski, Curr. Opin. Infect. Dis., 2013, 26:86-89). The risks in not picking up the presence of BV are a greater susceptibility to sexually transmitted infection (including HIV) and an associated risk of pre-term birth. The pre-term birth risk is believed to be linked to premature weakening of the cervical mucus barrier (Lewis et. al. , J. Biol. Chem., 2013, vol. 288, pages 12067-12079). Currently in the clinic, the test for BV is based on a vaginal sample smear, sent for analysis in the lab. Methods of analysis include looking for clue cells, the potassium hydroxide (KOH) whiff test, or applying set criteria such as the Nugent score (Nash, Jungmann, & Gubert, BMJ Learning, 2015, 1-29). The use of a rapid test at point of care would benefit the patient by delivering a quick answer, putting them on an immediate treatment plan and speed up the overall process for delivering outcomes. Likewise there would be potential benefit to the healthcare provider, by avoiding

outsourcing to a lab and the resource and time costs involved and keeping the patient time down to a single session in the first instance.

Two main types of products for diagnosing BV are currently available in the USA and Europe. One set of products is based on a colour changing swab that measures pH. The other set of products is also colourimetric and based on sialidase activity measurement.

The pH-based products are available at point of care and over the counter and include VS-SENSE PRO® (sold by Common Sense) and Canestest® (sold by Canesten). The main product currently on sale based on sialidase detection is BV Blue (sold by Sekisui

Diagnostics and Gryphus Diagnostics). BV Blue is a swab-based test; a sample swab is placed in an indicator solution which will change colour to indicate the presence of sialidase activity in the sample. The test takes approximately 15 minutes to run.

The pH-based tests lack accuracy since various conditions can cause a change in the local pH of the vagina (e.g. Candidiasis and Trichonomiasis as well as BV). The known tests such as BV Blue which detect sialidase activity lack sensitivity.

DESCRIPTION OF THE INVENTION

The present invention results from attempts to improve sensitivity and/or specificity of sialidase activity detection. The present inventors have now specifically designed peptides and corresponding indicator molecules useful for detecting cleavage activity of a sialidase enzyme. As described further herein, each peptide is conjugated to a sialyl group via a galactosyl group (and thus functions as a substrate for a sialidase enzyme) wherein the

peptide architecture is specifically designed so that, once the sialyl group has been cleaved from the peptide by one or more sialidase enzymes present in the sample, the de-sialylated derivative of the peptide is recognised and bound by specific binding molecules (e.g.

antibodies). In preferred embodiments, the peptide incorporates one or more amino acids which enhance the peptide’s resistance to protease cleavage.

Accordingly, in one aspect, the invention provides a peptide comprises a sequence according to the following formula:

Xi-X2-X3[Gal-Sial]-X4-X5

(Formula I) (SEQ ID NO: 9)

wherein:

(i) Sial is a sialyl group;

(ii) X3 is a natural or non-natural amino acid comprising a glycosyl acceptor group; and

(iii) X^ X2, X4 and X5 are independently selected from any amino acid provided that at least one of X^ X2, X4 and X5 is a D-amino acid and/or a non-standard amino acid or a non-natural amino acid.

The peptide may consist essentially of, or consist of, a sequence of Formula I.

According to all aspects of the invention, by“sialyl group” is meant a sialic acid substituent wherein a sialic acid is an N- or O-substituted derivative of neuraminic acid (e.g. N-acetylneuraminic acid; termed‘Neu5Ac’ or‘NANA’).

According to all aspects of the invention, by“Gal” is meant a galactosyl substituent. In preferred embodiments, the galactosyl substituent is a radical of the following structure:

In particular embodiments, the galactosyl subtituent is a radical of b-galactose. In such embodiments, the galactosyl substituent is O-linked to the sialyl group. All possible

regioisomers are encompassed by the invention. The skilled person will also appreciate that other saccharides may be used in place of a galactosyl group, such as radicals of glucose, fructose, lactose, maltose and sucrose for example. Thus, reference to a “galactosyl substituent” herein is to be interpreted accordingly.

The -Gal-Sial group is conjugated to X3 as shown above wherein X3 is a natural or non natural amino acid comprising a glycosyl acceptor group. That is to say the side-chain of the amino acid represented by X3 comprises a nucleophilic substituent that forms a bond with the Gal substituent (i.e. a glycosyl donor). For instance, the nucleophilic substituent may be a hydroxyl or amine substituent. Thus, in particular embodiments, X3 may be selected from serine (Ser), threonine (Thr), tyrosine (Tyr), hydroxylysine (Hyl),

hydroxyproline (Hyp), asparagine (Asn), arginine (Arg) and phosphoserine (SEP). In preferred embodiments, X3 is Ser.

It should be understood from Formula I that the -Gal-Sial group is bonded to X3 only (as indicated by the square brackets in Formula I). It does not bridge X3 to X4. For the avoidance of doubt, an alternative and equivalent formula (la) is shown below:

(Formula la)

Hence, the -Gal-Sial group is branched from the peptide backbone. Thus, the -Gal substituent is positioned centrally in the structure so that it is flanked by amino acids X^ X2, X4 and X5. Consequently, as described further herein, binding molecules (e.g. antibodies) can be generated with very high affinity and specificity for the de-sialylated derivative of each peptide and the generation of peripheral binding molecules (e.g. antibodies) which lack interaction with the Gal substituent is minimised.

X-] , X2, X4 and X5 are independently selected from any amino acid (natural and non-natural) provided that at least one of X^ X2, X4 and X5 is a D-amino acid and/or (i) a non-standard amino acid or (ii) a non-natural amino acid. Non-standard and non-natural amino acids add

to the sequence diversity and help promote an immune response in a host organism when generating binding molecules which are antibodies with high affinity for the de-sialylated derivative of the peptide. D-amino acids help to reduce susceptibility to proteases. In particular embodiments, at least two, three or all four of
X2, X4 and X5 are a D-amino acid and/or a non-standard amino acid or a non-natural amino acid.

The skilled person will be very familiar with the terms“natural” and“non-natural” amino acids. For the avoidance of doubt, a“natural” amino acid is one found in nature and includes both standard and non-standard amino acids. As known in the art, standard amino acids are those that are encoded directly by triplet codons in the universal genetic code. Conversely, non-standard amino acids are those which are found in nature but which are not encoded directly by triplet codons in the universal genetic code.“Non-natural” amino acids are those not found in nature but which only exist via artificial synthesis. As used herein, reference to an amino acid by name with no“D” or“L” prefix is used to mean both D and L stereoisomers. Use of a“D” or“L” prefix denotes that specific stereoisomer.

The“de-sialylated derivative” of a peptide or indicator molecule is the product produced by cleavage of a sialyl group from the peptide or indicator molecule, typically by a sialidase enzyme. Thus, the“de-sialylated derivative” of a peptide or indicator molecule typically differs from the sialylated peptide or indicator molecule only, or substantially only, with regard to the absence of the sialyl group. Any reference herein to a“de-sialylated derivative” of a peptide or indicator molecule should therefore be understood to mean the de-sialylated form of that peptide or indicator molecule.

Further increasing the diversity of the peptide sequence topology is beneficial as this has been shown to further promote the generation of binding molecules with very high affinity and specificity for the de-sialylated derivative of each peptide. This is particularly relevant for binding molecules which are antibodies. Thus, in preferred embodiments, X^ X2, X4 and X5 comprise at least two, three or four different amino acids. In some embodiments, at least one of Xi , X2, X4 and X5 is a hydrophobic amino acid. For instance, at least one of X^ X2,

X4 and X5 may be selected from alanine (Ala), valine (Val), isoleucine (lie), leucine (Leu), methionine (Met), phenylalanine (Phe), tyrosine (Tyr), tryptophan (Trp), proline (Pro), D-alanine (DAla), 1-aminocyclohexane-carboxylic acid (Cyc), b-alanine (bAIq), norleucine (Nle), norvaline (Nva), 2’-(aminomethyl)biphenyl-2-carboxylic acid (Bip) and

cyclohexylalanine (Cha). Preferably, the L-stereoisomer of Nle, Nva and Cha is employed.

