WO2020123798A1 - Bovine versions of fcmbl for the detection and treatment of bovine microbial infections - Google Patents

Abstract

Described herein are methods and compositions related to fusion polypeptides comprising a bovine mannose-binding lectin (MBL), or a fragment thereof, linked to a fragment of an antibody comprising a bovine C2 and/or C3 domain of IgG.

Description

BOVINE VERSIONS OF FCMBL FOR THE DETECTION AND TREATMENT OF BOVINE

MICROBIAL INFECTIONS

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims benefit under 35 U.S.C. § 119(e) of U.S. Provisional Application No. 62/778,578 filed December 12, 2018, the contents of which are incorporated herein by reference in their entirety.

TECHNICAL FIELD

[0002] The technology described herein relates to compositions and methods related to detecting, treating, and preventing bovine pathogenic and infectious diseases.

BACKGROUND

[0003] Infectious diseases of livestock such as cattle threaten the health and well-being of wildlife, livestock, and human populations. Bloodbome pathogens are microorganisms that cause disease when transferred from an infected animal to another animal through blood or other potentially infected body fluids. Specifically, bovine infectious diseases such as bovine respiratory syncytial virus (BRSV), Clostridium infections, or mastitis, detrimentally affect the health and fertility of cattle worldwide. A lack of preventative treatments for a number of bovine microbial diseases have led to a significant increase in veterinary costs among the dairy and beef industries. Furthermore, the current vaccines on the market for bovine microbial infections lack efficacy to stimulate protective and long- lasting immune responses against many bovine pathogens. Hence, there remains a need for improved techniques for diagnosis and treatment of animals and livestock with infectious diseases, blood-borne infections, sepsis, or systemic inflammatory response syndrome.

SUMMARY

[0004] In providing vaccines, identifying (or isolating) microorganisms, or portions thereof, which can successfully immunize a subject can be a challenge. Additionally, presenting the microorganism and/or portion thereof to the subject’s immune system in such a way as to induce an effective immune response can also be a point of failure in vaccination. Provided herein are engineered fusion polypeptides which can capture and display immunogenic material (e.g., microorganisms or microorganism-derived biomolecules) for the bovine immune system with surprisingly improved immunological behavior as compared to earlier technologies. The bovine version is designed to be less immunogenic in cows and related species, so it will not elicit an immune response when used as a therapeutic or vaccine component in cows. [0005] Additionally, the engineered fusion polypeptides can be used to bind biological pathogens or identify subclasses or specific pathogen species for use in the prevention, treatment, and diagnosis of livestock with infectious diseases, blood-borne infections, or in the identification of water- or food- borne pathogens. The compositions described herein are particularly, and surprisingly, efficacious in their ability to bind a wide variety of pathogens and/or pathogen-derived biomolecules while providing markedly improved immunological behavior in bovines.

[0006] In one aspect of any of the embodiments, described herein is a composition comprising: an engineered fusion polypeptide comprising: an antibody fragment comprising the C2 and/ or C3 domains of a bovine immunoglobulin G (IgG); and at least one bovine mannose binding lectin (MBL) domain.

[0007] In another aspect of any of the embodiments, described herein is a pharmaceutical or vaccine composition comprising the composition described herein and at least one of a

pharmaceutically acceptable carrier and an adjuvant.

[0008] In another aspect of any of the embodiments, described herein is a method of treating or preventing an infectious disease in a subject, the method comprises administering the composition described herein.

[0009] In another aspect of any of the embodiments, described herein is a method of stimulating an immune response in a subject or vaccinating a subject, the method comprises administering the composition described herein to the subject.

[0010] In another aspect of any of the embodiments, described herein is a method of preparing a vaccine for a bovine subject, the method comprises contacting the composition described herein with a microorganism or at least one microorganism-derived biomolecule, whereby the microorganism and/or biomolecule are bound by the composition.

[0011] In one embodiment of any of the aspects, the fusion polypeptide comprises at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten or more MBL domains.

[0012] In another embodiment of any of the aspects, the MBL domain binds to a microorganism or a microorganism-derived biomolecule.

[0013] In another embodiment of any of the aspects, the microorganism is a virus, a fungus, a bacterium, a parasite, or a yeast.

[0014] In another embodiment of any of the aspects, the fusion polypeptide further comprises a polypeptide linker domain between the antibody fragment and the MBL domain.

[0015] In another embodiment of any of the aspects, the bovine antibody fragment is linked to the N-terminus or C-terminus of the bovine MBL domain.

[0016] In another embodiment of any of the aspects, the fusion polypeptide comprises the sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6. [0017] In another embodiment of any of the aspects, the bovine antibody fragment is derived from Bos taurus.

[0018] In another embodiment of any of the aspects, the bovine antibody fragment is derived from bovine IgGl, IgG2, or IgG3.

[0019] In another embodiment of any of the aspects, the bovine antibody fragment is a polypeptide comprising the sequence of SEQ ID NO: 7, or SEQ ID NO: 8, or SEQ ID NO: 9, or SEQ ID NO: 10 or SEQ ID NO: 11, or SEQ ID NO: 12, or SEQ ID NO: 13, or SEQ ID NO: 14, or SEQ ID NO: 15.

[0020] In another embodiment of any of the aspects, one or more amino acids within the fusion polypeptide is substituted relative to a wild-type bovine sequence to prevent glycosylation.

[0021] In another embodiment of any of the aspects, the one or more substituted amino acids is substituted with an aspartate.

[0022] In another embodiment of any of the aspects, two or more amino acids within the fusion polypeptide are substituted relative to a wild-type bovine sequence to stimulate glycosylation.

[0023] In another embodiment of any of the aspects, the first amino acid of the fusion polypeptide is substituted with an aspartate and the terminal lysine of the fusion polypeptide is substituted with an alanine.

[0024] In another embodiment of any of the aspects, the composition further comprises at least one microorganism or microorganism -derived biomolecule bound to the fusion polypeptide.

[0025] In another embodiment of any of the aspects, the microorganism is a bovine pathogen.

[0026] In another embodiment of any of the aspects, the bovine pathogen is Clostridium tetani,,

Clostridium novyi, Clostridium perfringens, Clostridium perfringens, Clostridium perfringens, Clostridium septicum, Clostridium chauvoei, Clostridium sordellii, Clostridium hemolyticum, Steptococcus uberis, Staphylococcus aureus, Escherichia coli, Streptococcus agalactiae,

Streptococcus dysglalactiae, Coorynebacterium bovis, Mycoplasma bovis, Mannheimia haemolytica, Pasteurella multocida, Histophilus somni, Mycobacterium tuberculosis, bovine viral diarrhea virus, bovine respiratory syncyntial virus, rabies virus, bovine Herpes virus 1, Bovine Herpes virus 4, bovine rhinovirus, bovine enterovirus, bovine coronavirus, bovine reovirus, parainfluenza- 3 virus, or a round worm.

[0027] In another embodiment of any of the aspects, the fusion polypeptide is attached to a solid substrate.

[0028] In another embodiment of any of the aspects, the solid substrate is selected from a group consisting of: a magnetic microbead, a paramagnetic microbead, a microporous membrane, a hollow fiber, any other fluid filtration membrane, flow device, microtiter plate, cell culture plate, glass beads, latex beads, a living cell, an extracellular matrix of a biological tissue or organ, and a phagocyte.

[0029] In another embodiment of any of the aspects, the subject is a cow. [0030] In another embodiment of any of the aspects, the infectious disease is selected from the group consisting of: mastitis, ephemeral fever, actinomycosis, bovine respiratory disease, bovine respiratory syncytial virus, infectious bovine rhinotracheitis, balanoposthitis, influenza, tuberculosis, Clostridium infection, and pneumonia.

[0031] In another embodiment of any of the aspects, the immune response or vaccination is specific to a disease selected from the group consisting of: mastitis, ephemeral fever, actinomycosis, bovine respiratory disease, bovine respiratory syncytial virus, infectious bovine rhinotracheitis, balanoposthitis, influenza, tuberculosis, Clostridium infection, and pneumonia.

[0032] In another embodiment of any of the aspects, the method further comprises purifying the microorganism and/or biomolecule from the composition.

BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Fig. 1 demonstrates the amino acid sequence for a version of bovine FcMBL using an Fc fragment from bovine IgGl and a fragment of bovine MBL-C.

[0034] Fig. 2 demonstrates the amino acid sequence for a version of bovine FcMBL using an Fc fragment from bovine IgGl and a fragment of bovine MBL-C. The putative glycosylation site in IgGl has been mutated to aspartate to stop glycosylation.

[0035] Fig. 3 demonstrates the amino acid sequence for a version of bovine FcMBL using an Fc fragment from bovine IgG2 and a fragment of bovine MBL-C.

[0036] Fig. 4 demonstrates the amino acid sequence for a version of bovine FcMBL using an Fc fragment from bovine IgG2 and a fragment of bovine MBL-C. The putative glycosylation site in IgG2 has been mutated to aspartate to stop glycosylation.

[0037] Fig. 5 demonstrates the amino acid sequence for a version of bovine FcMBL using an Fc fragment from bovine IgG3 and a fragment of bovine MBL-C.

[0038] Fig. 6 demonstrates the amino acid sequence for a version of bovine FcMBL using an Fc fragment from bovine IgG3 and a fragment of bovine MBL-C. The putative glycosylation site in IgG3 has been mutated to aspartate to stop glycosylation.

[0039] Fig. 7 demonstrates the sequence for the portion of IgGl used in bovine FcMBL fragments. The glycosylated asparagine (underlined) can be mutated to aspartate to generate an aglycosylated version. The terminal lysine can be mutated to an alanine for a longer half-life.

[0040] Fig. 8 demonstrates the sequence for the portion of IgG2 used in bovine FcMBL fragments. The glycosylated asparagine (underlined) can be mutated to aspartate to generate an aglycosylated version. The terminal lysine can be mutated to an alanine.

[0041] Fig. 9 demonstrates the sequence for the portion of IgG3 used in bovine FcMBL fragments. The glycosylated asparagine (underlined) can be mutated to aspartate to generate an aglycosylated version. The terminal lysine can be mutated to an alanine.

[0042] Fig. 10 demonstrates the sequence for Fc Fragment 1 of bovine IgGl. [0043] Fig. 11 demonstrates the sequence for Fc Fragment 1 of bovine IgG2.

[0044] Fig. 12 demonstrates the sequence for Fc Fragment 2 of bovine IgG2.

[0045] Fig. 13 demonstrates the sequence for Fc Fragment 3 of bovine IgG2.

[0046] Fig. 14 demonstrates the sequence for Fc Fragment 4 of bovine IgG2.

[0047] Fig. 15 demonstrates the sequence for Fc Fragment 5 of bovine IgG2.

[0048] Fig. 16 demonstrates the SDS PAGE of supernatants from 293f cells transfected with DNA encoding bovine IgG Fc fragments. Lane 1 is negative control (no protein expressed in 293f cells). Bovine IgG2 Fc Fragment 4 did not express and is not included in the gel.

[0049] Fig. 17 demonstrates the bovine FcMBL variants were expressed in 293f cells and then purified using mannan conjugated agarose to identify FcMBL variants that are functional for binding mannan. The versions containing the IgG3 fragment did not express and were not included in this Coomassie stained SDS-PAGE.

[0050] Fig. 18 demonstrates multiple sequence alignments of Fc fragments from bovine IgG2.

[0051] Fig. 19 depicts the results of a screen for bovine FcMBL variants (BtFclN and BtFclD) vs. a human FcMBL control.

[0052] Fig. 20 depicts a screen for bovine FcMBL variants (BtFclN, BtFclD), vs. human FcMBL control. The TB isolates (irradiated) are: 1) H37Rv, 2) HN878, 3) CDC 1551, and 4) BCG Pasteur.

[0053] Fig. 21 depicts the results of a screen of human FcMBL binding to Mbovis AF2122 membrane fraction & irradiated whole cells was tested.

[0054] Fig. 22 depicts a screen for bovine FcMBL variants (BtFclN, BtFclD), vs. human FcMBL control. The TB isolates (irradiated) are: 1 ) Mbovis AF2122 membrane, T) Mbovis AF2122 irradiated cells.

[0055] Fig. 23 depicts a schematic of the Tb CAPS (Captured Antigen Presentation System) Vaccine which combines two technologies which are in clinical development. 1. Capture of inactivated pathogen antigens on particles coated with BtFcMBL 2. Biomaterial technology that forms an artificial lymph node that recruits and educates the immune response.

DETAILED DESCRIPTION

[0056] Described herein is an engineered fusion polypeptide comprising: a) an antibody fragment comprising the C2 and/ or C3 domains of a bovine immunoglobulin G (IgG); and b) at least one bovine mannose binding lectin (MBL) domain. These fusion polypeptides can be utilized in vaccination and/or immunization methods described herein, to bind biological pathogens, or identify subclasses or specific pathogen species for use in devices and systems for the treatment and diagnosis of livestock with infectious diseases, blood-bome infections, or sepsis. Treatment can be carried out in vivo or ex vivo.

[0057] The engineered fusion polypeptide(s) as described herein can bind or capture at least one microorganism, e.g., an intact microorganism, and/or microorganism-derived biomolecule. The terms "microbial matter,"“antigen,” or“microorganism -derived biomolecule” as used herein, refers to any matter or component that is derived, originated, or secreted from a microorganism. For example, a microorganism or a component derived or secreted from a microorganism that can bind to or be bound by an engineered fusion polypeptide or bovine mannose binding lectin as described herein. The microorganism-derived biomolecule can include, but is not limited to, a cell wall component, an outer membrane, a plasma membrane, a ribosome, a microbial capsule, a pili or flagella, any fragments of the aforementioned microbial components, any nucleic acid (e.g., DNA, including 16S ribosomal DNA, and RNA) derived from a microorganism, and microbial endotoxin (e.g., lipopolysaccharide). In addition, the microorganism-derived biomolecule(s) can encompass non-viable microorganisms or microorganism-derived biomolecule(s) that can cause an adverse effect (e.g., toxicity) to a host or an environment.

[0058] As used herein,“microbe” or“microorganism” refers to an organism which is microscopic. A microbe can be a single-celled organism. In some embodiments of any of the aspects, a microbe can be a bacterium. As used herein, the term“pathogen” refers to an organism or molecule that causes a disease or disorder in a subject. For example, pathogens include but are not limited to viruses, fungi, bacteria, parasites and other infectious organisms or molecules therefrom, as well as taxonomically related macroscopic organisms within the categories algae, fungi, yeast, protozoa, or the like. In some embodiments, the subject is a bovine or cow, e.g., the pathogen is pathogenic to a bovine or cow. In some embodiments of any of the aspects, the microorganism or pathogen is live, deactivated, killed, or a fragment thereof. In some embodiments of any of the aspects, the microorganism is a virus, bacterium, fungus, parasite, worm, yeast, or prion.

