Pathogen binding proteins

Abstract

The present invention relates to pathogen binding proteins, compositions and uses thereof wherein the protein comprises a first peptide having a first binding specificity, a second peptide having a second binding specificity and a linker wherein the first peptide and the second peptide bind to at least one pathogen surface component and / or at least one molecule produced by a pathogen.

Description

[0001] This application is a divisional application of the application with the application number 202080017074X, the application date of January 7, 2020, and the invention title of "Pathogen-binding Protein". Technical Field

[0002] The present invention relates to proteins, compositions and their uses, in particular proteins and compositions. The protein comprises a first peptide having a first binding specificity, a second peptide having a second binding specificity, and a linker, wherein the first peptide and the second peptide bind to at least one pathogen surface component and / or at least one molecule produced by a pathogen. Accordingly, the protein and / or composition can be used for preventing or treating an infection caused by a pathogen. Background Art

[0003] The article by Harmsen et al. 2006 (Selection and optimization of proteolytically stable llama single-domain antibody fragments for oral immunotherapy) relates to the oral application of the recombinant single-domain antibody (V H H fragment) clone K609. K609 is directed against the fimbriae of Escherichia Coli F4, more specifically the tip-adhesin FaeG, and has been shown to reduce Escherichia coli-induced diarrhea in piglets. However, since the orally applied K609 single-domain antibody is degraded by proteolysis in the stomach, it is only effective at high doses. By selection and DNA shuffling, four clones with increased in vitro stability were obtained. These single-domain antibodies differ by at most 10 amino acid residues from each other and from K609. The most stable clone, K922, retains 41% of its activity after incubation in gastric juice and 90% in jejunal fluid, and furthermore has a higher affinity for FaeG compared to the original K609 clone.

[0004] The article by Virdi et al. in 2013 (Orally fed seeds producing designer IgA protect weaned piglets against enterotoxigenic Escherichia coli infection) involves passive immunization based on oral feeding against post-weaning infections, such as post-weaning diarrhea in piglets caused by Enterotoxigenic Escherichia coli (ETEC). The anti-F4 + ETEC antibodies were designed by fusing the variable domain of the llama heavy chain-only antibody with the Fc portion of porcine immunoglobulin (IgG or IgA) and expressing them in Arabidopsis thaliana seeds. The article also describes monomeric, dimeric, and secretory IgA based on V H H-IgA.

[0005] The article by Lo et al. in 2014 (The molecular Mechanism of Shiga Toxin stx2e Neutralization by Single-domain Antibody Targeting the Cell Receptor-binding Domain) reported the discovery and characterization of a single-domain antibody isolated from a llama phage display library that has potent neutralizing ability against the Stx2e toxin. Edema disease is a severe disease characterized by neurological disorders, hemorrhagic lesions, and frequent fatal outcomes. Swine edema disease is caused by Escherichia coli producing Shiga toxin Stx2e. The article shows that the neutralizing NbStx2e1 could be used in the future to prevent or treat swine edema disease.

[0006] The article by Moonens et al. in 2014 (Nanobody Mediated Inhibition of Attachment of F18 Fimbriae Expressing Escherichia coli) involves nanobodies against the lectin domain of the F18 fimbrial adhesin FedF. These nanobodies that bind FedF were obtained from llamas.

[0007] The invention described in patent application WO 2014 / 033313 A1 relates to an antibody capable of protecting against ETEC infection in a passive immunization program. The anti-ETEC single-domain antibody is fused to the IgA Fc domain and produced in plant seeds. This patent application also relates to anti-ETEC, which is anti-F4 + ETEC single-domain antibody and anti-F18 + ETEC single-domain antibody. Summary of the Invention

[0008] Provided herein are proteins comprising a first peptide having a first binding specificity, a second peptide having a second binding specificity, and a linker, wherein the first peptide and the second peptide bind to at least one pathogen surface component and / or at least one molecule produced by a pathogen, such as a toxin. The proteins provided herein can be used for preventing and / or treating pathogen-induced infections.

[0009] In one aspect, the invention provides an isolated nucleic acid molecule encoding the protein described in the above aspect.

[0010] In another aspect, the invention provides a vector comprising the nucleic acid molecule described in the above aspect.

[0011] In another aspect, the invention provides a recombinant host cell comprising the nucleic acid molecule or vector described in the above aspect.

[0012] In one aspect, the invention provides a dietary composition comprising the protein described in the above aspect and optionally further comprising one or more prebiotics, probiotics, synbiotics, proteins, lipids, carbohydrates, vitamins, fibers, and / or nutrients, such as dietary minerals, for a living subject.

[0013] In one aspect, the invention provides a pharmaceutical composition comprising the protein, nucleic acid, vector, and / or recombinant host cell described in the above aspect and optionally one or more pharmaceutically acceptable excipients.

[0014] In another aspect, the invention provides the protein, nucleic acid, vector, recombinant host cell, or pharmaceutical composition described in the above aspect, for use as a medicament.

[0015] In one aspect, the invention provides the protein, nucleic acid, vector, recombinant host cell, or pharmaceutical composition described in the above aspect, for preventing or treating pathogen-induced infections associated with an inner surface and / or an outer surface.

[0016] In another aspect, the present invention provides the protein, nucleic acid, vector, recombinant host cell or pharmaceutical composition described in the above aspects for preventing or treating gastrointestinal infections and / or pulmonary infections.

[0017] In another aspect, the present invention provides a protein, wherein the protein comprises a first peptide and a second peptide, each comprising three complementary determining regions CDR1, CDR2 and CDR3.

[0018] In one aspect, the present invention provides a method for crosslinking pathogens, the method comprising administering to a subject a therapeutically effective amount of the protein, dietary composition and / or pharmaceutical composition described in any of the above aspects.

[0019] In one aspect, the present invention provides a method for preventing biofilms, the method comprising administering to a subject a therapeutically effective amount of the protein, dietary composition and / or pharmaceutical composition described in any of the above aspects.

[0020] In one aspect, the present invention provides a method for neutralizing pathogens and / or molecules secreted by pathogens, the method comprising administering to a subject a therapeutically effective amount of the protein, dietary composition and / or pharmaceutical composition described in any of the above aspects.

[0021] In one aspect, the present invention provides a method for treating or preventing an infection caused by a pathogen, such as a gastrointestinal infection and / or a pulmonary infection, in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the protein, dietary composition and / or pharmaceutical composition described in any of the above aspects.

[0022] In another aspect, the present invention provides a method for producing the protein described in any of the above aspects, the method comprising culturing the host cell described in any of the above aspects under conditions that permit the expression of the encoded protein.

[0023] According to one aspect, the present invention provides a protein comprising at least two single-domain antibodies, wherein the protein is specific for at least a first virulence factor and a second virulence factor.

[0024] According to another aspect, the present invention provides a composition comprising the protein of the present invention and optionally one or more excipients, diluents and / or binders.

[0025] According to one aspect, the present invention provides a composition comprising a first single-domain antibody specific for a first virulence factor and a second single-domain antibody specific for a second virulence factor.

[0026] According to one aspect, the present invention provides a method for treating or preventing a medical condition in an animal or a human, comprising administering the protein or composition of the present invention.

[0027] According to one aspect, the present invention provides a non-therapeutic use of the protein or composition of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 : Results of stability tests under conditions similar to gastrointestinal conditions are shown on a standard SDS gel. The product bands are marked by boxes.

[0029] Figure 2 : Results of stability tests under high protease concentration and low pH conditions are shown on a standard SDS gel. The product bands are marked by boxes.

[0030] Figure 3 : Results of stability tests under very high protease concentration and low pH conditions are shown on a standard SDS gel. The product bands are marked by boxes.

[0031] Figure 4 : Results of binding ability tests for homodivalente and heterodivalente protein constructs. When the product binds in an ELISA assay, the measured OD450 represents the signal, and the higher the signal, the more product is bound.

[0032] Figure 5 : Results of stability tests for monomeric and homodivalente protein constructs with and without exposure to citric acid. Figure A) shows the amount of unseparated monomeric and homodivalente protein constructs after citric acid shock, i.e., the amount of product remaining in the wells. Figure B) shows the amount of monomeric and homodivalente protein constructs eluted after citric acid shock.

[0033] Figure 6 : Results of flow-induced dispersion analysis (FIDA) of free LT-B and its interaction with LT1 monomer and LT1 homodivalente protein constructs.

[0034] Figure 7 : Figure A) shows the thermal stability results of the homodivalente protein construct. Figure B) shows the pH stability results of the homodivalente protein construct. Figure C) shows the bile salt stability results of the homodivalente protein construct.

[0035] Figure 8: Figure A) shows the results of the stability test of the homodimeric protein construct exposed to gastric juice. For one sample, the pH was adjusted to approximately 3 with HCl. The sample was incubated at 37 °C for 60 minutes or 120 minutes. Figure B) shows the results of the stability test of the homodimeric protein construct exposed to pig gall over time. Pig gall was mixed with bacteria and BSA to a final bile concentration of 50%, 10%, or 2%.

[0036] Figure 9 : Figure A) is a schematic diagram, and Figure B) shows an image taken using an optical microscope. Both Figure A) and Figure B) show F4 ETEC bacteria without the addition of any monomer or homodimeric protein construct, F4 ETEC bacteria with the F45 monomer, and F4 ETEC bacteria with the F45 homodimeric protein construct that links F4 ETEC bacteria together.

[0037] Figure 10 : The upper half of Figure A) shows enterocytes and their microvilli on the upper surface. The lower half of Figure A) shows isolated intestinal villous enterocytes from piglets with F4 ETEC bacteria, as indicated by the arrows in the left panel. The right panel shows that when the F45::(GGGS) 3 ::F45 homodimeric protein construct is present, F4 ETEC bacteria cannot bind to the microvilli of enterocytes. Figure B) shows the efficacy of blocking the binding of F4 ETEC to porcine villous enterocytes. The efficacy was quantified by counting enterocytes with 0, 1, or 2+ F4 ETEC cells bound compared to the relevant control. 1 was included because bacteria floating during microscopy may be trapped under the coverslip. The percentage of enterocytes with 1 or 2+ ETEC cells bound shows the efficacy of bacteria infecting enterocytes. The F183::(GGGS) 3 ::F183 homodimeric protein construct was used as a specific control.

[0038] Figure 11 : The homodimeric protein construct against F4 + In vivo treatment results of post-weaning diarrhea in piglets (N = 32). The x-axis shows the number of days after weaning. The y-axis shows the number of piglets with F4 bacteria in the feces (%). Four groups were tested: 1) control: no F45::(GGGGS) 3 ::F45 homodimeric protein construct and no F4, 2) control: with F45::(GGGGS) 3 ::F45 homodimeric protein construct but no F4, 3) no F45::(GGGGS) 3::F45 homodimeric protein construct but with F4, and 4) having F45::(GGGGS) 3 ::F45 homodimeric protein construct and having F4.

[0039] Figure 12 : The results of stability tests using porcine gastric juice under conditions similar to gastrointestinal conditions are shown on a standard SDS gel. The product bands are marked by boxes.

[0040] All cited references are incorporated by reference.

[0041] The accompanying drawings and examples are provided to explain rather than limit the invention. Those skilled in the art will appreciate that aspects, embodiments, claims, and any items of the present invention can be combined.

[0042] Unless otherwise indicated, all percentages are by weight / weight. Unless otherwise indicated, all measurements are made under standard conditions (ambient temperature and pressure). Unless otherwise indicated, the test conditions are in accordance with the European Pharmacopoeia 8.0. Detailed Description

[0043] The terms used in this specification generally have their ordinary meaning in the art in the context of the present disclosure and in the particular context in which each term is used. Certain terms are discussed below or elsewhere in the specification to provide additional guidance to the practitioner by describing the compositions and methods of the present disclosure and how to prepare and use them.

[0044] Definitions

[0045] As used herein, when used in conjunction with the term "comprising" in the claims and / or the specification, the use of the word "a" or "an" can mean "one", but it is also consistent with the meanings of "one or more", "at least one", and "one or more than one". Additionally, the terms "having", "including", "containing", and "comprising" are interchangeable, and those skilled in the art will appreciate that these terms are open-ended terms.

[0046] The terms "about" or "approximately" mean within an acceptable error range of a particular value as determined by a person of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measuring system. For example, in accordance with the practice in the art, "about" can mean within 3 or more standard deviations. Alternatively, "about" can mean a range of up to 20%, preferably up to 10%, more preferably up to 5%, and still more preferably up to 1% of a given value. Alternatively, especially for biological systems or methods, the term can mean within one order of magnitude of the value, preferably within 5-fold, more preferably within 2-fold.

[0047] As used herein, the term "subject" refers to any subject, such as a human or animal subject. Non-limiting examples thereof can be mammalian subjects and / or domestic animals. An "effective amount" of an agent is an amount that is effective to achieve a desired therapeutic or prophylactic result within the required dosage and time period.

[0048] As used herein, the term "combination" refers to the use of more than one therapeutic agent. The use of the term "combination" does not limit the order in which the therapeutic agents are administered to a subject. The first therapeutic agent can be administered before, simultaneously with, or after the second therapeutic agent is administered to the subject.

[0049] As used herein, "treatment" (and its grammatical variants, such as "treat" or "treating") refers to an intervention in an attempt to alter the natural course of an individual being treated, in an amount, manner, and / or mode that is effective to improve a condition, symptom, or parameter associated with a disease or to effectively prevent the development of a disease to a statistically significant degree or to a degree detectable by a person of ordinary skill in the art. Treatment can improve, cure a disease or condition of a subject, or reduce its duration.

[0050] As used herein, the term "monomer" or "monomeric protein" refers to a single peptide having a single binding specificity.

[0051] As used herein, the term "divalent protein construct" refers to a protein comprising a first peptide having a first binding specificity and a second peptide having a second binding specificity linked by a linker.

[0052] As used herein, the term "homodivalent protein construct" refers to a protein comprising a first peptide having a first binding specificity and a second peptide having a second binding specificity, wherein the peptides are the same and are linked by a linker. "Homodivalent protein construct" may also be referred to herein as "bivalent construct". Thus, the two terms are used interchangeably herein.

[0053] As used herein, the term "heterodivalent protein construct" refers to a protein comprising a first peptide having a first binding specificity and a second peptide having a second binding specificity, wherein the peptides are different and are linked by a linker. "Heterodivalent protein construct" may also be referred to herein as "heterovalent". Thus, the two terms are used interchangeably herein.

