WO2024121125A1

WO2024121125A1 - Single-domain antibodies to reduce the risk of cholera infection

Info

Publication number: WO2024121125A1
Application number: PCT/EP2023/084291
Authority: WO
Inventors: Sandra Wingaard Thrane; Marcus PETERSSON; Andreas Hougaard LAUSTSEN-KIEL
Original assignee: Bactolife A/S
Priority date: 2022-12-06
Filing date: 2023-12-05
Publication date: 2024-06-13

Abstract

The present disclosure relates to single-domain antibodies that bind to subunit B of Vibrio cholerae toxin and their use for reducing the risk, treatment, and/or prevention of cholera infection. Further disclosed are nucleic acids and vectors encoding the single- domain antibodies, host cells for expression of the single-domain antibodies, methods of manufacture and compositions.

Description

Single-domain antibodies to reduce the risk of Cholera infection

Technical field

The present disclosure relates to single-domain antibodies directed against Vibrio cholerae toxin, particularly subunit B of Vibrio cholerae toxin.

Background

Cholera is caused by the Gram-negative bacterium, Vibrio cholerae, often spreading via contaminated water or foods. The bacterium can colonize the small intestine and produce enterotoxins, such as the main virulence factor cholera toxin (CT), causing severe diarrhoea. There are more than a billion people living at risk of cholera worldwide and 140,000 associated deaths each year, a third of all deaths are children under the age of five.

CT consists of two subunits, the A-subunit (CTA) and B-subunit (CTB). CTB enables toxin transfer across the outer membrane of intestinal cells in the gut through interaction with specific ganglioside receptors (GM1). Once intracellular, CTA initiates a G-protein coupled cascade that ultimately causes fluid secretion into the intestinal lumen.

Treatments available to cholera endemic regions, which are not based on antibiotics, are killed whole-cell (WC) monovalent and bivalent oral vaccines. Monovalent WC vaccines are several strains of inactivated bacteria (one serotype) that can be administered together with recombinant cholera toxin B subunit (rCTB) to achieve a higher efficacy. In comparison to monovalent WC vaccines, bivalent WC vaccines are comprised of bacteria originating from two serogroups without supplementary rCTB. Vaccination is carried out as a two-dose regimen through administration of an oral suspension dissolved in water. However, oral cholera vaccines require a minimum of seven days to lapse to raise protective immunity and have shown limited efficiency in inducing protective immunity in young children. Furthermore, administration of these oral vaccines relies on trained medical staff.

Single-domain antibodies (sdAbs) are derived from the VH domain of naturally occurring immunoglobulins; the smallest antigen binding fragment. An sdAb can be of both natural (e.g., Camelidae species) and synthetic origin but is defined by a single polypeptide chain (12-15 kDa) with strong antigen binding affinity. Their small size and robustness, being able to retain functionality even after exposure to extreme pH, temperature, and proteolysis, make sdAbs suitable for oral delivery.

Summary

In the present disclosure, the inventors provide a novel option for the reduction of the risk of cholera infection and/or prevention/treatment of cholera infection by the provision of single-domain antibodies (sdAbs) and fusion proteins targeting Vibrio cholerae toxin. The sdAbs disclosed herein display unique properties making them particularly useful for reducing the risk of, preventing, and/or treating cholera infection and its clinical manifestations.

The unique properties of the herein disclosed sdAbs make them particularly suitable for oral delivery and for use in food, feed, and beverages, e.g., as a dietary supplement which can be used as a gut health/microbiome stabilizer, potentially reducing the risk of cholera infection.

The inventors have made the surprising discovery of sdAbs displaying high binding affinity towards Vibrio cholerae toxin B subunit (CTB), as shown in the examples. Moreover, the inventors found that the sdAbs disclosed herein showed high efficacy in blocking the interaction between CTB and the GM1 ganglioside receptor.

Description of Drawings

Figure 1. High-throughput screening of a large number of single-domain antibodies for their blocking capacity of CTB interaction with the GM1 receptor. Bars represent the decreased binding of CTB to GM1 in the presence of culture supernatants containing a single-domain antibody, in relation to a reference without any single-domain antibody. The screening showed that certain single-domain antibodies were more efficacious than others in blocking GM1-CTB interaction.

Figure 2. Monomeric single-domain antibody blocking capacity of GM 1 -CTB interaction. Bars represent the decreased binding of CTB to GM1 in the presence of each single-domain antibody, in relation to a reference containing a single-domain antibody without specific for CTB. 360 nM of each single-domain antibody was used, ten times the CTB concentration, in replicates of two. For the monomeric single-domain antibody M006_0008, only one datapoint could be included. The monomeric singledomain antibody M006_0001 displayed superior blocking capacity in comparison to the other binders.

Figure 3. Dimeric single-domain antibody blocking capacity of GM 1 -CTB interaction. Bars represent the decreased binding of CTB to GM1 in the presence of each singledomain antibody, in relation to a reference without any single-domain antibody. 36 nM of each single-domain antibody was used, in replicates of six. The dimeric single-domain antibody B006_0001 displayed superior blocking capacity in comparison to the other binders.

Figure 4. Dimeric single-domain antibody blocking capacity of GM 1 -CTB interaction. Bars represent the decreased binding of CTB to GM1 in the presence of each singledomain antibody, in relation to a reference without any single-domain antibody. 36 nM of each single-domain antibody was used, in replicates of six. Out of these two dimeric single-domain antibodies, B006_0004 showed a superior blocking capacity of CTB binding to GM1.

Figure 5. Sequence alignment of selected single-domain antibodies with a blocking capacity of CTB-GM1 interaction. All CDR regions of single-domain antibodies in a monomeric format are aligned against each other. The sequence analysis indicated that, in terms of binding to CTB in a position that hinder GM1 interaction, longer CDR3 regions (M006_0001 and M006_0004) are beneficial. Furthermore, an additional cysteine residue (CDR3 of M006_0001) can have a positive impact on blocking efficacy.

Figure 6. Phylogenetic tree of selected single-domain antibodies separated into clades based on protein sequence homology. This sequence homology analysis revealed that the superior blocker, of CTB interaction with GM1 , B006_0001 , belongs to an individual clade. Detailed description

Single-domain antibodies

A nanobody or single-domain antibody (sdAb), as used herein, refers to the smallest antigen binding fragment or single variable domain (“VHH”) derived from a naturally occurring heavy chain antibody and is known to the person skilled in the art.

Such single-domain antibodies can be derived from antibodies raised in Camelidae species, for example in camel, llama, dromedary, alpaca and guanaco. Single-domain antibodies may also be synthetically produced, such as by expression in bacteria. Single-domain antibodies are antibodies whose complementary determining regions are part of a single-domain polypeptide. Examples include, but are not limited to, heavy chain antibodies, antibodies naturally devoid of light chains, single-domain antibodies derived from conventional 4-chain antibodies, engineered antibodies and singledomain scaffolds other than those derived from antibodies.

The term single-domain antibody, in its broadest sense, is not limited to a specific biological source or to a specific method of preparation. For example, the singledomain antibodies of the disclosure can generally be obtained: (1) by isolating the VHH domain of a naturally occurring heavy chain antibody; (2) by expression of a nucleotide sequence encoding a naturally occurring VHH domain; (3) by “humanization” of a naturally occurring VHH domain or by expression of a nucleic acid encoding a such humanized VHH domain; (4) by “camelization” of a naturally occurring VH domain from any animal species, and in particular from a mammalian species, such as from a human being, or by expression of a nucleic acid encoding such a camelized VH domain; (5) by “camelization” of a “domain antibody” or “Dab,” as described in the art, or by expression of a nucleic acid encoding such a camelized VH domain; (6) by using synthetic or semi-synthetic techniques for preparing proteins, polypeptides or other amino acid sequences known per se: (7) by preparing a nucleic acid encoding a singledomain antibody using techniques for nucleic acid synthesis known per se, followed by expression of the nucleic acid thus obtained; and/or (8) by any combination of one or more of the foregoing.

M006 0001

In one embodiment, the single-domain antibody of the present disclosure is the singledomain antibody “M006_0001” (SEQ ID NO: 22) or a variant thereof. In one embodiment, the single-domain antibody of the present disclosure is a singledomain antibody comprising: i) a complementary-determining region 1 (CDR1) comprising or consisting of SEQ I D NO: 1 , or a variant thereof wherein one or more amino acids have been altered, with the proviso that no more than 3 amino acids have been so altered, for example wherein 2, or 1 amino acids have been so altered; and ii) a complementary-determining region 2 (CDR2) comprising or consisting of SEQ ID NO: 2, or a variant thereof wherein one or more amino acids have been altered, with the proviso that no more than 3 amino acids have been so altered, for example wherein 2, or 1 amino acids have been so altered; and iii) a complementary-determining region 3 (CDR3) comprising or consisting of SEQ ID NO: 3, or a variant thereof wherein one or more amino acids have been altered, with the proviso that no more than 3 amino acids have been so altered, for example wherein 2, or 1 amino acids have been so altered.

In one embodiment, no more than 2 amino acids have been altered in each CDR. In one embodiment, no more than 1 amino acid has been altered in each CDR.

In one embodiment, the single-domain antibody only has one or more amino acid alterations in CDR3. Thus, in one embodiment, the present disclosure relates to a single-domain antibody comprising: i) a complementary-determining region 1 (CDR1) comprising or consisting of SEQ ID NO: 1 ; and ii) a complementary-determining region 2 (CDR2) comprising or consisting of SEQ ID NO: 2; and iii) a complementary-determining region 3 (CDR3) comprising or consisting of SEQ ID NO: 3, or a variant thereof wherein one or more amino acids have been altered, with the proviso that no more than 3 amino acids have been so altered, for example wherein 2, or 1 amino acids have been so altered. In one embodiment, the single-domain antibody of the present disclosure is a singledomain antibody comprising: i) a complementary-determining region 1 (CDR1) comprising or consisting of an amino acid sequence according to SEQ ID NO: 1 ; ii) a complementary-determining region 2 (CDR2) comprising or consisting of an amino acid sequence according to SEQ ID NO: 2; and iii) a complementary-determining region 3 (CDR3) comprising or consisting of an amino acid sequence according to SEQ ID NO: 3.

In one embodiment, the single-domain antibody comprises or consists of the sequence as set forth in SEQ ID NO: 22, or a sequence having at least 90% sequence identity thereto. In one embodiment, the sequence identity is at least 95%, such as at least 96%, 97%, 98% or 99%. In one embodiment, the sequence variance is outside the CDRs.