[0059] As used herein, a“viral infection” refers to any infection caused by a virus. A viral infection as described herein can be caused by any virus type currently known, or yet to be discovered that results in a pathogenic disease. Exemplary viruses include, but are not limited to, coronavirus, respiratory syncytial virus, bovine diarrhea virus, rabies virus, Herpes virus, retrovirus, lentivirus, or any other virus known in the art.

[0060] As used herein, a“bacterial infection” refers to any infection caused by a bacterium. A bacterial infection as described herein can be caused by any bacteria type currently known, or yet to be discovered that results in a pathogenic disease. Exemplary bacteria listed by genus include, but are not limited to Clostridium, Staphalococcus, Streptococcus, Escherichia (e.g. E. coli), Mycobacterium, Pseudomonas, Burkholderiz, Trichomonas, Campylobacter, Shingella, Salmonella, their species, or any other bacteria known in the art. Pathogenic bacteria and diseases are well known in the art.

[0061] As used herein, a“fungal infection” refers to any infection caused by a fungus. A fungal infection as described herein can be caused by any fungi currently known, or yet to be discovered that results in a pathogenic disease. Exemplary fungi include, but are not limited to, Trichophyton, Microsporum, Epidermophyton known to cause ringworm, their species, or any other fungus known in the art. A fungal infection can be treated with antifungals, for example, fluconazole, ketoconazole, or amphotericin B.

[0062] As used herein, a“parasitic infection” refers to any infection caused by a parasite. A parasitic infection as described herein can be caused by any parasite currently known, or yet to be discovered that results in a pathogenic disease. Exemplary parasites that affect cattle include, but are not limited to, roundworms (nematodes), tapeworms (cestodes), flukes (trematodes), Cooperia, lice, their species, or any other parasite known in the art. A parasitic infection can be treated with, for example, a deworming agent such as oxfendazole.

[0063] As used herein, a“yeast infection” can be caused by any yeast or yeast spores currently known, or yet to be discovered that results in a pathogenic disease. Exemplary yeast that affect cattle include, but are not limited to, Cryptococcus, Candida, Rhodotorula, their species, or any other yeast known in the art. A yeast infection can be treated with oral antifungals, for example, ketoconazole, itraconazole, or fluconazole.

[0064] As used herein,“mad cow disease” or“bovine spongiform encephalopathy” refers to a fatal neurodegenerative disease in cattle that can be passed on to humans who have eaten beef from an affected cow. The cause of the infection is currently unknown. To not be bound by a particular theory, the disease can be associated with a prion infection. A prion is a misfolded protein that has the ability to replicate by causing other normally folded proteins of the same type to take on their misfolded shape. There is currently no known treatment for mad cow disease. The current treatments manage symptoms of the disease which include twitching, abnormal gait, hyper-responsiveness to certain stimuli, hindlimb ataxia, etc. In human subjects, the disease is also known as Creutzefeldt-Jakob disease. Similar symptoms may be present including anxiety, depression, memory loss, blurred vision, insomnia, or difficulty speaking.

[0065] In accordance with various embodiments described herein, the engineered fusion polypeptides described herein comprise a mannose binding lectin domain, directly or indirectly, conjugated to a bovine antibody fragment. In some embodiments, the fusion polypeptide can can further comprise a polypeptide linker, e.g., connecting the MBL domain and the antibody fragement. Thus, the engineered fusion polypeptide described herein can be used as soluble proteins, e.g., in therapeutic compositions, or be immobilized to a substrate for various applications ranging from diagnosis and/or treatment of a bovine microbial infection or disease, to microorganism-clearing compositions or devices, to drug delivery.

[0066] It is contemplated that MBL and/or antibody fragment sequences obtained from bovines other than Bos taurus can be used in the methods and compositions described herein, e.g., to engineer the fusion polypeptide as described herein. In some embodiments of any of the aspects, the fusion polypeptides described herein comprise MBL and/or antibody fragment sequences obtained from the same species of bovine as the subject. In some embodiments of any of the aspects, the fusion polypeptides described herein comprise MBL and/or antibody fragment sequences obtained from Bos taurus.

[0067] In some embodiments of any of the aspects, the MBL and antibody fragment sequences are obtained from the same species of bovine. In some embodiments of any of the aspects, the MBL and antibody fragment sequences are obtained from different species of bovine.

[0068] In one aspect of any of the embodiments, provided herein is a composition that comprises an engineered fusion polypeptide comprising an antibody fragment comprising the C2 and/ or C3 domains of a bovine immunoglobulin G (IgG); and at least one bovine mannose binding lectin (MBL) domain. The MBL domain as described herein can further bind to, or be bound to, a microorganism or a microorganism-derived biomolecule.

[0069] The terms“FcMBL” and“fusion polypeptide” are used interchangeably to refer to the engineered bovine polypeptide described herein that comprises bovine antibody domain or fragment thereof; and at least one mannose-binding lectin (MBL) domain.

[0070] As used herein, the terms “proteins” and “peptides” and “polypeptides” are used interchangeably herein to designate a series of amino acid residues connected to the other by peptide bonds between the alpha-amino and carboxy groups of adjacent residues. The terms“protein”, and “peptide”, which are used interchangeably herein, refer to amino acids, including modified amino acids (e.g., phosphorylated, glycated, etc.) and amino acid analogs, regardless of its size or function. Although “protein” is often used in reference to relatively large polypeptides, and“peptide” is often used in reference to small polypeptides, usage of these terms in the art overlaps and varies. The term“peptide” as used herein refers to peptides, polypeptides, proteins and fragments of proteins, unless otherwise noted. The terms“protein” and“peptide” are used interchangeably herein when referring to a gene product and fragments thereof. Thus, exemplary peptides or proteins include gene products, naturally occurring proteins, homologs, orthologs, paralogs, fragments and other equivalents, variants, fragments, and analogs of the foregoing.

[0071] The terms“immunoglobulin” or“Ig” refer to any class of proteins present in the serum and cells of the immune system that function as an antibody. The immunoglobulin can recognize pathogens that cause disease (e.g. bovine infectious diseases) such as bacteria, viruses, fungi, parasites, and the like. The immunoglobulin structure is known in the art to comprise a heavy chain, a light chain, and an antigen-binding domain. The heavy chain defines the class of the antibody (e.g. IgG) and comprises a variable region (VH), a constant domain (CHI), a hinge region, and two additional constant domains known as CH2 and CH3. As used herein,“C2” refers to the constant domain CH2 of the immunoglobulin. As used herein,“C3” refers to the constant domain CH3 of the immunoglobulin. The light chain of the antibody comprises one variable domain (VL), one constant domain (CL) antigen binding site, and hinge regions. The Fc fragment comprises the constant regions of the immunoglobulin and the Fab fragment comprises the variable region that binds to the antigen. [0072] Ig sequences, and the C2 and/or C3 domains thereof, are well known in the art. For example, Ig structures and domains are described in Janeway CA Jr, Travers P, Walport M, et al. Immunobiology: The Immune System in Health and Disease. 5th edition. New York: Garland Science; 2001. The structure of a typical antibody molecule. Available on the world wide web at ncbi.nlm.nih.gov/books/NBK27144/; which is incorporated by reference. The sequences of bovine Ig genes and polypeptides are readily available from publically available databases, e.g., IMGT maintains annotated sequence data for Bos taurus Ig genes on the world wide web at

imgt.org/IMGTrepertoire/index.php?section=LocusGenes&repertoire=genetable&species=bovine&gr oup=IGHC.

[0073] The terms“antibody” and“antibodies” and“antibody fragment” are used interchangeable with the term“immunoglobulin” that include polyclonal antibodies, monoclonal antibodies, humanized or chimeric antibodies, single chain Fv antibody fragments, Fab fragments, and F(ab)2 fragments. Antibodies having specific binding affinity for a bovine pathogen can be produced through standard methods known in the art. Alternatively, antibodies can be commercially available, for example, from R&D Systems, Inc., Minneapolis, Minn.

[0074] As used herein, the terms“antibody” and“antibodies” include intact antibody, or a binding fragment thereof that competes with the intact antibody for specific binding and includes chimeric, fully mammalian or bovine, and bispecific antibodies. In some embodiments of any of the aspects, binding fragments are produced by recombinant DNA techniques. In additional embodiments, binding fragments are produced by enzymatic or chemical cleavage of intact antibodies. Binding fragments include, but are not limited to, Fab, Fab', F(ab')2, Fv, and single-chain antibodies.

[0075] The antibody fragment as described herein can include but is not limited to full length sequences or fragments of immunoglobulin (IgG) 1, IgG2, IgG3, or IgG4. In some embodiments of any of the aspects, the C2 and the C3 domains of IgG that comprise the Fc portion of the antibody can be used. The structure of immunoglobulins is known in the art.

[0076] In some embodiments of any of the aspects, the antibody or antigen-binding fragment thereof is or is derived from bovine sequence. In some embodiments of any of the aspects, the bovine antibody fragment is or is derived from Bos taurus or Bos taurus taurus sequence.

[0077] In some embodiments of any of the aspects, the antibody fragment comprises the C2 and/or

C3 domain of the bovine immunoglobulin G (IgG). In some embodiments of any of the aspects, the bovine antibody fragment as described herein is from bovine IgGl, IgG2, or IgG3. In some embodiments of any of the aspects, the bovine antibody fragment is a polypeptide comprising the sequence of SEQ ID NO: 7, or SEQ ID NO: 8, or SEQ ID NO: 9, or SEQ ID NO: 10, or SEQ ID NO: 11, or SEQ ID NO: 12, or SEQ ID NO: 13, or SEQ ID NO: 14 or SEQ ID NO: 15. In some embodiments of any of the aspects, the bovine antibody fragment is a polypeptide consisting essentially of the sequence of SEQ ID NO: 7, or SEQ ID NO: 8, or SEQ ID NO: 9, or SEQ ID NO: 10, or SEQ ID NO: 11, or SEQ ID NO: 12, or SEQ ID NO: 13, or SEQ ID NO: 14 or SEQ ID NO: 15. In some embodiments of any of the aspects, the bovine antibody fragment is a polypeptide consisting of the sequence of SEQ ID NO: 7, or SEQ ID NO: 8, or SEQ ID NO: 9, or SEQ ID NO: 10, or SEQ ID NO: 11, or SEQ ID NO: 12, or SEQ ID NO: 13, or SEQ ID NO: 14 or SEQ ID NO: 15. These sequences are non-limiting and the antibody fragment can further comprise a fragment from another immunoglobulin class or sub-class.

[0078] In certain embodiments, the constant regions of bovine pathogen antibodies or fragments thereof for use in the methods disclosed herein are fully bovine. Fully bovine antibodies or fragements thereof can be made using techniques that are known in the art and as provided herein. For example, fully bovine antibodies against a specific antigen can be prepared by administering the antigen to a transgenic animal which has been modified to produce such antibodies in response to antigenic challenge, but whose endogenous loci have been disabled. Exemplary techniques that can be used to make such antibodies are described in U.S. Pat. Nos. 6, 150,584; 6,458,592; 6,420, 140. Other techniques are known in the art. Fully bovine antibodies and fragments thereof can likewise be produced by various display technologies, e.g., phage display or other viral display systems known in the art. Completely bovine antibodies or fragments theeof are particularly desirable for therapeutic treatment of bovine subjects. Bovine antibody fragments can be made by a variety of methods known in the art including phage display methods using antibody libraries derived from mammalian immunoglobulin sequences. See, for example, U.S. Pat. Nos. 9,644,021 B2, 4,716,111, and US 2014/0227723 Al .

[0079] In some embodiments of any of the aspects, an antibody or fragment thereof for use in the methods disclosed herein will not elicit a deleterious immune response in the mammal to be treated, e.g., in a cow. In some embodiments of any of the aspects, the antibodies or fragments thereof for use in the methods disclosed herein can be modified to reduce their immunogenicity using art-recognized techniques.

[0080] As used herein, the term“mannose binding lectin” or“mannan binding lectin” or“MBL” refers to a polypeptide that is responsible for innate immune responses and activation of the complement system. MBL can also signal the destruction or phagocytosis of microorganisms (e.g. bacteria or viruses) by immune cells (e.g. leukocytes or macrophages). These immune responses by MBL are well known in the art, for example, see Cedzyhski,et al. The Complement FactsBook 2018; which is incorporated by reference herein in its entirety. Mannose-binding lectin (MBL) is also known as mannose binding protein (MBP), or mannan-binding lectin or mannan-binding protein MBL is a calcium-dependent serum protein that can bind to carbohydrates on the surface of a wide range of microorganisms or pathogens (viruses, bacteria, fungi, protozoa) where it can activate the complement system. MBL can also serve as a direct fusion protein and mediate binding and uptake of pathogens by tagging the surface of a pathogen to facilitate recognition and ingestion by phagocytes. MBL is a member of the collectin family of proteins. A native MBL is a multimeric structure (e.g., about 650 kDa) composed of subunits, each of which contains three identical polypeptide chains. Each MBL polypeptide chain comprises a N-terminal cysteine rich region, a collagen-like region, a neck region, and a carbohydrate recognition domain (CRD). The sequence of each region has been identified and is well known in the art. The MBL described herein can comprise the full sequence of MBL or truncated sequence of the MBL. For example, the MBL can comprise the neck region and CRD domain but lack the N-terminal cysteine rich region. In some embodiments of any of the aspects, the MBL can comprise the neck region and CRD domain. In some embodiments of any of the aspects, the MBL can consist essentially of the neck region and CRD domain. In some embodiments of any of the aspects, the MBL can consist of the neck region and CRD domain. In some embodiments of any of the aspects, the MBL does not comprise the cysteine-rich region. In some embodiments of any of the aspects, the MBL does not comprise the collagen-like region. Any modification can be made to the MBL that permits binding to the bovine pathogen.

[0081] The MBL of the fusion polypeptides described herein is a bovine MBL. In some embodiments of any of the aspects, the bovine MBL is the MBL from Bos taurus. In some embodiments of any of the aspects, the bovine MBL can comprise an amino acid sequence of SEQ ID NO: 16 (GenBank: AAI09675.1) or SEQ ID NO: 17 (NCBI Reference Sequence: XP_005225409.1). In some embodiments of any of the aspects, the bovine MBL can comprise an amino acid sequence consisting essentially of SEQ ID NO: 16 (GenBank: AAI09675.1) or SEQ ID NO: 17 (NCBI Reference Sequence: XP_005225409.1). In some embodiments of any of the aspects, the bovine MBL can comprise an amino acid sequence consisting of SEQ ID NO: 16 (GenBank: AAI09675.1) or SEQ ID NO: 17 (NCBI Reference Sequence: XP_005225409.1). The MBL protein is translated from the MBL1 (NCBI Accession: NM_001010994.3) or MBL2 mRNA transcripts (NCBI Accession: NM_174107.2), e.g., from Bos taurus. In some embodiments of any of the aspects, the MBL as described herein can bind to a microorganism (e.g. virus, bacteria, etc) or a microorganism biomolecule as described herein.