[0054] As used herein, the term "surface component" refers to any surface component present on the surface of a pathogen, such as any cell surface component present on the surface of a pathogen. Thus, the cell surface component can be an adhesion factor, but is not limited thereto.

[0055] As used herein, the term "molecule" refers to any molecule produced by a pathogen. The molecule can be a secreted molecule or a molecule attached to a cell or surface. In some embodiments of the invention, the molecule is a toxin, protease, and / or binding molecule. In some embodiments of the invention, the molecule is an inhibitor that can bind to a drug component to inhibit the function of the drug component.

[0056] Properties of the protein

[0057] The present disclosure relates to a protein comprising

[0058] - a first peptide having a first binding specificity;

[0059] - a second peptide having a second binding specificity; and

[0060] - a linker,

[0061] wherein the first peptide and the second peptide bind to at least one pathogen surface component and / or at least one molecule produced by a pathogen.

[0062] The protein of the present disclosure is generally an isolated protein, i.e., it is not located in and does not provide within a cell, such as within a cell in a living subject.

[0063] In an embodiment of the present invention, the protein may comprise one or more further peptides that bind to at least one pathogen surface component and / or at least one molecule produced by a pathogen.

[0064] Single-domain antibodies, i.e., monomers, are typically degraded by proteolysis in the stomach. Thus, only very large amounts of single-domain antibodies are likely to have an effect in the gastrointestinal tract. Surprisingly, the inventors have found that by using a linker to connect at least two peptides as described in the present invention, the protein construct is stable in the gastrointestinal tract. Interestingly, the inventors have also found that the protein is capable of binding to pathogen surface components and / or molecules secreted by the pathogen. Thus, the present inventors have produced proteins that are effective in preventing and / or treating pathogen-induced infections, cross-linking pathogens, preventing biofilm formation, and / or neutralizing molecules secreted by pathogens.

[0065] Properties of the linker

[0066] The linker as described in the present invention refers to a linker that at least promotes dimerization. Thus, the present invention relates to a protein in which the first peptide and the second peptide are connected by a linker.

[0067] In an embodiment of the present invention, the linker is a stable linker and imparts stability to the protein of the present invention. Thus, under the exposures described below, the linker is not degraded and is thus able to connect the first peptide and the second peptide of the present invention. Preferably, the protein is stable in the gastrointestinal tract and / or respiratory system of a subject, i.e., the linker is capable of at least promoting dimerization of the protein in the gastrointestinal tract and / or respiratory system of a subject. In one embodiment, the protein is acid-stable and / or protease-stable. The protein may be stable in the presence of any acid such as bile acid and / or citric acid. The protein is also preferably stable in the presence of any protease such as pepsin and / or trypsin. In one embodiment, the protein is stable in the presence of bile, e.g., for 1 hour in 50%, 10%, or 2% bile. In another embodiment, the protein is stable in the presence of bile salt, e.g., for 1 hour in 0.2 mM, 1 mM, and 10 mM sodium deoxycholate (NaDeox).

[0068] In one embodiment of the present invention, the protein is temperature-stable, e.g., temperature-stable up to 70 °C. This indicates high protein stability. In another embodiment, the protein is pH-stable. Unexpectedly, the inventors have found that at pH 3 - 4, approximately 70 - 90% of the protein remains stable, and thus the protein is capable of passing through the gastrointestinal tract at least as a dimer protein and maintaining its activity.

[0069] According to one embodiment, the present invention relates to a protein in which a first peptide and a second peptide are linked together by a linker.

[0070] In some embodiments of the present invention, the first peptide and the second peptide are linked by a GS linker.

[0071] A non-limiting example herein is at least two single-domain antibodies linked together by a GS linker. The GS linker is a fusion protein linker mainly composed of segments of glycine and serine residues.

[0072] According to one embodiment, the present invention relates to a protein in which the GS linker is a linker having the structure (G x S) n where x refers to a plurality of consecutive Gs, where x can be between 1 and 10, for example 1, preferably 2, more preferably 3, preferably 4, preferably 5, more preferably 6, preferably 7, preferably 8, more preferably 9, preferably 10. n refers to the number of repetitions of the G x S sequence, where n can be between 1 and 10, for example 1, preferably 2, more preferably 3, preferably 4, preferably 5, more preferably 6, preferably 7, preferably 8, more preferably 9, preferably 10. Examples include but are not limited to the GGGGS linker (SEQ ID NO:20), the GGGGSGGGGS linker (SEQ ID NO:21), the GGGGSGGGGSGGGGS linker (SEQ ID NO:22), the GGGGSGGGGSGGGGSGGGGS linker (SEQ ID NO:23), the GGGGSGGGGSGGGGSGGGGSGGGGS linker (SEQ ID NO:24), or the GGGGSGGGGSGGGGSGGGGSGGGGSGGGGS linker (SEQ ID NO:25).

[0073] In one embodiment, the GS linker is the GGGGSGGGGSGGGGS (SEQ ID NO:22) linker.

[0074] The inventors have found that after adding proteases such as pepsin and trypsin, a protein comprising two monomers linked by an IgG3 linker is unstable (see Example 6). After adding the protease, the IgG3 linker is degraded and the two monomers are no longer linked.

[0075] According to one embodiment, preferably, the first peptide and the second peptide of the present invention are not linked by an IgG3 linker. Thus, the first peptide and the second peptide can be linked by any stable linker provided that the linker is not an IgG3 linker, such as the IgG3 hinge linker of SEQ ID NO:26.

[0076] Previous studies have linked the variable domain of the llama-only heavy chain antibody to the Fc portion of porcine immunoglobulins (IgG or IgA). A disadvantage of such constructs is that they may cause unwanted immune responses in the treated subject.

[0077] Thus, according to another embodiment of the invention, preferably, the linker does not contain a portion of an antibody, such as an Fc region or a portion of an Fc region. Thus, the first peptide and the second peptide may be linked by any stable linker, provided that the linker does not contain a portion of an antibody, such as an Fc region or a portion of an Fc region.

[0078] The pathogen surface component can be any component present on the surface of the pathogen. Thus, it can include, but is not limited to, any virulence factor, such as an adhesion factor.

[0079] Properties of the peptide

[0080] The first peptide and / or the second peptide can be any peptide having binding specificity for a pathogen surface component and / or a molecule produced by a pathogen.

[0081] The pathogen surface component can be any component present on the surface of the pathogen. Thus, the pathogen surface component can be a pathogen appendage and / or a virulence factor, such as an adhesion factor.

[0082] The molecule according to the invention can be a toxin, an inhibitor, and / or an enzyme.

[0083] Some pathogens are able to produce enzymes to protect themselves, resulting in, for example, antimicrobial resistance, such as bacterial antibiotic resistance. These secreted molecules can be proteases or other molecules that can bind and neutralize antibiotics or inhibitors against antibiotics. Thus, proteins against such molecules can neutralize pathogen antibiotic resistance.

[0084] In one embodiment, the molecule produced by the pathogen is beta-lactamase.

[0085] Preferably, the first peptide and the second peptide bind to non-pathogen surface components and / or molecules secreted by non-pathogens with very low affinity. Non-limiting examples can be when a protein is provided to a human subject, the protein has low binding specificity for human surface components (non-pathogen surface components).

[0086] In one embodiment of the invention, the first peptide and / or the second peptide is a single-domain antibody. In another embodiment, the protein is a DARPin.

[0087] According to one embodiment, the present invention relates to a protein, wherein the protein is homodivalent or heterodivalent.

[0088] The protein according to the present invention comprises the first peptide and the second peptide, which can bind to the following:

[0089] - The same surface component;

[0090] - Different surface components;

[0091] - Surface components and molecules;

[0092] - The same molecule; or

[0093] - Different molecules.

[0094] In one embodiment, the surface components are from the same pathogen.

[0095] In one embodiment, the surface components are from different pathogens.

[0096] In one embodiment, the protein according to the present invention is capable of crosslinking pathogens.

[0097] In one aspect of the present invention, wherein the first peptide and the second peptide bind to the same pathogen surface component, the protein is capable of crosslinking pathogens expressing the same surface component, such as the same pathogen species. In another aspect of the present invention, wherein the first peptide and the second peptide bind to the same pathogen surface component, the protein is capable of neutralizing pathogen adhesion and / or immobilizing the pathogen.

[0098] In one aspect of the present invention, wherein the first peptide and the second peptide bind to different pathogen surface components, the protein is capable of crosslinking pathogens expressing different surface components, such as crosslinking different pathogen species, or crosslinking different pathogen surface components present on the same pathogen species. In another aspect of the present invention, wherein the first peptide and the second peptide bind to different pathogen surface components, the protein is capable of neutralizing pathogen adhesion and / or immobilizing the pathogen.

[0099] In one embodiment, the protein is capable of crosslinking F4+ ETEC bacteria.

[0100] In another embodiment, the protein according to the present invention is capable of neutralizing pathogens and / or molecules produced by pathogens.

[0101] In one aspect of the present invention, wherein the first peptide and the second peptide bind to pathogen surface components and molecules secreted by pathogens, the protein is capable of crosslinking the molecules to the pathogens, such as neutralizing toxins secreted by pathogens.

[0102] In one aspect of the invention, wherein the first peptide and the second peptide bind to the same molecule, the protein is capable of neutralizing the molecule, such as neutralizing the action of secreted toxins and / or secreted proteases.

[0103] In one aspect of the invention, wherein the first peptide and the second peptide bind to different molecules, the protein is capable of neutralizing at least two different molecules, such as neutralizing the action of secreted toxins and / or secreted proteases (possibly in a combined manner).

[0104] In another embodiment, the protein according to the invention is capable of preventing biofilm formation.

[0105] In one embodiment, the protein in dimer form has an increased binding affinity compared to the monomeric protein. For example, a homodimeric protein construct comprising two identical single-domain antibodies has a binding affinity that is at least 1.5 times higher than that of the individual monomeric single-domain antibody.

[0106] In one embodiment, one of the first peptide or the second peptide binds to Escherichia coli bacteria, such as F4 + Escherichia coli bacteria and / or F18 + Escherichia coli bacteria.

[0107] In one embodiment, one of the first peptide or the second peptide binds to F4 + Escherichia coli bacteria, and wherein the other peptide binds to F18 + Escherichia coli bacteria.

[0108] In one embodiment, the surface component is selected from the group consisting of F18 + F4 + Stx2e, LT, and their animal and human variants.

[0109] According to one embodiment, the invention relates to a protein comprising at least two single-domain antibodies, wherein the protein is specific for at least a first virulence factor and a second virulence factor.

[0110] A virulence factor is a molecule produced by and / or part of a microorganism that causes disease.

[0111] A single-domain antibody is also referred to as "V" H"H fragment". A single-domain antibody is an antibody fragment consisting of a single monomeric variable antibody domain. Like a full antibody, it is capable of selectively binding to a specific antigen. The molecular weight of a single-domain antibody is only 12 - 15 kDa, much smaller than the common immunoglobulin G antibody (150 - 160 kDa) composed of two protein heavy chains and two light chains. A single-domain antibody is even smaller than the Fab fragment (~50 kDa, one light chain and half a heavy chain) and the single-chain variable fragment (~25 kDa, two variable domains, one from the light chain and one from the heavy chain). The first single-domain antibodies were engineered from heavy-chain antibodies found in camelids, which are called V H H fragment.

[0112] V H H can be derived from camelid heavy-chain antibodies and is naturally devoid of light chains.

[0113] The term "single-domain antibody construct" refers to at least one single-domain antibody linked to at least one other single-domain antibody or peptide by a linker.

[0114] The term "nanobody" refers to a single-domain antibody derived from natural sources such as camel, llama, or shark, selected and optimized for further use.

[0115] According to one embodiment, the present invention relates to a protein, wherein the second virulence factor is the same as or different from the first virulence factor.

[0116] According to one embodiment, the present invention relates to a protein, wherein the protein comprises a first single-domain antibody specific for the first virulence factor and a second single-domain antibody specific for the second virulence factor.

[0117] "Specific for..." generally means that a single-domain antibody binds to an epitope, and the binding requires some complementarity. By this definition, a single-domain antibody is said to "specifically bind" to an epitope when it binds to the epitope more readily than to a random unrelated epitope. The term "specificity" is used herein to quantify the relative affinity of a single-domain antibody for binding to an epitope.

[0118] As used herein, a protein "specific for a virulence factor" refers to a protein that binds to the virulence factor with a dissociation constant of 5 x 10 -7 M or less, 1 x 10 -7 M or less, 5 x 10 -8 M or less, 1 x 10 -8 M or less, 5 x 10 -9 M or less, 1 x 10 -9 M or less, 5 x 10 -10 M or less, 1 x 10 -10 M or less, 5 x 10 -11M or less or 1 x 10 -11 K of M or less d A protein that binds to a virulence factor. In certain embodiments, the protein has a K of 1 x 10 -8 M to 1 x 10 -10 K of M d binding. In certain other embodiments, the protein has a K of 1 - 5 x 10 -9 K of M d binding.

[0119] As used herein, "homobivalent" refers to the presence of two identical peptides, such as the V H H fragment.

[0120] As used herein, "heterobivalent" refers to the presence of two different peptides, such as the V H H fragment.

[0121] According to one embodiment, the present invention relates to a protein, wherein the protein is specific for 1, preferably 2, more preferably 3, preferably 4 pathogen surface components, such as virulence factors and / or molecules secreted by the pathogen. The virulence factors can be virulence factors from different groups or the same group of virulence factors of an organism.

[0122] According to one embodiment, the present invention relates to a protein, wherein the protein comprises a peptide specific for a virulence factor, such as a single - domain antibody, wherein the virulence factor forms part of or constitutes a target protein, and wherein the target protein is from the same protein sub - family. The protein sub - family can be defined according to the classification system of the Structural Classification of Proteins (SCOP) database.

[0123] According to one embodiment, the present invention relates to a protein, wherein at least one of the virulence factors is an adhesion factor.

[0124] As used herein, "adhesion factor" refers to a type of virulence factor that is a cell - surface component or appendage of a bacterium that promotes adhesion or binding to other cells or surfaces.

[0125] According to one embodiment, the present invention relates to a protein, wherein the first and second single - domain antibodies are specific for virulence factors from the same or two different microorganisms.

[0126] According to one embodiment, the present invention relates to a protein, wherein the first single - domain antibody is specific for an adhesion factor of a microorganism, and the second single - domain antibody is specific for a soluble virulence factor of the microorganism.

[0127] According to one embodiment, the present invention relates to a protein, wherein the one or more microorganisms are selected from Escherichia coli bacteria, Vibrio cholera bacteria, and Shigella bacteria.