In one embodiment, the single-domain antibody of the present disclosure is a humanised version of the single-domain antibody of M006_0001 as disclosed above.

M006 0002

In one embodiment, the single-domain antibody of the present disclosure is the singledomain antibody “M006_0002” (SEQ ID NO: 23) or a variant thereof.

In one embodiment, the single-domain antibody of the present disclosure is a singledomain antibody comprising: i) a complementary-determining region 1 (CDR1) comprising or consisting of SEQ ID NO: 4, or a variant thereof wherein one or more amino acids have been altered, with the proviso that no more than 3 amino acids have been so altered, for example wherein 2, or 1 amino acids have been so altered; and ii) a complementary-determining region 2 (CDR2) comprising or consisting of SEQ ID NO: 5, or a variant thereof wherein one or more amino acids have been altered for another amino acid, with the proviso that no more than 3 amino acids have been so altered, for example wherein 2, or 1 amino acids have been so altered; and iii) a complementary-determining region 3 (CDR3) comprising or consisting of SEQ ID NO: 6, or a variant thereof wherein one or more amino acids have been altered, with the proviso that no more than 3 amino acids have been so altered, for example wherein 2, or 1 amino acids have been so altered.

In one embodiment, the single-domain antibody only has one or more amino acid alterations in CDR3. Thus, in one embodiment, the present disclosure relates to a single-domain antibody comprising: i) a complementary-determining region 1 (CDR1) comprising or consisting of SEQ ID NO: 4; and ii) a complementary-determining region 2 (CDR2) comprising or consisting of SEQ ID NO: 5; and iii) a complementary-determining region 3 (CDR3) comprising or consisting of SEQ ID NO: 6, or a variant thereof wherein one or more amino acids have been altered, with the proviso that no more than 3 amino acids have been so altered, for example wherein 2, or 1 amino acids have been so altered.

In one embodiment, the single-domain antibody of the present disclosure is a singledomain antibody comprising: i) a complementary-determining region 1 (CDR1) comprising or consisting of an amino acid sequence according to SEQ ID NO: 4; ii) a complementary-determining region 2 (CDR2) comprising or consisting of an amino acid sequence according to SEQ ID NO: 5; and iii) a complementary-determining region 3 (CDR3) comprising or consisting of an amino acid sequence according to SEQ ID NO: 6.

In one embodiment, the single-domain antibody comprises or consists of the sequence as set forth in SEQ ID NO: 23, or a sequence having at least 90% sequence identity thereto. In one embodiment, the sequence identity is at least 95%, such as at least 96%, 97%, 98% or 99%. In one embodiment, the sequence variance is outside the CDRs.

In one embodiment, the single-domain antibody of the present disclosure is a humanized version of the single-domain antibody of M006_0002 as disclosed above.

M006 0003

In one embodiment, the single-domain antibody of the present disclosure is the singledomain antibody “M006_0003” (SEQ ID NO: 24) or a variant thereof.

In one embodiment, the single-domain antibody of the present disclosure is a singledomain antibody comprising: i) a complementary-determining region 1 (CDR1) comprising or consisting of SEQ ID NO: 7, or a variant thereof wherein one or more amino acids have been altered, with the proviso that no more than 3 amino acids have been so altered, for example wherein 2, or 1 amino acids have been so altered; and ii) a complementary-determining region 2 (CDR2) comprising or consisting of SEQ ID NO: 8, or a variant thereof wherein one or more amino acids have been altered, with the proviso that no more than 3 amino acids have been so altered, for example wherein 2, or 1 amino acids have been so altered; and iii) a complementary-determining region 3 (CDR3) comprising or consisting of SEQ ID NO: 9, or a variant thereof wherein one or more amino acids have been altered, with the proviso that no more than 3 amino acids have been so altered, for example wherein 2, or 1 amino acids have been so altered.

In one embodiment, the single-domain antibody only has one or more amino acid alterations in CDR3. Thus, in one embodiment, the present disclosure relates to a single-domain antibody comprising: i) a complementary-determining region 1 (CDR1) comprising or consisting of SEQ ID NO: 7; and ii) a complementary-determining region 2 (CDR2) comprising or consisting of SEQ ID NO: 8; and iii) a complementary-determining region 3 (CDR3) comprising or consisting of SEQ ID NO: 9, or a variant thereof wherein one or more amino acids have been altered, with the proviso that no more than 3 amino acids have been so altered, for example wherein 2, or 1 amino acids have been so altered.

In one embodiment, the single-domain antibody of the present disclosure is a singledomain antibody comprising: i) a complementary-determining region 1 (CDR1) comprising or consisting of an amino acid sequence according to SEQ ID NO: 7; ii) a complementary-determining region 2 (CDR2) comprising or consisting of an amino acid sequence according to SEQ ID NO: 8; and iii) a complementary-determining region 3 (CDR3) comprising or consisting of an amino acid sequence according to SEQ ID NO: 9.

In one embodiment, the single-domain antibody comprises or consists of the sequence as set forth in SEQ ID NO: 24, or a sequence having at least 90% sequence identity thereto. In one embodiment, the sequence identity is at least 95%, such as at least 96%, 97%, 98% or 99%. In one embodiment, the sequence variance is outside the CDRs.

In one embodiment, the single-domain antibody of the present disclosure is a humanised version of the single-domain antibody of M006_0003 as disclosed above.

M006 0004

In one embodiment, the single-domain antibody of the present disclosure is the singledomain antibody “M006_0004” (SEQ ID NO: 25) or a variant thereof.

In one embodiment, the single-domain antibody of the present disclosure is a singledomain antibody comprising: i) a complementary-determining region 1 (CDR1) comprising or consisting of SEQ ID NO: 10, or a variant thereof wherein one or more amino acids have been altered, with the proviso that no more than 3 amino acids have been so altered, for example wherein 2, or 1 amino acids have been so altered; and ii) a complementary-determining region 2 (CDR2) comprising or consisting of SEQ ID NO: 11 , or a variant thereof wherein one or more amino acids have been altered, with the proviso that no more than 3 amino acids have been so altered, for example wherein 2, or 1 amino acids have been so altered; and iii) a complementary-determining region 3 (CDR3) comprising or consisting of SEQ ID NO: 12, or a variant thereof wherein one or more amino acids have been altered, with the proviso that no more than 3 amino acids have been so altered, for example wherein 2, or 1 amino acids have been so altered.

In one embodiment, the single-domain antibody only has one or more amino acid alterations in CDR3. Thus, in one embodiment, the present disclosure relates to a single-domain antibody comprising: i) a complementary-determining region 1 (CDR1) comprising or consisting of SEQ ID NO: 10; and ii) a complementary-determining region 2 (CDR2) comprising or consisting of SEQ ID NO: 11 ; and iii) a complementary-determining region 3 (CDR3) comprising or consisting of SEQ ID NO: 12, or a variant thereof wherein one or more amino acids have been altered, with the proviso that no more than 3 amino acids have been so altered, for example wherein 2, or 1 amino acids have been so altered.

In one embodiment, the single-domain antibody of the present disclosure is a singledomain antibody comprising: i) a complementary-determining region 1 (CDR1) comprising or consisting of an amino acid sequence according to SEQ ID NO: 10; ii) a complementary-determining region 2 (CDR2) comprising or consisting of an amino acid sequence according to SEQ ID NO: 11 ; and iii) a complementary-determining region 3 (CDR3) comprising or consisting of an amino acid sequence according to SEQ ID NO: 12.

In one embodiment, the single-domain antibody comprises or consists of the sequence as set forth in SEQ ID NO: 25, or a sequence having at least 90% sequence identity thereto. In one embodiment, the sequence identity is at least 95%, such as at least 96%, 97%, 98% or 99%. In one embodiment, the sequence variance is outside the CDRs.

In one embodiment, the single-domain antibody of the present disclosure is a humanised version of the single-domain antibody of M006_0004 as disclosed above.

M006 0005

In one embodiment, the single-domain antibody of the present disclosure is the singledomain antibody “M006_0005” (SEQ ID NO: 26) or a variant thereof.

In one embodiment, the single-domain antibody of the present disclosure is a singledomain antibody comprising: i) a complementary-determining region 1 (CDR1) comprising or consisting of SEQ ID NO: 13, or a variant thereof wherein one or more amino acids have been altered, with the proviso that no more than 3 amino acids have been so altered, for example wherein 2, or 1 amino acids have been so altered; and ii) a complementary-determining region 2 (CDR2) comprising or consisting of SEQ ID NO: 14, or a variant thereof wherein one or more amino acids have been altered, with the proviso that no more than 3 amino acids have been so altered, for example wherein 2, or 1 amino acids have been so altered; and iii) a complementary-determining region 3 (CDR3) comprising or consisting of SEQ ID NO: 15, or a variant thereof wherein one or more amino acids have been altered, with the proviso that no more than 3 amino acids have been so altered, for example wherein 2, or 1 amino acids have been so altered. In one embodiment, no more than 2 amino acids have been altered in each CDR. In one embodiment, no more than 1 amino acid has been altered in each CDR.

In one embodiment, the single-domain antibody only has one or more amino acid alterations in CDR3. Thus, in one embodiment, the present disclosure relates to a single-domain antibody comprising: i) a complementary-determining region 1 (CDR1) comprising or consisting of SEQ ID NO: 13; and ii) a complementary-determining region 2 (CDR2) comprising or consisting of SEQ ID NO: 14; and iii) a complementary-determining region 3 (CDR3) comprising or consisting of SEQ ID NO: 15, or a variant thereof wherein one or more amino acids have been altered, with the proviso that no more than 3 amino acids have been so altered, for example wherein 2, or 1 amino acids have been so altered.

In one embodiment, the single-domain antibody of the present disclosure is a singledomain antibody comprising: i) a complementary-determining region 1 (CDR1) comprising or consisting of an amino acid sequence according to SEQ ID NO: 13; ii) a complementary-determining region 2 (CDR2) comprising or consisting of an amino acid sequence according to SEQ ID NO: 14; and iii) a complementary-determining region 3 (CDR3) comprising or consisting of an amino acid sequence according to SEQ ID NO: 15.

In one embodiment, the single-domain antibody comprises or consists of the sequence as set forth in SEQ ID NO: 26, or a sequence having at least 90% sequence identity thereto. In one embodiment, the sequence identity is at least 95%, such as at least 96%, 97%, 98% or 99%. In one embodiment, the sequence variance is outside the CDRs.