[0082] In some embodiments of any of the aspects, the fusion polypeptide as described herein further comprises a polypeptide linker domain between the antibody fragment and the MBL domain. As used herein, the term“linker” means a molecular moiety that connects two parts of a composition. Peptide linkers may affect folding of a given fusion protein, and may also react/bind with other proteins, and these properties can be screened for by known techniques. Example linkers, in addition to those described herein, include is a string of histidine residues, e.g., His6; sequences made up of Ala and Pro, varying the number of Ala-Pro pairs to modulate the flexibility of the linker; and sequences made up of charged amino acid residues e.g., mixing Glu and Lys. Flexibility can be controlled by the types and numbers of residues in the linker. See, e.g., Perham, 30 Biochem. 8501 (1991); Wriggers et ah, 80 Biopolymers 736 (2005). Chemical linkers may comprise a direct bond or an atom such as oxygen or sulfur, a unit such as NH, C(O), C(0)NH, SO, SO2, SO2NH, or a chain of atoms, such as substituted or unsubstituted Ci-Ce alkyl, substituted or unsubstituted C2-C6 alkenyl, substituted or unsubstituted C2- Ce alkynyl, substituted or unsubstituted C6-C12 aryl, substituted or unsubstituted C5-C12 heteroaryl, substituted or unsubstituted C5-C12 heterocyclyl, substituted or unsubstituted C3-C12 cycloalkyl, where one or more methylenes can be interrupted or terminated by O, S, S(O), SO2, NH, or C(O). The linker domain can be 1 amino acid or more, 5 amino acids or more, 10 amino acids or more, 15 amino acids or more, 20 amino acids or more, 25 amino acids or more, 30 amino acids or more, 35 amino acids or more, 40 amino acids or more, 45 amino acids or more, 50 amino acids or more and beyond.

[0083] In some embodiments of any of the aspects, the bovine antibody fragment is linked to the N-terminus or C-terminus of the bovine MBL domain. In some embodiments of any of the aspects, the bovine antibody fragment is separated from the MBL domain by a peptide linker or additional lectin. In some embodiments of any of the aspects, the MBL domain binds to a microorganism or a microorganism -derived biomolecule.

[0084] In some embodiments of any of the aspects, the fusion polypeptide comprises 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, or SEQ ID NO: 6. In some embodiments of any of the aspects, the fusion polypeptide consists essentially of 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, or SEQ ID NO: 6. In some embodiments of any of the aspects, the fusion polypeptide consists of 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, or SEQ ID NO: 6. The exemplary sequences provided herein for the fusion polypeptide as described herein are not construed to be limiting.

[0085] The term "conservative substitution," or“substitution” or“substituted” when describing a polypeptide, refers to a change in the amino acid composition of the polypeptide that does not substantially alter the polypeptide's activity, fore examples, a conservative substitution refers to substituting an amino acid residue for a different amino acid residue that has similar chemical properties. Conservative amino acid substitutions include replacement of a leucine with an isoleucine or valine, an aspartate with a glutamate, or a threonine with a serine. "Conservative amino acid substitutions" result from replacing one amino acid with another having similar structural and/or chemical properties, such as the replacement of a leucine with an isoleucine or valine, an aspartate with a glutamate, or a threonine with a serine. Thus, a "conservative substitution" of a particular amino acid sequence refers to substitution of those amino acids that are not critical for polypeptide activity or substitution of amino acids with other amino acids having similar properties (e.g., acidic, basic, positively or negatively charged, polar or non-polar, etc.) such that the substitution of even critical amino acids does not substantially alter activity. Conservative substitution tables providing functionally similar amino acids are well known in the art. For example, the following six groups each contain amino acids that are conservative substitutions for one another: 1) Alanine (A), Serine (S), Threonine (T); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W). (See also Creighton, Proteins, W. H. Freeman and Company (1984).) In addition, individual substitutions, deletions or additions that alter, add or delete a single amino acid or a small percentage of amino acids in an encoded sequence are also "conservative substitutions." Insertions or deletions are typically in the range of about 1 to 5 amino acids. [0086] In some embodiments of any of the aspects, the fusion polypeptide can comprise an antibody fragment and/or MBL sequence which has at least 80%, 85%, 90%, 95%, 98% or greater sequence homology with a reference sequences provided herein (e.g. SEQ ID NOs: 1-15). In some embodiments of any of the aspects, the fusion polypeptide can comprise an antibody fragment and/or MBL sequence which has at least 80%, 85%, 90%, 95%, 98% or greater sequence homology with a reference sequence provided herein and which retains the activity of the reference sequence, e.g., the ability of MBL to bind a microorganism and/or microorganism-derived biomolecule.

[0087] In some embodiments of any of the aspects, the fusion polypeptide can comprise an antibody fragment and/or MBL sequence which has at least 80%, 85%, 90%, 95%, 98% or greater sequence homology with a wild-type bovine sequence (SEQ ID NOs: 16 & 17). In some

embodiments of any of the aspects, the fusion polypeptide can comprise an antibody fragment and/or MBL sequence which has at least 80%, 85%, 90%, 95%, 98% or greater sequence homology with a wild-type bovine sequence and which retains the activity of the wild-type sequence, e.g., the ability of MBL to bind a microorganism and/or microorganism-derived biomolecule.

[0088] The fusion polypeptides as disclosed herein include derivatives that are modified, e.g., by the covalent attachment of any type of molecule to the polypeptide. Lor example, but not by way of limitation, the derivatives include polypeptides that have been modified, e.g., by glycosylation, acetylation, pegylation, phosphorylation, amidation, or derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to a cellular ligand or other protein. Any of numerous chemical modifications can be carried out by known techniques, including, but not limited to, specific chemical cleavage, acetylation, formylation, etc. Additionally, the derivative can contain one or more non- classical amino acids.

[0089] Lurther regarding modifications to the engineered fusion polypeptide as described herein, its binding characteristics can be manipulated by directed evolution for altered binding specificity.

The fusion polypeptide as described herein may be modified so that it binds to a more limited set of sugars or other molecular features, with the result that the modified fusion polypeptide will bind to a more limited set of microorganisms to provide a capability for pathogen class identification (e.g., one of virus, bacteria, fungi, or protozoan), subclass typing (e.g., gram negative or gram positive bacteria) or specific species determination. Numerous strategies are available in the art.

[0090] The fusion polypeptide as described herein can be derived from bovines, cattle, cows, Bos taurus, or Bos taurus taurus. Lor example, a straightforward directed evolution strategy visually examines an atomic structure of the fusion polypeptide or MBL domain complexed with a sugar, and then mutates appropriate amino acids that make contact in a sugar-specific manner, so that distinctive contacts are lost or particular types of steric hindrance are created. The three dimensional structure of rat MBL has been solved in a complex with a high-mannose oligosaccharide and with N

acetylglucosamine, a methylated fiicose, and so on. Hisl89Val and Ile207Val are examples of substitutions that modifications alter specificity. [0091] In another strategy of directed evolution, the fusion polypeptide can be subjected to random mutagenesis and the resulting polypeptides are screened for desired qualities. These methods are known in the art. See Wang et al. Cell, Volume 160, Issue 4, 2015, Pages 785-797; or Daugherty et al. Protein Engineering, Design and Selection, Volume 11, Issue 9, 1998, Pages 825-832.

[0092] The directed evolution paradigm can be applied to the antibody fragment, MBL, or the full length fusion polypeptide as described herein in order to select variants with specific binding to the microorganism or microorganism-derived biomolecules of viruses, yeast, gram-positive bacteria, gram-negative bacteria, coagulase negative, aerobic bacteria, etc. For this to work, however, the pattern and nature of the target sugars or related surface features on these target microorganisms can differ between the classes or species.

[0093] For example, derivatives of MBL with a particular specificity can be isolated by the following approach, which is a standard phage display strategy: First, express a set of MBL variants from a phagemid vector; then bind this library to a target of interest (e.g., Clostridium) and perform one or two rounds of selection; and then perform a round of negative selection against a related target (e.g., Candida), taking those phagemids that fail to bind. These cycles of positive and negative selection are then repeated until a population of phages that generally bind to the target and do not bind to the non target is generated. This method may be applied to any pair of microbial strains against which differential binding is desired, such as bacteria that are resistant and sensitive to a given antibiotic. This positive/negative enrichment strategy may also be used with an antibody-phage display library, which is an even more standard way to isolate such specific binders.

[0094] In some embodiments of any of the aspects, the fusion polypeptide preferentially binds to a bovine pathogen or microorganism. In some embodiments of any of the aspects, the MBL domain of the fusion polypeptide preferentially binds to a bovine pathogen selected from the group consisting of: Clostridium tetani,, Clostridium novyi, Clostridium perfringens, Clostridium perfringens, Clostridium perfringens, Clostridium septicum, Clostridium chauvoei, Clostridium sordellii, Clostridium hemolyticum, Steptococcus uberis, Staphylococcus aureus, Escherichia coli, Streptococcus agalactiae, Streptococcus dysglalactiae, Coorynebacterium bovis, Mycoplasma bovis, Mannheimia haemolytica, Pasteurella multocida, Histophilus somni, Mycobacterium tuberculosis, bovine viral diarrhea virus, bovine respiratory syncyntial virus, rabies virus, bovine Herpes virus 1, Bovine Herpes virus 4, bovine rhinovirus, bovine enterovirus, bovine coronavirus, bovine reovirus, parainfluenza- 3 virus, or round worm, or fragment thereof.

[0095] As described herein, the fusion polypeptide can comprise at least one mannose binding lectin domain, including at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten or more mannose binding lectin domains. In such embodiments, the distances between MBL domains can be engineered to match with the distance between the binding sites on the target microorganism surface. [0096] The fusion polypeptide as described herein can have each of the individual antibody fragment and/or MBL domains the same. Alternatively, a fusion polypeptide can have at least one, at least two, or at least three domains different from the rest. In such embodiments, fusion polypeptides that share a common binding specificity for microorganisms or microorganism biomolecules can be used.

[0097] In some embodiments of any of the aspects, the fusion polypeptide can have an amino acid sequence of about 10 to about 300 amino acid residues, or about 50 to about 150 amino acid residues. In some embodiments of any of the aspects, the fusion polypeptide can have an amino acid sequence of at least about 5, at least about 10, at least about 15, at least about 20, at least about 30, at least about 40, at least about 50, at least about 60, at least about 70, at least about 80, at least about 90, at least about 100, at least about 200, at least about 300, at least about 400, at least about 500, amino acid residues or more. For any known sequences of the fusion polypeptide, MBL, or antibody fragment as described herein, one of skill in the art can determine the optimum length of amino acid sequence for each domain.

[0098] In some embodiments of any of the aspects, one or more amino acids within the fusion polypeptide is substituted to prevent glycosylation. In some embodiments of any of the aspects, one or more substituted amino acids is substituted with an aspartate.

[0099] The term“glycosylation” as used herein refers to the addition of carbohydrate moieties found on a polypeptide sequence (e.g. a fusion polypeptide). Glycosylation is a post-translational modification that can occur in eukaryotic cells when a protein is extruded into the endoplasmic reticulum. Glycosylation variants may, for example, be prepared by removing, changing and/or adding one or more glycosylation sites in the nucleic acid or polypeptide sequence used to generate the fusion polypeptide. Methods of predicting and modifying sequences for glycosylation are well known in the art. For example, see US5047335A; US7338933B2; W01987005330A1;

W02003031464A2; Chuang et al. Bioinformatics, 2012; Steentoft et al. EMBO J, 2013; Li et al. Scientific Reports, 2016; Li et al. Bioinformatics, 2015; Gupta et al. Pacific Symposium on

Biocomputing, 2002, all of which have been incorporated herein in their entirety.

[00100] In general, glycosylation is either N-linked or O-linked. N-linked refers to the attachment of the carbohydrate moiety to the side chain of an asparagine residue. N-linked glycosylation can occur at polypeptide sequences such as Asparagine (N)-X-Threonine (T) or Asparagine (N)-X-Serine (S), with rare occurrences at Asparagine (N)-X-Cysteine (C), where X is any standard amino acid substitution except proline. Tools for predicting N-linked glycosylation sites of a polypeptide can be found on the world wide web at cbs.dtu.dk/services/NetNGlyc.

[00101] O-linked glycosylation refers to the attachment of one of the carbohydrates N- aceylgalactosamine, galactose, or xylose to a hydroxyamino acid, most commonly serine or threonine, although 5- hydroxyproline or 5-hydroxylysine may also be used. Tools for predicting O-linked glycosylation can be found on the world wide web at cbs.dtu.dk/services/NetOGlyc. Thus, the fusion polypeptide as described herein can be engineered with or without a glycosylation site. [00102] In some embodiments of any of the aspects, a fusion polypeptide described herein comprises N-linked and/or O-linked glycosylation. In some embodiments of any of the aspects, a fusion polypeptide described herein comprises N-linked glycosylation. In some embodiments of any of the aspects, a fusion polypeptide described herein comprises N-linked glycosylation but not O- linked glycosylation. In some embodiments of any of the aspects, a fusion polypeptide described herein comprises O-linked glycosylation. In some embodiments of any of the aspects, a fusion polypeptide described herein comprises O-linked glycosylation but not N-linked glycosylation.

[00103] In some embodiments of any of the aspects, a fusion polypeptide described herein does not comprise glycosylation. In some embodiments of any of the aspects, a fusion polypeptide described herein does not comprise glycosylation in an antibody fragment. In some embodiments of any of the aspects, a fusion polypeptide described herein does not comprise glycosylation in the MBL domain. A lack of glycosylation can reduce antibody effector function by decreased binding affinity to Fcgamma receptors.

[00104] In some embodiments of any of the aspects, the fusion polypeptide described herein comprises a mutation at a residue corresponding to residue 87 of the sequence of Fig. 1, such that the residue is not asparagine. In some embodiments of any of the aspects, the fusion polypeptide described herein comprises a mutation at a residue corresponding to residue 87 of the sequence of Fig. 1, such that the residue is aspartic acid.

[00105] In some embodiments of any of the aspects, the fusion polypeptide described herein comprises a mutation at a residue corresponding to residue 91 of the sequence of Fig. 3, such that the residue is not asparagine. In some embodiments of any of the aspects, the fusion polypeptide described herein comprises a mutation at a residue corresponding to residue 91 of the sequence of Fig. 3, such that the residue is aspartic acid.

[00106] In some embodiments of any of the aspects, the fusion polypeptide described herein comprises a mutation at a residue corresponding to residue 99 of the sequence of Fig. 5, such that the residue is not asparagine. In some embodiments of any of the aspects, the fusion polypeptide described herein comprises a mutation at a residue corresponding to residue 99 of the sequence of Fig. 5, such that the residue is aspartic acid.

[00107] In one aspect of any of the embodiments, described herein is a nucleic acid encoding a fusion polypeptide comprising a) an antibody fragment comprising the C2 and/ or C3 domains of a bovine immunoglobulin G (IgG); and b) a bovine mannose binding lectin (MBL) domain. In one aspect of any of the embodiments, described herein is a vector comprising a nucleic acid encoding a fusion polypeptide comprising a) an antibody fragment comprising the C2 and/ or C3 domains of a bovine immunoglobulin G (IgG); and b) a bovine mannose binding lectin (MBL) domain.