In further embodiments, at least two or three X2, X4 and X5 are a hydrophobic amino acid. All four of X^ X2, X4 and X5 may be a hydrophobic amino acid provided at least one of Xi , X2, X4 and X5 is a D-amino acid and/or a non-standard amino acid or a non-natural amino acid. In particular embodiments, the at least one hydrophobic amino acid is Ala. In further embodiments, X! and X2 are both Ala. In preferred embodiments, Ala is DAla or bAIq. In further embodiments, each hydrophobic amino acid is selected from DAla, bAIq, lie, Val, Pro, Nle, Nva, Cyc, Cha and Bip. Additionally or alternatively, in some embodiments at least one of X^ X2, X4 and X5 is a charged amino acid. For instance, at least one of X^ X2, X4 and X5 may be selected from arginine (Arg), histidine (His), lysine (Lys), aspartic acid (Asp), glutamic acid (Glu), ornithine (Orn) and phosphoserine (SEP). Preferably, the L-stereoisomer of Orn and the D-stereoisomer of Asp is employed. In further embodiments, at least two or three of X^ X2, X4 and X5 are a charged amino acid. All four of X^ X2, X4 and X5 may be a charged amino acid provided at least one of X^ X2, X4 and X5 is a D-amino acid and/or a non-standard amino acid or a non-natural amino acid. In particular embodiments, each charged amino acid is selected from Arg, Glu, DAsp, LOrn and SEP. Additionally or alternatively, in some embodiments at least one of X^ X2, X and X5 is a polar amino acid. For instance, at least one of X^ X2, X4 and X5 may be selected from serine (Ser), threonine (Thr), tyrosine (Tyr), asparagine (Asn), glutamine (Gin) and cysteine (Cys). Preferably, the D-stereoisomer of Ser is employed. In further embodiments, at least two or three of X! , X2, X4 and X5 are a polar amino acid. All four of X! , X2, X and X5 may be a polar amino acid provided at least one of X^ X2, X4 and X5 is a D-amino acid and/or a non-standard amino acid or a non-natural amino acid. In particular embodiments, each polar amino acid is selected from LSer, DSer and Thr. In preferred embodiments, to maximise the diversity of the peptide sequence topology, X^ X2, X4 and X5 are a

combination of hydrophobic, charged and polar amino acids. Pro and bAIq in particular are useful in the peptides of the invention as these function as“hinge groups” allowing additional degrees of freedom in the overall structural fold thereby further increasing the diversity of the peptide sequence topology.

In preferred embodiments, the peptide has a molecular weight of less than 5000 daltons. This facilitates stimulation of an immune response in the host organism for the generation of antibodies which bind to the de-sialylated derivative of the peptide. Thus, in particular embodiments, the peptide may be between 5-20 amino acids in length, more preferably 9-20 amino acids in length.

Thus, in a related aspect, the invention provides a peptide comprising a sequence according to the following formula:

Xi -X2-X3-X4-X5-X6-X7-X8-X9-XI O-XI 1 -Xi 2[Gal-Sial]-Xi 3-Xi 4-X15-X1 e-Xi rXi b-Ci 9-X20

(Formula II) (SEQ ID NO: 10)

wherein:

Sial is a sialyl group

Xi is absent or Thr

X2 is absent or DAla

X3 is absent or Nle

X4 is absent or Glu

X5 is absent or DAla

X6 is absent or Arg

X7 is absent or selected from Glu, Arg, Ser, Nva, BAIa

X8 is absent or selected from DSer, DAla, SEP, Cyc

X9 is absent or selected from Nva, BIP, DAla, BAIa, Orn

X10 is selected from Cyc, Ser, lie, DAla, DSer

Xu is selected from DAla, Pro, Orn, Nle

X12 is selected from Ser, Thr, Tyr, Hyl, Hyp, Asn, Arg or SEP

X13 is selected from DAla, BIP, BAIa

X14 is selected from Arg, DAsp, Nle, Orn, Nva

X15 is absent or selected from Phe, BIP, Ser, Glu, DAla, DSer

X16 is absent or selected from DSer, Glu

X17 is absent or selected from Val, Ser, Thr

X18 is absent or Cha

X19 is absent or DSer

X20 is absent or Val.

It should be understood from Formula II that the -Gal-Sial group is bonded to X12 only (as indicated by the square brackets in Formula II). It does not bridge X12 to X13. For the avoidance of doubt, an alternative and equivalent formula (I la) is shown below:

Gal

Sial

(Formula I la)

Hence, the -Gal-Sial group is branched from the peptide backbone. Thus, the -Gal substituent is positioned so that it is flanked by amino acids X10-11 and X13.14 as well as amino acids X^ and X15-20 (if present). Consequently, as described further herein, binding molecules (e.g. antibodies) can be generated with very high affinity and specificity for the de-sialylated derivative of each peptide and the generation of peripheral binding molecules (e.g. antibodies) which lack interaction with the Gal substituent is minimised.

In preferred embodiments, X12 is Ser.

In particular embodiments, according to all aspects of the invention, the peptide comprises, consists essentially of or consists of the following sequence:

(i) Cyc-DAla-Ser[Gal-Sial]-DAla-Arg (SEQ ID NO: 11)

(ii) Glu-DSer-Nva-Cyc-DAIa-Ser[Gal-Sial]- DAIa-Arg-Phe-DSer-Val (SEQ ID NO: 12)

(iii) Arg-DAla-Bip-Ser-Pro-Ser[Gal-Sial]-DAIa-DAsp-Ser (SEQ ID NO: 13)

(iv) Ser-Ser(P03)-DAIa-lle-Orn-Ser[Gal-Sial]-DAla-Nle-Glu (SEQ ID NO: 14)

(v) DAla-Arg-Nva-DSer-BAIa-DAla-Nle-Ser[Gal-Sial]-Bip-Orn-DAla-Glu-Ser (SEQ ID NO: 15); or

(vi) Thr-DAla-Nle-Glu-DAla-Arg-BAIa-Cyc-Orn-DSer-Pro-Ser[Gal-Sial]-BAIa-Nva-DSer-Glu-Thr-Cha-DSer-Val (SEQ ID NO: 16);

of which Cyc-DAla-Ser[Gal-Sial]-DAla-Arg is particularly preferred.

These specific peptides are been found by the inventors to be particularly useful for detecting sialidase activity and are described further in the Examples section.

Preferably, Nle, Nva, Orn and/or Cha (when present) are LNle, LNva, LOrn and LCha respectively.

In some embodiments, the peptide is biased for cleavage by one or more specific sialidases by constructing the peptide accordingly so that the amino acids flanking the -Gal Sial group ensure specificity and sensitivity of cleavage. Thus, specific sialidase enzymes may have differing affinities for the peptide. This permits the invention to be utilised in order to detect specific sialidase activity in the test sample. As described herein, the peptides of the invention are particularly useful for detecting bacterial vaginosis. Thus, in preferred embodiments, the one or more specific sialidases are of bacterial origin. In particular, the one or more specific sialidases may be from Prevotella, Bacteroides and/or Mobiluncus species and/or Gardnerella vaginalis.