[0128] According to one embodiment, the present invention relates to a protein, wherein the one or more pathogens are selected from F4+ Escherichia coli bacteria and F18+ Escherichia coli bacteria.

[0129] According to one embodiment, the present invention relates to a protein, wherein the first pathogen is F4 + Escherichia coli bacteria and the second pathogen is F18 + Escherichia coli bacteria.

[0130] Enterotoxigenic Escherichia coli (ETEC), Shigatoxigenic Escherichia coli (STEC), and Verotoxigenic Escherichia coli (VTEC) have two key virulence factors; an adhesion factor, usually fimbriae, and the production of one or more toxins that cause disease symptoms.

[0131] As used herein, "F4 + Escherichia coli" refers to an enterotoxigenic Escherichia coli strain (F4 + ETEC) carrying F4 fimbriae, which secretes one or more toxins, such as heat-labile toxin (LT), after colonization.

[0132] As used herein, "F18 + Escherichia coli" refers to an enterotoxigenic Escherichia coli strain (F18 + ETEC) carrying F18 fimbriae, which secretes one or more toxins, such as heat-labile toxin (LT) and / or heat-stable enterotoxin (ST), after colonization. F18 + STEC strains produce Shiga toxin Stx2e, and F18 + VTEC strains produce Shiga-like toxins.

[0133] According to one embodiment, the present invention relates to a protein, wherein the first pathogen produces heat-labile toxin (LT toxin).

[0134] According to one embodiment, the present invention relates to a protein, wherein the second pathogen produces Shiga toxin, such as porcine and human variants selected from stx2e and stx2e.

[0135] According to one embodiment, the present invention relates to a protein, wherein the first and / or second pathogen is ETEC Escherichia coli with fimbrial type or fimbrial adhesion, such as F5, F6, F41 or AIFA, human-specific variants, such as colonization factor antigen (CFA) or Escherichia coli surface antigen (CS), including but not limited to CFAI, CFAII, CFAIII and CFAIV.

[0136] According to one embodiment, the present invention relates to a protein, wherein the first and / or second pathogen produces Shiga toxin or Shiga-like toxin, such as Stx2e and human variants, including but not limited to stx, stx1a, stx1c, stxd, stx2a, stx2b, stx2c, stx2d, stx2f and stx2g.

[0137] According to one embodiment, the present invention relates to a protein, wherein the virulence factors are selected from ETEC fimbrial adhesins such as F4 and F18, heat-labile enterotoxin (LT), and Shiga toxin or Shiga-like toxin such as Stx2e, and porcine and human variants thereof.

[0138] According to one embodiment, the present invention relates to a protein, wherein the virulence factor is selected from the group consisting of: F18 + , F4 + , Stx2e, LT and porcine and human variants thereof.

[0139] In one embodiment of the present invention, the first peptide and / or the second peptide comprises an amino acid sequence having at least 90%, such as at least 95%, such as at least 98%, such as at least 99% sequence identity with the amino acid sequences of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9 and / or SEQ ID NO:10.

[0140] The present invention also encompasses the use of peptides having a certain degree of sequence identity or sequence homology with the amino acid sequences defined herein or with peptides having the specific properties defined herein. The present invention particularly encompasses peptides having a certain degree of sequence identity with SEQ ID NO:1-10 or homologues thereof. Here, the term "homologue" means an entity having sequence identity with the subject amino acid sequence.

[0141] In some embodiments of the present invention, the first peptide and / or the second peptide consist essentially of four "framework regions", which are referred to in the art and hereinafter as "framework region 1" or "FR1"; "framework region 2" or "FR2"; "framework region 3" or "FR3"; "framework region 4" or "FR4"; these framework regions are separated by three "complementary determining regions" or "CDRs", which are referred to in the art and hereinafter as "complementary determining region 1" or "CDR1"; "complementary determining region 2" or "CDR2"; "complementary determining region 3" or "CDR3". Thus, the overall structure or sequence of the first peptide and / or the second peptide can be represented as follows: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. The first peptide and / or the second peptide confer the first and / or second binding specificities of the first peptide and / or the second peptide.

[0142] In one embodiment, the first peptide and / or the second peptide comprise CDR1, CRD2 and / or CDR3 within the amino acid sequences of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9 and / or SEQ ID NO:10.

[0143] In one embodiment, any sequence variations are outside the CDRs.

[0144] In one embodiment, the sequence variations are within the framework regions of the first peptide and / or the second peptide.

[0145] In one embodiment, the first peptide and / or the second peptide comprise three complementary determining regions CDR1, CDR2 and CDR3, wherein CDR1 has an amino acid sequence comprising the amino acid sequence of SEQ ID NO:27, CDR2 has an amino acid sequence comprising the amino acid sequence of SEQ ID NO:29, and CDR3 has an amino acid sequence comprising the amino acid sequence of SEQ ID NO:31.

[0146] In one embodiment, the first peptide and / or the second peptide comprise three complementary determining regions CDR1, CDR2 and CDR3, wherein CDR1 has an amino acid sequence comprising the amino acid sequence of SEQ ID NO:28, CDR2 has an amino acid sequence comprising the amino acid sequence of SEQ ID NO:30, and CDR3 has an amino acid sequence comprising the amino acid sequence of SEQ ID NO:32.

[0147] In one embodiment, the protein comprises a first peptide and a second peptide, wherein the first peptide comprises three complementarity determining regions CDR1, CDR2, and CDR3, where CDR1 has an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 27, CDR2 has an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 29, and CDR3 has an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 31, and wherein the second peptide comprises three complementarity determining regions CDR1, CDR2, and CDR3, where CDR1 has an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 28, CDR2 has an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 30, and CDR3 has an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 32. In one aspect, homologous amino acid sequences should provide peptides that retain the functional activity of the peptides of SEQ ID NOs: 1-10.

[0148] In one embodiment, the first peptide and / or the second peptide of SEQ ID NOs: 1-4 and SEQ ID NO: 10 binds to FaeG on F4+ Escherichia coli bacteria.

[0149] In another embodiment, the first peptide and / or the second peptide of SEQ ID NO: 5 binds to the B subunit of Stx2e.

[0150] In another embodiment, the first peptide and / or the second peptide of SEQ ID NOs: 6-8 binds to FedF on F18+ Escherichia coli bacteria.

[0151] In another embodiment, the first peptide and / or the second peptide of SEQ ID NO: 9 binds to the B subunit of LT.

[0152] In this context, homologous sequences are considered to include amino acid sequences that are at least 90%, at least 95%, at least 98%, or at least 99% identical to the subject sequence. Generally, homologs will contain the same active sites and the like as the subject amino acid sequence. Although homology can also be considered similarity (i.e., amino acid residues with similar chemical properties / functions), in the context of the present invention, homology preferably denotes sequence identity.

[0153] In one embodiment, the protein according to the present invention comprises a first peptide and a second peptide, wherein both the first peptide and the second peptide comprise an amino acid sequence having at least 90%, such as at least 95%, such as at least 98%, such as at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 8.

[0154] In another embodiment, the protein of the present invention comprises a first peptide and a second peptide, wherein both the first peptide and the second peptide comprise an amino acid sequence having at least 90%, such as at least 95%, such as at least 98%, such as at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 10.

[0155] In another embodiment, the protein of the present invention comprises a first peptide and a second peptide, wherein both the first peptide and the second peptide comprise an amino acid sequence having at least 90%, such as at least 95%, such as at least 98%, such as at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 5.

[0156] In another embodiment, the protein of the present invention comprises a first peptide and a second peptide, wherein both the first peptide and the second peptide comprise an amino acid sequence having at least 90%, such as at least 95%, such as at least 98%, such as at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 9.

[0157] In another embodiment, the protein of the present invention comprises a first peptide and a second peptide, wherein the first peptide comprises an amino acid sequence having at least 90%, such as at least 95%, such as at least 98%, such as at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 10, and wherein the second peptide comprises an amino acid sequence having at least 90%, such as at least 95%, such as at least 98%, such as at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 9.

[0158] In another embodiment, the protein of the present invention comprises a first peptide and a second peptide, wherein the first peptide comprises an amino acid sequence having at least 90%, such as at least 95%, such as at least 98%, such as at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 9, and wherein the second peptide comprises an amino acid sequence having at least 90%, such as at least 95%, such as at least 98%, such as at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 10.

[0159] Nucleic acid

[0160] In one aspect, the present invention provides an isolated nucleic acid molecule encoding the protein described herein.

[0161] "Nucleic acid molecule" includes DNA (such as genomic DNA or complementary DNA) and mRNA molecules, which may be single-stranded or double-stranded. "Isolated" means that the nucleic acid molecule is not located within a cell or otherwise provided within a cell.

[0162] Vector

[0163] In one aspect, the present invention provides a vector comprising the nucleic acid molecule described herein. In one embodiment, the vector is an expression vector.

[0164] Host cell

[0165] In one aspect, the present invention provides a recombinant host cell comprising the nucleic acid molecule or vector described herein. In one embodiment, the host cell is a bacterium, a fungus such as yeast and / or a mammalian cell.

[0166] In one embodiment, the bacterium according to the present invention may be Bacillus, such as Bacillus licheniformis, Bacillus subtilis and / or Bacillus lactobacillus.

[0167] In another embodiment, the yeast according to the present invention may be any species selected from the genus Pichia, the genus Hansenula and the genus Saccharomyces.

[0168] In another embodiment, the fungus according to the present invention may be Aspergillus oryzae and Aspergillus niger.

[0169] Composition

[0170] Dietary composition

[0171] The dietary composition according to the present invention may comprise the protein as described above. The dietary composition may further comprise one or more of the following: prebiotics, probiotics, synbiotics, proteins, lipids, carbohydrates, vitamins, fibers and / or nutrients, such as dietary minerals, for a living subject.

[0172] In one aspect, the dietary composition is a food additive.

[0173] In another aspect, the present invention relates to the use of the dietary composition in promoting the growth of a subject.

[0174] In another aspect, the present invention relates to the use of the dietary composition as an additive to an infant formula product.

[0175] In another aspect, the present invention relates to the use of the dietary composition as a protective aid against pathogen infection in a subject during travel.

[0176] In another aspect, the present invention relates to the use of a dietary composition as a protective adjuvant against pathogen infections in subjects with impaired immune efficiency, such as the elderly.

[0177] In various aspects of the present invention, the use of the dietary composition is non-therapeutic.

[0178] Pharmaceutical composition

[0179] The present invention also provides a pharmaceutical composition comprising the proteins, nucleic acids, vectors, and / or recombinant host cells described herein and further comprising one or more compounds. The compounds can be active compounds and / or excipients.

[0180] In one embodiment, the active compound and / or excipient is one or more complex carbohydrates, such as cellulose and / or alginate, enzymes, activated carbon, surfactants, antibiotics, antiadherents, binders, coating agents, disintegrants, fillers, solvents / cosolvents, flavoring agents, pigments, lubricants, glidants, preservatives, adsorbents, sweeteners, carriers, polymers, buffers, antioxidants, wetting agents, antifoaming agents, thickening agents, humectants, and mixtures thereof.

[0181] According to one embodiment, the present invention relates to a composition comprising a protein and optionally one or more excipients, diluents, proteins, and / or binders. According to one embodiment, the composition according to the present invention may further or optionally comprise one or more of the following: adhiron, adnectin, affibody, affitin, anticalin, armadillo repeat protein, avimer, beta-hairpin mimetics, bicyclic peptide, DARPin, and / or fynomer.

[0182] The present invention also provides the proteins described above, as well as a dietary composition comprising one or more of the proteins, and / or a pharmaceutical composition comprising one or more of the proteins.

[0183] According to one embodiment, the present invention relates to a composition comprising a first single-domain antibody specific for a first virulence factor and a second single-domain antibody specific for a second virulence factor.

[0184] According to one embodiment, the present invention relates to a composition wherein the second single-domain antibody is the same as or different from the first single-domain antibody.

[0185] According to one embodiment, the present invention relates to a composition, wherein the second virulence factor is the same as or different from the first virulence factor.

[0186] According to one embodiment, the present invention relates to a composition comprising single domain antibodies specific for 2, preferably 3, more preferably 4, preferably 5, more preferably 6, preferably 7, more preferably 8, preferably 9, more preferably 10, preferably 11, more preferably 12 different virulence factors. According to one embodiment, the single domain antibodies can form part of one or more, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 proteins.

[0187] According to one embodiment, the present invention relates to a composition comprising a protein of the present invention and one or more additional single domain antibodies, preferably 1 - 5, more preferably 2 - 3 different single domain antibodies, wherein the different single domain antibodies are each specific for at least one virulence factor.

[0188] According to one embodiment, the present invention relates to a protein or composition, wherein the protein or composition comprises a first single domain antibody specific for the first virulence factor and a second single domain antibody specific for the second virulence factor.

[0189] According to one embodiment, the present invention relates to a composition, wherein at least one of the virulence factors is an adhesion factor.

[0190] As used herein, "adhesion factor" refers to a virulence factor that is a cell surface component or appendage of a bacterium that promotes adhesion or binding to other cells or surfaces.

[0191] According to one embodiment, the present invention relates to a composition, wherein the first and second single domain antibodies are specific for virulence factors from the same or two different microorganisms.

[0192] According to one embodiment, the present invention relates to a composition, wherein the first single domain antibody is specific for an adhesion factor of a microorganism and the second single domain antibody is specific for a soluble virulence factor of the microorganism.

[0193] According to one embodiment, the present invention relates to a composition, wherein the one or more microorganisms are selected from Escherichia coli bacteria, Vibrio cholera and Shigella bacteria.

[0194] According to one embodiment, the present invention relates to a composition, wherein the virulence factors are selected from ETEC fimbrial adhesins such as F4 and F18, heat-labile enterotoxin (LT), and Shiga toxin or Shiga-like toxin such as Stx2e, and their porcine and human variants.

[0195] According to one embodiment, the present invention relates to a composition, wherein the virulence factors are selected from the group consisting of: F18 + , F4 + , Stx2e, LT and their porcine and human variants.

[0196] Disease

[0197] In one aspect, the present invention provides the proteins, nucleic acids, vectors, recombinant host cells or pharmaceutical compositions described herein for use as a medicament.

[0198] In another aspect, the present invention provides the proteins, nucleic acids, vectors, recombinant host cells or pharmaceutical compositions described herein for preventing or treating pathogen-induced infections associated with the inner surface and / or outer surface of a subject. In one embodiment, the infection is a gastrointestinal infection. In another embodiment, the infection is a pulmonary infection.