[00108] Expression of the fusion polypeptide as described herein can be under the control of an inducible promoter or introduced to a subject, cell, or system by a vector. The term "vector", as used herein, refers to a nucleic acid construct designed for delivery to a host cell or for transfer between different host cells. As used herein, a vector can be viral or non-viral. The term“vector” encompasses any genetic element that is capable of replication when associated with the proper control elements and that can transfer gene sequences to cells. A vector can include, but is not limited to, a cloning vector, an expression vector, a plasmid, phage, transposon, cosmid, artificial chromosome, virus, virion, etc. Such promoters and vector are known to those of skill in the art and, thus, are not described in detail herein.

[00109] In one aspect of any of the embodiments, described herein is a pharmaceutical or vaccine composition comprising the engineered fusion polypeptide as described herein, optionally bound to a microorganism and/or microorganism-derived biomolecule and at least one of a pharmaceutically acceptable carrier and an adjuvant.

[00110] As used herein, the term“pharmaceutical composition” refers to the fusion polypeptide as described herein in combination with a pharmaceutically acceptable carrier e.g. a carrier commonly used in the pharmaceutical industry. The phrase "pharmaceutically acceptable" is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio. In some embodiments of any of the aspects, a pharmaceutically acceptable carrier can be a carrier other than water. In some embodiments of any of the aspects, a pharmaceutically acceptable carrier can be a cream, emulsion, gel, liposome, nanoparticle, and/or ointment. In some embodiments of any of the aspects, a pharmaceutically acceptable carrier can be an artificial or engineered carrier, e.g., a carrier that the active ingredient would not be found to occur in in nature.

[00111] The term "vaccine composition" used herein is defined as composition used to elicit an immune response against an antigen within the composition in order to protect or treat an organism against disease.

[00112] The term "pharmaceutically acceptable" can refer to compounds and compositions which can be administered to a subject (e.g., a mammal or a cow) without undue toxicity. As used herein, the term "pharmaceutically acceptable carrier" can include any material or substance that, when combined with an active ingredient, allows the ingredient to retain biological activity and is non reactive with the subject's immune system. Examples include, but are not limited to, any of the standard pharmaceutical carriers such as a phosphate buffered saline solution, emulsions such as oil/water emulsion, and various types of wetting agents. Non-limiting examples of pharmaceutical carriers include particle or polymer-based vehicles such as nanoparticles, microparticles, polymer microspheres, or polymer-drug conjugates.

[00113] In some embodiments of any of the aspects, other ingredients can be added to vaccine formulations, including antioxidants, e.g., ascorbic acid; low molecular weight (less than about ten residues) polypeptides, e.g., polyarginine or tripeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids, such as glycine, glutamic acid, aspartic acid, or arginine; monosaccharides, disaccharides, and other carbohydrates including cellulose or its derivatives, glucose, mannose, or dextrins; chelating agents such as EDTA; and sugar alcohols such as mannitol or sorbitol.

[00114] As used herein, the term“adjuvant” can include any material or substance that, when combined with an active ingredient, that modifies the effect of the agent (e.g. fusion polypeptide). Adjuvants can be added to increase the immune response to a microorgansim or pathogen. Non limiting examples of adjuvants used in pharmaceutical or vaccine compositions include aluminum salts (e.g. aluminum hydroxide), paraffin oil, squalene, MF59® (Novartis Vaccines and Diagnostics Inc., MA, USA), or any other adjuvant known in the art.

[00115] The pharmaceutical compositions and vaccines described herein can be formulated for any of a variety of routes of administration as discussed further below. For example, the compositions or vaccines can be formulated as a spray for intranasal inhalation, nose drops, swabs for tonsils, etc. The compositions or vaccines can be formulated for oral delivery in the form of capsules, tablets, gels, thin films, liquid suspensions and/or elixirs, etc. In some embodiments of any of the aspects, the composition or vaccine is formulated for sublingual administration.

[00116] In some embodiments of any of the aspects, the pharmaceutical or vaccine compositions as described herein can be administered intravenously, intranasally, intramuscularly, subcutaneously, intraperitoneally, sublingually, vaginal, rectal or orally. In some embodiments of any of the aspects, the route of administration is oral, intranasal, subcutaneous, or intramuscular. In some embodiments of any of the aspects, the route of administration is sublingual, nasal, or oral administration.

[00117] When oral preparations are desired, the vaccine compositions can be combined with typical carriers, such as lactose, sucrose, starch, talc magnesium stearate, crystalline cellulose, methyl cellulose, carboxymethyl cellulose, glycerin, sodium alginate or gum arabic among others.

[00118] For some formulations (i.e., intravenous injection), the pharmaceutical or vaccine compositions as described herein for administration must be sterile. Sterility is readily accomplished by filtration through sterile filtration membranes, or by gamma radiation.

[00119] In some embodiments of any of the aspects, the vaccine composition is administered in a pure or substantially pure form, but it is preferable to present it as a pharmaceutical composition, formulation or preparation. Such formulation comprises deactivated or killed microorganisms or microorganism-derived biomolecule (e.g. bacteria or viruses described in TABLE 1 or TABLE 2) together with one or more pharmaceutically acceptable carriers and optionally other therapeutic ingredients.

[00120] In some embodiments of any of the aspects, the fusion polypeptide makes contact with microorganism as described herein or a microorganism-derived biomolecule. As used herein, the term “contacting” when used in reference to a cell or molecule, encompasses both introducing the polypeptide to a cell/molecule in a manner that permits physical contact of the cell/molecule with the fusion polypeptide and introducing an element, such as a genetic construct or vector, that permits the expression of the fusion polypeptide, in the cell.

[00121] In one aspect of any of the embodiments, provided herein is a method of treating or preventing an infectious disease in a subject (e.g. a cow). The term“animal”,“cow,”“cattle,” “livestock,” and“subject” are used interchangeably herein, and refer to an animal, particularly a cow (e.g. Bos taurus taurus), to whom treatment, including prophylactic treatment is provided (e.g., vaccination). The term“subject” as used herein refers to non-human animals. The term“non-human animals” and“non-human mammals” are used interchangeably herein includes all vertebrates, e.g., mammals, such as non-human primates, (particularly higher primates), sheep, dog, rodent (e.g. mouse or rat), guinea pig, goat, pig, cat, rabbits, cows, and non-mammals such as chickens, amphibians, reptiles etc. In some embodiments of any of the aspects, the subject is a cow. In some embodiments of any of the aspects, the subject is Bos taurus. In some embodiments of any of the aspects, the subject is a Bos inidicus (Zebu). In some embodiments of any of the aspects, the subject is Bos taurus primigenius, Bos taurus indicus, Bos taurus africanus, or Bos taurus taurus. However, it is contemplated that other forms of related livestock can be the subject such as yaks or buffalo.

[00122] In some embodiments of any of the aspects, the subject is an experimental animal or animal substitute as a disease model. In some embodiments of any of the aspects, the subject is a domesticated animal including companion animals, dairy, or beef cattle. A subject can have previously received a treatment for an infectious disease, or has never received treatment for an infectious disease. A subject can have previously been diagnosed with having an infectious disease, or has never been diagnosed with an infectious disease.

[00123] As used herein "preventing" or "prevention" refers to any methodology where the disease state does not occur due to the actions of the methodology (such as, for example, administration of a composition as described herein, e.g., vaccination). In one aspect of any of the embodiments, it is understood that prevention can also mean that the disease is not established to the extent that occurs in untreated controls. Accordingly, prevention of a disease encompasses a reduction in the likelihood that a subject can develop the disease, relative to an untreated subject (e.g. a subject who is not treated with the methods or compositions described herein).

[00124] As used herein, the terms "treat,” "treatment," "treating,” or“amelioration” refer to therapeutic treatments, wherein the object is to reverse, alleviate, ameliorate, inhibit, slow down or stop the progression or severity of a condition associated with having an infectious disease, e.g., bovine respiratory syncytial virus. The term“treating" includes reducing or alleviating at least one adverse effect or symptom of having an infectious disease, for example, dehydration, vomiting, diarrhea. Treatment is generally“effective" if one or more symptoms or clinical markers are reduced. Alternatively, treatment is“effective" if the progression of a disease is reduced or halted. That is, “treatment" includes not just the improvement of symptoms or markers, but also a cessation of, or at least slowing of, progress or worsening of symptoms compared to what would be expected in the absence of treatment. Beneficial or desired clinical results include, but are not limited to, alleviation of one or more symptom(s), diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, remission (whether partial or total), and/or decreased mortality, whether detectable or undetectable. The term "treatment" of a disease also includes providing relief from the symptoms or side-effects of the disease (including palliative treatment).

[00125] As used herein, the term“infectious disease” refers to any disease that is caused by a microorganism or toxin released from a microorganism that can lead to the spread of an infection among other animals or subjects. The microorganism can be for example, a virus, bacterium, fungus, parasite, worm, or prion. The symptoms of the infectious disease can include, for example, fever, dehydration, diarrhea, redness, or swelling of a wound. Specifically, cattle with an infectious disease can further exhibit a loss of milk production or infertility.

[00126] In some embodiments of any of the aspects, the infectious disease is selected from the group consisting of: mastitis, ephemeral fever, actinomycosis, bovine respiratory disease, bovine respiratory syncytial virus, infectious bovine rhinotracheitis, balanoposthitis, influenza, tuberculosis, Clostridium infection, and pneumonia.

[00127] In some embodiments of any of the aspects, the methods described relate to administering to the subject a fusion polypeptide as described herein bound to a microorganism described herein, e.g., a microorganism of TABLE 1 or any other bovine pathogen described herein. In some embodiments of any of the aspects, the methods described relate to administering to the subject a fusion polypeptide as described herein bound to a microorganism-derived biomolecule derived from a microorganism described herein, e.g., a microorganism of TABLE 1 or any other bovine pathogen described herein.

[00128] Other exemplary bovine infectious diseases and their corresponding pathogens include but are not limited to those listed in TABLE 1 below, and any other bovine diseases known in the art.

TABLE 1: BOVINE DISEASE AND PATHOGENS

[00129] The fusion polypeptide as described herein can be engineered to bind microorganisms or microorganism-derived biomolecules as described herein. In some embodiments of any of the aspects, the microorganism is selected from the group of Clostridium tetani,, Clostridium novyi, Clostridium perfringens, Clostridium perfringens, Clostridium perfringens, Clostridium septicum, Clostridium chauvoei, Clostridium sordellii, Clostridium hemolyticum, Steptococcus uberis, Staphylococcus aureus, Escherichia coli, Streptococcus agalactiae, Streptococcus dysglalactiae, Coorynebacterium bovis, Mycoplasma bovis, Mannheimia haemolytica, Pasteurella multocida, Histophilus somni, Mycobacterium tuberculosis, bovine viral diarrhea virus, bovine respiratory syncyntial virus, rabies virus, bovine Herpes virus 1, Bovine Herpes virus 4, bovine rhinovirus, bovine enterovirus, bovine coronavirus, bovine reovirus, parainfluenza- 3 virus, round worm, any of the microorganisms described in TABLE 1 or TABLE 2, or any other bovine pathogen known in the art. Examples of bovine pathogens and diseases can be found, for example, in Radostits et al. Veterinary Medicine, 10^th edition, 2007 or Blowery et al. Color Atlas of Diseases and Disorders of Cattle, 3^rd edition, 2011.

[00130] The compositions described herein can comprise multiple copies or versions of the fusion polypeptides described herein, e.g., in a mixture of physically-separate fusion polypeptides or in a multivalent molecule which comprises multiple copies or versions of the fusion polypeptides described herein in a single amino-acid chain, conjugated to each other, or conjugated to a scaffold molecule. Multivalent can include, e.g., bivalent, trivalent, quadvalent, or greater valency. Such compositions can be used to bind multiple different strain or species of microorganisms or biomolecules derived from different strains or species of microorganisms. Such compositions permit immunization against multiple strains or species using a single vaccine composition.

[00131] In another aspect of any of the embodiments, described herein is a method of stimulating an immune response in a subject or vaccinating a subject with the method comprising administering the composition as described herein to the subject.

[00132] As used herein, the terms "administering," and "injecting" are used interchangeably in the context of a therapeutic agent or vaccine. In some embodiments of any of the aspects, the term “administering” refers to the administration of a pharmaceutical composition comprising one or more agents or cells. The administering can be done by direct injection (e.g., directly administered to a target cell), subcutaneous injection, muscular injection, oral, or nasal delivery to the subject in need thereof. Administering can be local or systemic.

[00133] In some embodiments of any of the aspects, the agent or compositions described herein is used as a monotherapy. In some embodiments of any of the aspects, the agents described herein can be used in combination with other known agents and therapies for an infectious disease. Administered "in combination," as used herein, means that two (or more) different treatments are delivered to the subject during the course of the subject's affliction with the disorder, e.g., the two or more treatments are delivered after the subject has been diagnosed with the disorder (e.g. an infectious disease) and before the disorder has been cured or eliminated or treatment has ceased for other reasons. Current treatments for bovine infectious diseases are listed in TABLE 2. The pharmaceutical compositions of TABLE 2 can be used in combination with the fusion polypeptide described herein.

[00134] In some embodiments of any of the aspects, the methods described herein can further comprise administering an additional treatment for the relevant disease or condition.

[00135] Current therapeutic treatments for infectious veterinary diseases that affect cattle include (1) vaccines comprising inactivated bacterial cells, (ii) a live attenuated vaccine containing the genetically manipulated viruses, (iii) conjugates of pathogen lipopolysaccharides within a fusion protein or polypeptide, (iv) antibiotics and antiviral medications administered following infection.

[00136] The term "therapeutic agents" is art-recognized and refers to any chemical moiety that is a biologically, physiologically, or pharmacologically active substance that acts locally or systemically in a subject. Examples of therapeutic agents, also referred to as "drugs", are described in well-known literature references such as the Merck Index, the Physicians’ Desk Reference, and The

Pharmacological Basis of Therapeutics, and they include, without limitation, medications; vitamins; mineral supplements; substances used for the treatment, prevention, diagnosis, cure or mitigation of a disease or illness; substances which affect the structure or function of the body; or pro-drugs, which become biologically active or more active after they have been placed in a physiological environment. Various forms of a therapeutic agent may be used which are capable of being released from the subject composition into adjacent tissues or fluids upon administration to a subject.

[00137] Exemplary therapeutic agents and vaccines for infectious diseases in cattle include but are not limited to those in TABLE 2.