In a related aspect, the peptides of the invention are incorporated into an indicator molecule for use in detecting the presence in a test sample of cleavage activity of a sialidase enzyme. The indicator molecule is a core component of the enzyme detection devices, enzyme detection kits, enzyme detection compositions of matter and methods for detecting the presence in a test sample of cleavage activity of a sialidase enzyme described further herein. The indicator molecules described herein have been specifically developed by the inventors in the context of detecting sialidase activity for diagnosing BV based on the inventors’ earlier disclosure of the general concept of an indicator molecule for use in detecting cleavage activity of an enzyme and binding molecules which

specifically bind to the cleaved product (see PCT/GB2014/053171 which is incorporated herein by reference). Thus, the invention provides an indicator molecule for use in detecting the presence in a test sample of cleavage activity of a sialidase enzyme, the indicator molecule comprising:

a) a peptide of the invention as described herein; and

b) a capture site which remains intact following cleavage of the sialyl group from the indicator molecule by a sialidase enzyme present in the sample.

The capture site is a discrete region of the indicator molecule which mediates binding of the indicator molecule to a capture molecule present within a capture zone (as described in greater detail below). Thus, the capture site is the portion of the indicator molecule responsible for retaining or localising the indicator molecule within the capture zone.

Following cleavage of the indicator molecule, the capture site may remain intact or substantially intact, such that the site is still recognised and bound by a capture molecule present within the capture zone of the device. Under these circumstances, both intact indicator molecules and the part of the indicator molecules comprising the capture site

following cleavage will be bound to capture molecules within the capture zone. The capture site may comprise any suitable molecule, for example a biotin molecule or an oxime moiety. The capture site may be at the N- or C-terminus of the peptide. Key to

effectiveness of the indicator molecule is immobilization via the interaction between capture site and a capture molecule at the capture zone and simultaneous binding by a binding molecule after cleavage has occurred.

Thus, the intact indicator molecule may comprise a capture site moiety linked to a peptide of the invention, which peptide comprises a first sialyl group. Cleavage of the indicator molecule by a sialidase enzyme yields a fragment comprising the capture site moiety and the de-sialylated form of the peptide. The fragment (i.e. the cleaved indicator molecule) differs structurally from the intact indicator molecule in that it lacks the first sialyl group that is present in the intact indicator molecule. This lack of the first sialyl group reveals or exposes on the fragment an epitope (a novel binding site) that is absent, hidden or inaccessible in the intact indicator molecule. The intact indicator molecule may therefore be considered to comprise a cryptic epitope. As discussed below, the binding molecules of the devices, kits, compositions of matter and methods of the invention are specific for this epitope and therefore bind only, or preferentially, to the indicator fragment compared to the intact indicator.

The peptide of the indicator molecule and the capture site may be associated by any means known to one of skill in the art. In a preferred embodiment, the peptide and capture site may be associated via a direct covalent linkage. The peptide and capture site may be immediately adjacent or may be separated by a linker or spacer, for example, a

polyethylene glycol (PEG) moiety. In some embodiments, the peptide is linked to a biotin group at its N- or C- terminus via a linker comprising, consisting essentially of or consisting of a polyethylene glycol moiety. One or more additional amino acids may be present at the N- or C-terminus of the peptide to link the linker group (such as PEG) to the peptide. The one or more additional amino acids may comprise Asp in some embodiments. The PEG moiety may further comprise a functional group (e.g. an amine group, optionally an alkylamine group) at one or both ends of the PEG moiety that can react with the terminal residue of the peptide and/or the capture site (e.g. a biotin molecule) during synthesis to link the peptide and the capture site to one another. Thus, in particular embodiments, the indicator molecule comprises, consists essentially of or consists of the following structure:

(i) Cyc-DAla-Ser[Gal-Sial]-DAla-Arg-PEG-Biotin (SEQ ID NO: 11-PEG-Biotin)

(ii) Biotin-PEG-Asp-Glu-DSer-Nva-Cyc-DAla-Ser[Gal-Sial]- DAla-Arg-Phe-DSer-Val (Biotin-PEG-Asp-SEQ ID NO: 12)

(iii) Biotin-PEG-Asp-Arg-DAla-BIP-Ser-Pro-Ser[Gal-Sial]-DAla-DAsp-Ser (Biotin-PEG-Asp-SEQ ID NO: 13)

(iv) Biotin-PEG-Asp-Ser-SEP-DAla-lle-Orn-Ser[Gal-Sial]-DAla-Nle-Glu (Biotin-PEG-Asp-SEQ ID NO: 14)

(v) Biotin-PEG-Asp-DAla-Arg-Nva-DSer-BAIa-DAla-Nle-Ser[Gal-Sial]-BIP-Orn-DAla-Glu-Ser (Biotin-PEG-Asp-SEQ ID NO: 15); or

(vi) Biotin-PEG-Asp-Thr-DAla-Nle-Glu-DAla-Arg-BAIa-Cyc-Orn-DSer-Pro-Ser[Gal-Sial]-BAIa-Nva-DSer-Glu-Thr-Cha-DSer-Val (Biotin-PEG-Asp-SEQ ID NO: 16);

of which Cyc-DAla-Ser[Gal-Sial]-DAla-Arg-PEG-Biotin (SEQ ID NO: 11-PEG-Biotin) is particularly preferred.

Within the context of the present invention the indicator molecules (via the capture site) may bind to the capture molecules in a capture zone (as described in greater detail below) with relatively high affinity. In some embodiments, the dissociation constant (kd) for the indicator molecule will be relatively low and preferably between 0M and 1 x 107M

(depending on the sensitivity required of the assay). In certain embodiments of the invention, the dissociation constant for the indicator molecule will be between 1 x 10 15M and 1 x 109M.

In certain embodiments of the invention, such a binding interaction may be achieved as a result of direct binding of the capture site of the indicator molecule to the capture molecule present in the capture zone. In this context, direct binding means binding of the indicator molecule (via the capture site) to the capture molecule without any intermediary.

In preferred embodiments of the invention, the capture site of the indicator molecule and the capture molecule present in the capture zone are two halves of a binding pair. In this context, a binding pair consists of two molecules or entities capable of binding to each other. In certain embodiments of the invention, the binding interaction is specific such that each member of the binding pair is only able to bind its respective partner, or a limited number of binding partners. Moreover, as detailed above, it is preferable for the binding pair to exhibit relatively high affinity. The binding pair may be a binding pair found in nature or an artificially generated pair of interacting molecules or entities.

In some embodiments of the invention, the capture site of the indicator molecule and the capture molecule are two halves of a binding pair wherein the binding pair is selected from the following:- an antigen and an antibody or antigen binding fragment thereof; biotin and avidin, streptavidin, neutravidin or captavidin; an immunoglobulin (or appropriate domain thereof) and protein A or G; a carbohydrate and a lectin; complementary nucleotide sequences; a ligand and a receptor molecule; a hormone and hormone binding protein; an enzyme cofactor and an enzyme; an enzyme inhibitor and an enzyme; a cellulose binding domain and cellulose fibres; immobilised aminophenyl boronic acid and cis-diol bearing molecules; and xyloglucan and cellulose fibres and analogues, derivatives and fragments thereof.

In particular embodiments of the invention, the binding pair consists of biotin and streptavidin. In a further embodiment of the invention, the capture site of the indicator molecule comprises an epitope and the capture molecule comprises an antibody, which specifically binds to the epitope present at the first capture site. In the context of the present invention, the term antibody covers native immunoglobulins from any species, chimeric antibodies, humanised antibodies, F(ab')2 fragments, Fab fragments, Fv fragments, sFv fragments and highly related molecules such as those based upon antibody domains which retain specific binding affinity (for example, single domain antibodies). The antibodies may be monoclonal or polyclonal. Thus, in specific embodiments, the capture molecule comprises an antibody. In other embodiments, the capture site comprises a biotin molecule and the capture zone comprises a streptavidin molecule.