In one embodiment, the single-domain antibody of the present disclosure is a humanised version of the single-domain antibody of M006_0005 as disclosed above. M006 0007

In one embodiment, the single-domain antibody of the present disclosure is the singledomain antibody “M006_0007” (SEQ ID NO: 27) or a variant thereof.

In one embodiment, the single-domain antibody of the present disclosure is a singledomain antibody comprising: i) a complementary-determining region 1 (CDR1) comprising or consisting of SEQ ID NO: 16, or a variant thereof wherein one or more amino acids have been altered, with the proviso that no more than 3 amino acids have been so altered, for example wherein 2, or 1 amino acids have been so altered; and ii) a complementary-determining region 2 (CDR2) comprising or consisting of SEQ ID NO: 17, or a variant thereof wherein one or more amino acids have been altered, with the proviso that no more than 3 amino acids have been so altered, for example wherein 2, or 1 amino acids have been so altered; and iii) a complementary-determining region 3 (CDR3) comprising or consisting of SEQ ID NO: 18, or a variant thereof wherein one or more amino acids have been altered, with the proviso that no more than 3 amino acids have been so altered, for example wherein 2, or 1 amino acids have been so altered.

In one embodiment, the single-domain antibody only has one or more amino acid alterations in CDR3. Thus, in one embodiment, the present disclosure relates to a single-domain antibody comprising: i) a complementary-determining region 1 (CDR1) comprising or consisting of SEQ ID NO: 16; and ii) a complementary-determining region 2 (CDR2) comprising or consisting of SEQ ID NO: 17; and iii) a complementary-determining region 3 (CDR3) comprising or consisting of SEQ ID NO: 18, or a variant thereof wherein one or more amino acids have been altered, with the proviso that no more than 3 amino acids have been so altered, for example wherein 2, or 1 amino acids have been so altered.

In one embodiment, the single-domain antibody of the present disclosure is a singledomain antibody comprising: i) a complementary-determining region 1 (CDR1) comprising or consisting of an amino acid sequence according to SEQ ID NO: 16; ii) a complementary-determining region 2 (CDR2) comprising or consisting of an amino acid sequence according to SEQ ID NO: 17; and iii) a complementary-determining region 3 (CDR3) comprising or consisting of an amino acid sequence according to SEQ ID NO: 18.

In one embodiment, the single-domain antibody comprises or consists of the sequence as set forth in SEQ ID NO: 27, or a sequence having at least 90% sequence identity thereto. In one embodiment, the sequence identity is at least 95%, such as at least 96%, 97%, 98% or 99%. In one embodiment, the sequence variance is outside the CDRs.

In one embodiment, the single-domain antibody of the present disclosure is a humanised version of the single-domain antibody of M006_0007 as disclosed above.

M006 0008

In one embodiment, the single-domain antibody of the present disclosure is the singledomain antibody “M006_0008” (SEQ ID NO: 28) or a variant thereof.

In one embodiment, the single-domain antibody of the present disclosure is a singledomain antibody comprising: i) a complementary-determining region 1 (CDR1) comprising or consisting of SEQ ID NO: 19, or a variant thereof wherein one or more amino acids have been altered, with the proviso that no more than 3 amino acids have been so altered, for example wherein 2, or 1 amino acids have been so altered; and ii) a complementary-determining region 2 (CDR2) comprising or consisting of SEQ ID NO: 20, or a variant thereof wherein one or more amino acids have been altered, with the proviso that no more than 3 amino acids have been so altered, for example wherein 2, or 1 amino acids have been so altered; and iii) a complementary-determining region 3 (CDR3) comprising or consisting of SEQ ID NO: 21 , or a variant thereof wherein one or more amino acids have been altered, with the proviso that no more than 3 amino acids have been so altered, for example wherein 2, or 1 amino acids have been so altered.

In one embodiment, the sdAb only has one or more amino acid alterations in CDR3. Thus, in one embodiment, the present disclosure relates to a single-domain antibody comprising: i) a complementary-determining region 1 (CDR1) comprising or consisting of SEQ ID NO: 19; and ii) a complementary-determining region 2 (CDR2) comprising or consisting of SEQ ID NO: 20; and iii) a complementary-determining region 3 (CDR3) comprising or consisting of SEQ ID NO: 21 , or a variant thereof wherein one or more amino acids have been altered, with the proviso that no more than 3 amino acids have been so altered, for example wherein 2, or 1 amino acids have been so altered.

In one embodiment, the single-domain antibody of the present disclosure is a singledomain antibody comprising: i) a complementary-determining region 1 (CDR1) comprising or consisting of an amino acid sequence according to SEQ ID NO: 19; ii) a complementary-determining region 2 (CDR2) comprising or consisting of an amino acid sequence according to SEQ ID NO: 20; and iii) a complementary-determining region 3 (CDR3) comprising or consisting of an amino acid sequence according to SEQ ID NO: 21.

In one embodiment, the single-domain antibody comprises or consists of the sequence as set forth in SEQ ID NO: 28, or a sequence having at least 90% sequence identity thereto. In one embodiment, the sequence identity is at least 95%, such as at least 96%, 97%, 98% or 99%. In one embodiment, the sequence variance is outside the CDRs.

In one embodiment, the single-domain antibody of the present disclosure is a humanised version of the single-domain antibody of M006_0008 as disclosed above.

Sequence modifications

In one embodiment, the present disclosure allows for minor variations in the CDRs since such CDR variants can retain the activity of and in some cases even improve the activity of the sdAb. The data of the inventors indicate that it may be beneficial to have a cysteine residue included in the CDR(s) of the single-domain antibody, particularly in CDR3. Thus, in some embodiments the CDR sequence of any sdAb disclosed herein may be altered, with the proviso that no more than three amino acids have been so altered, for example wherein two or one amino acids have been so altered in each CDR.

In one embodiment the alteration of one or more amino acids comprises a substitution, a deletion or an insertion.

In one embodiment the alteration of one or more amino acids comprises or is a substitution.

In one embodiment, the alteration comprises or is a deletion.

In one embodiment, the alteration comprises or is an insertion.

In one embodiment, the alteration increases the number of cysteine residues in the CDR(s).

For example, the alteration may comprise a substitution and/or an insertion of one or more cysteines.

In one embodiment, the alteration of one or more amino acids is in CDR3. In a preferred embodiment, the alteration comprise a substitution and/or an insertion of one or more cysteines in CDR3.

In some embodiments, CDR3 comprises at least 12 amino acids, such as at least 13 amino acids, such as at least 14 amino acids, such as at least 15 amino acids, such as at least 16 amino acids, such as at least 17 amino acids, such as at least 18 amino acids, such as at least 19 amino acids, such as at least 20 amino acids.

In some embodiments, CDR3 comprises between 12 and 20 amino acids, such as between 13 and 19 amino acids, such as between 14 and 19 amino acids.

Single-domain antibody features

The sdAbs of the present disclosure are useful in ameliorating and/or preventing cholera infection, reducing the risk of cholera infection, as well as in the treatment of cholera infection. To be useful for oral delivery, sdAbs of the present disclosure should be capable of blocking the interaction of cholera toxin B subunit (CTB) with the human GM1 receptor following passage through the Gl tract. Thus, blocking the cytotoxic effect of cholera toxin on mammalian cells. Furthermore, sdAbs of the disclosure should demonstrate a high binding affinity towards CTB (e.g., Kd < 10 nM) under complex conditions in the gut lumen.

In one embodiment, the sbAbs disclosed herein efficiently bind to their target CTB and have a Kd < 10 nM.

The sdAbs disclosed herein preferably have one or more of the following features: a) prevents and/or reduces Vibrio cholerae toxin cytotoxicity; b) prevents and/or reduces toxin activity of Vibrio cholerae toxin ; c) prevents and/or reduces one or more Vibrio crto/erae-mediated symptoms, such as diarrhea, vomiting, muscle cramps, abdominal discomfort, and/or dehydration; d) reduces the risk of Vibrio cholerae infection; e) is stable in the gastrointestinal tract of a subject; f) is protease stable; g) is pH stable; h) is storage stable; i) is temperature stable; and/or j) improves the gut microbiome.

In one embodiment, the single-domain antibody prevents and/or reduces Vibrio cholerae toxin cytotoxicity.

In one embodiment, the single-domain antibody prevents and/or reduces toxin activity of Vibrio cholerae toxin.

In one embodiment, the single-domain antibody prevents and/or reduces one or more Vibrio cholerae-mediated symptoms, such as diarrhea, vomiting, muscle cramps, seizures, abdominal discomfort, and/or dehydration.

In another embodiment, the provided single-domain antibody reduces the risk of Vibrio cholerae infection. In other words, said single-domain antibody reduces the risk of cholera.

Cholera is an infection of the small intestine caused by the bacterium Vibrio cholerae. The classic symptom is watery diarrhoea lasting for a few days. The diarrhoea can be so severe that it within hours leads to severe dehydration and electrolyte imbalance. Other symptoms include vomiting, muscle cramps, seizures and coma. Severe cholera, without treatment, kills about half of infected individuals. Children are often more severely affected than adults.

Cholera spreads via unsafe water and unsafe food that has been contaminated with Vibrio cholerae. Once the bacteria reach the intestinal wall, they start producing the toxin that gives the infected person the watery diarrhoea. The cholera toxin (CT) comprise six protein subunits; a single copy of subunit A (CTA) and five copies of subunit B (CTB). The five B subunits form a ring that binds to GM1 (monosialotetrahexosylganglioside) on the surface of the intestinal epithelium cells. This results in secretion of water, sodium, potassium and bicarbonate into the lumen of the small intestine. The chloride and sodium ions create a salt-water environment, which through osmosis can pull up to six litres of water per day through the intestinal cells, creating the massive amounts of diarrhoea of the infected individual. In one embodiment, the single-domain antibody prevents and/or reduces the interaction between GM1 and subunit B of Vibrio cholerae toxin.

In another embodiment, the single-domain antibody blocks the interaction between subunit B of Vibrio cholerae toxin and the GM1 receptor.

The single-domain antibody disclosed herein is able to block the interaction between the GM1 receptor and toxin subunit B of any type of Vibrio cholerae. In particular, the single-domain antibody disclosed herein is able to block the interaction between the GM1 receptor and toxin subunit B of any type of Vibrio cholerae serogroup. In other words, the single-domain antibody as disclosed herein is effective for prevention, treatment and/or reducing the risk of cholera infection caused by any type of Vibrio cholerae serogroup, such as Vibrio cholerae 01 and/or Vibrio cholerae 0139.