[0199] In one aspect, the route of administration of the protein, nucleic acid, vector, host cell or pharmaceutical composition is oral, pulmonary and / or topical. In one embodiment, it is administered by a nebulizer.

[0200] In one embodiment, the protein, nucleic acid, vector, host cell or pharmaceutical composition is administered to a subject one or more times.

[0201] In one aspect, the present invention provides the proteins, nucleic acids, vectors, recombinant host cells or pharmaceutical compositions described herein for reducing the time span of active infection in a subject. In one embodiment, the time span of active infection is reduced by at least 20%, such as at least 30%, such as at least 40%, such as at least 50%.

[0202] In one embodiment, the protein is capable of reducing the time span of active infection of F4 ETEC infection in a subject such as a piglet.

[0203] In another aspect, the present invention provides the proteins, nucleic acids, vectors, recombinant host cells or pharmaceutical compositions described herein for reducing the virulence in a subject.

[0204] In one embodiment, the subject is a human. In another embodiment, the subject is a domestic animal, such as an animal selected from the group consisting of pigs, cows, sheep, goats, horses, chickens, donkeys, mules, ducks, geese, and turkeys. Preferably, the subject is a pig.

[0205] In another aspect, the present invention provides the protein, nucleic acid, vector, recombinant host cell, or pharmaceutical composition described herein for preventing or treating post-weaning diarrhea (PWD).

[0206] In another aspect, the present invention provides the protein, nucleic acid, vector, recombinant host cell, or pharmaceutical composition described herein for preventing or treating edema disease.

[0207] According to one embodiment, the present invention relates to a protein or composition for use as a medicament.

[0208] According to one embodiment, the present invention relates to a protein or composition for treating an animal or a human.

[0209] According to one embodiment, the present invention relates to a protein or composition for prophylactic use in an animal or a human.

[0210] According to one embodiment, the present invention relates to a protein or composition for non-systemic use, and / or wherein the protein does not interact with the stomach lining, and preferably does not interact with epithelial cells.

[0211] According to one embodiment, the present invention relates to a protein or composition, wherein the protein is stable in the gastrointestinal tract.

[0212] "Stable in the gastrointestinal tract" means that a product containing the single-domain antibody retains 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more of its binding activity after passing through the gastrointestinal tract.

[0213] According to one embodiment, the present invention relates to a protein or composition, wherein the protein or composition is used as an additive to an infant formula product, will be used in combination with an infant formula product, or is formulated as part of an infant formula for animals or humans.

[0214] According to one embodiment, the present invention relates to a protein or composition, wherein the protein or composition is used as a component of artificial colostrum.

[0215] According to one embodiment, the present invention relates to a protein or composition for oral or pulmonary administration.

[0216] According to one embodiment, the present invention relates to a method of treating or preventing a medical condition in an animal or a human, comprising administering the protein or composition.

[0217] According to one embodiment, the present invention relates to the use of the protein or composition in the treatment or prevention of post-weaning diarrhea (PWD).

[0218] In the farming industry, enterotoxigenic Escherichia coli (ETEC) and Shiga toxin-producing Escherichia coli (STEC) are important pathogens, causing high mortality and severe production losses. In piglets, ETEC, VTEC, and STEC strains expressing F18 + fimbriae are associated with post-weaning diarrhea. After the initial adhesion step facilitated by F4 or F18 fimbriae, ETEC strains produce and secrete LT and / or Stx2e, thus stimulating the secretion of electrolytes and water. This results in dehydration of intestinal epithelial cells and watery diarrhea. F18 + STEC strains produce the Shiga toxin Stx2e, and F18 + VTEC strains produce Shiga-like toxins. Damage to vascular endothelium ultimately leads to edema, hemorrhage, and microthrombosis, and this is fatal in 90% of all animals infected with VTEC.

[0219] Use of the protein or composition as claimed in any of the preceding claims in the treatment or prevention of edema disease. Edema disease is a severe disease characterized by neurological disorders, hemorrhagic lesions, and frequent fatal outcomes. Swine edema disease is caused by enterotoxigenic and verotoxin-producing Stx2e-producing Escherichia coli.

[0220] According to one embodiment, the present invention relates to non-therapeutic uses of the protein or composition.

[0221] According to one embodiment, the present invention relates to non-therapeutic uses of the protein or composition for promoting the growth of livestock.

[0222] According to one embodiment, the present invention relates to the use of the protein or composition as a mammalian feed or feed additive.

[0223] According to one embodiment, the present invention relates to the use of the protein or composition, wherein the protein or composition is dissolved or dispersed in water for mammals, such as drinking water or water administered in combination with feed.

[0224] According to one embodiment, the present invention relates to the use of the protein or composition in combination with at least one antimicrobial agent.

[0225] According to one embodiment, the present invention relates to a use, wherein the antimicrobial agent is zinc oxide.

[0226] According to one embodiment, the present invention relates to the use of a protein or a composition in stabilizing the microbiome of a human or an animal, preferably for use in the preparation of an agent for stabilizing the microbiome of a human or an animal.

[0227] According to one embodiment, the present invention relates to the use of a protein or a composition as a non-therapeutic supplement for a human or an animal.

[0228] According to one embodiment, the present invention relates to the use of a protein or a composition as a protective aid against bacterial intestinal infections during travel.

[0229] According to one embodiment, the present invention relates to the use of a protein or a composition as a protective aid against bacterial intestinal infections in subjects with impaired immune efficacy, such as the elderly, people with down-regulated immune efficacy including HIV patients, organ recipients, patients receiving rheumatism treatment, patients receiving cancer treatment, and / or patients receiving treatment for bacterial infections (such as pneumonia and sepsis).

[0230] According to one embodiment, the present invention relates to the use of a protein or a composition in the prevention or treatment of an infection caused by Vibrio cholerae.

[0231] According to one embodiment, the present invention relates to the medical use of a protein or a composition.

[0232] According to one embodiment, the present invention relates to a method for preparing a protein or a composition, wherein at least one single-domain antibody is expressed by a fungal host.

[0233] According to one embodiment, the present invention relates to a method for preparing a protein or a composition, wherein the fungal host is a yeast strain.

[0234] According to one embodiment, the present invention relates to a method for preparing a protein or a composition, wherein the fungal host is selected from the group consisting of Aspergillus, Pichia, Hansenula, and Saccharomyces.

[0235] According to one embodiment, the present invention relates to the use of a protein or a composition in the preparation of a food ingredient for animal or human food.

[0236] Use of a protein, a dietary composition, and / or a pharmaceutical composition

[0237] In one embodiment, the present invention relates to a method of cross-linking a pathogen, the method comprising administering to a subject an effective amount of the protein, dietary composition, and / or pharmaceutical composition of any one of the preceding claims. The protein, dietary composition, and pharmaceutical composition are as described herein.

[0238] In another embodiment, the present invention relates to a method of preventing biofilm formation, the method comprising administering to a subject an effective amount of the protein, dietary composition, and / or pharmaceutical composition of any one of the preceding claims. The protein, dietary composition, and pharmaceutical composition are as described herein.

[0239] In another embodiment, the present invention relates to a method of neutralizing a pathogen and / or a molecule secreted by a pathogen, the method comprising administering to a subject an effective amount of the protein, dietary composition, and / or pharmaceutical composition of any one of the preceding claims. The protein, dietary composition, and pharmaceutical composition are as described herein.

[0240] The present invention also relates to the use of the protein, nucleic acid, vector, or host cell of any one of the preceding claims in the preparation of a medicament for preventing or treating a pathogen-induced infection associated with the inner surface and / or outer surface of a subject.

[0241] Treatment methods

[0242] In one aspect, the present invention provides a method of treating or preventing a pathogen-induced infection, such as a gastrointestinal infection and / or a pulmonary infection, the method comprising administering to a subject a therapeutically effective amount of the protein, nucleic acid, vector, recombinant host cell, or pharmaceutical composition described herein.

[0243] An "effective amount" or "therapeutically effective amount" of a compound is an amount of the compound sufficient to provide a beneficial effect to a subject to which the compound is administered. As used herein, the phrase "therapeutically effective amount" may refer to an amount of a protein sufficient to treat (delay or prevent onset, prevent development, inhibit, reduce, or reverse) an infection.

[0244] Pathogen

[0245] The pathogen can be any pathogen, such as bacteria, viruses, fungi, protozoa, and / or worms.

[0246] In one embodiment, the pathogen is Escherichia coli bacteria, Vibrio cholera bacteria, Salmonella, Campylobacter bacteria, Staphylococcus bacteria, Listeria bacteria, Shigella bacteria, Mycoplasma, and / or Clostridium difficile.

[0247] In another embodiment, the pathogen is F4 + Escherichia coli bacteria and / or F18 + Escherichia coli bacteria.

[0248] In another embodiment, one of the first peptide or the second peptide binds to F4 + Escherichia coli bacteria, and the other peptide binds to F18 + Escherichia coli bacteria.

[0249] In another embodiment, the pathogen produces a heat-labile toxin (LT toxin).

[0250] In another embodiment, the pathogen produces Shiga toxin, such as porcine and human variants selected from stx2e and stx2e, or Shiga-like toxin.

[0251] In one embodiment, the pathogen is enterotoxigenic Escherichia coli (ETEC) with a fimbrial type or fimbrial adhesion, such as F5, F6, F41, or AIFA, human-specific variants such as colonization factor antigen (CFA) or Escherichia coli surface antigen (CS), including but not limited to CFAI, CFAII, CFAIII, and CFAIV.

[0252] In one embodiment, the pathogen produces Shiga toxin or Shiga-like toxin, such as Stx2e and human variants, including but not limited to stx, stx1a, stx1c, stxd, stx2a, stx2b, stx2c, stx2d, stx2f, and stx2g.

[0253] Production method

[0254] The present invention provides a method for producing the protein described in any of the above aspects, the method comprising culturing the host cell as defined herein under conditions that permit the expression of the encoded protein.

[0255] Accordingly, the present invention relates to a method for producing the protein described herein, the method comprising:

[0256] a) transfecting the nucleic acid molecule described above and / or the vector described above into a host cell to obtain the recombinant host cell described above;

[0257] b) culturing the recombinant host cell under suitable conditions;

[0258] c) collecting and purifying the protein expressed by the recombinant host cell.

[0259] Accordingly, the recombinant host cell can be cultured under conditions that permit the expression of the encoded protein.

[0260] Item

[0261] 1. A protein comprising at least two single-domain antibodies, wherein the protein is specific for at least a first virulence factor and a second virulence factor.

[0262] 2. The protein according to item 1, wherein the second virulence factor is the same as or different from the first virulence factor.

[0263] 3. The protein according to any one of the preceding items, wherein the at least two single-domain antibodies are linked together by a linker.

[0264] 4. The protein according to any one of the preceding items, wherein the at least two single-domain antibodies are linked together by a GS linker.

[0265] 5. The protein according to item 4, wherein the GS linker has the structure (G x S) n where x can be a number between 1 and 10, preferably 2 to 5, and n refers to the number of repetitions of the GS sequence, where n can be between 1 and 10, preferably 2 to 5. x 6. The protein according to item 4 or 5, wherein the GS linker is the GGGGSGGGGSGGGGS linker (SEQ ID NO: 22).

[0266] 7. The protein according to any one of the preceding items, wherein the protein comprises a first single-domain antibody specific for the first virulence factor and a second single-domain antibody specific for the second virulence factor.

[0267] 8. The protein according to any one of the preceding items, wherein the protein is homodivalent or heterodivalent.

[0268] 9. The protein according to any one of the preceding items, wherein the protein is specific for 1, preferably 2, more preferably 3, preferably 4 virulence factors.

[0269] 9. The protein according to any one of the preceding items, wherein the protein is specific for 1, preferably 2, more preferably 3, preferably 4 virulence factors.

[0270] 10. The protein according to any one of the preceding items, wherein the protein comprises a single-domain antibody specific for a virulence factor, wherein the virulence factor forms part of or constitutes a target protein, and wherein the target protein is from the same protein subfamily.

[0271] 11. The protein according to item 10, wherein the protein subfamily is defined according to the SCOP system.

[0272] 12. A composition comprising the protein according to any one of the preceding items and optionally one or more excipients, diluents, proteins, and / or binders.

[0273] 13. The composition according to item 12, the composition comprising a plurality of different proteins, each of the proteins being as described in any one of the preceding items, wherein the number of proteins is selected from the group consisting of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12.

[0274] 14. The composition according to item 13, wherein the proteins are all specific for one or two virulence factors.

[0275] 15. A composition comprising a first single-domain antibody specific for a first virulence factor and a second single-domain antibody specific for a second virulence factor.

[0276] 16. The composition according to item 15, wherein the second single-domain antibody is the same as or different from the first single-domain antibody.

[0277] 17. The composition according to item 15 or 16, wherein the second virulence factor is the same as or different from the first virulence factor.

[0278] 18. The composition according to any one of items 12 to 17, comprising single-domain antibodies specific for 2, preferably 3, more preferably 4, preferably 5 different virulence factors.

[0279] 19. The composition according to any one of items 12 to 18, comprising the protein according to any one of items 1 to 11, and one or more additional single-domain antibodies, preferably 1 to 5, more preferably 2 to 3 different single-domain antibodies, wherein the different single-domain antibodies are all specific for at least one virulence factor.

[0280] 20. The protein or composition according to any one of the preceding items, wherein the protein or composition comprises a first single-domain antibody specific for the first virulence factor and a second single-domain antibody specific for the second virulence factor.

[0281] 21. The protein or composition according to any one of the preceding items, comprising at least one single-domain antibody that neutralizes a toxin.

[0282] 22. The protein or composition according to any one of the preceding items, wherein at least one of the virulence factors is an adhesion factor.

[0283] 23. The protein or composition according to any one of the preceding items, wherein the first and second single-domain antibody pairs are specific for virulence factors from the same or two different microorganisms.

[0284] 24. The protein or composition according to any one of the preceding items, wherein the first single-domain antibody is specific for an adhesion factor of a microorganism, and the second single-domain antibody is specific for a soluble virulence factor of the same or a different microorganism.

[0285] 25. The protein or composition according to any one of items 23 to 24, wherein the one or more microorganisms are selected from Escherichia coli bacteria, Vibrio cholerae bacteria, and Shigella bacteria.

[0286] 26. The protein according to any one of items 23 to 25, wherein the one or more microorganisms are selected from F4 + Escherichia coli bacteria and F18 + Escherichia coli bacteria.