Another core component of the enzyme detection devices, enzyme detection kits, enzyme detection compositions of matter and methods for detecting the presence in a test sample of cleavage activity of a sialidase enzyme described further herein is the binding molecule. The binding molecule is designed to specifically bind to the de-sialylated derivative of the indicator molecule formed following cleavage of the sialyl group from the indicator molecule by sialidase activity present in the sample. Formation of the de-sialylated derivative reveals a novel binding site to which the binding molecule can specifically bind. Thus, in preferred embodiments, the binding molecule cannot bind to the indicator molecule (at any appreciable or detectable level) unless and until cleavage of the sialyl group from the indicator molecule has occurred. In alternative embodiments, the binding molecule preferentially binds to the de-sialylated derivative over the sialylated peptide or indicator molecule. Thus, the binding molecule has a higher affinity for the de-sialylated derivative when compared with the sialylated form.

Alternatively viewed, in preferred embodiments, the binding molecule is capable of specifically binding to the de-sialylated indicator molecule (fragment) and is incapable of binding to the intact (sialylated) indicator molecule (at any appreciable or detectable level).

The skilled person is well able to determine whether or not a particular binding molecule binds to the de-sialylated derivative in preference over the sialylated form. This can, for instance, be expressed in terms of the relative dissociation constants. For example, in particular embodiments, the dissociation constant for the binding interaction between the binding molecule and the de-sialylated derivative may be 2-, 3-, 4-, 5-, 10-, 20-, 30-, 40-,

50-, 100-, 200-, 500-, 1000-fold (or more) lower than the dissociation constant for the binding interaction between the binding molecule and the sialylated form.

In specific embodiments, the binding molecule comprises an antibody. For the avoidance of doubt, the term antibody covers native immunoglobulins from any species, chimeric antibodies, humanised antibodies, F(ab')2 fragments, Fab fragments, Fv fragments, sFv fragments and highly related molecules such as those based upon antibody domains which retain specific binding affinity (for example, single domain antibodies). The antibodies may be monoclonal or polyclonal. The inventors have produced antibodies which only recognise the de-sialylated derivative of the indicator molecule and will therefore not bind to the indicator molecule (to any significant degree) unless and until cleavage of the sialyl group has occurred. Antibodies may be produced according to techniques known in the art. This may rely upon immunisation of an animal, such as a sheep, rabbit or goat, with the de-sialylated derivatives of the indicator molecules. Alternatively, the animal may be immunised with the de-sialylated peptides (i.e. without the capture site being attached). Polyclonal antibodies may be isolated from serum and affinity purified. Monoclonal antibodies may be produced using well-known and characterised hybridoma technology.

Thus, in a related aspect, the invention also provides an antibody capable of specifically binding to the de-sialylated derivative of a peptide of the invention as defined herein or an indicator molecule of the invention as defined herein. As the skilled person will appreciate based on the disclosure herein, the antibody may binds preferentially to the de-sialylated derivative over the sialylated peptide or indicator molecule. Thus, the antibody has a higher affinity for the de-sialylated derivative when compared with the sialylated form. The skilled person is well able to determine whether or not a particular antibody binds to the de-sialylated derivative in preference over the sialylated form. This can, for instance, be expressed in terms of the relative dissociation constants. For example, in particular embodiments, the dissociation constant for the binding interaction between the antibody and the de-sialylated derivative may be 2-, 3-, 4-, 5-, 10-, 20-, 30-, 40-, 50-, 100-, 200-,

500-, 1000-fold (or more) lower than the dissociation constant for the binding interaction between the antibody and the sialylated form. In particular embodiments, the antibody is incapable of binding to the sialylated peptide or indicator molecule. That is to say, it can only bind to the de-sialylated derivative after cleavage of the sialyl group has occurred.

Therefore, in a preferred embodiment, the antibody can specifically bind to the epitope: Cyc-DAla-Ser[Gal]-DAla-Arg-PEG-Biotin (de-sialylated form of SEQ ID NO: 1 1-PEG-Biotin); particularly to an epitope present in this molecule, more particularly an epitope present in the Gal peptide moiety of this molecule, Cyc-DAla-Ser[Gal]-DAla-Arg (de-sialylated form of SEQ ID NO: 11). Preferably, the antibody cannot bind (at any appreciable or detectable level) to the sialylated form of this molecule (Cyc-DAla-Ser[Gal-Sial]-DAla-Arg, i.e. SEQ ID NO: 1 1).

In another embodiment, an antibody is provided that can specifically bind to the epitope: Biotin-PEG-Asp-Glu-DSer-Nva-Cyc-DAla-Ser[Gal]-DAIa-Arg-Phe-DSer-Val (de-sialylated form of Biotin-PEG-Asp-SEQ ID NO: 12); particularly to an epitope present in this molecule, more particularly an epitope present in the Gal peptide moiety of this molecule. Preferably, the antibody cannot bind (at any appreciable or detectable level) to the sialylated form of this molecule.

In a further embodiment, an antibody is provided that can specifically bind to the epitope: Biotin-PEG-Asp-Arg-DAla-BIP-Ser-Pro-Ser[Gal]-DAla-DAsp-Ser (de-sialylated form of Biotin-PEG-Asp-SEQ ID NO: 13); particularly to an epitope present in this molecule, more particularly an epitope present in the Gal peptide moiety of this molecule. Preferably, the antibody cannot bind (at any appreciable or detectable level) to the sialylated form of this molecule.

In a further embodiment, an antibody is provided that can specifically bind to the epitope:

Biotin-PEG-Asp-Ser-SEP-DAIa-lle-Orn-Ser[Gal]-DAla-Nle-Glu (de-sialylated form of Biotin-PEG-Asp-SEQ ID NO: 14); particularly to an epitope present in this molecule, more particularly an epitope present in the Gal peptide moiety of this molecule. Preferably, the antibody cannot bind (at any appreciable or detectable level) to the sialylated form of this molecule.

In a further embodiment, an antibody is provided that can specifically bind to the epitope: Biotin-PEG-Asp-DAla-Arg-Nva-DSer-BAIa-DAla-Nle-Ser[Gal]-BIP-Orn-DAla-Glu-Ser (de-sialylated form of Biotin-PEG-Asp-SEQ ID NO: 15); particularly to an epitope present in this molecule, more particularly an epitope present in the Gal peptide moiety of this molecule. Preferably, the antibody cannot bind (at any appreciable or detectable level) to the sialylated form of this molecule.

In a further embodiment, an antibody is provided that can specifically bind to the epitope: Biotin-PEG-Asp-Thr-DAla-Nle-Glu-DAIa-Arg-BAIa-Cyc-Orn-DSer-Pro-Ser[Gal]-BAIa-Nva-□Ser-Glu-Thr-Cha-oSer-Val (de-sialylated form of Biotin-PEG-Asp-SEQ ID NO: 16);

particularly to an epitope present in this molecule, more particularly an epitope present in the Gal peptide moiety of this molecule. Preferably, the antibody cannot bind (at any appreciable or detectable level) to the sialylated form of this molecule.

In a further embodiment, an antibody is provided that has a heavy chain with 3 CDRs and a light chain with 3 CDRs, wherein the heavy chain CDR1 has SEQ ID NO: 1 ; the heavy chain CDR2 has SEQ ID NO: 2; the heavy chain CDR3 has SEQ ID NO: 3; the light chain CDR1 has SEQ ID NO: 4; the light chain CDR2 has SEQ ID NO: 5; and/or the light chain CDR3 has SEQ ID NO: 6.