In one embodiment, the single-domain antibody disclosed herein is able to block the interaction between the GM1 receptor and toxin subunit B of Vibrio cholerae 01.

In one embodiment, the single-domain antibody disclosed herein is able to block the interaction between the GM1 receptor and toxin subunit B of Vibrio cholerae 0139.

In one embodiment, the Vibrio cholerae toxin subunit B has an amino acid sequence according to SEQ ID NO: 35.

In another embodiment, the single-domain antibody inhibits the Vibrio cholerae toxin subunit B biological activity of causing secretion of water, sodium, potassium and bicarbonate into the lumen of the small intestine.

In another embodiment, the single-domain antibody disclosed herein improves the gut microbiome.

The single-domain antibodies provided herein are preferably stable in the gastrointestinal tract of a subject. Thus, they are usually both protease stable and pH stable and can withstand the harsh conditions of the gastrointestinal tract while retaining their biological activity. By “stable” herein is meant that at least 50% of the original binding activity is retained.

More preferably, at least 60%, 70% or 80% of the original binding activity is retained.

In one embodiment, at least 60% of the original binding activity is retained.

In one embodiment, at least 70% of the original binding activity is retained.

In one embodiment, at least 80% of the original binding activity is retained.

In one embodiment, at least 90% of the original binding activity is retained.

To determine whether a sdAb is stable in the gastrointestinal tract, one can measure the stability in simulated gastric fluid (SGF) and/or simulated intestinal fluid (SIF).

The term "simulated gastric fluid" or "SGF" used herein refers to an aqueous solution utilized in dissolution testing to mimic the conditions of the stomach. On the other hand, the term "simulated intestinal fluid" or "SIF" used herein refers to an aqueous solution utilized in dissolution testing to mimic the conditions of the intestines.

In one embodiment, the single-domain antibody is stable in SGF e.g. in a solution comprising 0.05-0.15 M NaCI, such as 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11 , 0.12, 0.13, 0.14 or 0.15 M NaCI and 0.02-0.013 M HCI, such as 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.12, or 0.013 M, pH ~1.5 for at least 0.5 hours, such as at least for 1 hour.

In one embodiment, the single-domain antibody is stable in SIF, e.g. in a solution comprising 0.011-0.021 M NaOH, such as 0.011 , 0.012, 0.013, 0.014, 0.015, 0.016, 0.017, 0.018, 0.019, 0.020, or 0.021 M NaOH and 0.027-0.036 M K₃PO₄„ such as 0.027, 0.028, 0.029, 0.030, 0.031 , 0.032, 0.033, 0.034, 0.035, 0.036 M K₃PO₄, pH ~7 for at least 0.5 hours, such as at least for 1 hour.

In one embodiment the single-domain antibody is protease stable. In one embodiment, the single-domain is stable in 50-550 U/ml pepsin, such as 50, 100, 220, 500, or 550 U/ml pepsin in SGF and 12-150 U/ml of pancreatin, such as 12, 25, 50, 100, and 150 U/ml of pancreatin in SIF for at least 0.5 hours, such as at least for 1 hour. In one embodiment the single-domain antibody is pH stable. In one embodiment, the single-domain antibody is stable at a pH of 1.5 for at least 0.5 hours, such as at least for 1 hour.

In one embodiment the single-domain antibody is stable at a pH of 4 for at least 0.5 hours, such as at least for 1 hour.

In another embodiment the single-domain antibody is storage stable. By storage stable is meant that the sdAb can be stored for extended periods of time under various conditions as explained further herein below while retaining at least 50% of the original binding activity. More preferably at least 60%, 70% or 80% of the original binding activity is retained.

In a particular embodiment, the single-domain antibody is stable at negative degrees, for example between -15 and -25 °C, such as being stable at -15°C, -16°C, -17°C, - 18°C, -19°C, -20 °C, -21 °C, -22°C, -23°C, -24°C and -25°C, such as for at least 10 days, more preferably at least 15 days, 20 days, 25 days, 30 days, 60 days or more.

In another embodiment, the single-domain antibody is stable at positive degrees, for example between 0°C and 4°C, such as at 0°C, 1 °C, 2°C, 3°C, and 4°C and at room temperatures, such as between 20°C and 30°C, such as at 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C and 30°C for at least 10 days, more preferably at least 15 days, 20 days, 25 days, 30 days or more.

In one embodiment, the single-domain antibody is stored and is stable in saline phosphate solution or in dry form.

In one embodiment the single-domain antibody is stable at elevated temperatures, such as at 25°C, 30°C, 37°C, 45°C, 50°C, 60°C, 75°C and 80°C for 1h and at 85°C, 86°C, 87°C, 88°C, 89°C, 90°C, 91 °C, 92°C, 93°C, 95°C for at least 5 seconds, such as 10 or 15 seconds. Thus, the sdAb is able to withstand harsh conditions that may be involved in food, feed and beverage processing, such as able to tolerate pasteurization, which may be performed at 72°C for 15 seconds. In one embodiment, the sdAb of the present disclosure is stable in dry form, e.g. as a dry powder, such as in freeze-dried form for at least 4 months at room temperature (25°C), such as for at least 5 months or 6 months.

In one embodiment, the sdAb of the present disclosure is stable as a solid with medium water activity for 2 months at room temperature (25°C).

In one embodiment, the sdAb of the present disclosure is stable in a liquid with 3<pH<7 for at least 4 days, more preferably for at least 5, 6 or 7 days at 4°C.

In one embodiment, the sdAb of the present disclosure is stable in different food products or beverages such as in a low moisture food matrix (LMF; water activity <0.6), an intermediate moisture food matrix (IMF; 0.6<a_w<0.85) and/or in a high moisture food matrix (HMF; a_w>0.85) as shown in the below table so that the sdAb of the present disclosure can be used in such foods or beverages as a dietary supplement/food ingredient and can retain sufficient activity, i.e. it is stable, throughout the normal shelflife of such products.

Fusion proteins

The present disclosure also provides fusion proteins comprising at least one of the single-domain antibodies as described herein. Such fusion protein can be assembled by methods known to the person skilled in the art. Preferably, the fusion protein is recombinantly designed by fusing gene sequences in vitro. In some embodiments, the fusion protein further comprises a linker connecting the sbAbs. In one embodiment, a fusion protein comprising a single-domain antibody as described herein and one or more further single-domain antibodies, and optionally one or more linkers is provided. In one embodiment, the fusion protein is a homodimer, i.e. containing two identical sdAbs, or a heterodimer, i.e. containing two different sdAbs.

In one embodiment, the one or more further single-domain antibodies bind to Vibrio cholerae toxin subunit B and/or Vibrio cholerae toxin subunit A.

In a preferred embodiment, the one or more further single-domain antibodies are single-domain antibodies as provided in the present disclosure.

In one embodiment, the fusion protein comprises any combination of the sdAbs as disclosed herein (including variants thereof) according to the below table.

Thus, in one embodiment, the fusion protein comprises the single-domain antibody M006_0001 (SEQ ID NO: 22) and at least one of M006_0001, M006_0002, M006_0003, M006_0004, M006_0005, M006_0007 or M006_0008.

In a particular embodiment, the fusion protein is a homodimer of M006_0001 as disclosed herein. In a particular embodiment the fusion protein is B006_001 (SEQ ID NO: 29).

In one embodiment, the fusion protein comprises the single-domain antibody M006_0002 (SEQ ID NO: 23) and at least one of M006_0001, M006_0002, M006_0003, M006_0004, M006_0005, M006_0007 or M006_0008.

In a particular embodiment, the fusion protein is a homodimer of M006_0002 as disclosed herein. In a particular embodiment, the fusion protein is B006_002 (SEQ ID NO: 30).

In one embodiment, the fusion protein comprises the single-domain antibody M006_0003 (SEQ ID NO: 24) and at least one of M006_0001, M006_0002, M006_0003, M006_0004, M006_0005, M006_0007 or M006_0008.

In a particular embodiment, the fusion protein is a homodimer of M006_0003 as disclosed herein. In a particular embodiment, the fusion protein is B006_003 (SEQ ID NO: 31).

In one embodiment, the fusion protein comprises the single-domain antibody M006_0004 (SEQ ID NO: 25) and at least one of M006_0001, M006_0002, M006_0003, M006_0004, M006_0005, M006_0007 or M006_0008. In a particular embodiment, the fusion protein is a homodimer of M006_0004 as disclosed herein. In a particular embodiment, the fusion protein is B006_004 (SEQ ID NO: 32).

In one embodiment, the fusion protein comprises the single-domain antibody M006_0005 (SEQ ID NO: 26) and at least one of M006_0001, M006_0002, M006_0003, M006_0004, M006_0005, M006_0007 or M006_0008.

In a particular embodiment, the fusion protein is a homodimer of M006_0005 as disclosed herein. Thus, in a particular embodiment the fusion protein is B006_005 (SEQ ID NO: 33).

In one embodiment, the fusion protein comprises the single-domain antibody M006_0007 (SEQ ID NO: 27) and at least one of M006_0001, M006_0002, M006_0003, M006_0004, M006_0005, M006_0007 or M006_0008.

In a particular embodiment, the fusion protein is a homodimer of M006_0007 as disclosed herein. Thus, in a particular embodiment, the fusion protein is B006_007 (SEQ ID NO: 34).

In one embodiment, the fusion protein comprises the single-domain antibody M006_0008 (SEQ ID NO: 28) and at least one of M006_0001, M006_0002, M006_0003, M006_0004, M006_0005, M006_0007 or M006_0008.

In a particular embodiment, the fusion protein is a homodimer of M006_0008 as disclosed herein. Thus, in a particular embodiment the fusion protein is B006_008 (SEQ ID NO: 35).

The fusion protein of the present disclosure is usually at least as stable and active as described herein in relation to the individual sdAbs of the present disclosure.

In a preferred embodiment the fusion protein comprises a linker connecting the sdAbs. In one embodiment the linker is a GS linker, i.e. a linker which comprises or consists of glycine and serine residues. Such linkers are well known in the art.

In one embodiment the linker is a GS linker of the structure (G_xS)_n, where x may be a number between 1 to 10, preferably 2 to 5, and n refers to a number of repeats of the GxS sequence, where n may be between 1 to 10, preferably 2 to 5.

Examples of suitable linkers according to the present disclosure include a GGGGS linker (SEQ ID NO: 37), a GGGGSGGGGS linker (SEQ ID NO: 38), a GGGGSGGGGSGGGGS linker (SEQ ID NO: 39), a GGGGSGGGGSGGGGSGGGGS linker (SEQ ID NO: 40), a GGGGSGGGGSGGGGSGGGGSGGGGS linker (SEQ ID NO: 41), or a GGGGSGGGGSGGGGSGGGGSGGGGSGGGGS linker (SEQ ID NO: 42).