TABLE 2: THERAPEUTIC AGENTS AND VACCINES FOR BOVINE INFECTIOUS DISEASES

[00138] In some embodiments of any of the aspects, the delivery of one treatment is still occurring when the delivery of the second begins, so that there is overlap in terms of administration. This is sometimes referred to herein as "simultaneous" or "concurrent delivery." In other embodiments, the delivery of one treatment ends before the delivery of the other treatment begins. In some

embodiments of either case, the treatment is more effective because of combined administration. For example, the second treatment is more effective, e.g., an equivalent effect is seen with less of the second treatment, or the second treatment reduces symptoms to a greater extent, than would be seen if the second treatment were administered in the absence of the first treatment, or the analogous situation is seen with the first treatment. In some embodiments of any of the aspects, delivery is such that the reduction in a symptom, or other parameter related to the disorder is greater than what would be observed with one treatment delivered in the absence of the other. The effect of the two treatments can be partially additive, wholly additive, or greater than additive. The delivery can be such that an effect of the first treatment delivered is still detectable when the second is delivered. The agents described herein and the at least one additional therapy can be administered simultaneously, in the same or in separate compositions, or sequentially. For sequential administration, the agent described herein can be administered first, and the additional agent can be administered second, or the order of administration can be reversed. The agent and/or other therapeutic agents, procedures or modalities can be administered during periods of active disorder, or during a period of remission or less active disease. The agent can be administered before another treatment, concurrently with the treatment, post-treatment, or during remission of the disorder.

[00139] As will be appreciated by one of skill in the art, appropriate dosing regimens for a given composition or vaccine can comprise a single administration/immunization or multiple ones. For example, vaccines can be given as a primary immunization followed by one or more boosters. Boosters may be delivered via the same and/or different route as the primary immunization. Boosters are generally administered after a time period following the primary immunization or the previously administered booster. For example, a booster can be given about two weeks or more after a primary immunization, and/or a second booster can be given about two weeks or more after the first boosters. Boosters may be given repeatedly at time periods, for example, about two weeks or greater throughout up through the entirety of a subject's life. Boosters can be spaced, for example, about two weeks, about three weeks, about four weeks, about one month, about two months, about three months, about four months, about five months, about six months, about seven months, about eight months, about nine months, about ten months, about eleven months, about one year, about one and a half years, about two years, about two and a half years, about three years, about three and a half years, about four years, about four and a half years, about five years, about ten years, about 20 years, about 30 years or more after a primary immunization or after a previous booster.

[00140] The precise dose to be employed in the formulation will also depend on the route of administration and should be decided according to the judgment of the practitioner and each patient's circumstances. Ultimately, the veterinarian will decide the amount of protein or vaccine composition to administer to particular subjects. Doses generally each comprise between about 1 x 10⁶ and lx 10¹⁰ cells or more, for example, at least 5 x 10⁶, at least 1 x 10⁷, at least 5 x 10⁷, at least lxl 0⁸, at least 5 x 10⁸, at least 1 x 10⁹, at least 5 x 10⁹, at least lx 10¹⁰, at least 5x 10¹⁰ or more.

[00141] Vaccination can be conducted by conventional methods. For example, a displayed polypeptide can be used in a suitable diluent such as saline or water, or complete or incomplete adjuvants. The vaccine can be administered by any route appropriate for eliciting an immune response (e.g., sublingual, nasal, oral or intramuscular injection). The vaccine can be administered once or at periodic intervals until an immune response is elicited. Immune responses can be detected by a variety of methods known to those skilled in the art, including but not limited to, antibody production, cytotoxicity assay, proliferation assay and cytokine release assays. For example, samples of blood can be drawn from the immunized mammal, and analyzed for the presence of antibodies against the antigens of the immunogenic composition by ELISA (see de Boer GF, et. al, 1990, Arch Virol.

115:47-61) and the titer of these antibodies can be determined by methods known in the art.

[00142] In some embodiments of any of the aspects, efficacy is determined by measuring the immunogenicity of the administered composition or vaccine, for example, by assessing immunity to the individual to which the composition is administered or immunity conferred to one or more offspring of the individual to which the composition is administered. For example, the individual (e.g. a cow) being administered the composition can be a pregnant female, whose future or current offspring benefit from immune protection. Such immunity can be passed from mother to calf, for example, through lactation and/or through blood exchanged between from mother and fetus via the placenta. [00143] In some embodiments of any of the aspects, antibody titer can be used as a measure of the humoral immunogenicity of a given composition or vaccine. As used herein, antibody titer is a measurement of how much antibody an organism, such as, for example, a cow, has produced that recognizes a particular epitope, expressed as the greatest dilution that still gives a positive result. ELISA is a common means of determining antibody titers, but other assays known to one of skill in the art can be used as well.

[00144] In other embodiments, a pharmaceutical or vaccine composition as described herein can elicit increased T cell responses or cell-mediated immunogenicity relative to the T cell immune response or cell-mediated immune response elicited when a live microorganism (e.g. bacterium or virus) is administered.

[00145] In other embodiments, efficacy can be determined by assessing a variety of clinical measures including, but not limited to, fewer cases of bovine infectious diseases than expected in a given population, a reduction in mortality, a reduction in the severity of the infectious disease (e.g., reduction in symptoms or pathogen load).

[00146] The efficacy of a given composition for inducing immunity to the bovine pathogenic disease can be determined by the skilled clinician. However, a composition is considered“effective," as the term is used herein, if any one or all of the signs or symptoms of the disease (e.g., Clostridium infection) is/are altered in a beneficial manner, or other clinically accepted symptoms or markers of disease are improved, or ameliorated, e.g., by at least 10% in animals administered the composition compared to a substantially similar animal that has not been administered or immunized as described herein. Efficacy can also be measured by failure of an individual to worsen as assessed by stabilization of the disease (e.g., shorter duration, less intense symptoms), or the need for medical interventions (i.e., progression of the disease is halted or at least slowed). Methods of measuring these indicators are known to those of skill in the art and/or described herein.

[00147] In some embodiments of any of the aspects, the subject is further evaluated using one or more additional diagnostic procedures, for example, by medical imaging, physical exam, laboratory test(s), clinical history, genetic tests, blood and fecal tests, and the like. Medical imaging is well known in the art. As such, the medical imaging can be selected from any known method of imaging, including, but not limited to, ultrasound, computed tomography scan, positron emission tomography, photon emission computerized tomography, and magnetic resonance imaging.

[00148] The fusion polypeptide as described herein can be used to detect or screen for bovine pathogens. The fusion polypeptide can be further used to screen for test agents that prevent bovine microbial infections.

[00149] In some embodiments of any of the aspects, the fusion polypeptide described herein is attached to a solid substrate. As used herein, a“solid substrate” is a structure upon which one or more fusion polypeptides can be displayed for contact with a target microorganism or microorganism- derived biomolecule. A solid substrate provides a ready means for isolating or removing bound target microorganisms or microorganism-derived biomolecules from a mixture or suspension.

[00150] In some embodiments of any of the aspects, the solid substrate may comprise magnetic beads or other structured materials, which then pull microorganisms out from fluids, including biological fluids such as blood, and concentrate and collect the microorganisms, including living microorganisms. This approach is advantageous because the beads can then be examined for the presence of the microorganism, or be used to transfer the collected microorganisms to conventional pathogen culture and sensitivity testing assays, or for use in a pharmaceutical or vaccine composition. In other words, the engineered fusion polypeptide may be used in diagnostics as a means of collecting potential pathogens for identification; not only in the diagnosis of disease, but in the identification of water- or food-borne pathogens, particulates or other contaminants. Alternatively, the solid substrate may comprise a hollow-fiber reactor or any other blood filtration membrane or flow device (e.g., a simple dialysis tube) or other resins, fibers, or sheets to selective bind and sequester the biological pathogens.

[00151] In some embodiments of any of the aspects, the solid substrate is selected from a group consisting of: a magnetic microbead, a paramagnetic microbead, a microporous membrane, a hollow fiber, any other fluid filtration membrane, flow device, microtiter plate, cell culture plate, glass beads, latex beads, a living cell, an extracellular matrix of a biological tissue or organ, and a phagocyte.

[00152] The magnetic beads can be of any shape, including but not limited to spherical, rod, elliptical, cylindrical, disc, and the like. In some embodiments of any of the aspects, magnetic beads having a true spherical shape and defined surface chemistry are used to minimize chemical agglutination and non-specific binding. As used herein, the term“magnetic beads” refers to a nano- or micro-scale particle that is attracted or repelled by a magnetic field gradient or has a non-zero magnetic susceptibility. The magnetic bead can be paramagnetic or super-paramagnetic. In some embodiments of any of the aspects, magnetic beads are super-paramagnetic. Magnetic beads are also referred to as magnetic particles herein. In some embodiments of any of the aspects, magnetic beads having a polymer shell are used to protect the pathogen from exposure to iron. For example, polymer- coated magnetic beads can be used to protect pathogens from exposure to iron.

[00153] The magnetic beads can range in size from 1 nm to 1 mm. For example, magnetic beads are about 250 nm to about 250 pm in size. In some embodiments of any of the aspects, magnetic bead is 0.1 pm to 100 pm in size. In some embodiments of any of the aspects, magnetic bead is 0.1 pm to 50 pm in size. In some embodiments of any of the aspects, magnetic bead is 0.1 pm to 10 pm in size. In some embodiments of any of the aspects, the magnetic bead is a magnetic nano-particle or magnetic micro-particle. Magnetic nanoparticles are a class of nanoparticle which can be manipulated using magnetic field or magnetic field gradient. Such particles commonly consist of magnetic elements such as iron, nickel and cobalt and their chemical compounds. Magnetic nano-particles are well-known and methods for their preparation have been described in the art. See, e.g. , U.S. Patents No. 6,878,445; No. 5,543,158; No. 5,578,325; No. 6,676,729; No. 6,045,925; and No. 7,462,446; and U.S. Patent Publications No. 2005/0025971; No. 2005/0200438; No. 2005/0201941;

No. 2005/0271745; No. 2006/0228551; No. 2006/0233712; No. 2007/01666232;

and No. 2007/0264199.

[00154] Magnetic beads are easily and widely available commercially, with or without functional groups capable of binding to affinity molecules. Suitable magnetic beads are commercially available such as from Dynal Inc. (Lake Success, NY); PerSeptive Diagnostics, Inc. (Cambridge, MA);

Invitrogen Corp. (Carlsbad, CA); Cortex Biochem Inc. (San Leandro, CA); and Bangs Laboratories (Fishers, IN). In particular embodiments, magnetic particles are MyOne™ Dynabeads® magnetic beads (Dynal Inc.).

[00155] The solid substrate can be fabricated from or coated with a biocompatible material. As used herein, the term“biocompatible material” refers to any material that does not deteriorate appreciably and does not induce a significant immune response or deleterious tissue reaction, e.g., toxic reaction or significant irritation, over time when implanted into or placed adjacent to the biological tissue of a subject, or induce blood clotting or coagulation when it comes in contact with blood. Suitable biocompatible materials include, for example, derivatives and copolymers of a polyimides, polyethylene glycol), polyvinyl alcohol, polyethyleneimine, and polyvinylamine, polyacrylates, polyamides, polyesters, polycarbonates, and polystyrenes.

[00156] In some embodiments of any of the aspects, the solid substrate is fabricated or coated with a material selected from the group consisting of polydimethylsiloxane, polyimide, polyethylene terephthalate, polymethylmethacrylate, polyurethane, polyvinylchloride, polystyrene polysulfone, polycarbonate, polymethylpentene, polypropylene, a polyvinylidine fluoride, polysilicon, polytetrafluoroethylene, polysulfone, acrylonitrile butadiene styrene, polyacrylonitrile, polybutadiene, poly(butylene terephthalate), poly(ether sulfone), poly(ether ether ketones), poly(ethylene glycol), styrene -acrylonitrile resin, poly(trimethylene terephthalate), polyvinyl butyral,

polyvinylidenedifluoride, poly(vinyl pyrrolidone), and any combination thereof.

[00157] In an aspect of the invention, the engineered fusion polypeptide described herein can be conjugated with the solid substrate by methods well known in the art for conjugating peptides with other molecules. For example, Hermanson, Bioconjugate Techniques (2nd Ed., Academic Press (2008)) and Niemeyr, Bioconjugation Protocols: Strategies & Methods, in Methods in Molecular Biology (Humana Press, 2004), provide a number of methods and techniques for conjugating peptides to other molecules. For example, de Graaf, et al., 20 Biocojugate Chem. 1281 (2009) provides a review of site -specific introduction of non-natural amino acids into peptides for conjugation.

[00158] Alternatively, the surface of the solid substrate can be functionalized to include binding molecules that bind selectively with the fusion polypeptide. These binding molecules are also referred to as affinity molecules herein. The binding molecule can be bound covalently or non-co valently on the surface of the solid substrate. As used herein, the term“binding molecule” or“affinity molecule” refers to any molecule that is capable of specifically binding a fusion polypeptide described herein. Representative examples of affinity molecules include, but are not limited to, antibodies, antigens, lectins, proteins, peptides, nucleic acids (DNA, RNA, PNA and nucleic acids that are mixtures thereof or that include nucleotide derivatives or analogs); receptor molecules, such as the insulin receptor; ligands for receptors; and biological, chemical or other molecules that have affinity for another molecule, such as biotin and avidin. The binding molecules need not comprise an entire naturally occurring molecule but may consist of only a portion, fragment or subunit of a naturally or non- naturally occurring molecule, as for example the Fab fragment of an antibody. The binding molecule may further comprise a marker that can be detected.

[00159] The binding molecule can be conjugated to surface of the solid substrate using any of a variety of methods known to those of skill in the art. The binding molecule can be coupled or conjugated to surface of the solid substrate covalently or non-co valently. Covalent immobilization may be accomplished through, for example, silane coupling. See, e.g., Weetall, 15 Adv. Mol. Cell Bio. 161 (2008); Weetall, Meths. Enzymol. 134 (1976). The covalent linkage between the binding molecule and the surface can also be mediated by a linker. The non-covalent linkage between the affinity molecule and the surface can be based on ionic interactions, van der Waals interactions, dipole-dipole interactions, hydrogen bonds, electrostatic interactions, and/or shape recognition interactions.

[00160] In some embodiments of any of the aspects, the substrate to which the fusion polypeptide binds is a living cell or extracellular matrix of a tissue or organ. For example, the substrate may be the surface of a cell, tissue or organ associated with the immune response. For example, the cell may be a phagocyte (macrophage, neutrophil, and dendritic cell), mast cell, eosinophil, basophil, and/or natural killer cell. The cell may be the cell of tissues or organs of the immune system, such as spleen, lymph nodes, lymphatic vessels, tonsils, thymus, bone marrow, Peyer’s patches, connective tissues, mucous membranes, the reticuloendothelial system, etc. The surface to which the fusion polypeptide binds may also be the extracellular matrix of one or more of these tissues or organs.

[00161] The fusion polypeptide can be conjugated with surface of the solid substrate by an affinity binding pair. The term“affinity binding pair” or“binding pair” refers to first and second molecules that specifically bind to each other. One member of the binding pair is conjugated with the solid substrate while the second member is conjugated with the fusion polypeptide. As used herein, the term“specific binding" refers to binding of the first member of the binding pair to the second member of the binding pair with greater affinity and specificity than to other molecules.