The antibody may have a heavy chain that has SEQ ID NO: 7 and/or a light chain that has SEQ ID NO: 8.

Preferably, the antibody can specifically bind to an epitope present in the molecule Cyc-DAla-Ser[Gal]-DAla-Arg. Preferably, the antibody does not (at any appreciable or detectable level) bind to the molecule Cyc-DAla-Ser[Gal-Sial]-DAla-Arg.

For the (de-sialidated) molecule Cyc-DAla-Ser[Gal]-DAla-Arg-PEG-Oxime (de-sialylated form of SEQ ID NO: 11-PEG-Oxime) the antibody may have a KD of less than 100 nM,

preferably less than 80, 60, 50, 40, 30, 20 or 10 nM, for example about 7.9 nM. For this molecule it may have a Kon of about 54080M V1;

and a K0ff of about 0.000427 s 1.

The binding molecule may be directly or indirectly conjugated to (i.e. labelled with) a reporter molecule to permit detection of binding of the binding molecule to the indicator molecule. The reporter molecule may be any substance or moiety suitable for detection by any means available to those skilled in the art. Thus, the reporter molecule is typically capable of signal generation or production. In certain embodiments of the invention, the reporter molecule is selected from the following: - a gold particle; a chromogen; a luminescent compound; a fluorescent molecule; a radioactive compound; a visible compound; a liposome or other vesicle containing signal producing substances; an electroactive species; or a combination of enzyme and its substrate. A suitable enzyme-substrate combination for use as a reporter moiety may be the enzyme alkaline

phosphatase and the substrate nitro blue tetrazolium-5-bromo-4-chloro-3-indolyl phosphate. In a particular embodiment of the invention, the reporter molecule is a gold particle.

Indirect labelling of the binding molecule with a reporter molecule is also envisaged within the present invention. Thus, the reporter molecule may be attached to a further binding molecule which in turn binds to the binding molecule to provide the label. This indirect binding may be mediated by an adaptor capable of simultaneously binding the binding molecule and the reporter molecule. As an illustrative embodiment, where the binding molecule is an antibody, indirect labelling could be mediated by a further antibody that binds to the antibody binding molecule in specific fashion. The further antibody may be directly labelled with a reporter molecule such as a gold particle; a chromogen; a luminescent compound; a fluorescent molecule; a radioactive compound; a visible compound; a liposome or other vesicle containing signal producing substances; an electroactive species; or a combination of enzyme and its substrate. A suitable enzyme-substrate combination for use as a reporter moiety may be the enzyme alkaline

phosphatase and the substrate nitro blue tetrazolium-5-bromo-4-chloro-3-indolyl phosphate. In a particular embodiment of the invention, the reporter moiety is a gold particle.

In embodiments where the reporter is a gold particle, the gold particle-binding molecule conjugate should be used an optical density of at least 4, preferably at least 5, 6 or 7, most preferably at least or about 8, 9 or 10.

In embodiments of the invention wherein the reporter molecule binds to the binding molecule by virtue of an adaptor molecule, the adaptor may be pre-complexed with the binding molecule prior to the addition of the test sample to the indicator molecule, provided that the adaptor does not prevent binding of the binding molecule to the cleaved indicator molecule.

The adaptor may be any material or molecule capable of mediating the indirect interaction of the binding molecule with the reporter molecule. In some embodiments, the adaptor is streptavidin and the binding molecule comprises a biotin molecule. The adaptor may also be an“adaptor binding pair” wherein said binding pair comprises:

(i) a first member capable of binding to the binding molecule; and

(ii) a second member capable of binding to the first member of the pair and to the reporter molecule. In certain embodiments of the invention, the detection region of the indicator molecule comprises biotin, the first member of the adaptor binding pair is avidin or streptavidin, the second member of the adaptor binding pair is biotin, and the reporter molecule comprises a moiety capable of binding biotin.

In view of the foregoing, in a complementary aspect, the de-sialylated derivatives of the peptides of the invention also form part of the invention and are used in the generation of the binding molecules, in particular antibodies. Thus, the invention provides a peptide for use in generating an antibody as defined herein wherein the peptide is a de-sialylated derivative of a peptide according to the invention and the antibody is capable of specifically binding to the de-sialylated derivative of a peptide of the invention. Thus, in particular embodiments, the peptide is:

(i) Cyc-DAla-Ser[Gal]-DAla-Arg (de-sialylated form of SEQ ID NO: 11)

(ii) Asp-Glu-DSer-Nva-Cyc-DAla-Ser[Gal]-DAla-Arg-Phe-DSer-Val (de-sialylated form of SEQ ID NO: 12)

(iii) Asp-Arg-DAla-BIP-Ser-Pro-Ser[Gal]-DAIa-DAsp-Ser (de-sialylated form of SEQ ID NO: 13)

(iv) Asp-Ser-SEP-DAla-lle-Orn-Ser[Gal]-DAla-Nle-Glu (de-sialylated form of SEQ ID NO: 14)

(v) Asp-DAIa-Arg-Nva-DSer-BAIa-DAIa-Nle-Ser[Gal]-BIP-Orn-DAla-Glu-Ser (de-sialylated form of SEQ ID NO: 15); or

(vi) Asp-Thr-DAla-Nle-Glu-DAIa-Arg-BAIa-Cyc-Orn-DSer-Pro-Ser[Gal]-BAIa-Nva-DSer-Glu-Thr-Cha-DSer-Val (de-sialylated form of SEQ ID NO: 16).

A peptide comprising, consisting essentially of, or consisting of the sequence Cyc-DAla-Ser[Gal]-DAla-Arg is particularly preferred.

In particular embodiments, the peptide may be conjugated to a carrier protein in order to improve immunogenicity and thus antibody production in the host organism. For instance, the peptide may be conjugated to keyhole limpet hemocyanin. The carrier protein may be at the N- or C-terminus of the peptide.

In view of the foregoing, the invention further provides enzyme detection devices, enzyme detection kits, enzyme detection compositions of matter and methods for detecting the presence in a test sample of cleavage activity of a sialidase enzyme incorporating an indicator molecule, capture molecules and binding molecules as defined above. The use of binding molecules, such as antibodies, that bind only to the de-sialylated derivative of the indicator molecule but not to the uncleaved indicator molecule, enable detection of sialidase activity at low concentrations in test samples.

Thus, in a further aspect, the invention provides an enzyme detection device, enzyme detection kit or enzyme detection composition of matter for detecting the presence in a test sample of cleavage activity of a sialidase enzyme, the device comprising:

(i) an indicator molecule as defined herein;

(ii) a capture zone to receive the test sample, wherein the capture zone comprises capture molecules as defined herein capable of binding to the capture site of the indicator molecule, irrespective of whether or not the indicator molecule has been cleaved, in order to immobilise the indicator molecule; and

(iii) binding molecules as defined herein capable of binding to the de-sialylated derivative of the indicator molecule, wherein the binding molecules are incapable of binding to the indicator molecule unless and until cleavage of the sialyl group from the indicator molecule by sialidase enzyme present in the sample has occurred.

The invention further provides a method for detecting the presence or absence in a test sample of cleavage activity of a sialidase enzyme, the method comprising:

(i) bringing an indicator molecule as defined herein into contact with the test sample;

(ii) adding to the test sample binding molecules as defined herein capable of binding to the de-sialylated derivative of the indicator molecule, wherein the binding molecules are incapable of binding to the indicator molecule unless and until cleavage of the sialyl group from the indicator molecule by sialidase enzyme present in the sample has occurred;

(iii) capturing the de-sialylated derivative of the indicator molecule at a capture zone through binding of capture molecules in the capture zone to the capture site, said capture molecules being able to bind to the capture site irrespective of whether or not the indicator molecule has been cleaved; and

(iv) detecting cleavage of the sialyl group from the indicator molecule by determining binding of the binding molecules to the de-sialylated derivative of the indicator molecule captured in the capture zone.