[0287] 27. The protein according to any one of items 23 to 26, wherein the first microorganism is F4 + Escherichia coli bacteria and the second microorganism is F18 + Escherichia coli bacteria.

[0288] 28. The protein according to any one of items 23 to 27, wherein the first microorganism produces a heat-labile toxin (LT toxin).

[0289] 29. The protein according to any one of items 23 to 28, wherein the second microorganism produces a Shiga toxin, such as porcine and human variants selected from stx2e and stx2e, or a Shiga-like toxin.

[0290] 30. The protein according to any one of items 23 to 29, wherein the first and / or second microorganism is ETEC Escherichia coli with a fimbrial type or fimbrial adhesion: such as F5, F6, F41, or AIFA, human-specific variants such as colonization factor antigen (CFA) or Escherichia coli surface antigen (CS), including but not limited to CFAI, CFAII, CFAIII, and CFAIV.

[0291] 31. The protein according to any one of items 23 to 30, wherein the first and / or second microorganism produces Shiga toxin or Shiga-like toxin, such as Stx2e and human variants, including but not limited to stx, stx1a, stx1c, stxd, stx2a, stx2b, stx2c, stx2d, stx2f, and stx2g.

[0292] 32. The protein or composition according to any one of the preceding items, wherein the virulence factor is selected from ETEC fimbrial adhesins such as F4 and F18, heat-labile enterotoxin (LT), and Shiga toxin or Shiga-like toxin such as Stx2e and its porcine and human variants.

[0293] 33. The protein or composition according to any one of the preceding items, wherein the virulence factor is selected from the group consisting of + F18 + , F4

[0294] , Stx2e, LT, and their porcine and human variants.

[0295] 34. The protein or composition according to any one of the preceding items, used as a medicine.

[0296] 35. The protein or composition according to item 34, for treating animals or humans.

[0297] 36. The protein or composition according to any one of the preceding items, for prophylactic use in animals or humans.

[0298] 37. The protein or composition according to any one of the preceding items, for non-systemic use, and / or wherein the protein does not interact with the gastric mucosa and preferably does not interact with epithelial cells.

[0299] 38. The protein or composition according to any one of the preceding items, wherein the protein or composition is stable in the gastrointestinal tract.

[0300] 39. The protein or composition according to any one of the preceding items, wherein the protein or composition is used as an additive to an infant formula product, used in combination with an infant formula product, or formulated as part of an infant formula for animals or humans.

[0301] 40. The protein or composition according to any one of the preceding items, wherein the protein or composition is used as a component of artificial colostrum and / or natural colostrum or used therewith.

[0302] 41. The protein or composition according to any one of the preceding items, for oral or pulmonary administration.

[0303] 42. A method for treating or preventing a medical condition in an animal or a human, comprising administering the protein or composition according to any one of the preceding items.

[0303] 43. Use of the protein or composition according to any one of the preceding items in the treatment or prevention of post-weaning diarrhea (PWD).

[0304] 44. Use of the protein or composition according to any one of the preceding items in the treatment or prevention of edema disease.

[0305] 45. Non-therapeutic use of the protein or composition according to any one of items 1 to 33, 36 to 41.

[0306] 46. Therapeutic or non-therapeutic use of the protein or composition according to any one of the preceding items to promote the growth of livestock.

[0307] 47. Use of the protein or composition according to any one of the preceding items as a feed or feed additive for mammals.

[0308] 48. Use of the protein or composition according to any one of the preceding items, wherein the protein or composition is dissolved or dispersed in water for mammals, such as drinking water or water administered in combination with feed.

[0309] 49. Use of the protein or composition according to any one of the preceding items in combination with at least one antimicrobial agent.

[0310] 50. Use according to item 49, wherein the antimicrobial agent is zinc oxide.

[0311] 51. Use of the protein or composition according to any one of the preceding items in stabilizing the microbiota of humans or animals, preferably for use in the preparation of an agent for stabilizing the microbiota of humans or animals.

[0312] 52. Use of the protein or composition according to any one of the preceding items as a therapeutic or non-therapeutic supplement for humans or animals.

[0313] 53. Use of the protein or composition according to any one of the preceding items as a protective aid against bacterial intestinal infections during travel.

[0314] 54. Use of the protein or composition according to any one of the preceding items as a protective aid against bacterial intestinal infections in persons with impaired immune efficacy, such as the elderly, persons with down-regulated immune efficacy including HIV patients, organ recipients, patients receiving rheumatism treatment, patients receiving cancer treatment, and / or patients receiving medical treatment for bacterial infections (such as pneumonia and sepsis).

[0315] 55. Use of the protein or composition according to any one of the preceding items for the prevention or treatment of infections caused by Vibrio cholerae.

[0316] 56. Medical use of the protein or composition according to any one of the preceding items.

[0317] 57. A method for preparing the protein or composition according to any one of the preceding items, wherein at least one protein comprising a single-domain antibody is expressed by a fungal host.

[0318] 58. The method for preparing a protein or composition according to item 57, wherein the fungal host is a yeast strain.

[0319] 59. A method for preparing the protein or composition according to items 57 to 58, wherein the fungal host is selected from the group consisting of Aspergillus, Pichia, Hansenula, and Saccharomyces.

[0320] 60. Use of the protein or composition according to any one of the preceding items in the preparation of a food ingredient for animal or human food, preferably wherein the protein or composition is used together with probiotics, prebiotics, and / or synbiotics.

[0321] Examples

[0322] The following examples are intended to illustrate the invention and should in no way be construed as limiting.

[0323] Example 1: Construction of a single-domain antibody construct for expression in Aspergillus oryzae

[0324] All single-domain antibodies used in this study were generated, subsequently selected and optimized in published studies; Lo et al. 2014 (The molecular Mechanism of Shiga Toxin stx2e Neutralization by Single-domain Antibody Targeting the Cell Receptor-binding Domain); Moonens et al. 2014 (Nanobody Mediated Inhibition of Attachment of F18 Fimbriae Expressing Escherichia coli); Virdi et al. 2013 (Orally fed seeds producing designer IgA protect weaned piglets against enterotoxigenic Escherichia coli infection) and Harmsen et al. 2006 (Selection and optimization of proteolytically stable llama single-domain antibody fragments for oral immunotherapy). However, what all single-domain antibodies have in common is that they were all obtained by immunizing healthy llamas with F4, F18, stx4e or LT antigens, and at specific time points after immunization, blood samples were collected from the animals. Blood lymphocytes, which are the genetic source of the single-domain antibodies, were isolated from the blood samples and total RNA was extracted. According to standard methods of nucleotide PCR, cloning and ligation, the nanobody gene sequences were cloned into suitable vectors and transformed into Escherichia coli to generate a clone library. To express single-domain antibodies in Aspergillus oryzae, synthetic genes with different single-domain antibodies were constructed as described by Christensen et al. 1988 (High Level Expression of Recombinant Genes in Aspergillus Oryzae). For bivalent single-domain antibody constructs, a linker region was introduced, the linker (GGGGS) 3 or the porcine IgG3 hinge region.

[0325] The single-domain antibody constructs and specific targets are described in Table 1.

[0326] Table 1: Single-domain antibody constructs

[0327]

[0328]

[0329] Example 2: Selection and production of Aspergillus oryzae expressing single-domain antibody constructs

[0330] The plasmid was transformed into Aspergillus oryzae, a filamentous fungus capable of producing and secreting proteins during fermentation. The transformation was carried out as described by Christensen et al. 1988 (High Level Expression of Recombinant Genes in Aspergillus Oryzae). The transformants producing the single-domain antibody constructs were identified by an additional band of approximately 15 kD appearing on the SDS gel compared to the supernatant from the untransformed parental strain. The cultures were filtered and the resulting supernatant containing the single-domain antibody constructs was used for further analysis.

[0331] Example 3: In vitro testing of the binding ability of homodimeric and heterodimeric protein constructs

[0332] Methods and materials

[0333] To detect the binding of the protein to bacteria, an ELISA assay was designed. Briefly, Nunc MaxiSorp TM plates F96 were coated overnight (O / N) at 4 °C with approximately 5 - 25 ng / mL of the unlabeled F45::(GGGGS) 3 ::F45 homodimeric protein construct (SEQ ID NO:13) in phosphate-buffered saline (PBS) pH 7.4. After blocking with milk or bovine serum albumin (3%), heat-inactivated F4+ Escherichia coli (0149:F4) with OD600 = 0.2 was added to the milk or BSA and incubated on the plates at room temperature. After washing in PBS and heat-inactivating the bacteria in a 56 °C water bath for 1 hour. To detect Escherichia coli, FLAG-tagged protein constructs encoding F45::(GGGGS) 3 ::F45 (SEQ ID NO:13) or F45::(GGGGS) 3 ::LT1 (SEQ ID NO:16) were added after washing. The homodimeric FLAG or heterodimeric FLAG protein constructs were detected using anti-FLAG-HRP (A8592) from SIGMA. The substrate solution (a 1:1 mixture of H 2 O 2 and tetramethylbenzidine) was developed, and an equal volume of 2M H 3 PO 4Stop the reaction. Measure the optical density at 450 nm using a microplate reader.

[0334] Results

[0335] When the product binds in the ELISA assay, the measured OD450 represents the signal, and the higher the signal, the more product is bound. Thus, F45::(GGGGS) 3 ::F45 homodimeric protein construct and F45::(GGGGS) 3 ::LT1 heterodimeric protein construct binding was confirmed in an ELISA experiment for binding F4 ETEC bacteria (see Figure 4 ). The included negative control F183 (SEQ ID NO:8) is specific for FedF and not for FaeG.

[0336] Conclusions

[0337] The homodimeric and heterodimeric protein constructs are active and bind to F4 ETEC bacteria.

[0338] Example 4: In vitro testing of the citric acid stability of single-domain antibodies and homodimeric protein constructs Methods and materials

[0339] Compare the citric acid stability between monomeric single-domain antibodies and homodimeric protein constructs. First, coat the wells of a plate with the unlabeled F45::(GGGGS) 3 ::F45 homodimeric protein construct, then add bacteria for binding, wash away the excess bacteria, and then add either the FLAG-tagged F45 single-domain antibody (SEQ ID NO:10) or the F45::(GGGGS) 3 ::F45 homodimeric protein construct (SEQ ID NO:13). After washing, add 0.2 M citric acid to the wells. Transfer the citric acid eluate to another ELISA plate that is also coated with the unlabeled F45::(GGGGS) 3 ::F45 homodimeric protein construct, and bind to the bacteria. Then neutralize the citric acid with NaOH and allow the single-domain antibody-FLAG to rebind to the bacteria. Then incubate both plates with anti-FLAG and develop as described in Example 3 (ELISA). The OD450 signal shows the amount of product bound.

[0340] Add a standard curve to each plate to ensure that the values from the product are within the linear range of the ELISA assay.

[0341] Results

[0342] At Figure 5In A, the high signal corresponds to a higher binding affinity, which is attributed to the inability of the product to be separated by citric acid. After citric acid shock, compared with the F45 single-domain antibody, F45::(GGGGS) 3 ::F45 homodimeric protein construct retained a higher amount in the wells. Therefore, it was demonstrated that the homodimeric protein construct bound better to bacteria after citric acid shock compared with the single-domain antibody ( Figure 5 A).

[0343] Figure 5 B shows the quantification of the F45 single-domain antibody and F45::(GGGGS) 3 ::F45 homodimeric protein construct eluted after citric acid shock, and thus it is an indicator of the ease with which the product is washed off from ETEC bacteria. A lower amount of the homodimeric protein construct was detected in the ELISA assay compared with the single-domain antibody, demonstrating that less of the homodimeric protein construct was eluted compared with the single-domain antibody.

[0344] Conclusions

[0345] We can conclude that the homodimeric protein construct binds with a higher affinity than the single-domain antibody (i.e., monomer) because more product was retained after acid shock and less of the homodimeric protein construct was eluted.

[0346] Therefore, after citric acid exposure, the binding affinity of the homodimeric protein construct is at least 1.5-fold higher than that of the monomer.

[0347] Example 5: In vitro testing of the thermal stability, pH stability, bile salt stability, stability over time in porcine gastric juice, and stability over time in porcine bile of the homodimeric protein construct

[0348] Materials and methods

[0349] ELISA was performed as described in Example 3.

[0350] A standard curve was added to each plate to ensure that the values from the product were within the linear range of the ELISA assay.

[0351] The relative values show how much signal was detected, and thus the product was bound after incubation, which means that a high signal indicates high retention and high stability of the signal.

[0352] Thermal stability

[0353] Based on F45::(GGGGS) 3The F45::F45 homodimeric protein construct was incubated for 1 hour at different temperatures and then product binding relative to the control was evaluated in an ELISA setup to assess thermal stability (see Figure 7 A).

[0354] We can conclude that the F45::F45 homodimeric protein construct remains completely stable up to 70 °C, indicating high product stability.

[0355] pH stability

[0356] Based on incubating the F45::(GGGGS) 3 ::F45 homodimeric protein construct for 1 hour at 37 °C at different pH values and then evaluating product binding relative to the control in an ELISA experiment to assess pH stability (see Figure 7 B).

[0357] We can conclude that the F45::F45 homodimeric protein construct gradually loses stability as the pH value decreases, but ~70 - 90% of its activity is retained between pH 3 - 4, and the dimeric protein construct can pass through the stomach of a subject such as an animal and retain biologically relevant activity.

[0358] Bile salt stability

[0359] Sodium deoxycholate (NaDeox) is a bile salt that is added in the step of binding to the F45::(GGGGS) 3 ::F45 homodimeric protein construct coated in the pore with bacteria. NaDeox is added at concentrations of 0.2 mM, 1 mM, and 10 mM to evaluate its effect on bacteria - product binding. After incubation, the plates are washed and processed as described above.

[0360] Based on incubating the F45::(GGGGS) 3 ::F45 construct with bacteria for 1 hour at different concentrations of NaDeox to evaluate the effect on product - bacteria binding in bile salts, values are represented relative to the control (see Figure 7 C).

[0361] We can conclude that the F45::F45 homodimeric protein construct remains completely stable and active in the presence of bile salts (also outside the biologically relevant range), and will thus be active in the intestine.

[0362] Stability over time in porcine gastric juice

[0363] Gastric juice was extracted from the stomach of euthanized pigs (Yorkshire Landrace pigs weighing approximately 30 kg). Within 1 - 2 hours after extraction, the gastric juice was clarified by centrifugation and then kept at -80 °C until further use. When the pretreated unlabeled F45::(GGGGS) 3 ::F45 homodimeric protein construct sample was diluted 1:200 in gastric juice, the pH was measured, and the pH of one sample was adjusted to approximately 3 using HCl. Subsequently, the sample was incubated at 37 °C for 1 hour, then further diluted and used to coat ELISA plates. For all treatments, all subsequent steps (ELISA) were processed as described in Example 3.