[00162] Exemplary binding pairs include any haptenic or antigenic compound in combination with a corresponding antibody or binding portion or fragment thereof (e.g., digoxigenin and anti- digoxigenin; mouse immunoglobulin and goat antimouse immunoglobulin) and nonimmunological binding pairs (e.g., biotin-avidin, biotin-streptavidin), hormone (e.g., thyroxine and cortisol-hormone binding protein), receptor-receptor agonist, receptor-receptor antagonist (e.g., acetylcholine receptor- acetylcholine or an analog thereof), IgG-protein A, lectin-carbohydrate, enzyme-enzyme cofactor, enzyme-enzyme inhibitor, and complementary oligonucleoitde pairs capable of forming nucleic acid duplexes), and the like. The binding pair can also include a first molecule that is negatively charged and a second molecule that is positively charged.

[00163] One example of using binding pair conjugation is the biotin-sandwich method. See, e.g., Davis et al., 103 PNAS 8155 (2006). The two molecules to be conjugated together are biotinylated and then conjugated together using tetravalent streptavidin as a linker. A peptide can be coupled to the 15- amino acid sequence of an acceptor peptide for biotinylation (referred to as AP; Chen et al, 2 Nat. Methods 99 (2005)). The acceptor peptide sequence allows site-specific biotinylation by the E. coli enzyme biotin ligase (BirA; Id.).

[00164] The FcMBL as described herein can be similarly biotinylated for conjugation with a solid substrate. Many commercial kits are also available for biotinylating proteins. Another example for conjugation to a solid surface would be to use PLP -mediated bioconjugation. See, e.g. , Witus et al., 132 JACS 16812 (2010). In this example, an AKT sequence on the Fc N terminal allows conjugation to the solid surface and orientation of the lectin binding domain in the optimal orientation pointing away from the solid surface.

[00165] In some embodiments of any aspects described herein, the engineered fusion polypeptide can further comprise a detectable label, e.g., to facilitate detection of the presence or absence of a microorganism and/or microbial matter. As used herein, the term “detectable label” refers to a composition capable of producing a detectable signal indicative of the presence of a target. Detectable labels include any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means. Suitable labels include fluorescent molecules, radioisotopes, nucleotide chromophores, enzymes, substrates, chemiluminescent moieties, biolumine scent moieties, and the like. As such, a label is any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means needed for the methods and devices provided herein.

[00166] Detectable labels suitable for conjugation to some embodiments of the engineered fusion polypeptide can include any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, magnetic, optical or chemical means, as well as any examples of detectable labels described herein and any equivalent thereof. In some embodiments of any of the aspects, the detectable labels also encompass any imaging agent (e.g., but not limited to, a bubble, a liposome, a sphere, a contrast agent, or any detectable label described herein) that can facilitate imaging or visualization of a tissue or an organ in a subject, e.g., for diagnosis of an infection.

[00167] A wide variety of fluorescent reporter dyes are known in the art. Typically, the fluorophore is an aromatic or heteroaromatic compound and can be a pyrene, anthracene, naphthalene, acridine, stilbene, indole, benzindole, oxazole, thiazole, benzothiazole, cyanine, carbocyanine, salicylate, anthranilate, coumarin, fluorescein, rhodamine or other like compound. [00168] Exemplary fluorophores include, but are not limited to, 1,5 IAEDANS; 1,8-ANS ; 4- Methylumbelliferone; 5-carboxy-2,7-dichlorofluorescein; 5-Carboxyfluorescein (5-FAM); 5- Carboxynapthofluorescein (pH 10); 5-Carboxytetramethylrhodamine (5-TAMRA); 5-FAM (5- Carboxyfluorescein); 5-Hydroxy Tryptamine (HAT); 5-ROX (carboxy-X-rhodamine); 5-TAMRA (5- Carboxytetramethylrhodamine); 6-Carboxyrhodamine 6G; 6-CR 6G; 6-JOE; 7-Amino-4- methylcoumarin; 7-Aminoactinomycin D (7-AAD); 7-Hydroxy-4-methylcoumarin; 9-Amino-6- chloro-2-methoxyacridine; ABQ; Acid Fuchsin; ACMA (9-Amino-6-chloro-2-methoxyacridine); Acridine Orange; Acridine Red; Acridine Yellow; Acriflavin; Acriflavin Feulgen SITSA; Aequorin (Photoprotein); Alexa Fluor 350™; Alexa Fluor 430™; Alexa Fluor 488™; Alexa Fluor 532™; Alexa Fluor 546™; Alexa Fluor 568™; Alexa Fluor 594™; Alexa Fluor 633™; Alexa Fluor 647™; Alexa Fluor 660™; Alexa Fluor 680™; Alizarin Complexon; Alizarin Red; Allophycocyanin (APC); AMC, AMCA-S; AMCA (Aminomethylcoumarin); AMCA-X; Aminoactinomycin D; Aminocoumarin; Anilin Blue; Anthrocyl stearate; APC-Cy7; APTS; Astrazon Brilliant Red 4G; Astrazon Orange R; Astrazon Red 6B; Astrazon Yellow 7 GFF; Atabrine; ATTO-TAG™ CBQCA; ATTO-TAG™ FQ; Auramine; Aurophosphine G; Aurophosphine; BAO 9 (Bisaminophenyloxadiazole); BCECF (high pH); BCECF (low pH); Berberine Sulphate; Beta Factamase; BFP blue shifted GFP (Y 66H); BG-647; Bimane; Bisbenzamide; Blancophor FFG; Blancophor SV; BOBO™ -1; BOBO™ -3; Bodipy 492/515; Bodipy 493/503; Bodipy 500/510; Bodipy 505/515; Bodipy 530/550; Bodipy 542/563; Bodipy 558/568; Bodipy 564/570; Bodipy 576/589; Bodipy 581/591; Bodipy 630/650-X; Bodipy 650/665-X; Bodipy 665/676; Bodipy FI; Bodipy FF ATP; Bodipy Fl-Ceramide; Bodipy R6G SE; Bodipy TMR; Bodipy TMR-X conjugate; Bodipy TMR-X, SE; Bodipy TR; Bodipy TR ATP; Bodipy TR-X SE; BO-PRO™ -1; BO-PRO™ -3; Brilliant Sulphoflavin FF; Calcein; Calcein Blue; Calcium Crimson™; Calcium Green; Calcium Green- 1 Ca2+ Dye; Calcium Green-2 Ca2+; Calcium Green-5N Ca2+; Calcium Green-C18 Ca2+; Calcium Orange; Calcofluor White; Carboxy-X-rhodamine (5- ROX); Cascade Blue™; Cascade Yellow; Catecholamine; CFDA; CFP - Cyan Fluorescent Protein; Chlorophyll; Chromomycin A; Chromomycin A; CMFDA; Coelenterazine ; Coelenterazine cp;

Coelenterazine f; Coelenterazine fcp; Coelenterazine h; Coelenterazine hep; Coelenterazine ip;

Coelenterazine O; Coumarin Phalloidin; CPM Methylcoumarin; CTC; Cy2™; Cy3.1 8; Cy3.5™; Cy3™; Cy5.1 8; Cy5.5™; Cy5™; Cy7™; Cyan GFP; cyclic AMP Fluorosensor (FiCRhR); d2;

Dabcyl; Dansyl; Dansyl Amine; Dansyl Cadaverine; Dansyl Chloride; Dansyl DHPE; Dansyl fluoride; DAPI; Dapoxyl; Dapoxyl 2; Dapoxyl 3; DCFDA; DCFH (Dichlorodihydrofluorescein Diacetate); DDAO; DHR (Dihydorhodamine 123); Di-4-ANEPPS; Di-8-ANEPPS (non-ratio); DiA (4-Di- 16-ASP); DIDS; Dihydorhodamine 123 (DHR); DiO (DiOC18(3)); DiR; DiR (DiIC18(7)); Dopamine; DsRed; DTAF; DY-630-NHS; DY-635-NHS; EBFP; ECFP; EGFP; EFF 97; Eosin;

Erythrosin; Erythrosin ITC; Ethidium homodimer-1 (EthD-1); Euchrysin; Europium (III) chloride; Europium; EYFP; Fast Blue; FDA; Feulgen (Pararosaniline); FITC; FF-645; Flazo Orange; Fluo-3; Fluo-4; Fluorescein Diacetate; Fluoro-Emerald; Fluoro-Gold (Hydroxystilbamidine); Fluor-Ruby; FluorX; FM 1-43™; FM 4-46; Fura Red™ (high pH); Fura-2, high calcium; Fura-2, low calcium; Genacryl Brilliant Red B; Genacryl Brilliant Yellow 10GF; Genacryl Pink 3G; Genacryl Yellow 5GF; GFP (S65T); GFP red shifted (rsGFP); GFP wild type, non-UV excitation (wtGFP); GFP wild type, UV excitation (wtGFP); GFPuv; Gloxalic Acid; Granular Blue; Haematoporphyrin; Hoechst 33258; Hoechst 33342; Hoechst 34580; HPTS; Hydroxycoumarin; Hydroxystilbamidine (FluoroGold);

Hydroxytryptamine; Indodicarbocyanine (DiD); Indotricarbocyanine (DiR); Intrawhite Cf; JC-1; JO- JO-1; JO-PRO-1; LaserPro; Laurodan; LDS 751; Leucophor PAF; Leucophor SF; Leucophor WS; Lissamine Rhodamine; Lissamine Rhodamine B; LOLO-1; LO-PRO-1; Lucifer Yellow; Mag Green; Magdala Red (Phloxin B); Magnesium Green; Magnesium Orange; Malachite Green; Marina Blue; Maxilon Brilliant Flavin 10 GFF; Maxilon Brilliant Flavin 8 GFF; Merocyanin; Methoxycoumarin; Mitotracker Green FM; Mitotracker Orange; Mitotracker Red; Mitramycin; Monobromobimane; Monobromobimane (mBBr-GSH); Monochlorobimane; MPS (Methyl Green Pyronine Stilbene); NBD; NBD Amine; Nile Red; Nitrobenzoxadidole; Noradrenaline; Nuclear Fast Red; Nuclear Yellow; Nylosan Brilliant Iavin E8G; Oregon Green™; Oregon Green 488-X; Oregon Green™ 488; Oregon Green™ 500; Oregon Green™ 514; Pacific Blue; Pararosaniline (Feulgen); PE-Cy5; PE-Cy7; PerCP; PerCP-Cy5.5; PE-TexasRed (Red 613); Phloxin B (Magdala Red); Phorwite AR; Phorwite BKL; Phorwite Rev; Phorwite RPA; Phosphine 3R; PhotoResist; Phycoerythrin B [PE];

Phycoerythrin R [PE]; PKH26 ; PKH67; PMIA; Pontochrome Blue Black; POPO-1; POPO-3; PO- PRO-1; PO-PRO-3; Primuline; Procion Yellow; Propidium Iodid (PI); PyMPO; Pyrene; Pyronine; Pyronine B; Pyrozal Brilliant Flavin 7GF; QSY 7; Quinacrine Mustard; Resorufm; RH 414; Rhod-2; Rhodamine; Rhodamine 110; Rhodamine 123; Rhodamine 5 GLD; Rhodamine 6G; Rhodamine B 540; Rhodamine B 200 ; Rhodamine B extra; Rhodamine BB; Rhodamine BG; Rhodamine Green; Rhodamine Phallicidine; Rhodamine Phalloidine; Rhodamine Red; Rhodamine WT; Rose Bengal; R- phycoerythrin (PE); red shifted GFP (rsGFP, S65T); S65A; S65C; S65L; S65T; Sapphire GFP;

Serotonin; Sevron Brilliant Red 2B; Sevron Brilliant Red 4G; Sevron Brilliant Red B; Sevron Orange; Sevron Yellow L; sgBFP™; sgBFP™ (super glow BFP); sgGFP™; sgGFP™ (super glow GFP);

SITS; SITS (Primuline); SITS (Stilbene Isothiosulphonic Acid); SPQ (6-methoxy-N-(3-sulfopropyl)- quinolinium); Stilbene; Sulphorhodamine B can C; Sulphorhodamine G Extra; Tetracycline;

Tetramethylrhodamine ; Texas Red™; Texas Red-X™ conjugate; Thiadicarbocyanine (DiSC3); Thiazine Red R; Thiazole Orange; Thioflavin 5; Thioflavin S; Thioflavin TCN; Thiolyte; Thiozole Orange; Tinopol CBS (Calcofluor White); TMR; TO-PRO-1; TO-PRO-3; TO-PRO-5; TOTO-1; TOTO-3; TriColor (PE-Cy5); TRITC (TetramethylRodaminelsoThioCyanate); True Blue; TruRed; Ultralite; Uranine B; Uvitex SFC; wt GFP; WW 781; XL665; X-Rhodamine; XRITC; Xylene Orange; Y66F; Y66H; Y66W; Yellow GFP; YFP; YO-PRO-1; YO-PRO-3; YOYO-1; and YOYO-3. Many suitable forms of these fluorescent compounds are available and can be used.

[00169] Other exemplary detectable labels include luminescent and biolumine scent markers (e.g., biotin, luciferase (e.g., bacterial, firefly, click beetle and the like), luciferin, and aequorin), radiolabels (e.g., 3H, 1251, 35S, 14C, or 32P), enzymes (e.g., galactosidases, glucorinidases, phosphatases (e.g., alkaline phosphatase), peroxidases (e.g., horseradish peroxidase), and cholinesterases), and calorimetric labels such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, and latex) beads. Patents teaching the use of such labels include U.S. Pat. Nos. 3,817,837, 3,850,752, 3,939,350, 3,996,345, 4,277,437, 4,275,149, and 4,366,241, each of which is incorporated herein by reference.

[00170] Means of detecting such labels are well known to those of skill in the art. Thus, for example, radiolabels can be detected using photographic fdm or scintillation counters, fluorescent markers can be detected using a photo-detector to detect emitted light. Enzymatic labels are typically detected by providing the enzyme with an enzyme substrate and detecting the reaction product produced by the action of the enzyme on the enzyme substrate, and calorimetric labels can be detected by visualizing the colored label.

[00171] The present invention provides for engineered bovine fusion polypeptides, e.g., engineered FcMBL or MBL, for use in devices and systems for pathogen detection and clearance. The feasibility of using combined micromagnetic and microfluidic techniques to clear living pathogens from flowing fluids are known in the art, such as biological fluids, such as blood. Xia et al, 8 Biomed. Dev. Biomed. Microdev. 299 (2006); Yung et al., Lab on a Chip DOI: 10.1039/b816986a (2009). In these microdevices (magnetic microbeads that are coated with molecules that bind specifically to surface markers on pathogen cells), are allowed to bind to these cells in whole human blood, and then are pulled free from blood flowing through microfluidic channels using an applied magnetic field gradient. See WO/2008/130618; WO/2007/044642.