The devices, kits, compositions of matter and methods of the invention have been shown by the inventors to have specific application in the field of diagnosis of BV. Thus, the invention further provides a method for diagnosing bacterial vaginosis in a test sample by detecting cleavage activity of a sialidase enzyme in the sample, the method comprising:

(i) bringing an indicator molecule as defined herein into contact with the test sample;

(ii) adding to the test sample binding molecules as defined herein capable of binding to the de-sialylated derivative of the indicator molecule, wherein the binding molecules are incapable of binding to the indicator molecule unless and until cleavage of the sialyl group from the indicator molecule by sialidase enzyme present in the sample has occurred;

(iii) capturing the de-sialylated derivative of the indicator molecule at a capture zone through binding of capture molecules as defined herein in the capture zone to the capture site, said capture molecules being able to bind to the capture site irrespective of whether or not the indicator molecule has been cleaved; and

(iv) detecting cleavage of the sialyl group from the indicator molecule by determining binding of the binding molecules to the de-sialylated derivative of the indicator molecule captured in the capture zone wherein an increased level of cleavage compared to a control diagnoses bacterial vaginosis.

In particular embodiments, the indicator molecule may be (pre-)immobilised in the capture zone via the capture molecules (i.e. prior to contact with the test sample). Thus, the

invention also provides a method for detecting the presence or absence in a test sample of cleavage activity of a sialidase enzyme, the method comprising:

(i) adding the test sample to a capture zone comprising capture molecules, said capture molecules as defined herein being bound to the capture site of an indicator molecule as defined herein wherein said capture molecules remain bound to the capture site

irrespective of whether or not cleavage of the sialyl group from the indicator molecule by sialidase enzyme present in the sample occurs;

(ii) adding binding molecules as defined herein capable of binding to the de-sialylated derivative of the indicator molecule, wherein the binding molecules are incapable of binding to the indicator molecule unless and until cleavage of the sialyl group from the indicator molecule by sialidase enzyme present in the sample has occurred;

(iii) detecting cleavage of the sialyl group from the indicator molecule by determining binding of the binding molecules to the de-sialylated derivative of the indicator molecule captured in the capture zone.

Similarly, the invention also provides a method for diagnosing bacterial vaginosis in a test sample by detecting cleavage activity of a sialidase enzyme in the sample, the method comprising:

(i) adding the test sample to a capture zone comprising capture molecules as defined herein, said capture molecules being bound to the capture site of an indicator molecule as defined herein wherein said capture molecules remain bound to the capture site

irrespective of whether or not cleavage of the sialyl group from the indicator molecule by sialidase enzyme present in the sample occurs;

(ii) adding binding molecules as defined herein capable of binding to the de-sialylated derivative of the indicator molecule, wherein the binding molecules are incapable of binding to the indicator molecule unless and until cleavage of the sialyl group from the indicator molecule by sialidase enzyme present in the sample has occurred;

(iii) detecting cleavage of the sialyl group from the indicator molecule by determining binding of the binding molecules to the de-sialylated derivative of the indicator molecule captured in the capture zone wherein an increased level of cleavage compared to a control diagnoses bacterial vaginosis.

Consequently, for those embodiments in which the indicator molecule is (pre-)immobilised in the capture zone via the capture molecules (i.e. prior to contact with the test sample), prior to step (i) of the method, the indicator molecule may be added to the capture zone such that the indicator molecule is bound in the capture zone via the capture molecules. Addition of the binding molecules may occur simultaneously with or after the test sample has been added to the capture zone. Thus, steps (i) and (ii) of the method may occur sequentially or simultaneously.

In the context of the methods provided herein, the step of“adding” binding molecules to the test sample should be understood to encompass any step that brings the binding molecules into contact with the test sample. Thus, this step may encompass a step of applying the test sample to a device comprising the binding molecules. The binding molecules may be in solution, or may be on a carrier. For example, the binding molecules may be dried onto or otherwise impregnated into or onto a solid support, which may be the “conjugate pad” discussed elsewhere herein. In preferred embodiments the test sample is brought into contact with a solid support onto which the binding molecules have been dried. Liquid comprised in the test sample and/or added to the carrier allows the binding molecules to resolvate.

In embodiments where the binding molecules are dried onto or otherwise impregnated into or onto a solid support, the solid support preferably comprises or consists of fibreglass, polyester fibres, or a material having similar properties. The solid support should preferably have one or more of the following properties: basis weight around 75g/m2; caliper about 0.38 - 0.43 mm; wicking rate about 3-5 (s/2 cm); and/or water absorption about 63-79 mg/cm2. Thus, the conjugate pad may have these properties. Similarly, the sample pad may have these properties.

Whilst in preferred embodiments the binding molecules are incapable of binding to the indicator molecule unless and until cleavage of the sialyl group from the indicator molecule by sialidase enzyme present in the sample has occurred, in some embodiments, the binding molecules preferentially bind to the de-sialylated derivative of the indicator molecule over the sialylated form as described elsewhere herein. Thus, some degree of binding to the sialylated indicator molecule may occur (this may be considered background signal in some cases). However, because the binding molecules will preferentially bind to the de-sialylated derivative a much greater signal is generated in samples comprising the de-sialylated derivative relative to samples in which the sialylated indicator molecule has not been cleaved or no indicator molecule is present.

In order to take into account background levels of sialidase activity (if present), the methods typically involve comparing measured levels of cleavage in the test sample to a control. Typically, the control represents corresponding levels of sialidase activity in a healthy subject. By“healthy subject” is meant a subject not suffering from BV. The control may be in a corresponding test sample taken from a matched healthy control. Alternatively, the control may be a threshold level of sialidase activity set by determining sialidase activity in a range of healthy and diseased patients. Suitable methods for setting a threshold are well known to those skilled in the art. The threshold may be mathematically derived from a training set of patient data. The score threshold thus separates the test samples according to presence or absence of BV. The interpretation of this quantity, i.e. the cut-off threshold may be derived in a development or training phase from a set of patients with known outcome. The threshold may therefore be fixed prior to performance of the claimed methods from training data by methods known to those skilled in the art.

For example, a cube reader may be used, for example the Optricon reader from OpTricon GmbH (Chembio Diagnostic systems) of Schwarzschildstrasse 1 , D-12489 Berlin,

Germany), as demonstrated in the Examples. By way of example, in the assays used in the examples a cube reading of less than 10 correlates with the absence of a visual signal and is considered to be a negative result and therefore indicative of the absence of bacterial vaginosis; and a cube reading of at least 30 units correlates with a strong visual signal and is considered to be a positive result and therefore indicative of the presence of bacterial vaginosis.

A cube reading of 10-20 units correlates with a faint visual signal and is considered to be a result that may be indicative of the absence of established bacterial vaginosis, but may be indicative of a low level of infection, for example the early-stage of bacterial vaginosis.

In particular embodiments, the sialidase enzyme to be detected originates from Prevotella, Bacteroides and/or Mobiluncus species and/or Gardnerella vaginalis.