Nucleic acids and vectors

The present disclosure also provides isolated nucleic acids and vectors encoding the sbAbs and fusion proteins disclosed herein.

In one embodiment, the isolated nucleic acid molecule comprises or consists of SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48 and/or SEQ ID NO: 49.

In one embodiment, the isolated nucleic acid molecule comprises or consists of SEQ ID NO: 50, SEQ ID NO: 51 , SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55 and/or SEQ ID NO: 56.

In one embodiment, the provided isolated nucleic acid is codon-optimized for a host cell wherein said nucleic acid molecule is expressed.

In one embodiment, an expression vector comprising the nucleic acid molecule encoding the sbAbs and fusion proteins disclosed herein is provided. The expression vector may be any vector suitable for expression of the herein disclosed nucleic acids. In one embodiment, the vector is a viral vector, such as an adenoviral vector. The vector may comprise a promoter to enhance expression of the nucleic acid molecule in at least some host cells.

In one embodiment, the nucleic acid molecule is operably linked to one or more control sequences, such as an inducible promoter, to direct its expression.

Host cells and methods of manufacture

In one aspect, the present disclosure relates to a recombinant host cell, which is a cultured cell that has been transformed or transfected with a polypeptide-encoding nucleic acid, which can then be expressed in the host cell.

The phrase "recombinant host cell" can be used to denote a host cell that has been transformed or transfected with a nucleic acid to be expressed. Constructs comprising the sequences of interest may be introduced into a host cell by standard techniques. These techniques include transfection, infection, holistic impact, electroporation, microinjection, scraping, or any other method that introduces the sequences of interest into the host cell as known to a person of skill. A host cell that has been manipulated by any method to take up a DNA sequence, construct or vector will be referred to as "transformed" or "recombinant" herein.

In one embodiment, the present disclosure provides a recombinant host cell comprising the nucleic acid molecule or the expression vector as disclosed herein.

In one embodiment, the host cell is a bacterium, a plant, a fungus, such as a yeast, or a mammalian cell.

In one embodiment, the host cell is a bacterium, such as a bacillus, such as Bacillus licheniformis, Bacillus subtilis, or Bacillus lactobacillus, Lactobacillus spp., or a Bifidobacterium spp. In one embodiment, the host cell is Escherichia coli.

In one embodiment, the host cell is a yeast, such as a yeast selected from the genus of Pichia, Hansenula and Saccharomyces.

In one embodiment, the host cell is a fungus selected from Aspergillus oryzae and Aspergillus niger. In one embodiment, the present disclosure relates to a method of producing the provided single-domain antibody or the fusion protein, the method comprising culturing the host cell as disclosed herein under conditions wherein the single-domain antibody or fusion protein is expressed. The method may further comprise a step of purifying and/or isolating the single-domain antibody molecule or fusion protein.

Compositions and pharmaceutical uses

The present disclosure further provides a composition comprising a single-domain antibody, a fusion protein, a nucleic acid, a vector, and/or a host cell as disclosed herein, optionally further comprising one or more excipients. The composition may further comprise one more buffers, diluents, carriers and/or adjuvants adjusted to the intended use of the composition.

For pharmaceutical compositions, all components of the composition should be pharmaceutically acceptable. By “pharmaceutically acceptable" we mean a non-toxic material that does not decrease the effectiveness of the sbAb. Such pharmaceutically acceptable buffers, carriers or excipients are well-known in the art (see Remington's Pharmaceutical Sciences, 18th edition, A.R Gennaro, Ed., Mack Publishing Company (1990) and handbook of Pharmaceutical Excipients, 3rd edition, A. Kibbe, Ed., Pharmaceutical Press (2000)).

The composition may further comprise one or more further active components such as an antibiotic, fecal matter transfer and/or monoclonal antibodies.

In one embodiment, the composition is a pharmaceutical composition.

In one embodiment, the present disclosure provides a single-domain antibody, a fusion protein, a nucleic acid, a vector, a host cell or a pharmaceutical composition as described herein for use as a medicament.

In one embodiment, the present disclosure provides the use of a single-domain antibody, a fusion protein, a nucleic acid, a vector, a host cell or a pharmaceutical composition as described herein for the manufacture of a medicament for therapeutic application. In one embodiment, the present disclosure provides a single-domain antibody, a fusion protein, a nucleic acid, a vector, a host cell or a pharmaceutical composition as disclosed herein for use in the prevention or treatment of Vibrio cholerae infection, i.e. cholera, in a subject.

In one embodiment, the present disclosure provides the use of a single-domain antibody, a fusion protein, a nucleic acid, a vector, a host cell or a pharmaceutical composition as described herein for the manufacture of a medicament for the prevention or treatment of Vibrio cholerae infection, i.e. cholera, in a subject.

Treatment encompasses both curative and ameliorative treatment. By ameliorative treatment is meant a treatment that results in the improvement of one or more symptoms of cholera infection in a subject.

In one embodiment, the subject is a human, e.g. a child, such as a child of less than 10 years of age, and/or an immunocompromised subject. In one embodiment the subject is a child of between 0 and 10 years of age, such as a child of between 1 and 6 years of age, such as a child of between 1 and 5 years of age, such as a child of between 1 and 4 years of age, such as a child of between 2 and 5 years of age, such as a child of between 2 and 4 years of age.

In one embodiment, the present disclosure provides a method for prevention and/or treatment of Vibrio cholerae infection in a subject in need thereof, said method comprising administering to the subject a single-domain antibody, a fusion protein, a nucleic acid, a vector, a host cell or a composition as disclosed herein.

In one embodiment the present disclosure provides a method for reducing the risk of Vibrio cholerae infection in a subject in need thereof, said method comprising administering to the subject a single-domain antibody, a fusion protein, a nucleic acid, a vector, a host cell or a composition as disclosed herein.

The single-domain antibody, fusion protein, nucleic acid, vector, host cell or composition as disclosed herein may be administered in any manner deemed suitable by a person of skill. Enteral administration is particularly preferred, such as orally as a food supplement, as a tablet or a gel, or via gastric intubation.

Dietary compositions and dietary uses

In one embodiment, the present disclosure provides a dietary/nutraceutical composition comprising a single-domain antibody, a fusion protein, a nucleic acid, a vector, a host cell or a composition as disclosed herein. Such dietary compositions are considered useful for improving the gut microbiome and for reducing the risk of Vibrio cholerae infection, i.e. reducing the risk of cholera.

In one embodiment, the dietary composition comprises one or more of prebiotics, probiotics, synbiotics, proteins, lipids, carbohydrates, vitamins, fibers, and/or nutrients, such as dietary minerals.

In one embodiment, the dietary composition is an oral rehydration solution or yoghurt.

In a further embodiment, the disclosure relates to the use of a single-domain antibody, a fusion protein, a nucleic acid, a vector, and/or a host cell as disclosed herein as a food ingredient or dietary supplement.

In a further embodiment, the disclosure relates to the use of a single-domain antibody, a fusion protein, a nucleic acid, a vector, and/or a host cell as disclosed herein as a food or beverage additive

In a further embodiment, the disclosure relates to the use of a single-domain antibody, a fusion protein, a nucleic acid, a vector, and/or a host cell as disclosed herein as a food or beverage preservative.

Detections methods

The single-domain antibodies provided herein may also find use in a detection method for detecting Vibrio cholerae in a sample.

Thus, in one aspect, the present disclosure provides a method for detecting Vibrio cholerae, wherein the method comprises the steps of: a) providing a sample; b) contacting the sample with one of more single-domain antibodies of the present disclosure; and c) detecting the complex between the sample and the one or more singledomain antibodies of the present disclosure.

In one embodiment, the sample is an isolated sample.

Step b) of the method may further comprises a step of washing the sample, thereby removing any unbound antibody.

In a further embodiment, the method comprises detecting the complex of step c) e.g. by western blotting, ELISA, LFA, microscopy, flow cytometry; TRF, and/or by immunocytochemistry.

In one embodiment, the single-domain antibody comprises a detection label, such as a colorimetric, a fluorescent, a luminescent, a magnetic, or a paramagnetic label, or is biotinylated.

The detection method is usually an in vitro method and is performed on samples isolated from a subject.

Examples

Example 1. Screening of single-domain antibodies

Numerous single-domain antibodies in a monomeric format were screened for their capacity for blocking cholera toxin B subunit (CTB) interaction with the GM1 receptor. The recombinant CTB (Sigma-Aldrich #C9903) used during screening shares the three residues (Trp88, Gly33, and Tyr12) critical for GM1 interaction with strains of the serotype responsible for all cholera pandemics to date (Sanchez and Holmgren, 2011).

In short, black 96-well Immuno Plates were coated with 5 ug/ml of GM1 receptor (SIGMA #G7641) in phosphate buffer saline (PBS), with a pH of 7.4, overnight (O/N) at 4°C. Individual supernatants of single-domain antibody cultures were incubated with 36 nM of biotinylated CTB, in a 6% milk-blocking solution (PBS) at 37 °C for 30 minutes. After blocking with 3% milk (in PBS) for 1 hour at room temperature, the mixture of singledomain antibody cultures and biotinylated CTB was added and incubated at room temperature for 1 hour. Biotinylated CTB (36 nM) incubated at 37 °C was used as a reference for the blocking effect.

After washing, streptavidin-conjugated europium (Perkin Elmer, #1244-360) diluted 1/500 in DELFIA assay buffer (Perkin Elmer #1244-111) was added and the plate was incubated for 30 minutes at room temperature. DELFIA enhancement solution 20 (Perkin Elmer #4001-0010) was used to activate europium fluorescence, followed by intensity measurements at 615 nm.

In total, several hundred single-domain antibodies were screened based on the blocking capacity of CTB interaction with the GM1 receptor. A subset of the screened singledomain antibodies are depicted in Fig. 1. Based on the reduced fluorescence intensity compared to the reference (CTB without single-domain antibody culture), a dozen singledomain antibodies with greater blocking capacity were selected for further characterization. Based on this characterization, the seven best-performing singledomain antibodies in terms of blocking capacity were further characterized. Said seven single-domain antibodies were M006_0001 (SEQ ID NO: 22), M006_0002 (SEQ ID NO: 23), M006_0003 (SEQ ID NO: 24), M006_0004 (SEQ ID NO: 25), M006_0005 (SEQ ID NO: 26), M006_0007 (SEQ ID NO: 27) and M006_0008 (SEQ ID NO: 28).