[00172] Among other uses, these devices have great promise to rapidly clear blood of infected subjects of toxin-producing pathogens, and hence greatly increase response to conventional antibiotic therapies. The ability to rapidly (within minutes) bind, detect and isolate living pathogens circulating in blood, or present within other biological fluids, using a potentially inexpensive and easy-to-use microdevice also circumvents the major limitations of current pathogen detection and sensitivity testing assays that require multiple days of microbial culture in hospital or commercial laboratories.

[00173] Biological fluids from cattle that may be used in the present invention include, for example, blood, cerebrospinal fluid, joint fluid, urine, semen, saliva, tears, and fluids collected by insertion of a needle. Additionally, fluids may be collected from food or water samples for rapid, general contamination assays according to the present invention: such fluid can be collected and analyzed for natural microbial contamination.

[00174] It should be understood that this invention is not limited to the particular methodology, protocols, and reagents, etc., described herein and as such may vary. The terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which is defined solely by the claims. [00175] As used herein and in the claims, the singular forms include the plural reference and vice versa unless the context clearly indicates otherwise. Other than in the operating examples, or where otherwise indicated, all numbers expressing quantities of ingredients or reaction conditions used herein should be understood as modified in all instances by the term“about.”

[00176] All patents and other publications identified are expressly incorporated herein by reference for the purpose of describing and disclosing, for example, the methodologies described in such publications that might be used in connection with the present invention. These publications are provided solely for their disclosure prior to the filing date of the present application. Nothing in this regard should be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention or for any other reason. All statements as to the date or representation as to the contents of these documents is based on the information available to the applicants and does not constitute any admission as to the correctness of the dates or contents of these documents.

[00177] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as those commonly understood to one of ordinary skill in the art to which this invention pertains. Although any known methods, devices, and materials can be used in the practice or testing of the invention, the methods, devices, and materials in this regard are provided herein.

[00178] Unless stated otherwise, or implicit from context, the following terms and phrases include the meanings provided below. Unless explicitly stated otherwise, or apparent from context, the terms and phrases below do not exclude the meaning that the term or phrase has acquired in the art to which it pertains. The definitions are provided to aid in describing particular embodiments of the aspects provided herein, and are not intended to limit the claimed invention, because the scope of the invention is limited only by the claims. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.

[00179] As used herein the term“comprising” or“comprises” is used in reference to compositions, methods, and respective component(s) thereof, that are essential to the invention, yet open to the inclusion of unspecified elements, whether essential or not.

[00180] As used herein the term“consisting essentially of’ refers to those elements required for a given embodiment. The term permits the presence of additional elements that do not materially affect the basic and novel or functional characteristic(s) of that embodiment of the invention.

[00181] The term“consisting of’ refers to compositions, methods, and respective components thereof as provided herein, which are exclusive of any element not recited in that description of the embodiment.

[00182] Other than in the operating examples, or where otherwise indicated, all numbers expressing quantities of ingredients or reaction conditions used herein should be understood as modified in all instances by the term“about.” The term“about” when used in connection with percentages can mean ±1%. [00183] The terms“decrease”,“reduced”,“reduction”,“decrease” or“inhibit” are all used herein generally to mean a decrease by a statistically significant amount. However, for avoidance of doubt, ““reduced”,“reduction” or“decrease” or“inhibit” means a decrease by at least 10% as compared to a reference level, for example a decrease by at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% decrease (e.g. absent level as compared to a reference sample), or any decrease between 10-100% as compared to a reference level.

[00184] The terms “increased” ’increase” or“enhance” or“activate” are all used herein to generally mean an increase by a statically significant amount; for the avoidance of any doubt, the terms “increased”,“increase” or“enhance” or“activate” means an increase of at least 10% as compared to a reference level, for example an increase of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% increase or any increase between 10-100% as compared to a reference level, or at least about a 2-fold, or at least about a 3 -fold, or at least about a 4-fold, or at least about a 5-fold or at least about a 10-fold increase, or any increase between 2-fold and 10-fold or greater as compared to a reference level.

[00185] The term“statistically significant” or“significantly” refers to statistical significance and generally means at least two standard deviations (2SD) away from a reference level. The term refers to statistical evidence that there is a difference. It is defined as the probability of making a decision to reject the null hypothesis when the null hypothesis is actually true.

[00186] As used herein, a“reference level” refers to a normal, otherwise unaffected cell population or tissue (e.g., a biological sample obtained from a healthy subject, or a biological sample obtained from the subject at a prior time point, e.g., a biological sample obtained from a subject prior to being diagnosed with an infectious disease, or a biological sample that has not been contacted with the composition disclosed herein).

[00187] As used herein, an“appropriate control” or“control” refers to an untreated, otherwise identical cell or population (e.g., a biological sample that was not contacted by a composition provided herein, or not contacted in the same manner, e.g., for a different duration, as compared to a non-control cell).

[00188] The singular terms“a,”“an,” and“the” include plural referents unless context clearly indicates otherwise. Similarly, the word“or” is intended to include“and” unless the context clearly indicates otherwise. Thus for example, references to“the method” includes one or more methods, and/or steps of the type provided herein and/or which will become apparent to those persons skilled in the art upon reading this disclosure and so forth.

[00189] As used herein, the term“corresponding to” refers to an amino acid or nucleotide at the enumerated position in a first polypeptide or nucleic acid, or an amino acid or nucleotide that is equivalent to an enumerated amino acid or nucleotide in a second polypeptide or nucleic acid. Equivalent enumerated amino acids or nucleotides can be determined by alignment of candidate sequences using degree of homology programs known in the art, e.g., BLAST.

[00190] Although methods and materials similar or equivalent to those provided herein can be used in the practice or testing of this disclosure, suitable methods and materials are described below. The term“comprises” means“includes.” The abbreviation,“e.g.” is derived from the Latin exempli gratia, and is used herein to indicate a non-limiting example. Thus, the abbreviation“e.g.” is synonymous with the term“for example.”

[00191] Although preferred embodiments have been depicted and described in detail herein, it will be apparent to those skilled in the relevant art that various modifications, additions, substitutions, and the like can be made without departing from the spirit of the invention and these are therefore considered to be within the scope of the invention as defined in the claims which follow further, to the extent not already indicated, it will be understood by those of ordinary skill in the art that any one of the various embodiments herein described and illustrated can be further modified to incorporate features shown in any of the other embodiments disclosed herein.

[00192] The following examples illustrate some embodiments and aspects of the invention. It will be apparent to those skilled in the relevant art that various modifications, additions, substitutions, and the like can be performed without altering the spirit or scope of the invention, and such modifications and variations are encompassed within the scope of the invention as defined in the claims which follow. The following examples do not in any way limit the invention.

[00193] Some embodiments of the various aspects described herein can be described as in the following numbered paragraphs:

1. A composition comprising:

an engineered fusion polypeptide comprising:

a. an antibody fragment comprising the C2 and/ or C3 domains of a bovine

immunoglobulin G (IgG); and

b. at least one bovine mannose binding lectin (MBL) domain.

2. The composition of paragraph 1, wherein the fusion polypeptide comprises at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten or more MBL domains.

3. The composition of paragraph 1, wherein the MBL domain binds to a microorganism or a microorganism -derived biomolecule.

4. The composition of paragraph 3, wherein the microorganism is a virus, a fungus, a bacterium, a parasite, or a yeast.

5. The composition of any of paragraphs 1-4, wherein the fusion polypeptide further comprises a polypeptide linker domain between the antibody fragment and the MBL domain.

6. The composition of any of paragraphs 1-5, wherein the bovine antibody fragment is linked to the N-terminus or C-terminus of the bovine MBL domain. The composition of any of paragraph 1-6, wherein the fusion polypeptide comprises the sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6.

The composition of any of paragraphs 1-7, wherein the bovine antibody fragment is derived from Bos taurus.

The composition of any of paragraphs 1-8, wherein the bovine antibody fragment is derived from bovine IgGl, IgG2, or IgG3.

The composition of any of paragraphs 1-9, wherein the bovine antibody fragment is a polypeptide comprising the sequence of SEQ ID NO: 7, or SEQ ID NO: 8, or SEQ ID NO: 9, or SEQ ID NO: 10 or SEQ ID NO: 11, or SEQ ID NO: 12, or SEQ ID NO: 13, or SEQ ID NO: 14, or SEQ ID NO: 15. The composition of any of paragraphs 1-10, wherein one or more amino acids within the fusion polypeptide is substituted relative to a wild-type bovine sequence to prevent glycosylation.

The composition of paragraph 11, wherein the one or more substituted amino acids is substituted with an aspartate.

The composition of any of paragraphs 1-12, wherein two or more amino acids within the fusion polypeptide are substituted relative to a wild-type bovine sequence to stimulate glycosylation.

The composition of any of paragraphs 1-13, wherein the first amino acid of the fusion polypeptide is substituted with an aspartate and the terminal lysine of the fusion polypeptide is substituted with an alanine.

The composition of any of paragraphs 1-14, further comprising at least one microorganism or microorganism -derived biomolecule bound to the fusion polypeptide.

The composition of paragraph 15, wherein the microorganism is a bovine pathogen.

The composition of paragraph 16, wherein the bovine pathogen is Clostridium tetani,, Clostridium novyi, Clostridium perfringens, Clostridium perfringens, Clostridium perfringens, Clostridium septicum, Clostridium chauvoei, Clostridium sordellii, Clostridium hemolyticum, Steptococcus uberis, Staphylococcus aureus, Escherichia coli, Streptococcus agalactiae, Streptococcus dysglalactiae, Coorynebacterium bovis, Mycoplasma bovis, Mannheimia haemolytica, Pasteurella multocida, Histophilus somni, Mycobacterium tuberculosis, bovine viral diarrhea virus, bovine respiratory syncyntial virus, rabies virus, bovine Herpes virus 1, Bovine Herpes virus 4, bovine rhinovirus, bovine enterovirus, bovine coronavirus, bovine reovirus, parainfluenza- 3 virus, or a round worm.

The composition of any of paragraphs 1-17, wherein the fusion polypeptide is attached to a solid substrate. The composition of paragraph 18, wherein the solid substrate is selected from a group consisting of: a magnetic microbead, a paramagnetic microbead, a microporous membrane, a hollow fiber, any other fluid filtration membrane, flow device, microtiter plate, cell culture plate, glass beads, latex beads, a living cell, an extracellular matrix of a biological tissue or organ, and a phagocyte.

A pharmaceutical or vaccine composition comprising the composition of any of paragraphs 1- 19 and at least one of a pharmaceutically acceptable carrier and an adjuvant.

A method of treating or preventing an infectious disease in a subject, the method comprising administering the composition of any of paragraphs 1-20.

The method of paragraph 21, wherein the subject is a cow.

The method of any of paragraphs 20-22, wherein the infectious disease is selected from the group consisting of: mastitis, ephemeral fever, actinomycosis, bovine respiratory disease, bovine respiratory syncytial virus, infectious bovine rhinotracheitis, balanoposthitis, influenza, tuberculosis, Clostridium infection, and pneumonia.

A method of stimulating an immune response in a subject or vaccinating a subject, the method comprising administering the composition of any of paragraphs 1-20 to the subject. The method of paragraph 24, wherein the subject is a cow.

The method of any of paragraphs 24-25, wherein the immune response or vaccination is specific to a disease selected from the group consisting of: mastitis, ephemeral fever, actinomycosis, bovine respiratory disease, bovine respiratory syncytial virus, infectious bovine rhinotracheitis, balanoposthitis, influenza, tuberculosis, Clostridium infection, and pneumonia.

A method of preparing a vaccine for a bovine subject, the method comprising contacting the composition of any of paragraphs 1-14 and 18-19 with a microorganism or at least one microorganism-derived biomolecule, whereby the microorganism and/or biomolecule are bound by the composition.

The method of paragraph 27, further comprising purifying the microorganism and/or biomolecule from the composition.

The method of any of paragraphs 27-28, wherein the microorganism is a bovine pathogen. The method of paragraph 29, wherein the bovine pathogen is Clostridium tetani,, Clostridium novyi, Clostridium perfringens, Clostridium perfringens, Clostridium perfringens,

Clostridium septicum, Clostridium chauvoei, Clostridium sordellii, Clostridium hemolyticum, Steptococcus uberis, Staphylococcus aureus, Escherichia coli, Streptococcus agalactiae, Streptococcus dysglalactiae, Coorynebacterium bovis, Mycoplasma bovis, Mannheimia haemolytica, Pasteurella multocida, Histophilus somni, Mycobacterium tuberculosis, bovine viral diarrhea virus, bovine respiratory syncyntial virus, rabies virus, bovine Herpes virus 1, Bovine Herpes virus 4, bovine rhinovirus, bovine enterovirus, bovine coronavirus, bovine reovirus, parainfluenza- 3 virus, or a round worm.

31. The method of any of paragraphs 27-30, wherein the vaccine is specific to a disease selected from the group consisting of: mastitis, ephemeral fever, actinomycosis, bovine respiratory disease, bovine respiratory syncytial virus, infectious bovine rhinotracheitis, balanoposthitis, influenza, tuberculosis, Clostridium infection, and pneumonia.

[00194] Some embodiments of the various aspects described herein can be described as in the following numbered paragraphs:

1. A composition comprising:

an engineered fusion polypeptide comprising:

a. an antibody fragment comprising the C2 and/ or C3 domains of a bovine

immunoglobulin G (IgG); and

b. at least one bovine mannose binding lectin (MBL) domain.

2. The composition of paragraph 1, wherein the fusion polypeptide comprises at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten or more MBL domains.

3. The composition of paragraph 1, wherein the MBL domain binds to a microorganism or a microorganism -derived biomolecule.

4. The composition of paragraph 3, wherein the microorganism is a virus, a fungus, a bacterium, a parasite, or a yeast.

5. The composition of any of paragraphs 1-4, wherein the fusion polypeptide further comprises a polypeptide linker domain between the antibody fragment and the MBL domain.

6. The composition of any of paragraphs 1-5, wherein the bovine antibody fragment is linked to the N-terminus or C-terminus of the bovine MBL domain.

7. The composition of any of paragraphs 1-6, wherein the fusion polypeptide comprises the sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6.

8. The composition of any of paragraphs 1-7, wherein the bovine antibody fragment is derived from Bos taurus.

9. The composition of any of paragraphs 1-8, wherein the bovine antibody fragment is derived from bovine IgGl, IgG2, or IgG3.

10. The composition of any of paragraphs 1-9, wherein the bovine antibody fragment is a

polypeptide comprising the sequence of SEQ ID NO: 7, or SEQ ID NO: 8, or SEQ ID NO: 9, or SEQ ID NO: 10 or SEQ ID NO: 11, or SEQ ID NO: 12, or SEQ ID NO: 13, or SEQ ID NO: 14, or SEQ ID NO: 15. The composition of any of paragraphs 1-10, wherein one or more amino acids within the fusion polypeptide is substituted relative to a wild-type bovine sequence to prevent glycosylation.