The enzyme detection devices and compositions of matter of the invention may be supplied in a format ready for immediate use. Alternatively, the essential components may be provided as a kit of parts, optionally together with suitable reagents and/or instructions for assembly of the enzyme detection device. Accordingly, in another aspect, the invention

provides an enzyme detection kit for detecting the presence in a test sample of cleavage activity of a sialidase enzyme, the kit comprising:

(i) an indicator molecule as defined herein for adding to the test sample;

(ii) capture molecules as defined herein capable of binding to the capture site of the indicator molecule, irrespective of whether or not the indicator molecule has been cleaved;

(iii) a solid support to which the capture molecules can be attached to form a capture zone to receive the test sample; and

(iv) binding molecules as defined herein capable of binding to the de-sialylated derivative of the indicator molecule, wherein the binding molecules are incapable of binding to the indicator molecule unless and until cleavage of the sialyl group from the indicator molecule by sialidase enzyme present in the sample has occurred.

CLAIMS

1. An enzyme detection device, enzyme detection kit or enzyme detection composition of matter for detecting the presence in a test sample of cleavage activity of a sialidase enzyme, the device, kit or composition of matter comprising:

(i) an indicator molecule comprising

(a) a peptide comprising the following sequence:

Xi-X2-X3[Gal-Sial]-X4-X5 (SEQ ID NO: 17)

wherein:

Sial is a sialyl group;

X3 is an amino acid comprising a glycosyl acceptor group and is selected from Ser, Thr, Tyr, Hyl, Hyp, Asn, Arg or phosphoserine (SEP), preferably wherein X3 is Ser; and

Xi, X2, X4 and X5 are independently selected from any amino acid provided that at least one, preferably at least two or three, of X^ X2, X4 and X5 is a D- amino acid and/or a non-standard amino acid or a non-natural amino acid; and

b) a capture site which remains intact following cleavage of the sialyl group from the indicator molecule by a sialidase enzyme present in the sample;

(ii) a capture zone to receive the test sample, wherein the capture zone comprises capture molecules capable of binding to the capture site of the indicator molecule, irrespective of whether or not the indicator molecule has been cleaved, in order to immobilise the indicator molecule; and

(iii) binding molecules capable of binding to the de-sialylated derivative of the indicator molecule, wherein the binding molecules are incapable of binding to the indicator molecule unless and until cleavage of the sialyl group from the indicator molecule by sialidase enzyme present in the sample has occurred.

2. The device, kit or composition of matter of claim 1 wherein at least one of X! , X2, X4 and X5 is Ala, preferably wherein X! and X2 are both Ala, and/or preferably wherein Ala is DAla or bAIq.

3. The device, kit or composition of matter of any one of claims 1-2 wherein at least one of X^ X2, X4 and X5 is a charged amino acid, optionally wherein each charged amino acid is selected from Arg, DAsp and LOrn.

4. The device, kit or composition of matter of any one of claims 1-3 wherein at least one X2, X4 and X5 is a polar amino acid, optionally wherein each polar amino acid is selected from LSer, DSer and Thr.

5. The device, kit or composition of matter of any one of claims 1-4 wherein the peptide comprises the following sequence:

X! -X2-X3-X4-X5-X8-X7-X8-X9-Xi o-Xi 1 -Xi 2[Gal-Sial]-X13-Xi 4-Xi 5-Xi 6-Xi rXi 8-Xi 9-X20 (SEQ ID NO: 10) wherein:

Sial is a sialyl group

X1 is absent or Thr

X2 is absent or DAla

X3 is absent or Nle

X4 is absent or Glu

X5 is absent or DAla

X6 is absent or Arg

X7 is absent or selected from Glu, Arg, Ser, Nva, BAIa

X8 is absent or selected from DSer, DAla, SEP, Cyc

X9 is absent or selected from Nva, BIP, DAla, BAIa, Orn

X10 is selected from Cyc, Ser, lie, DAla, DSer

Xu is selected from DAla, Pro, Orn, Nle

X12 is selected from Ser, Thr, Tyr, Hyl, Hyp, Asn, Arg or SEP, preferably Ser

X13 is selected from DAla, BIP, BAIa

X14 is selected from Arg, DAsp, Nle, Orn, Nva

X15 is absent or selected from Phe, BIP, Ser, Glu, DAla, DSer

X16 is absent or selected from DSer, Glu

X17 is absent or selected from Val, Ser, Thr

X18 is absent or Cha

X19 is absent or DSer

X20 is absent or Val.

6. The device, kit or composition of matter of any one of claims 1-5 wherein the peptide comprises, consists essentially of, or consists of, the following sequence:

(i) Cyc-DAla-Ser[Gal-Sial]-DAla-Arg (SEQ ID NO: 1 1)

(ii) Glu-DSer-Nva-Cyc-DAla-Ser[Gal-Sial]- DAIa-Arg-Phe-DSer-Val (SEQ ID NO:

12)

(iii) Arg-DAIa-BIP-Ser-Pro-Ser[Gal-Sial]-DAIa-DAsp-Ser (SEQ ID NO: 13)

(iv) Ser-SEP-DAla-lle-Orn-Ser[Gal-Sial]-DAla-Nle-Glu (SEQ ID NO: 14)

(v) DAIa-Arg-Nva-DSer-BAIa-DAIa-Nle-Ser[Gal-Sial]-BIP-Orn-DAla-Glu-Ser (SEQ ID NO: 15); or

(vi) Thr-DAla-Nle-Glu-DAla-Arg-BAIa-Cyc-Orn-DSer-Pro-Ser[Gal-Sial]-BAIa-Nva- DSer-Glu-Thr-Cha-DSer-Val (SEQ ID NO: 16)

7. The device, kit or composition of matter of any one of claims 1-6 wherein the peptide is biased for cleavage by one or more specific sialidases, optionally wherein the one or more specific sialidases are of bacterial origin, optionally wherein the bacteria are Prevotella, Bacteroides and/or Mobiluncus species and/or Gardnerella vaginalis.

8. The device, kit or composition of matter of any one of claims 1-7 wherein the binding molecule is specific for the de-sialylated form of the peptide defined in claim 6, preferably is specific for an epitope that is present in the peptide motif Cyc-DAla-Ser[Gal]-□Ala-Arg and that is absent or cryptic in the corresponding sialylated peptide motif Cyc-DAla-Ser[Gal-Sial]-DAla-Arg.

9. The device, kit or composition of matter of any one of claims 1-8 wherein the binding molecule is an antibody having a heavy chain with 3 CDRs and a light chain with 3 CDRs, wherein the heavy chain CDR1 has SEQ ID NO:1 ; the heavy chain CDR2 has SEQ ID NO:2; the heavy chain CDR3 has SEQ ID NO:3; the light chain CDR1 has SEQ ID NO: 4; the light chain CDR2 has SEQ ID NO: 5; and the light chain CDR3 has SEQ ID NO: 6, preferably wherein the heavy chain has SEQ ID NO: 7 and/or the light chain has SEQ ID NO: 8.

10. The device, kit or composition of matter of any one of claims 1-9 wherein the binding molecule is labelled with a reporter molecule, preferably a gold particle.

11. The device, kit or composition of matter of any one of claims 1-10 wherein the capture site of the indicator molecule comprises, consists essentially of, or consists of a biotin molecule or an oxime moiety; and/or is at the N- or C-terminus of the peptide.

12. The device, kit or composition of matter of any one of claims 1-11 wherein the capture site of the indicator molecule is attached to the peptide by a linker, optionally wherein the linker comprises a polyethylene glycol (PEG) moiety, optionally wherein the peptide is linked to a biotin group at its N- or C- terminus via a linker comprising, consisting essentially of, or consisting of, a polyethylene glycol moiety.