[0364] Based on incubating the F45::(GGGGS) 3 ::F45 homodimeric protein construct with gastric contents from three different piglets at 37 °C for 1 hour, and then evaluating product binding relative to the control in an ELISA experiment, the product stability in porcine gastric juice was evaluated. pH = 4 / 5, and the pH of the last sample was adjusted to 3 using HCl (see Figure 8 A).

[0365] We can conclude that the homodimeric protein construct remains stable in the gastric juice of piglets and thus remains active upon oral administration.

[0366] Stability over time in porcine bile

[0367] Bile was extracted from the gallbladder of pigs (Yorkshire Landrace pigs obtained from a specific pathogen - free herd and euthanized at a weight of approximately 30 kg). The bile was stored on ice / refrigerated until further use. Before use, the bile was slightly heated at 37 °C to dissolve lipids. High levels of bile cause milk to precipitate, so for these assays, BSA (final concentration 3%) was used in the buffer and blocking buffer. Bile was mixed with bacteria and BSA to a final bile concentration of 50%, 10%, or 2%. After bile / bacteria incubation, the plates were washed and processed as described above.

[0368] Based on incubating the F45::(GGGGS) 3 ::F45 with bacteria (F4 ETEC) at different bile concentrations from 3 separate piglets for 1 hour, and then evaluating product - bacteria binding relative to the control in an ELISA setup, the effect on product - bacteria binding in porcine bile was evaluated (see Figure 8 B).

[0369] We can conclude that the homodimeric protein construct remains stable and active in bile and will thus remain functional in the intestine.

[0370] General conclusions

[0371] We can conclude that the homodimeric protein construct is completely stable within the temperature range relevant to humans and animals. The homodimeric protein construct retains its biologically relevant activity upon a decrease in pH, and it remains completely stable and active in the presence of bile / gall, and thus it will be active in the intestine. The homodimeric protein construct also remains stable in the gastric juice of piglets and thus retains its activity upon oral administration.

[0372] Example 6: In vitro testing of the protease stability of single-domain antibody constructs at specific concentrations and pH

[0373] To ensure the stability of the single-domain antibody constructs upon oral administration, a stability test with added protease was conducted. The following monomeric single-domain antibodies were used: F41, F45, F183, and the following dimeric single-domain antibody constructs: F183::(GGGGS) 3 ::F183, F183::IgG3::F183. The latter linker sequence has previously been used and described as stable by Virdi et al. 2013 (Orally fed seeds producing designer IgAs protect weaned piglets against enterotoxigenic Escherichia coli infection). The single-domain antibody constructs were each incubated at 37 °C for 1 hour with the following:

[0374] · Pepsin: An endopeptidase found in the digestive system

[0375] · Trypsin: A serine protease found in the digestive system

[0376] · Pancreatin: A mixture of amylase, lipase, and protease obtained from porcine pancreas

[0377] · Gastric juice from pigs

[0378] When the single-domain antibody constructs are administered orally at a later stage, the proteases and gastric juice were selected to determine the possible stability in the gastrointestinal tract.

[0379] The stability test was conducted with proteases at different concentrations and pH. Samples containing the single-domain antibody constructs were analyzed using SDS-page before and after incubation with the protease to evaluate possible protein degradation. Bovine serum albumin (BSA) was used as a reference for normal protein content.

[0380] The specific protease concentrations and pH used are as follows:

[0381] Stability test under normal activity ( Figure 1 ):

[0382] · Pepsin 250 μg / mL (incubation pH 3)

[0383] · Trypsin 125 μg / mL

[0384] · Pancreatin 100 μg / mL

[0385] Stability test under high activity ( Figure 2 ):

[0386] · Pepsin 500 μg / mL (incubation pH 1 - 2)

[0387] · Trypsin 250 μg / mL

[0388] · Pancreatin 100 μg / mL

[0389] Stability test under very high activity ( Figure 3 ):

[0390] · Pepsin 50 μg / mL (incubation pH 1 - 2)

[0391] · Trypsin 500 μg / mL

[0392] · Pancreatin 150 μg / mL

[0393] In the stability test under conditions similar to gastrointestinal conditions, all individual single - domain antibody constructs and the homodivalent single - domain antibody construct F183::(GGGGS) 3 ::F183 were stable. However, another homodivalent single - domain antibody construct F183::IgG3::F183 was degraded by trypsin and pancreatin, and lighter products were visible on the SDS gel. Thus, surprisingly, the IgG3 linker was degraded by the two proteases trypsin and pancreatin, while the (GGGGS) 3 linker remained completely stable. A similar surprising pattern was observed after incubation in porcine gastric juice, where the homodivalent construct with the IgG3 linker was cleaved, while the homodivalent construct with the (GGGGS) 3 linker remained stable ( Figure 12 ).

[0394] At high and very high protease concentrations and lower pH, generally a greater amount of single-domain antibody constructs are degraded. When comparing individual single-domain antibody constructs, differences in stability were observed: F41 was less degraded by pepsin and more degraded by trypsin and pancreatin, while the opposite was observed for F45 at high protease concentrations. Additionally, at very high concentrations, F45 was more stable than F41. F45 and F183 had similar stabilities under all conditions.

[0395] Example 7: In vitro testing of bacterial aggregation

[0396] Materials and methods

[0397] The F4 Escherichia coli (0149:F4) O / N culture was grown O / N in LB with shaking at 37 °C at 180 rpm. Subsequently, the culture was washed once in PBS and the OD600 was adjusted to 1. 1 mL of the bacterial pellet was resuspended in 40 μl of either the labeled or unlabeled F45 single-domain antibody or the homodivalent F45::(GGGGS) 3 ::F45 homodivalent protein construct. The samples were allowed to sediment for a few minutes and then mounted on a glass slide. Images were taken on an optical microscope using a 100X oil immersion lens.

[0398] Results

[0399] Microscopic pictures of F4 ETEC bacterial cultures with and without the F45 monomer or the F45::(GGGGS) 3 ::F45 product showed how the product affected the bacteria. Although F45 did not affect bacterial aggregation or motility, the homodivalent protein construct F45::(GGGGS) 3 ::F45 interlinked and aggregated F4 ETEC with each other, which was visible as bacterial clumps when observed under the microscope (see Figure 9 ).

[0400] Conclusions

[0401] Based on these pictures, we can conclude that the homodivalent protein construct is active, binds at both ends, and has a cumulative effect greater than the two individual single-domain antibodies it contains, which is attributed to its ability to promote aggregate formation and block far more binding sites.

[0402] Example 8: In vitro testing of the binding of single-domain antibody constructs to ETEC bacteria and competition with cell receptors

[0403] An adhesion assay can be established to test the binding of single-domain antibody monomers or constructs and their competition with native receptors. This assay can demonstrate the efficacy of the product in sequestering ETEC bacteria from binding to porcine epithelial cells. The Intestinal Porcine Epithelial Cell line-1 (IPEC-1) can be inoculated, cultured, and subsequently infected with F18 + ETEC bacteria to mimic the binding between cells and bacteria in the intestine. The ETEC bacterial cells can be stained with DAPI for visualization and measured using flow cytometry. To quantify the bacterial load, unstained bacterial cells can be incubated with porcine cells for colony-forming unit assays. To investigate whether the single-domain antibody construct can block the adhesion of ETEC bacterial cells to IPEC-1 cells and thus compete with the cell receptor, the adhesion assay can be performed with or without the single-domain antibody construct. Single-domain antibody monomers, constructs, mixtures of monomers and / or constructs, or any of the above can be tested after treatment with protease, low pH, or porcine gastric juice (as described in Example 3). The single-domain antibody monomer or construct binds to F18 + ETEC bacteria, preventing binding to IPEC-1 cells. Another analysis can also be performed to study the adhesion of F4 + ETEC bacteria to the IPEC-J2 cell line expressing F4 in the presence or absence of the single-domain antibody monomer or construct.

[0404] Assays are performed to determine one or more of the following effects:

[0405] 1. Compared to single single-domain antibody monomers or constructs alone, the use of mixtures of multiple single-domain antibody constructs has potentially higher efficacy in sequestering ETEC from adhering to porcine epithelial cells.

[0406] 2. Compared to single-domain antibody monomers, bivalent constructs have potentially similar or higher efficacy.

[0407] 3. After treatment with biologically relevant proteases, low pH, or porcine gastric juice from the digestive tract (as described in Example 3), compared to single-domain antibody monomers, bivalent constructs have potentially similar or higher stability, measured by the activity retained by the constructs in the adhesion assay after treatment.

[0408] Example 9: Cytotoxicity Assay for Determining the Toxicity of LT and stx2e and the Effect of Toxin-Specific Single-Domain Antibody Constructs in Sequestering Toxin Binding

[0409] Cytotoxicity assays can be performed to determine the toxicity of the ETEC toxins LT and stx2e, and the ability of the single-domain antibody constructs to eliminate this toxicity through toxin sequestration. Cells exposed to cytotoxic compounds can respond in a variety of ways; if the toxin is lethal, the cells may undergo necrosis, apoptosis, or autophagy, or if the damage is less severe, the cells may reduce or stop their growth and proliferation. During in vitro cytotoxicity assays, any of these responses can be measured in the presence of LT and stx2e. Vero cells, a cell line established from kidney epithelial cells extracted from African green monkeys, can be used in this study. Cells can be exposed to different / increasing concentrations of LT and stx2e, and then the cell responses can be measured to determine the severity of the two toxins. In this study, single-domain antibody monomers, constructs, mixtures thereof, or any of the above can be tested after treatment with products such as proteases, low pH, or gastric juice (as described in Example 3). The single-domain antibody constructs are expected to sequester the toxins and thus inhibit the cytotoxic effects of the ETEC toxins LT and stx2e. Cytotoxicity assays can be performed using toxin mixtures obtained from ETEC bacterial supernatants. The effects can be dose-dependent.

[0410] Assays are performed to determine one or more of the following effects:

[0411] 1. Compared to individual single-domain antibody monomers or constructs, the use of mixtures of multiple single-domain antibody constructs potentially has a higher efficacy in sequestering the toxic effects of the toxin.

[0412] 2. Compared to single-domain antibody monomers, bivalent constructs potentially have a similar or higher efficacy.

[0413] 3. After treatment with biologically relevant proteases, low pH, or porcine gastric juice from the digestive tract (as described in Example 3), compared to single-domain antibody monomers, bivalent constructs potentially have a similar or higher stability, determined by measuring the activity retained by the constructs in cytotoxicity assays after treatment.

[0414] Example 10: In vitro testing of the binding of single-domain antibody constructs to ETEC bacteria and competition with cell receptors on porcine intestinal villi

[0415] To show the effect of single-domain antibodies on ETEC villus binding, small intestinal villus enterocytes can be isolated from the intestinal mucosa of newly slaughtered piglets. The villi can be incubated with ETEC bacteria in the presence and absence of single-domain antibody constructs, and the number of adherent bacteria will be determined using phase contrast microscopy and immunohistochemistry. Single-domain antibody monomers, constructs, mixtures thereof, or any of the above can be tested after treatment with products such as proteases, low pH, or gastric juice from pigs (as described in Example 3). Addition of single-domain antibody monomers or constructs prior to incubation with ETEC bacteria can sequester the ETEC bacteria from adhering and will show the efficacy of the product.

[0416] Assays are performed to determine one or more of the following effects:

[0417] 1. A mixture of multiple single-domain antibody constructs has potentially higher efficacy in sequestering adhesion to porcine intestinal villi compared to a single single-domain antibody monomer or construct.

[0418] 2. A bivalent construct has potentially similar or higher efficacy compared to a single-domain antibody monomer.

[0419] 3. A bivalent construct has potentially similar or higher stability after treatment with biologically relevant proteases, low pH, or gastric juice from pigs (see Example 3) from the digestive tract, as measured by the activity of the construct remaining after treatment in sequestering the adhesion of ETEC bacteria to porcine intestinal villi.

[0420] Example 11: In vitro effect of homodimeric protein constructs on ETEC binding to porcine villus enterocytes

[0421] Materials and methods

[0422] Intestinal villus enterocytes were isolated from piglets and subsequent adhesion assays were performed as described by Van den Broeck et al. (1999). Briefly, a 15 - 20 cm long intestinal segment was excised from the mid-jejunum of pigs (Yorkshire Landrace pigs obtained from a specific pathogen-free herd and euthanized at 3 - 4 months of age). The segment was opened and washed in Krebs-Henseleit buffer (120 mM NaCl, 14 mM KCl, 25 mM NaHCO3, 1 mM KH2PO4 [pH 7.4]), and then kept in Krebs Henseleit buffer containing 1% formaldehyde for approximately 1 hour. Subsequently, the buffer was replaced with sterile PBS (pH 7.4), and the villi were gently scraped off the mucosa with a coverslip and suspended and washed in sterile PBS (pH 7.4) buffer until the supernatant was clear. The samples were stored in PBS and kept at 4 °C until further use.

[0423] F4+ Escherichia coli (0149:F4) was inoculated from a single colony and cultured overnight with shaking in LB at 37 °C. The bacteria were washed once in PBS (pH 7.4) and pretreated with a solution containing either unlabeled or labeled F45::(GGGS) 3 ::F45 homodimeric protein construct, F183::(GGGS) 3 ::F183 homodimeric protein construct. The bacteria / F45::(GGGS) 3 ::F45 mixture or bacteria / F183::(GGGS) 3 ::F183 mixture was added to the slurry of villous material and allowed to adhere by gentle rotation at room temperature. After one hour, the samples were fixed on slides and images were taken using a 100X oil immersion lens on an optical microscope, and quantification was performed manually based on the microscope images.

[0424] Results

[0425] To quantify the efficacy of the product in blocking the binding of F4 ETEC to porcine villous enterocytes, compared to the relevant control, intestinal epithelial cells bound to 0, 1, or 2+ F4 ETEC cells were counted compared to the relevant control. 1 was included because floating bacteria might be trapped under the coverslip during microscopy. The percentage of intestinal epithelial cells bound to 1 or 2+ ETEC cells showed the efficacy of bacterial infection of intestinal epithelial cells. F4 ETEC+F183::(GGGS) 3 ::F183 was used as a control. See Figure 10 。

[0426] Conclusions

[0427] It was concluded that with the addition of the F45::(GGGS) 3 ::F45 homodimeric protein construct, the adhesion of F4 ETEC to intestinal epithelial cells almost completely ceased, which was attributed to the homodimeric protein construct blocking the binding of bacteria to intestinal epithelial cells.