Example 2. Monomeric single-domain antibody blocking capacity of the GM1-CTB interaction.

Monomeric single-domain antibodies, purified from cultures that previously demonstrated the highest blocking capacity during high-throughput screening, were further evaluated based on their blocking capacity but this time using a defined singledomain antibody to CTB ratio. In a similar manner as before, a DELFIA assay was utilized to determine the level of CTB binding to the GM1 receptor. However, this time, 360 nM of each single-domain antibody was incubated (37 °C) with 36 nM biotinylated CTB prior to adding the mixture to a black 96-well Immuno Plate coated with 5 ug/ml of G 1 receptor. Biotinylated CTB (36 nM) incubated with a single-domain antibody, in the same format, without specificity for cholera toxin was used as a reference for the blocking effect.

Streptavidin-conjugated europium (Perkin Elmer, #1244-360) diluted 1/500 in DELFIA assay buffer (Perkin Elmer #1244-111) was added and the plate was incubated for 30 minutes at room temperature. DELFIA enhancement solution 20 (Perkin Elmer #4001- 0010) was used to activate europium fluorescence, followed by intensity measurements at 615 nm.

Once purified, from bacterial cultures, the selected monomeric single-domain antibodies demonstrated a high blocking capacity of GM1-CTB interaction at a 10x single-domain antibody to toxin ratio. At 360 nM, M006_0001 blocked the great majority of all GM1 and CTB interactions.

Example 3. Dimeric single-domain antibody blocking capacity of the GM1-CTB interaction

The blocking capacity of selected single-domain antibodies in a homo-dimer format for blocking CTB interaction with the GM1 receptor was determined using a similar DELFIA assay as previously described. Briefly, 36 nM of each single-domain antibody was incubated with 36 nM biotinylated CTB, before being added to a black 96-well Immuno Plate previously coated with 5 ug/ml of GM1 receptor. Biotinylated CTB (36 nM) incubated at 37 °C was used as a reference for the blocking effect.

Almost all of the previously investigated monomeric single-domain antibodies, now in a dimeric format, demonstrated a significant blocking capacity of CTB interaction with the G 1 receptor (Fig. 3 and Fig. 4). Interestingly, the transition from a monomeric to a dimeric format, increased the blocking effect of the single-domain antibodies to a level comparable to the monomeric format when in a 10x single-domain antibody to toxin ratio. In particular, single-domain antibody B006_0001 was highly efficacious compared to the other single-domain antibodies investigated, showed as a greatly reduced fluorescent signal compared to the GM 1 -CTB reference. Example 4. CDR homology analysis

The amino acid sequence of selected single-domain antibodies with a blocking capacity of the GM1-CTB interaction was analyzed based on homology. Analysis was carried out using the Alignment (Fig. 5) and Tree functions (Fig. 6) of CLC Main Workbench 22.0. The alignment of all CDR regions of the selected single-domain antibodies shows that the main sequence diversity, in relation to each other, can be found in the CDR3 region (Fig. 5). More specifically, the two single-domain antibodies that were most efficacious in blocking CTB interaction with GM1 (B006_0001 and B006_0004) displayed longer CDR3 regions. Interestingly, the superior blocker B006_0001 have an additional cysteine residue (CDR3) in comparison to all other selected single-domain antibodies which might contribute to the increased efficacy. Furthermore, B006_0001 , was identified to belong to an individual clade (Fig. 6) whereas B006_0001 showed sequence homology with another single-domain antibody (M006_0002).

Example 5. Further B006_0001 positive characteristics

Gastrointestinal stability

Physiological (biochemical) conditions representative of passage through the stomach and small intestine, the site of V. cholerae colonization and CTX secretion, were generated through incubation (37 °C) of B006_0001 in simulated gastric fluid (SGF; pH 1.2) and small intestinal fluid (SIF; pH 6.8). At first, CTXB-binding activity of B006_0001 was investigated following incubation in either PBS, SGF, or SIF for a maximum of four (SGF) to five (SIF) hours depending on the time documented for in vivo gut transit.

Four hours after incubation in SGF, B006_0001 displayed a maintained CTXB-binding activity relative to a B006_0001 control (4 °C; in PBS) of 96%, which is identical to the CTXB-binding activity (96%) of B006_0001 incubated (4 h) in PBS (see Table below). Similarly, incubation of B006_0001 in SIF (5 h) indicated to have little effect on CTXB- binding activity, as B006_0001 maintained a blocking activity of 86%, comparable to the B006_0001 control (85%) subjected to PBS at 4 °C (see Table below).

Table. CTXB-binding activity of B006_0001 , relative to a B006_0001 control (stored at 4 °C in PBS), after incubation in either simulated gastric fluid (SGF; pH 1.2), simulated intestinal fluid (SIF; pH 6.8), or PBS (pH 7.0) at 37 °C. B006_0001 was incubated for a maximum of four (SGF) to five (SIF) hours and the average CTXB-binding activity was determined from duplicate measurements.

Sequence overview

References

Sanchez and Holmgren (2011) Cholera toxin - A foe & a friend. Indian J Med Res; 133(2): 153-163.

Items

1 . A single-domain antibody which binds to subunit B of Vibrio cholerae toxin, wherein said single-domain antibody is: a) a single-domain antibody comprising: i. a complementary-determining region 1 (CDR1) comprising or consisting of SEQ I D NO: 1 , or a variant thereof wherein one or more amino acids have been altered, with the proviso that no more than 3 amino acids have been so altered, for example wherein 2, or 1 amino acids have been so altered; and ii. a complementary-determining region 2 (CDR2) comprising or consisting of SEQ ID NO: 2, or a variant thereof wherein one or more amino acids have been altered, with the proviso that no more than 3 amino acids have been so altered, for example wherein 2, or 1 amino acids have been so altered; and iii. a complementary-determining region 3 (CDR3) comprising or consisting of SEQ ID NO: 3, or a variant thereof wherein one or more amino acids have been altered, with the proviso that no more than 3 amino acids have been so altered, for example wherein 2, or 1 amino acids have been so altered; b) a single-domain antibody comprising or consisting of the sequence as set forth in SEQ ID NO: 22, or a sequence having at least 90% sequence identity thereto; or c) a humanised version of the single-domain antibody of a) or b). A single-domain antibody which binds to subunit B of Vibrio cholerae toxin, wherein the single-domain antibody is: a) a single-domain antibody comprising: i. a complementary-determining region 1 (CDR1) comprising or consisting of SEQ ID NO: 4, or a variant thereof wherein one or more amino acids have been altered, with the proviso that no more than 3 amino acids have been so altered, for example wherein 2, or 1 amino acids have been so altered; and ii. a complementary-determining region 2 (CDR2) comprising or consisting of SEQ ID NO: 5, or a variant thereof wherein one or more amino acids have been altered for another amino acid, with the proviso that no more than 3 amino acids have been so altered, for example wherein 2, or 1 amino acids have been so altered; and iii. a complementary-determining region 3 (CDR3) comprising or consisting of SEQ ID NO: 6, or a variant thereof wherein one or more amino acids have been altered, with the proviso that no more than 3 amino acids have been so altered, for example wherein 2, or 1 amino acids have been so altered; b) a single-domain antibody comprising or consisting of the sequence as set forth in SEQ ID NO: 23, or a sequence having at least 90% sequence identity thereto, or c) a humanised version of the single-domain antibody of a) or b). 3. A single-domain antibody which binds to subunit B of Vibrio cholerae toxin, wherein the single-domain antibody is: a) a single-domain antibody comprising: i. a complementary-determining region 1 (CDR1) comprising or consisting of SEQ ID NO: 7, or a variant thereof wherein one or more amino acids have been altered, with the proviso that no more than 3 amino acids have been so altered, for example wherein 2, or 1 amino acids have been so altered; and ii. a complementary-determining region 2 (CDR2) comprising or consisting of SEQ ID NO: 8, or a variant thereof wherein one or more amino acids have been altered, with the proviso that no more than 3 amino acids have been so altered, for example wherein 2, or 1 amino acids have been so altered; and iii. a complementary-determining region 3 (CDR3) comprising or consisting of SEQ ID NO: 9, or a variant thereof wherein one or more amino acids have been altered, with the proviso that no more than 3 amino acids have been so altered, for example wherein 2, or 1 amino acids have been so altered; b) a single-domain antibody comprising or consisting of the sequence as set forth in SEQ ID NO: 24, or a sequence having at least 90% sequence identity thereto, or c) a humanised version of the single-domain antibody of a) or b).