13. The device, kit or composition of matter of any one of claims 1-12 wherein the indicator molecule comprises, consists essentially of, or consists of, the following structure:

(i) Cyc-DAla-Ser[Gal-Sial]-DAla-Arg-PEG-Biotin

(ii) Biotin-PEG-Asp-Glu-DSer-Nva-Cyc-DAla-Ser[Gal-Sial]- DAla-Arg-Phe-DSer- Val

(iii) Biotin-PEG-Asp-Arg-DAla-BIP-Ser-Pro-Ser[Gal-Sial]-DAla-DAsp-Ser

(iv) Biotin-PEG-Asp-Ser-Ser(P03)-DAIa-lle-Orn-Ser[Gal-Sial]-DAla-Nle-Glu

(v) Biotin-PEG-Asp-DAla-Arg-Nva-DSer-BAIa-DAla-Nle-Ser[Gal-Sial]-BIP-Orn- □Ala-Glu-Ser; or

(vi) Biotin-PEG-Asp-Thr-DAla-Nle-Glu-DAIa-Arg-BAIa-Cyc-Orn-DSer-Pro-Ser[Gal- Sial]-BAIa-Nva-DSer-Glu-Thr-Cha-DSer-Val.

14. The device, kit or composition of matter of any one of claims 1-13 wherein the indicator molecule comprises (i) a peptide comprising, consisting essentially of, or consisting of the sequence Cyc-DAla-Ser[Gal-Sial]-DAla-Arg; and (ii) a capture site that comprises, consists essentially of, or consists of a biotin molecule or an oxime moiety which is optionally linked to the N- or C- terminus of the peptide via a linker comprising, consisting essentially of or consisting of a polyethylene glycol moiety; and wherein the binding molecule is an antibody as defined in claim 8 or 9 and preferably is labelled with a reporter molecule, most preferably a gold particle.

15. An antibody capable of specifically binding to (i) the de-sialylated derivative of a peptide as defined in any one of claims 1-7; or to (ii) an indicator molecule as defined in any one of claims 1-7 or 11-14, wherein the antibody binds preferentially to the de-sialylated derivative over the sialylated peptide or indicator molecule.

16. The antibody according to claim 15, wherein the antibody is as defined in claim 8, 9 and/or 10.

17. An indicator molecule suitable for use in detecting the presence in a test sample of cleavage activity of a sialidase enzyme, the indicator molecule comprising:

a) a peptide as defined in any one of claims 1-7; and

b) a capture site which remains intact following cleavage of the sialyl group from the indicator molecule by a sialidase enzyme present in the sample, wherein the indicator molecule is preferably as defined in any one of claims 1-7 or 1 1-14.

18. The indicator molecule according to claim 17, wherein the indicator molecule is as defined in claim 13.

19. A peptide comprising, consisting essentially of, or consisting of, the following sequence:

Xi-X2-X3[Gal-Sial]-X4-X5

wherein:

Sial is a sialyl group;

X3 is an amino acid comprising a glycosyl acceptor group and is selected from Ser, Thr, Tyr, Hyl, Hyp, Asn, Arg or phosphoserine (SEP), preferably wherein X3 is Ser; and

Xi , X2, X4 and X5 are independently selected from any amino acid provided that at least one, preferably at least 2 or 3, of X^ X2, X4 and X5 is a D-amino acid and/or a non-standard amino acid or a non-natural amino acid, wherein the peptide is preferably as defined in any one of claims 1-7.

20. The peptide according to claim 19, wherein the peptide is as defined in claim 6, and preferably is a peptide comprising, consisting essentially of, or consisting of the sequence Cyc-DAla-Ser[Gal-Sial]-DAla-Arg.

21. A peptide for use in generating an antibody according to claim 15 or 16 wherein the peptide is a de-sialylated derivative of the peptide as defined in any one of claims 1-7.

22. A method for detecting the presence or absence in a test sample of cleavage activity of a sialidase enzyme, the method comprising:

(i) bringing an indicator molecule as defined in any one of claims 1-7 or 11-14 into contact with the test sample;

(ii) adding to the test sample binding molecules capable of binding to the de-sialylated derivative of the indicator molecule, wherein the binding molecules are incapable of binding to the indicator molecule unless and until cleavage of the sialyl group from the indicator molecule by sialidase enzyme present in the sample has occurred and wherein the binding molecules are preferably as defined in claims 8, 9 and/or 10;

(iii) capturing the de-sialylated derivative of the indicator molecule at a capture zone through binding of capture molecules in the capture zone to the capture site, said capture molecules being able to bind to the capture site irrespective of whether or not the indicator molecule has been cleaved; and

(iv) detecting cleavage of the sialyl group from the indicator molecule by determining binding of the binding molecules to the de-sialylated derivative of the indicator molecule captured in the capture zone.

23. A method for detecting the presence or absence in a test sample of cleavage activity of a sialidase enzyme, the method comprising:

(i) adding the test sample to a capture zone comprising capture molecules, said capture molecules being bound to the capture site of an indicator molecule as defined in any one of claims 1-7 or 11-14 wherein said capture molecules remain bound to the capture site irrespective of whether or not cleavage of the sialyl group from the indicator molecule by sialidase enzyme present in the sample occurs;

(ii) adding binding molecules capable of binding to the de-sialylated derivative of the indicator molecule, wherein the binding molecules are incapable of binding to the indicator molecule unless and until cleavage of the sialyl group from the indicator molecule by sialidase enzyme present in the sample has occurred and wherein the binding molecules are preferably as defined in claims 8, 9 and/or 10;

(iii) detecting cleavage of the sialyl group from the indicator molecule by determining binding of the binding molecules to the de-sialylated derivative of the indicator molecule captured in the capture zone.

24. A method for diagnosing bacterial vaginosis in a test sample by detecting cleavage activity of a sialidase enzyme in the sample, the method comprising:

(i) bringing an indicator molecule as defined in any one of claims 1-7 or 11-14 into contact with the test sample;

(ii) adding to the test sample binding molecules capable of binding to the de-sialylated derivative of the indicator molecule, wherein the binding molecules are incapable of binding to the indicator molecule unless and until cleavage of the sialyl group from the indicator molecule by sialidase enzyme present in the sample has occurred and wherein the binding molecules are preferably as defined in claims 8, 9 and/or 10;

(iii) capturing the de-sialylated derivative of the indicator molecule at a capture zone through binding of capture molecules in the capture zone to the capture site, said capture molecules being able to bind to the capture site irrespective of whether or not the indicator molecule has been cleaved; and

(iv) detecting cleavage of the sialyl group from the indicator molecule by determining binding of the binding molecules to the de-sialylated derivative of the indicator molecule captured in the capture zone wherein an increased level of cleavage compared to a control diagnoses bacterial vaginosis.

25. An enzyme detection kit for detecting the presence in a test sample of cleavage activity of a sialidase enzyme, the kit comprising:

(i) an indicator molecule as defined in any one of claims 1-7 wherein the indicator molecule is preferably freeze-dried onto a carrier;

(ii) capture molecules capable of binding to the capture site of the indicator molecule, irrespective of whether or not the indicator molecule has been cleaved, wherein the capture molecules preferably comprise streptavidin;

(iii) a solid support to which the capture molecules can be attached, or are attached, to form a capture zone to receive the test sample, wherein the solid support preferably comprises nitrocellulose; and

(iv) binding molecules capable of binding to the de-sialylated derivative of the indicator molecule, wherein the binding molecules are incapable of binding to the indicator molecule unless and until cleavage of the sialyl group from the indicator molecule by sialidase enzyme present in the sample has occurred and wherein the binding molecules are preferably as defined in claims 8, 9 and/or 10; and optionally

(v) an extraction tube and/or a buffer; and/or

(vi) control detection molecules and control detection binders.

26. Use of an enzyme detection device, enzyme detection kit or enzyme detection composition of matter according to any one of claims 1-14, method according to any one of claims 22-24 or the enzyme detection kit according to claim 25 for diagnosing bacterial vaginosis in a test sample.