[0428] Example 12: FIDA was performed to determine the binding of single domain antibodies and their bivalent constructs

[0429] To determine the binding and complex formation between single-domain antibodies and their bivalent single-domain antibody constructs and an analyte, flow induced dispersion analysis (FIDA) can be performed as described in Poulsen et al. 2015 (Flow Induced Dispersion Analysis Rapidly Quantifies Proteins in Human Plasma Samples). All of the single-domain antibodies and their bivalent single-domain antibody constructs mentioned can be analyzed to record and quantify analyte binding, including K d and complex formation. The single-domain antibody monomers, constructs, mixtures thereof, or any of the foregoing can be tested after treatment with protease, low pH, or gastric juice (as described in Example 5). A change in the hydrodynamic radius is expected to be observed and thereby confirm analyte binding. Multiple changes in the hydrodynamic range indicate binding of multiple analytes to the single-domain antibody construct, which confirms the formation of higher-order complexes. Similarly, this analysis can show how the bivalent construct achieves increased binding through an increase in avidity.

[0430] Example 13: Flow Induced Dispersion Analysis (FIDA)

[0431] Materials and methods

[0432] FIDA is capable of characterizing and quantifying proteins under native conditions. FIDA is based on measuring the change in the size of a ligand as it selectively interacts with a target protein. The unbound ligand has a relatively small apparent hydrodynamic radius (size), while its apparent hydrodynamic radius increases due to binding to the analyte in the presence of the analyte. Prior to FIDA measurement, VH antigen (LT-B or FaeG) was fluorescently labeled using the Atto 488 Protein Labeling Kit (Sigma-Aldrich) using the standard protocol. H H antigen (LT-B or FaeG) was fluorescently labeled.

[0433] FIDA quantification of the binding between FaeG and F45 monomer and between FaeG and F45::(GGGGS) 3 ::F45 homodimeric protein construct

[0434] FIDA quantification was performed using 39 nL of Atto-labeled FaeG (100 nM) and 7 μL of fermentation medium containing the monomer and homodimeric protein construct. The hydrodynamic radius was detected at 488 nm by detecting the fluorescence of FaeG-atto488. For the mixed samples, both the monomer and the bivalent construct were pre-incubated with different concentrations of fermentation medium (0 and 50% v / v).

[0435] The binding of LT-B to LT1 monomers and the binding of LT-B to LT1::(GGGGS) 3 ::LT1 homodimeric protein constructs were quantitatively analyzed by FIDA

[0436] FIDA quantification was performed using 39 nL of Atto-labeled LT-B (100 nM) and 7 μL of fermentation medium containing monomers and homodimeric protein constructs. The hydrodynamic radius was detected at 488 nm by detecting the fluorescence of LT-B-atto488. For the mixed samples, both the monomers and the dimeric constructs were pre-incubated with different concentrations of fermentation medium (0 - 12.5% v / v). The LT-specific LT1 monomers and homodimeric protein constructs had been treated with endoH (NEB, P0702) before the experiment to remove known glycosylation.

[0437] The FIDA results were analyzed using FIDA-tech software to calculate the binding affinity and visualize the binding curves.

[0438] Results

[0439] FaeG

[0440] The hydrodynamic radius of free FaeG was measured, and monomeric F45 (SEQ ID NO:10) or F45::(GGGGS) 3 ::F45 homodimeric protein construct (SEQ ID NO:13) was added to evaluate the binding affinity. The hydrodynamic range was determined by measuring the width of the peak generated when the sample was measured using FIDA. A narrow peak is the antigen without product binding, and the peak becomes wider (and the hydrodynamic range increases) when the product binds. Therefore, a higher number indicates product binding, and the higher the value, the higher the affinity of the product (see Table 1 below).

[0441] Table 1

[0442] Fermentation medium content (v / v) Hydrodynamic radius (nm) 0％ 2.40+ / -0.09 50％F45 3.02+ / -0.06 <![CDATA[50% F45::(GGGGS) 3 ::F45]]> 2.97+ / -0.20

[0443] Based on the FIDA measurements, we can conclude that the F45 single-domain antibody and F45::(GGGGS) 3 ::F45 homodimeric protein constructs both bind to FaeG (see Table 1). Interestingly, when correlated with the concentration difference, it can be concluded that the homodimeric protein construct binds with a higher affinity compared to the single-domain antibody.

[0444] LT-B

[0445] The hydrodynamic radius of free LT-B was also determined, and LT1 monomer (SEQ ID NO:9) or LT1::(GGGGS) 3 ::LT1 homodimeric protein construct (SEQ ID NO:15) was added to evaluate the binding affinity (see Figure 6 ). The homodimeric protein construct showed a higher hydrodynamic radius, and thus the homodimeric protein construct showed a higher binding affinity compared to the single-domain antibody.

[0446] Conclusions

[0447] In these two experiments, the homodimeric protein constructs F45::(GGGGS) 3 ::F45 and LT1::(GGGGS) 3 ::LT1 showed a higher binding affinity compared to the corresponding F45 and LT1 monomers.

[0448] Example 14: In vivo treatment of post-weaning diarrhea in piglets +

[0449] In vivo studies can be conducted to determine the effect of single-domain antibody constructs on treating post-weaning diarrhea (PWD) in piglets. Piglets + can be challenged with F4 + ETEC bacteria to induce PWD. In the study, 3 technical replicates of 8 piglets can be performed, and each technical replicate is divided into 4 groups of 2 piglets each; one group is the control (no treatment), one group will be treated with the single-domain antibody construct product, one group will be challenged with F4 + ETEC bacteria, and the last group will be treated with the single-domain antibody construct product and challenged with F4 + ETEC bacteria. The single-domain antibody monomers, constructs, mixtures thereof, or any of the above can be tested after treatment with protease, low pH, or gastric juice (as described in Example 3). The treatment can be from two days before weaning to 12 days after weaning, and the challenge will be performed on the first and second days after weaning. Disease progression can be determined by measuring parameters including the degree of diarrhea, body weight, food intake, fever, clinical manifestations, and analysis of inflammatory biomarkers from blood samples. It is expected that treatment with the single-domain antibody construct can reduce or inhibit the development of PWD in all or some of the measured parameters and may show a curative effect in piglets. This study can be conducted multiple times to compare the effects of different combinations of single-domain antibody constructs. Therefore, we can show the in vivo efficacy of the product by improvements in parameters such as the degree of diarrhea, weight gain, food intake, and other disease markers relative to piglets challenged with F4 + ETEC bacteria without treatment.

[0450] Example 15: Treatment of F4 with homodimeric protein constructs​+ In Vivo Treatment of Post-Weaning Diarrhea in Piglets Materials and methods

[0451] Five sows genetically characterized as susceptible to F4+ Escherichia coli from Aarhus University, Foulum were used in this study and fed a standard Danish sow diet (diet based on wheat, barley, and soybean meal). Piglets were born at AU-Foulum on January 26, 2019 (litter 1 = 11 piglets; litter 2 = 8 piglets, litter 3 = 5 piglets), 27th (litter 4 = 10 piglets), and 31st. At weaning (28 days of age), piglets (total N = 32) were enrolled in a challenge study with F4+ Escherichia coli inoculation. From 3 weeks of age (lactation) to the end of the experiment (5 weeks of age), all pigs were provided with the same feed. Pigs were fed twice a day (semi-free) with feed (standard commercial mixture) and provided with free access to water.

[0452] The experimental design involved four treatments:

[0453] 1) Control: no F45::(GGGGS) 3 ::F45 homodimeric protein construct and no F4,

[0454] 2) Control: with F45::(GGGGS) 3 ::F45 homodimeric protein construct but no F4,

[0455] 3) no F45::(GGGGS) 3 ::F45 homodimeric protein construct but with F4, and

[0456] 4) with F45::(GGGGS)3::F45 homodimeric protein construct and with F4.

[0457] At weaning, piglets were housed in two fenced rooms (similar climatic conditions), 2 pigs per pen, for a total of 16 pens, or 4 replicates per group.

[0458] Sixteen pens were randomly assigned to 2 treatments: namely, a solution of F45::(GGGGS) 3 ::F45 homodimeric protein construct provided by Bactolife and a control (egg white protein / Novo). These solutions were provided to each pig twice a day from the afternoon of the day of weaning until the end of 14 days. The solutions were mixed with non-alcoholic beer (from Hancock ). The trial lasted for 21 weeks (until 49 days of age).

[0459] On the 1st and 2nd days after weaning, piglets were orally challenged with Escherichia coli (F4, 0149). The CFU of the inoculum at the time of inoculation was 1.05x10 9 and 1.72x10 9 CFU on the 1st and 2nd days, respectively. Control pigs were inoculated with buffer (sodium bicarbonate). On days 0, 1, 2, 3, 4, 5, and 7 (and every two days in the 2nd week and every three days in the 3rd week), fecal samples (taken directly from the rectum) were collected from each pig, and the hemolytic and non-hemolytic bacteria in the samples were analyzed, and Escherichia coli was verified by serotyping. At the end of the experiment, the piglets were killed (no samples were taken).

[0460] Results

[0461] An in vivo challenge experiment was conducted on 30 weaned piglets (6 - 8 in each group) in a post-weaning diarrhea model system. The results showed that F4 ETEC was present in the feces of piglets throughout the experiment. The presence of F4 ETEC in the feces indicated active infection (especially in large numbers). The data generated showed that after the piglets were challenged (days 1 + 2), F4 ETEC bacteria were present, but in piglets treated with the F45::(GGGGS) 3 ::F45 homodimeric protein construct, infection did not form but was flushed out compared to untreated piglets. See Figure 11 .

[0462] Conclusions

[0463] Therefore, it can be concluded that the homodimeric protein construct prevented F4 ETEC bacteria from adhering to the gastrointestinal tract of newly weaned piglets and significantly reduced the infection time compared to no treatment.

[0464] References

[0465] Gonzales,L.;Ali,Z.B.;Nygren,E.;Wang,Z.;Karlsson,S.;Zhu,B.;

[0466] Quiding- M.; Alkaline pH Is a Signal for OptimalProduction and Secretion of the Heat Labile Toxin,LT in EnterotoxigenicEscherichia Coli(ETEC).PLoS One 2013,8,e74069

[0467] Harmsen, M.M.; Solt, C.B. Van; Bemmel, A.M. van Z.; Niewold, T.A.; Zijderveld, F.G. van Selection and optimization of proteolytically stable llama single-domain antibody fragments for oral immunotherapy. Appl. Microbiol. Biotechnol. 2006, 72, 544–551, doi:10.1007 / s00253-005-0300-7.

[0468] Harmsen, M.M.; Solt, C.B. Van; Fijten, H.P.D. Enhancement of toxin- and virus-neutralizing capacity of single-domain antibody fragments by N-glycosylation. Appl. Microbiol. Biotechnol. 2009, 84, 1087–1094, doi:10.1007 / s00253-009-2029-1.

[0469] Lo, A.W.H.; Moonens, K.; De Kerpel, M.; Brys, L.; Pardon, E.; Remaut, H.; De Greve, H. The molecular mechanism of Shiga toxin Stx2e neutralization by a single-domain antibody targeting the cell receptor-binding domain. J. Biol. Chem. 2014, 289, 25374–25381, doi:10.1074 / jbc.M114.566257.

[0470] Moonens, K.; Kerpel, M. De; Coddens, A.; Cox, E.; Pardon, E.; Remaut, H.; Greve, H. De Nanobody Mediated Inhibition of Attachment of F18 FimbriaeExpressingEscherichia coli. PLoS One 2014, 9, e114691, doi:10.1371 / journal.pone.0114691.

[0471] Van den Broeck, W.; Cox, E.; Godderiis, E. M. Receptor-DependentImmuneResponses in Pigs after Oral Immunization with F4Fimbriae. Infect. Immun. 1999, 67, 520 - 526

[0472] Virdi, V.; Coddens, A.; Buck, S. De; Millet, S.; Maria, B.; Cox, E.; de Greve, H.; de Picker, A. Orally fed seeds producing designer IgAs protect weanedpigletsagainst enterotoxigenic Escherichia coli infection. PNAS 2013, 110, 11809–11814, doi:10.1073 / pnas.1301975110.

Claims

1. An isolated protein comprising - a first peptide having a first binding specificity; - a second peptide having a second binding specificity; and - a linker, wherein the first peptide and the second peptide bind to at least one pathogen surface component and / or at least one molecule produced by a pathogen, wherein the first peptide and / or the second peptide comprises CDR1, CDR2, and / or CDR3 within the amino acid sequence of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, and / or SEQ ID NO:

10.

2. An isolated protein comprising - a first peptide having a first binding specificity; - a second peptide having a second binding specificity; and - a linker, wherein the first peptide and the second peptide bind to at least one pathogen surface component and / or at least one molecule produced by a pathogen, wherein the protein comprises the amino acid sequence as shown in any one of SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, or SEQ ID NO:19, or an amino acid sequence having at least 90%, such as at least 95%, such as at least 98%, such as at least 99% sequence identity with SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, or SEQ ID NO:

19.

3. An isolated protein comprising - a first peptide having a first binding specificity; - a second peptide having a second binding specificity; and - a linker, wherein the first peptide and the second peptide bind to at least one pathogen surface component and / or at least one molecule produced by a pathogen, wherein, the first peptide comprises three complementarity-determining regions CDR1, CDR2, and CDR3, wherein CDR1 has an amino acid sequence comprising the amino acid sequence of SEQ ID NO:27, CDR2 has an amino acid sequence comprising the amino acid sequence of SEQ ID NO:29, and CDR3 has an amino acid sequence comprising the amino acid sequence of SEQ ID NO:31, and wherein the second peptide comprises three complementarity-determining regions CDR1, CDR2, and CDR3, CDR1 having an amino acid sequence comprising the amino acid sequence of SEQ ID NO:28, CDR2 having an amino acid sequence comprising the amino acid sequence of SEQ ID NO:30, and CDR3 having an amino acid sequence comprising the amino acid sequence of SEQ ID NO:

32.