4. A single-domain antibody which binds to subunit B of Vibrio cholerae toxin, wherein the single-domain antibody is: a) a single-domain antibody comprising: i. a complementary-determining region 1 (CDR1) comprising or consisting of SEQ ID NO: 10, or a variant thereof wherein one or more amino acids have been altered, with the proviso that no more than 3 amino acids have been so altered, for example wherein 2, or 1 amino acids have been so altered; and ii. a complementary-determining region 2 (CDR2) comprising or consisting of SEQ ID NO: 11, or a variant thereof wherein one or more amino acids have been altered, with the proviso that no more than 3 amino acids have been so altered, for example wherein 2, or 1 amino acids have been so altered; and iii. a complementary-determining region 3 (CDR3) comprising or consisting of SEQ ID NO: 12, or a variant thereof wherein one or more amino acids have been altered, with the proviso that no more than 3 amino acids have been so altered, for example wherein 2, or 1 amino acids have been so altered; b) a single-domain antibody comprising or consisting of the sequence as set forth in SEQ ID NO: 25, or a sequence having at least 90% sequence identity thereto, or c) a humanised version of the single-domain antibody of a) or b). A single-domain antibody which binds to subunit B of Vibrio cholerae toxin, wherein the single-domain antibody is: a) a single-domain antibody comprising: i. a complementary-determining region 1 (CDR1) comprising or consisting of SEQ ID NO: 13, or a variant thereof wherein one or more amino acids have been altered, with the proviso that no more than 3 amino acids have been so altered, for example wherein 2, or 1 amino acids have been so altered; and ii. a complementary-determining region 2 (CDR2) comprising or consisting of SEQ ID NO: 14, or a variant thereof wherein one or more amino acids have been altered, with the proviso that no more than 3 amino acids have been so altered, for example wherein 2, or 1 amino acids have been so altered; and iii. a complementary-determining region 3 (CDR3) comprising or consisting of SEQ ID NO: 15, or a variant thereof wherein one or more amino acids have been altered, with the proviso that no more than 3 amino acids have been so altered, for example wherein 2, or 1 amino acids have been so altered; b) a single-domain antibody comprising or consisting of the sequence as set forth in SEQ ID NO: 26, or a sequence having at least 90% sequence identity thereto, or c) a humanised version of the single-domain antibody of a) or b). A single-domain antibody which binds to subunit B of Vibrio cholerae toxin, wherein the single-domain antibody is: a) a single-domain antibody comprising: i. a complementary-determining region 1 (CDR1) comprising or consisting of SEQ ID NO: 16, or a variant thereof wherein one or more amino acids have been altered, with the proviso that no more than 3 amino acids have been so altered, for example wherein 2, or 1 amino acids have been so altered; and ii. a complementary-determining region 2 (CDR2) comprising or consisting of SEQ ID NO: 17, or a variant thereof wherein one or more amino acids have been altered, with the proviso that no more than 3 amino acids have been so altered, for example wherein 2, or 1 amino acids have been so altered; and iii. a complementary-determining region 3 (CDR3) comprising or consisting of SEQ ID NO: 18, or a variant thereof wherein one or more amino acids have been altered, with the proviso that no more than 3 amino acids have been so altered, for example wherein 2, or 1 amino acids have been so altered; b) a single-domain antibody comprising or consisting of the sequence as set forth in SEQ ID NO: 27, or a sequence having at least 90% sequence identity thereto, or c) a humanised version of the single-domain antibody of a) or b). A single-domain antibody which binds to subunit B of Vibrio cholerae toxin, wherein the single-domain antibody is: a) a single-domain antibody comprising: i. a complementary-determining region 1 (CDR1) comprising or consisting of SEQ ID NO: 19, or a variant thereof wherein one or more amino acids have been altered, with the proviso that no more than 3 amino acids have been so altered, for example wherein 2, or 1 amino acids have been so altered; and ii. a complementary-determining region 2 (CDR2) comprising or consisting of SEQ ID NO: 20, or a variant thereof wherein one or more amino acids have been altered, with the proviso that no more than 3 amino acids have been so altered, for example wherein 2, or 1 amino acids have been so altered; and iii. a complementary-determining region 3 (CDR3) comprising or consisting of SEQ ID NO: 21, or a variant thereof wherein one or more amino acids have been altered, with the proviso that no more than 3 amino acids have been so altered, for example wherein 2, or 1 amino acids have been so altered; b) a single-domain antibody comprising or consisting of the sequence as set forth in SEQ ID NO: 28, or a sequence having at least 90% sequence identity thereto, or c) a humanised version of the single-domain antibody of a) or b). The single-domain antibody according to any one of the preceding items, wherein the single-domain antibody has one or more of the following features: a) prevents and/or reduces Vibrio cholerae toxin cytotoxicity; b) prevents and/or reduces toxin activity of Vibrio cholerae toxin; c) prevents and/or reduces one or more Vibrio cho/erae-mediated symptoms, such as diarrhea, vomiting, muscle cramps, abdominal discomfort, and/or dehydration; d) reduces the risk of Vibrio cholerae infection; e) is stable in the gastrointestinal tract of a subject; f) is protease stable; g) is pH stable; h) is storage stable; i) is temperature stable; and/or j) improves the gut microbiome. The single-domain antibody according to any one of the preceding items, wherein any sequence variance is outside the complementary-determining regions (CDRs). The single-domain antibody according to any one of the preceding items, wherein the alteration of one or more amino acids comprises a substitution, a deletion or an insertion. 11. The single-domain antibody according to item 10, wherein the alteration is in CDR3.

12. The single-domain antibody according to any one of items 10-11 , wherein the alteration increases the number of cysteine residues in the CDR(s).

13. The single-domain antibody according to any one of the preceding items, wherein the single-domain antibody is able to block the interaction between subunit B of Vibrio cholerae toxin and the GM1 (monosialotetrahexosylganglioside) receptor.

14. The single-domain antibody according to item 13, wherein subunit B of Vibrio cholerae toxin has an amino acid sequence according to SEQ ID NO: 36.

15. The single-domain antibody according to any one of the preceding items, wherein the single-domain antibody comprises a detection label, such as a colorimetric, a fluorescent, a luminescent, a magnetic, or a paramagnetic label, or is biotinylated.

16. A fusion protein comprising a single-domain antibody as defined in any one of the preceding items and one or more further single-domain antibodies, and optionally one or more linkers.

17. The fusion protein according to item 16, wherein the fusion protein is a homodimer or a heterodimer.

18. The fusion protein according to any one of items 16-17, wherein the one or more further single-domain antibodies bind to subunit B of Vibrio cholerae toxin and/or to subunit A of Vibrio cholerae toxin.

19. The fusion protein according to any one of items 16-18 comprising: a) a single-domain antibody as defined in item 2 and a further singledomain antibody as defined in item 2; b) a single-domain antibody as defined in item 2 and a further singledomain antibody as defined in item 3; c) a single-domain antibody as defined in item 2 and a further singledomain antibody as defined in item 4; d) a single-domain antibody as defined in item 2 and a further singledomain antibody as defined in item 5; e) a single-domain antibody as defined in item 2 and a further singledomain antibody as defined in item 6; f) a single-domain antibody as defined in item 2 and a further singledomain antibody as defined in item 7; g) a single-domain antibody as defined in item 2 and a further singledomain antibody as defined in item 8; h) a single-domain antibody as defined in item 3 and a further singledomain antibody as defined in item 3; i) a single-domain antibody as defined in item 3 and a further singledomain antibody as defined in item 4; j) a single-domain antibody as defined in item 3 and a further singledomain antibody as defined in item 5; k) a single-domain antibody as defined in item 3 and a further singledomain antibody as defined in item 6; l) a single-domain antibody as defined in item 3 and a further singledomain antibody as defined in item 7; m) a single-domain antibody as defined in item 3 and a further singledomain antibody as defined in item 8; n) a single-domain antibody as defined in item 4 and a further singledomain antibody as defined in item 4; o) a single-domain antibody as defined in item 4 and a further singledomain antibody as defined in item 5; p) a single-domain antibody as defined in item 4 and a further singledomain antibody as defined in item 6; q) a single-domain antibody as defined in item 4 and a further singledomain antibody as defined in item 7; r) a single-domain antibody as defined in item 4 and a further singledomain antibody as defined in item 8; s) a single-domain antibody as defined in item 5 and a further singledomain antibody as defined in item 5; t) a single-domain antibody as defined in item 5 and a further singledomain antibody as defined in item 6; u) a single-domain antibody as defined in item 5 and a further singledomain antibody as defined in item 7; v) a single-domain antibody as defined in item 5 and a further singledomain antibody as defined in item 8; w) a single-domain antibody as defined in item 6 and a further singledomain antibody as defined in item 6; x) a single-domain antibody as defined in item 6 and a further singledomain antibody as defined in item 7; y) a single-domain antibody as defined in item 6 and a further singledomain antibody as defined in item 8; z) a single-domain antibody as defined in item 7 and a further singledomain antibody as defined in item 7; aa) a single-domain antibody as defined in item 7 and a further singledomain antibody as defined in item 8; bb) a single-domain antibody as defined in item 8 and a further singledomain antibody as defined in item 8.

20. The fusion protein according to any one of items 16-19, wherein the linker is a GS linker of the structure (G_xS)_n, where x may be a number between 1 to 10, preferably 2 to 5, and n refers to a number of repeats of the G_XS sequence, where n may be between 1 to 10, preferably 2 to 5.

21. The fusion protein according to item 20, wherein said GS linker is a GGGGS linker (SEQ ID NO: 37), a GGGGSGGGGS linker (SEQ ID NO: 38), a GGGGSGGGGSGGGGS linker (SEQ ID NO: 39), a GGGGSGGGGSGGGGSGGGGS linker (SEQ ID NO: 40), a GGGGSGGGGSGGGGSGGGGSGGGGS linker (SEQ ID NO: 41), or a GGGGSGGGGSGGGGSGGGGSGGGGSGGGGS linker (SEQ ID NO: 42).

22. The fusion protein according to any one of items 20-21 , wherein said GS linker is a GGGGSGGGGSGGGGS linker (SEQ ID NO: 39). 23. The fusion protein according to any one of items 16-22, wherein said fusion protein comprises or consists of SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31 , SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34 and/or SEQ ID NO: 35.

24. An isolated nucleic acid molecule encoding the single-domain antibody according to any one of items 1-15 or the fusion protein according to any one of items 16-23.

25. The isolated nucleic acid molecule according to item 24, wherein the nucleic acid molecule comprises or consists of SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51 , SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55 and/or SEQ ID NO: 56.

26. The isolated nucleic acid molecule according to any one of items 24-25, wherein the nucleic acid molecule is codon-optimized for a host cell wherein said nucleic acid molecule is expressed.

27. An expression vector comprising the nucleic acid molecule according to any one of items 24-26.

28. The expression vector according to item 27, wherein the nucleic acid molecule is operably linked to one or more control sequences, such as an inducible promoter to direct its expression.

29. A recombinant host cell comprising the nucleic acid molecule according to any one of items 24-26 or the expression vector according to any one of items 27- 28.

30. The recombinant host cell according to item 29, wherein the host cell is a bacterium, a plant, a fungus, such as a yeast, or a mammalian cell.

31. The recombinant host cell according to item 30, wherein the bacterium is a bacillus, such as a Bacillus licheniformis, Bacillus subtilis. Bacillus lactobacillus, Lactobacillus spp. or Bifidobacterium spp. 32. The recombinant host cell according to item 30, wherein the host cell is a yeast, such as a yeast selected from the genus of Pichia, Hansenula and Saccharomyces.

33. The recombinant host cell according to item 30, wherein the fungus is selected from Aspergillus oryzae and Aspergillus niger.

34. A method of producing the single-domain antibody according to any one of items 1-15 or the fusion protein according to any one of items 16-23, the method comprising culturing the host cell according to any one of items 29-33 under conditions wherein the single-domain antibody or fusion protein is expressed.

35. The method according to item 34, wherein the method further comprises a step of purifying and/or isolating the single-domain antibody molecule or fusion protein.

36. A composition comprising the single-domain antibody according to any one of items 1-15, the fusion protein according to any one of items 16-23, the nucleic acid according to any one of items 24-26, the vector according to any one of items 27-28, and/or the host cell according to any one of items 29-33, optionally further comprising one or more excipients.

37. The composition according to item 36, wherein the composition comprises one or more further compounds selected from antibiotics, fecal matter transfer and/or monoclonal antibodies.

38. A single-domain antibody according to any one of items 1-15, the fusion protein according to any one of items 16-23, the nucleic acid according to any one of items 24-26, the vector according to any one of items 27-28, the host cell according to any one of items 29-33 and/or the composition according to any one of items 36-37 for use as a medicament. 39. A single-domain antibody according to any one of items 1-15, the fusion protein according to any one of items 16-23, the nucleic acid according to any one of items 24-26, the vector according to any one of items 27-28, the host cell according to any one of items 29-33 and/or the composition according to any one of items 36-37 for use in the reduction of risk, prevention or treatment of Vibrio cholerae infection in a subject.

40. The single-domain antibody, the fusion protein, the nucleic acid, the vector, the host cell or the composition for use according to any one of items 38-39, wherein the subject is a human.

41. The single-domain antibody, the fusion protein, the nucleic acid, the vector, the host cell or the composition for use according to any one of items 38-40, wherein the subject is a child, such as a child of between 0 and 10 years of age, such as a child of between 1 and 6 years of age, such as a child of between 1 and 5 years of age, such as a child of between 1 and 4 years of age, such as a child of between 2 and 5 years of age, such as a child of between 2 and 4 years of age.

42. The single-domain antibody, the fusion protein, the nucleic acid, the vector, the host cell or the composition for use according to any one of items 38-41 , wherein said subject is an immunocompromised subject.

43. The single-domain antibody, the fusion protein, the nucleic acid, the vector, the host cell or the composition for use according to any one of items 38-42, wherein the single-domain antibody, the fusion protein, the nucleic acid, the vector, the host cell or the composition is administered enterally, such as orally, such as a food supplement, as a tablet or a gel, or via gastric intubation.

44. Use of the single-domain antibody according to any one of items 1-15, the fusion protein according to any one of items 16-23, the nucleic acid according to any one of items 24-26, the vector according to any one of items 27-28, the host cell according to any one of items 29-33 and/or the composition according to any one of items 36-37 for the manufacture of a medicament for therapeutic application, such as for the reduction of risk, prevention or treatment of Vibrio cholerae infection in a subject, optionally wherein the subject is as defined in any one of items 40-42.

45. A method for prevention and/or treatment of Vibrio cholerae infection in a subject in need thereof, said method comprising administering to the subject a single-domain antibody according to any one of items 1-15, the fusion protein according to any one of items 16-23, the nucleic acid according to any one of items 24-26, the vector according to any one of items 27-28, the host cell according to any one of items 29-33 and/or the composition according to any one of items 36-37.

46. A method for reducing the risk of Vibrio cholerae infection in a subject in need thereof, said method comprising administering to the subject a single-domain antibody according to any one of items 1-15, the fusion protein according to any one of items 16-23, the nucleic acid according to any one of items 24-26, the vector according to any one of items 27-28, the host cell according to any one of items 29-33 and/or the composition according to any one of items 36-37.

47. A dietary composition comprising the single-domain antibody according to any one of items 1-15, the fusion protein according to any one of items 16-23, the nucleic acid according to any one of items 24-26, the vector according to any one of items 27-28, the host cell according to any one of items 29-33 and/or the composition according to any one of items 36-37.

48. The dietary composition according to item 47, wherein said dietary composition comprises one or more of prebiotics, probiotics, synbiotics, proteins, lipids, carbohydrates, vitamins, fibers, and/or nutrients, such as dietary minerals.

49. The dietary composition according to any one of items 47 to 48, wherein the dietary composition is an oral rehydration solution or a yoghurt.

50. Use of the single-domain antibody according to any one of items 1-15, the fusion protein according to any one of items 16-23, the nucleic acid according to any one of items 24-26, the vector according to any one of items 27-28, the host cell according to any one of items 29-33, the composition according to any one of items 36-37 and/or the dietary composition according to any one of items 47-48 as a food or beverage ingredient, as a food or beverage additive, or as food or beverage preservative.

51. A method for detecting Vibrio cholerae, wherein the method comprises the steps of: a) providing a sample; b) contacting the sample with one of more single-domain antibodies according to any one of the items 1-15; and c) detecting the complex between the sample and the one or more singledomain antibodies.

52. The method according to item 51 wherein step b) further comprises a step of washing the sample, thereby removing any unbound antibody.

53. The method according to any one of items 51-52 wherein the method comprises detecting the complex of step c) by western blotting, ELISA, LFA, microscopy, flow cytometry; TRF, or immunocytochemistry.

Claims

1 . A single-domain antibody which binds to subunit B of Vibrio cholerae toxin, wherein said single-domain antibody is: a) a single-domain antibody comprising: i. a complementary-determining region 1 (CDR1) comprising or consisting of SEQ I D NO: 1 , or a variant thereof wherein one or more amino acids have been altered, with the proviso that no more than 3 amino acids have been so altered, for example wherein 2, or 1 amino acids have been so altered; and ii. a complementary-determining region 2 (CDR2) comprising or consisting of SEQ ID NO: 2, or a variant thereof wherein one or more amino acids have been altered, with the proviso that no more than 3 amino acids have been so altered, for example wherein 2, or 1 amino acids have been so altered; and iii. a complementary-determining region 3 (CDR3) comprising or consisting of SEQ ID NO: 3, or a variant thereof wherein one or more amino acids have been altered, with the proviso that no more than 3 amino acids have been so altered, for example wherein 2, or 1 amino acids have been so altered; b) a single-domain antibody comprising or consisting of the sequence as set forth in SEQ ID NO: 22, or a sequence having at least 90% sequence identity thereto; or c) a humanised version of the single-domain antibody of a) or b).

2. The single-domain antibody according to claim 1, wherein said single-domain antibody is: a) a single-domain antibody comprising: i. a complementary-determining region 1 (CDR1) comprising or consisting of SEQ I D NO: 1 , or a variant thereof wherein one or more amino acids have been altered, with the proviso that no more than 2 amino acids have been so altered; and ii. a complementary-determining region 2 (CDR2) comprising or consisting of SEQ ID NO: 2, or a variant thereof wherein one or more amino acids have been altered, with the proviso that no more than 2 amino acids have been so altered; and iii. a complementary-determining region 3 (CDR3) comprising or consisting of SEQ ID NO: 3, or a variant thereof wherein one or more amino acids have been altered, with the proviso that no more than 2 amino acids have been so altered. The single-domain antibody according to claim 2, wherein said single-domain antibody is: a) a single-domain antibody comprising: i. a complementary-determining region 1 (CDR1) comprising or consisting of SEQ ID NO: 1 , or a variant thereof wherein one or more amino acids have been altered, with the proviso that no more than 1 amino acid has been so altered; and ii. a complementary-determining region 2 (CDR2) comprising or consisting of SEQ ID NO: 2, or a variant thereof wherein one or more amino acids have been altered, with the proviso that no more than 1 amino acid has been so altered; and iii. a complementary-determining region 3 (CDR3) comprising or consisting of SEQ ID NO: 3, or a variant thereof wherein one or more amino acids have been altered, with the proviso that no more than 1 amino acid has been so altered. The single-domain antibody according to claim 1 , wherein any sequence variance is outside the complementary-determining regions (CDRs). A single-domain antibody which binds to subunit B of Vibrio cholerae toxin, wherein said single-domain antibody is: a) a single-domain antibody comprising: i. a complementary-determining region 1 (CDR1) comprising or consisting of SEQ ID NO: 1 ; and ii. a complementary-determining region 2 (CDR2) comprising or consisting of SEQ ID NO: 2; and iii. a complementary-determining region 3 (CDR3) comprising or consisting of SEQ ID NO: 3; b) a single-domain antibody comprising or consisting of the sequence as set forth in SEQ ID NO: 22; or a humanised version of the single-domain antibody of a) or b).

6. A fusion protein comprising a single-domain antibody as defined in any one of the preceding claims and one or more further single-domain antibodies, and optionally one or more linkers, such as one or more GS linkers.

7. The fusion protein according to claim 6, wherein the fusion protein is a homodimer or a heterodimer.

8. An isolated nucleic acid molecule encoding the single-domain antibody according to any one of claims 1-5 or the fusion protein according to any one of claims 6-7.

9. An expression vector comprising the nucleic acid molecule according to claim 8.

10. A recombinant host cell comprising the nucleic acid molecule according to claim 8 or the expression vector according to claim 9.

11. A method of producing the single-domain antibody according to any one of claims 1-5 or the fusion protein according to any one of claims 6-7, the method comprising culturing the host cell according to claim 10 under conditions wherein the single-domain antibody or fusion protein is expressed.

12. A composition comprising the single-domain antibody according to any one of claims 1-5, the fusion protein according to any one of claims 6-7, the nucleic acid according to claim 8, the vector according to claim 9, and/or the host cell according to claim 10, optionally further comprising one or more excipients.

13. A single-domain antibody according to any one of claims 1-5, the fusion protein according to any one of claims 6-7, the nucleic acid according to claim 8, the vector according to claim 9, the host cell according to claim 10 and/or the composition according to claim 12 for use as a medicament, such as for use in the reduction of risk, prevention or treatment of Vibrio cholerae infection in a subject. The single-domain antibody, the fusion protein, the nucleic acid, the vector, the host cell or the composition for use according to claim 12, wherein the singledomain antibody, the fusion protein, the nucleic acid, the vector, the host cell or the composition is administered enterally, such as orally, such as a food supplement, as a tablet or a gel, or via gastric intubation. A dietary composition comprising the single-domain antibody according to any one of claims 1-5, the fusion protein according to any one of claims 6-7, the nucleic acid according to claim 8, the vector according to claim 9, the host cell according to claim 10 and/or the composition according to claim 11 , optionally wherein the dietary composition is an oral rehydration solution or a yoghurt. Use of the single-domain antibody according to any one of claims 1-5, the fusion protein according to any one of claims 6-7, the nucleic acid according to claim 8, the vector according to claim 9, the host cell according to claim 10, the composition according to claim 12 and/or the dietary composition according to claim 15 as a food or beverage ingredient, as a food or beverage additive, or as food or beverage preservative, such as wherein the food or beverage is an oral rehydration solution or yoghurt. A method for detecting Vibrio cholerae, wherein the method comprises the steps of: a) providing a sample; b) contacting the sample with one of more single-domain antibodies according to any one of claims 1-5; and c) detecting the complex between the sample and the one or more singledomain antibodies.