4. An isolated protein comprising - a first peptide having a first binding specificity; - a second peptide having a second binding specificity; and - a linker, wherein the first peptide and the second peptide bind to at least one pathogen surface component and / or at least one molecule produced by a pathogen, wherein the first peptide comprises the amino acid sequence of SEQ ID NO:5 or an amino acid sequence having at least 90%, such as at least 95%, such as at least 98%, such as at least 99% sequence identity with the amino acid sequence of SEQ ID NO:5, and wherein the second peptide comprises the amino acid sequence of SEQ ID NO:5 or an amino acid sequence having at least 90%, such as at least 95%, such as at least 98%, such as at least 99% sequence identity with the amino acid sequence of SEQ ID NO:5, wherein any sequence variation is outside the CDR.

5. An isolated protein comprising - a first peptide having a first binding specificity; - a second peptide having a second binding specificity; and - a linker, wherein the first peptide and the second peptide bind to at least one pathogen surface component and / or at least one molecule produced by a pathogen, wherein the first peptide comprises the amino acid sequence as shown in SEQ ID NO:10 or an amino acid sequence having at least 90%, such as at least 95%, such as at least 98%, such as at least 99% sequence identity with SEQ ID NO:10, and wherein the second peptide comprises the amino acid sequence as shown in SEQ ID NO:9 or an amino acid sequence having at least 90%, such as at least 95%, such as at least 98%, such as at least 99% sequence identity with SEQ ID NO:

9.

6. An isolated protein comprising - a first peptide having a first binding specificity; - a second peptide having a second binding specificity; and - a linker, wherein the first peptide and the second peptide bind to at least one pathogen surface component and / or at least one molecule produced by a pathogen, wherein the first peptide comprises the amino acid sequence as shown in SEQ ID NO:9 or an amino acid sequence having at least 90%, such as at least 95%, such as at least 98%, such as at least 99% sequence identity with SEQ ID NO:9, and wherein the second peptide comprises the amino acid sequence as shown in SEQ ID NO:10 or an amino acid sequence having at least 90%, such as at least 95%, such as at least 98%, such as at least 99% sequence identity with SEQ ID NO:

10.

7. An isolated protein comprising - a first peptide having a first binding specificity; - a second peptide having a second binding specificity; and - a linker, wherein the first peptide and the second peptide bind to at least one pathogen surface component and / or at least one molecule produced by a pathogen, wherein the first peptide comprises the amino acid sequence as shown in SEQ ID NO:8 or an amino acid sequence having at least 90%, such as at least 95%, such as at least 98%, such as at least 99% sequence identity with SEQ ID NO:8, and wherein the second peptide comprises the amino acid sequence as shown in SEQ ID NO:5 or an amino acid sequence having at least 90%, such as at least 95%, such as at least 98%, such as at least 99% sequence identity with SEQ ID NO:

5.

8. An isolated protein comprising - a first peptide having a first binding specificity; - a second peptide having a second binding specificity; and - a linker, wherein the first peptide and the second peptide bind to at least one pathogen surface component and / or at least one molecule produced by a pathogen, wherein the first peptide comprises the amino acid sequence as shown in SEQ ID NO:5 or an amino acid sequence having at least 90%, such as at least 95%, such as at least 98%, such as at least 99% sequence identity with SEQ ID NO:5, and wherein the second peptide comprises the amino acid sequence as shown in SEQ ID NO:8 or an amino acid sequence having at least 90%, such as at least 95%, such as at least 98%, such as at least 99% sequence identity with SEQ ID NO:

8.

9. An isolated protein comprising - a first peptide having a first binding specificity; - a second peptide having a second binding specificity; and - a linker, wherein the first peptide and the second peptide bind to at least one pathogen surface component and / or at least one molecule produced by a pathogen.

10. The protein according to any one of claims 1-9, wherein the first and / or the second peptide is a single-domain antibody.

11. The protein according to any one of the preceding claims, wherein the protein is stable in the gastrointestinal tract and / or respiratory system of a subject.

12. The protein according to any one of the preceding claims, wherein the protein is acid-stable and / or protease-stable.

13. The protein according to any one of the preceding claims, wherein the protein is pH-stable and / or temperature-stable.

14. The protein according to any one of the preceding claims, wherein the linker is a GS linker.

15. The protein according to claim 14, wherein the GS linker has (G x S) n structure, where x can be a number between 1 and 10, preferably 2 to 5, and n refers to the number of repetitions of the G x S sequence, where n can be between 1 and 10, preferably 2 to 5.

16. The protein according to any one of claims 14 to 15, wherein the GS linker is a GGGGS linker (SEQ ID NO:20), a GGGGSGGGGS linker (SEQ ID NO:21), a GGGGSGGGGSGGGGS linker (SEQ ID NO:22), a GGGGSGGGGSGGGGSGGGGS linker (SEQ ID NO:23), a GGGGSGGGGSGGGGSGGGGSGGGGS linker (SEQ ID NO:24), or a GGGGSGGGGSGGGGSGGGGSGGGGSGGGGS linker (SEQ ID NO:25).

17. The protein according to any one of the preceding claims, provided that the linker does not comprise a part of an antibody, such as an Fc region or a part of an Fc region.

18. The protein according to any one of the preceding claims, provided that the linker is not an IgG3 linker, such as the IgG3 hinge linker of SEQ ID NO:

26.

19. The protein according to any one of the preceding claims, wherein the binding affinity of the protein is at least 1.5-fold higher than the binding affinity of a monomeric single-domain antibody.

20. The protein according to any one of the preceding claims, wherein the protein comprises one or more further peptides that bind to at least one pathogen surface component and / or at least one molecule produced by a pathogen.

21. The protein according to any one of the preceding claims, wherein the peptide binds with low affinity to non-pathogen surface components and / or molecules secreted by non-pathogens.

22. The protein according to any one of the preceding claims, wherein the protein is homodivalent or heterodivalent.

23. The protein according to any one of the preceding claims, wherein the first peptide and the second peptide bind to the following: a) the same surface component; b) different surface components; c) a surface component and a molecule; d) the same molecule; or e) different molecules.

24. The protein according to any one of the preceding claims, wherein the surface component is from the same or different pathogens.

25. The protein according to any one of the preceding claims, wherein the surface component is a pathogen appendage and / or a virulence factor, such as an adhesion factor.

26. The protein according to any one of the preceding claims, wherein the molecule is a toxin, an inhibitor, and / or an enzyme, such as β-lactamase.

27. An isolated nucleic acid molecule encoding the protein according to any one of claims 1 to 26.

28. A vector comprising the nucleic acid molecule according to claim 27.

29. The vector according to claim 28, wherein the vector is an expression vector.

30. A recombinant host cell comprising the nucleic acid molecule according to claim 27 or the vector according to any one of claims 28 to 29.

31. The recombinant host cell according to claim 30, wherein the host cell is a bacterium, a fungus such as yeast, and / or a mammalian cell.

32. The recombinant host cell according to any one of claims 30 to 31, wherein the bacterium is a Bacillus, such as Bacillus licheniformis, Bacillus subtilis, and / or Bacillus lactobacillus.

33. The recombinant host cell according to any one of claims 30 to 32, wherein the yeast is selected from the genera Pichia, Hansenula, and Saccharomyces.

34. The recombinant host cell according to any one of claims 30 to 33, wherein the fungus is selected from Aspergillus oryzae and Aspergillus niger.

35. A dietary composition comprising the protein according to any one of claims 1 to 26.

36. The dietary composition according to claim 35, wherein the composition further comprises one or more of the following: prebiotics, probiotics, synbiotics, proteins, lipids, carbohydrates, vitamins, fibers, and / or nutrients, such as dietary minerals for a living subject.

37. The dietary composition according to any one of claims 35 to 36, wherein the composition is a food additive.

38. Use of the protein according to any one of claims 1 to 26 in the production of a dietary composition.

39. Use of the protein according to any one of claims 1 to 26 in promoting the growth of a subject.

40. Use of the protein according to any one of claims 1 to 26 as an additive in an infant formula product.

41. Use of the protein according to any one of claims 1 to 26 as a protective aid against pathogen infection in a subject during travel.

42. Use of the protein according to any one of claims 1 to 26 as a protective aid against pathogen infection in an immunodeficient subject, such as an elderly person.

43. The use according to any one of claims 38 to 42, wherein the use is non-therapeutic.

44. A pharmaceutical composition comprising the protein according to any one of claims 1 to 26, the nucleic acid according to claim 27, the vector according to any one of claims 28 to 29, and / or the recombinant host cell according to any one of claims 30 to 34, and further comprising one or more excipients.

45. The pharmaceutical composition according to claim 44, wherein the excipients are selected from the group consisting of surfactants, anti-adhesives, binders, coating agents, disintegrants, fillers, solvents / cosolvents, flavoring agents, pigments, lubricants, glidants, preservatives, adsorbents, sweeteners, carriers, polymers, buffers, antioxidants, wetting agents, antifoaming agents, thickening agents, humectants, and mixtures thereof.

46. The pharmaceutical composition according to any one of claims 44 to 45, further comprising one or more carbohydrates, such as cellulose and / or alginate, enzymes, activated carbon, antibiotics, and mixtures thereof.

47. The protein according to any one of claims 1 to 26, the nucleic acid according to claim 27, the vector according to any one of claims 28 to 29, and / or the recombinant host cell according to any one of claims 30 to 34, or the pharmaceutical composition according to any one of claims 44 to 46, for use as a medicament.

48. The protein according to any one of claims 1 to 26, the nucleic acid according to claim 27, the vector according to any one of claims 28 to 29, and / or the recombinant host cell according to any one of claims 30 to 34, or the pharmaceutical composition according to any one of claims 44 to 46, for preventing or treating pathogen-induced infections associated with the inner surface and / or outer surface of a subject.

49. The protein, nucleic acid, vector, host cell, or pharmaceutical composition according to claim 48, wherein the infection is a gastrointestinal infection, a pulmonary infection, or a skin infection.

50. The protein, nucleic acid, vector, host cell, or pharmaceutical composition according to any one of claims 48 to 49, wherein the infection is a gastrointestinal infection.

51. The protein, nucleic acid, vector, host cell, or pharmaceutical composition according to any one of claims 47 to 50, wherein the route of administration is oral, pulmonary, and / or topical.

52. The protein, nucleic acid, vector, host cell, or pharmaceutical composition according to claim 48, wherein the pulmonary administration is carried out by a nebulizer.

53. The protein, nucleic acid, vector, host cell, or pharmaceutical composition according to any one of claims 48 to 52, wherein the use reduces the time span of active infection.

54. The protein, nucleic acid, vector, host cell, or pharmaceutical composition according to any one of claims 48 to 53, wherein the use reduces virulence.

55. The protein, nucleic acid, vector, host cell, or pharmaceutical composition according to any one of claims 47 to 54, wherein the subject is a human subject.

56. The protein, nucleic acid, vector, host cell, or pharmaceutical composition according to any one of claims 47 to 55, wherein the subject is a domestic animal, such as an animal selected from the group consisting of pigs, cattle, sheep, goats, horses, chickens, donkeys, mules, ducks, geese, and turkeys.

57. The protein, nucleic acid, vector, host cell, or pharmaceutical composition according to any one of claims 47 to 56, wherein the subject is a pig.

58. The use of the protein according to any one of claims 1 to 26, the nucleic acid according to claim 27, the vector according to any one of claims 28 to 29, the recombinant host cell according to any one of claims 30 to 34, or the pharmaceutical composition according to any one of claims 44 to 46, in the preparation of a medicament for preventing or treating post-weaning diarrhea (PWD).

59. Use of the protein according to any one of claims 1 to 26, the nucleic acid according to claim 27, the vector according to any one of claims 28 to 29, and the recombinant host cell according to any one of claims 30 to 34 in the preparation of a medicament for preventing or treating oedema disease.

60. The protein according to any one of the preceding claims, or the protein, nucleic acid, vector, host cell or pharmaceutical composition according to any one of the preceding claims, wherein the pathogen is a bacterium, virus, fungus, protozoan and / or worm.

61. The protein according to any one of the preceding claims, or the protein, nucleic acid, vector, host cell or pharmaceutical composition according to any one of the preceding claims, wherein the pathogen is Escherichia coli bacterium, Vibrio cholera bacterium, Salmonella, Campylobacter bacterium, Staphylococcus bacterium, Listeria bacterium, Shigella bacterium, Mycoplasma and / or Clostridium difficile. The protein according to any one of the preceding claims, or the protein, nucleic acid, vector, host cell or pharmaceutical composition according to any one of the preceding claims, wherein the pathogen is F4 + Escherichia coli (F4 + Escherichia coli) bacteria and / or F18 + Escherichia coli (F18 + Escherichia coli) bacteria.

63. The protein according to any one of the preceding claims, or the protein, host cell or pharmaceutical composition according to any one of the preceding claims, wherein one of the first peptide or the second peptide binds to F4 + Escherichia coli bacteria, and wherein the other peptide binds to F18 + Escherichia coli bacteria.

64. The protein according to any one of the preceding claims, or the protein, nucleic acid, vector, host cell or pharmaceutical composition according to any one of the preceding claims, wherein the pathogen produces a heat-labile toxin (LT toxin).

65. The protein according to any one of the preceding claims, or the protein, nucleic acid, vector, host cell or pharmaceutical composition according to any one of the preceding claims, wherein the pathogen produces Shiga toxin, such as selected from stx2e and porcine and human variants of stx2e or Shiga-like toxin.

66. The protein according to any one of the preceding claims, or the protein, nucleic acid, vector, host cell or pharmaceutical composition according to any one of the preceding claims, wherein the pathogen is enterotoxigenic Escherichia coli (ETEC) with fimbrial type or fimbrial adhesion: such as F5, F6, F41 or AIFA, human-specific variants such as colonization factor antigen (CFA) or Escherichia coli surface antigen (CS), including but not limited to CFAI, CFAII, CFAIII and CFAIV.

67. The protein according to any one of the preceding claims, or the protein, nucleic acid, vector, host cell or pharmaceutical composition according to any one of the preceding claims, wherein the pathogen produces Shiga toxin or Shiga-like toxin, such as Stx2e and human variants, including but not limited to stx, stx1a, stx1c, stxd, stx2a, stx2b, stx2c, stx2d, stx2f and stx2g.

68. The protein according to any one of the preceding claims, or the protein, nucleic acid, vector, host cell or pharmaceutical composition according to any one of the preceding claims, wherein the surface component is selected from the group consisting of: F18 + , F4 + , Stx2e, LT and their animal and human variants. Use of the protein, nucleic acid, vector or host cell according to any one of the preceding claims in the preparation of a medicament for preventing or treating a pathogen-induced infection associated with the inner surface and / or outer surface of a subject.

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