The composition of paragraph 11, wherein the one or more substituted amino acids is substituted with an aspartate.

The composition of any of paragraphs 1-12, wherein two or more amino acids within the fusion polypeptide are substituted relative to a wild-type bovine sequence to stimulate glycosylation.

The composition of any of paragraphs 1-13, wherein the first amino acid of the fusion polypeptide is substituted with an aspartate and the terminal lysine of the fusion polypeptide is substituted with an alanine.

The composition of any of paragraphs 1-14, further comprising at least one microorganism or microorganism -derived biomolecule bound to the fusion polypeptide.

The composition of paragraph 15, wherein the microorganism is a bovine pathogen.

The composition of paragraph 16, wherein the bovine pathogen is Clostridium tetani,, Clostridium novyi, Clostridium perfringens, Clostridium perfringens, Clostridium perfringens, Clostridium septicum, Clostridium chauvoei, Clostridium sordellii, Clostridium hemolyticum, Steptococcus uberis, Staphylococcus aureus, Escherichia coli, Streptococcus agalactiae, Streptococcus dysglalactiae, Coorynebacterium bovis, Mycoplasma bovis, Mannheimia haemolytica, Pasteurella multocida, Histophilus somni, Mycobacterium tuberculosis, bovine viral diarrhea virus, bovine respiratory syncyntial virus, rabies virus, bovine Herpes virus 1, Bovine Herpes virus 4, bovine rhinovirus, bovine enterovirus, bovine coronavirus, bovine reovirus, parainfluenza- 3 virus, or a round worm.

The composition of any of paragraphs 1-17, wherein the composition is multivalent.

The composition of paragraph 18, wherein the composition is multivalent and comprises multiple microorganisms or microorganism-derived biomolecules bound to the multivalent fusion polypeptide.

The composition of paragraph 19, wherein the multiple microorganisms or microorganism- derived biomolecules are or are derived from different strains of the same species.

The composition of any of paragraphs 1-20, wherein the fusion polypeptide is attached to a solid substrate.

The composition of paragraph 21, wherein the solid substrate is selected from a group consisting of: a magnetic microbead, a paramagnetic microbead, a microporous membrane, a hollow fiber, any other fluid filtration membrane, flow device, microtiter plate, cell culture plate, glass beads, latex beads, a living cell, an extracellular matrix of a biological tissue or organ, and a phagocyte. A pharmaceutical or vaccine composition comprising the composition of any of paragraphs 1- 22 and at least one of a pharmaceutically acceptable carrier and an adjuvant.

A method of treating or preventing an infectious disease in a subject, the method comprising administering the composition of any of paragraphs 1-23.

The method of paragraph 24, wherein the subject is a cow.

The method of any of paragraphs 24-25, wherein the infectious disease is selected from the group consisting of: mastitis, ephemeral fever, actinomycosis, bovine respiratory disease, bovine respiratory syncytial virus, infectious bovine rhinotracheitis, balanoposthitis, influenza, tuberculosis, Clostridium infection, and pneumonia.

A method of stimulating an immune response in a subject or vaccinating a subject, the method comprising administering the composition of any of paragraphs 1-23 to the subject. The method of paragraph 27, wherein the subject is a cow.

The method of any of paragraphs 27-28, wherein the immune response or vaccination is specific to a disease selected from the group consisting of: mastitis, ephemeral fever, actinomycosis, bovine respiratory disease, bovine respiratory syncytial virus, infectious bovine rhinotracheitis, balanoposthitis, influenza, tuberculosis, Clostridium infection, and pneumonia.

A method of preparing a vaccine for a bovine subject, the method comprising contacting the composition of any of paragraphs 1-23 with a microorganism or at least one microorganism- derived biomolecule, whereby the microorganism and/or biomolecule are bound by the composition.

The method of paragraph 30, further comprising purifying the microorganism and/or biomolecule from the composition.

The method of any of paragraphs 30-31, wherein the microorganism is a bovine pathogen. The method of paragraph 32, wherein the bovine pathogen is Clostridium tetani,, Clostridium novyi, Clostridium perfringens, Clostridium perfringens, Clostridium perfringens,

Clostridium septicum, Clostridium chauvoei, Clostridium sordellii, Clostridium hemolyticum, Steptococcus uberis, Staphylococcus aureus, Escherichia coli, Streptococcus agalactiae, Streptococcus dysglalactiae, Coorynebacterium bovis, Mycoplasma bovis, Mannheimia haemolytica, Pasteurella multocida, Histophilus somni, Mycobacterium tuberculosis, bovine viral diarrhea virus, bovine respiratory syncyntial virus, rabies virus, bovine Herpes virus 1, Bovine Herpes virus 4, bovine rhinovirus, bovine enterovirus, bovine coronavirus, bovine reovirus, parainfluenza- 3 virus, or a round worm.

The method of any of paragraphs 30-33, wherein the vaccine is specific to a disease selected from the group consisting of: mastitis, ephemeral fever, actinomycosis, bovine respiratory disease, bovine respiratory syncytial virus, infectious bovine rhinotracheitis, balanoposthitis, influenza, tuberculosis, Clostridium infection, and pneumonia. EXAMPLE

EXAMPLE 1: BOVINE VERSIONS OF FCMBL

Advantages of current invention/discovery

[00195] Described herein is an antigen presenting platform that addresses the problem that the presentation of antigens to the immune system via capture by FcMBL or other proteins in species other than humans can cause an additional immune response against the presenting protein (e.g. the human version of FcMBL). To overcome these issues, bovine versions of FcMBL have been generated that can be used on a vaccine platform for developing vaccines specifically for cattle.

[00196] The figures provided herein show the sequences generated as well as data demonstrating the ability to make and demonstrate functionality.

[00197] In Fig. 1, amino acid sequence for a version of bovine FcMBL using an Fc fragment from bovine IgGl and a fragment of bovine MBL-C.

[00198] In Fig. 2, the amino acid sequence for a version of bovine FcMBL using an Fc fragment from bovine IgGl and a fragment of bovine MBL-C is shown. The putative glycosylation site in IgGl has been mutated to aspartate to stop glycosylation.

[00199] In Fig. 3, the amino acid sequence for a version of bovine FcMBL using an Fc fragment from bovine IgG2 and a fragment of bovine MBL-C is shown.

[00200] In Fig. 4, Amino acid sequence for a version of bovine FcMBL using an Fc fragment from bovine IgG2 and a fragment of bovine MBL-C is shown. The putative glycosylation site in IgG2 has been mutated to aspartate to stop glycosylation.

[00201] In Fig. 5, the amino acid sequence for a version of bovine FcMBL using an Fc fragment from bovine IgG3 and a fragment of bovine MBL-C is shown.

[00202] In Fig. 6, the amino acid sequence for a version of bovine FcMBL using an Fc fragment from bovine IgG3 and a fragment of bovine MBL-C is shown. The putative glycosylation site in IgG3 has been mutated to aspartate to stop glycosylation.

[00203] In Fig. 7, the sequence for the portion of IgGl used in bovine FcMBL fragments is shown. The glycosylated asparagine (underlined) can be mutated to aspartate to generate a glycosylated version. The terminal lysine can be mutated to an alanine.

[00204] In Fig. 8, the sequence for the portion of IgG2 used in bovine FcMBL fragments. The glycosylated asparagine (underlined) can be mutated to aspartate to generate a glycosylated version. The terminal lysine can be mutated to an alanine.

[00205] In Fig. 9, the sequence for the portion of IgG3 used in bovine FcMBL fragments is shown. The glycosylated asparagine (underlined) can be mutated to aspartate to generate a glycosylated version. The terminal lysine can be mutated to an alanine.

[00206] In Fig. 10, the sequence for Fc Fragment 1 of bovine IgGl is shown.

[00207] In Fig. 11, the sequence for Fc Fragment 1 of bovine IgG2 is shown. [00208] In Fig. 12, the sequence for Fc Fragment 2 of bovine IgG2 is shown.

[00209] In Fig. 13, the sequence for Fc Fragment 3 of bovine IgG2 is shown.

[00210] In Fig. 14, the sequence for Fc Fragment 4 of bovine IgG2 is shown.

[00211] In Fig. 15, the sequence for Fc Fragment 5 of bovine IgG2 is shown.

[00212] In Fig. 16, the SDS PAGE of supematents from 293f cells transfected with DNA encoding bovine IgG Fc fragments are shown. Lane 1 is a negative control (no protein expressed in 293f cells). Bovine IgG2 Fc Fragment 4 did not express and is not included in the gel.

[00213] In Fig. 17, the bovine FcMBL variants were expressed in 293f cells and then purified using mannan conjugated agarose to identify FcMBL variants that are functional for binding mannan are shown. The versions containing the IgG3 fragment did not express and were not included in this Coomassie stained SDS-PAGE.

[00214] In Fig. 18, the sequence alignments of Fc fragments from bovine IgG2 are shown for comparison.

[00215] Fig. 19 depicts the results of a screen for bovine FcMBL variants (BtFclN and BtFclD) vs. a human FcMBL control. Both BtFclN and btFclD bound the mannan standard curve, though not quite as well as human FcMBL. Fig. 20 depicts a screen for bovine FcMBL variants (BtFclN, BtFclD), vs. human FcMBL control. The TB isolates (irradiated) are: 1) H37Rv, 2) HN878, 3) CDC 1551, and 4) BCG Pasteur. Both BtFclN and btFclD bound the TB, not quite as well as human FcMBL. For BCG, the binding of the BtFclD was much better.

[00216] Human FcMBL binding to Mbovis AF2122 membrane fraction & irradiated whole cells was tested (Fig. 21). FcMBL binds to his Mbovis, both to the membrane fraction & irradiated whole cells. Fig. 22 depicts a screen for bovine FcMBL variants (BtFclN, BtFclD), vs. human FcMBL control. The TB isolates (irradiated) are: 1 ) Mbovis AF2122 membrane, 2) Mbovis AF2122 irradiated cells. Both BtFclN and btFclD bound th Q Mbovis AF2122 fractions, not quite as well as human FcMBL.

[00217] Described herein is a Captured Antigen Presentation System (Tb CAPS) as a vaccine against bovine tuberculosis (Fig. 23). The bovine version of the FcMBL has been manufactured and characterized. The vaccine was successfully formulated to demonstrate the capture and incorporation of the antigen lysate into the vaccine construct. A safety study evaluating the implantation technique and biocompatibility in the calf model were successfully completed.

[00218] The Tb CAPS Vaccine was produced using bovine FcMBL (BtFclD-MBLC) cloned, purified, & coupled to myOne Dynabeads. PAMPs from 4 mycobacteria species were captured on bovine (Bt) FcMBL beads: irradiated M. bovis AF2122, M. tb H37Rv, M. tb HN878 & BCG Pasteur. TbCAPS were produced: MPS, Bt GMCSF, BtCpG & the quadrivalent FcMBL-TbPAMPs

[00219] Trial 1: Tested the Scaffold - MPS, bovine GMCSF & bovine CpG for immunogenicity in 5 Holstein calves (8 weeks old). Trial 2: Immunized complete Tb CAPS VAX into 35 Holstein calves (8 weeks old). Experimental groups included: 1) naive 2) BCG vaccine control 3) low dose Tb CAPS VAX 4) high dose Tb CAPS VAX. Booster with low & high dose Tb CAPS VAX groups only at day 28. Sera & whole blood were collected at several time-points post-immunization for anti-Tb CAPS VAX antibody testing.

[00220] Validation can be further conducted in a TB challenge model. Quadrivalent vaccines, e.g., using scaffold, are contemplated herein and have been produced. Such vaccines were tested, e.g., by vaccinating animals with a TB MPS quad vaccine (BCG, H37Rv, AF2122, HN878). Blood for serum isolation is collected before vaccination and 7, 14, 21, 28- and 41-days post vaccination.

Animals are primed 12 days post vaccination. To measure IgG antibody titers in serum from vaccinated animals, plates are coated with lug/mF of each antigen used for the study in TBST buffer. ChromPure Bovine IgG, whole molecule is used for the standard curve starting at lOOng/mF in lx PBS and diluted down at 2-fold dilutions (7 dilutions). Peroxidase AffmiPure F(ab Fragment Goat Anti-Bovine IgG (H+F) is used as II antibody at the concentration of 1: 10000 in blocking buffer (1% NGS in TBST buffer). Serum samples are diluted at 1: 100, 1: 1000 and 1: 10000 dilutions in blocking buffer (1% NGS in TBST buffer). Sera from baseline, 14, 28- and 41-days post vaccination is used, with TMB substrate as detection buffer and an EFISA readout.

SEQUENCES:

[00221] SEQ ID NO: 1 (Fig. 1: BtFcMBL-1)

AKTVDKAVDPRSKTTCDCCPPPEFPGGPSVFIFPPKPKDTFTISGTPEVTCVVVDVGHDDPEV

KFSWFVDDVEVNTATTKPREEQFN STYRVV S ALRIQHQDWTGGKEFKCKVHNEGLPAPIVR

TISRTKGPAREPQVYVFAPPQEEFSKSTVSFTCMVTSFYPDYIAVEWQRNGQPESEDKYGTTP

PQLDADGSYFLYSRLRVDRNSWQEGDTYTCVVMHEALHNHYTQKSTSKSAGKGDPGENMG

DYIRLATSERATLQSELNQIKNWLIFSLGKRVGKKAFFTNGKKMPFNEVKTLCAQFQGRVAT

PMNAEENRAFKDFVTEEAFFGITDQETEGKFVDFTGKGVTYQNWNDGEPNNASPGEHCVTF

FSDGTWNDIACSASFFTVCEFSF

[00222] SEQ ID NO: 2 (Fig. 2: BtFcMBF-2)

AKTVDKAVDPRSKTTCDCCPPPEFPGGPSVFIFPPKPKDTFTISGTPEVTCVVVDVGHDDPEV

KFSWFVDDVEVNTATTKPREEQFDSTYRVVSAFRIQHQDWTGGKEFKCKVHNEGFPAPIVR

TISRTKGPAREPQVYVFAPPQEEFSKSTVSFTCMVTSFYPDYIAVEWQRNGQPESEDKYGTTP

PQFDADGSYFFYSRFRVDRNSWQEGDTYTCVVMHEAFHNHYTQKSTSKSAGKGDPGENMG

DYIRFATSERATFQSEFNQIKNWFIFSFGKRVGKKAFFTNGKKMPFNEVKTFCAQFQGRVAT

PMNAEENRAFKDFVTEEAFFGITDQETEGKFVDFTGKGVTYQNWNDGEPNNASPGEHCVTF

FSDGTWNDIACSASFFTVCEFSF

[00223] SEQ ID NO: 3 (Fig. 3: BtFcMBF-3)

AKTVDKAVGVSIDCSKCHNQPCVREPSVFIFPPKPKDTFMITGTPEVTCVVVNVGHDNPEVQ

FSWFVDDVEVHTARSKPREEQFNSTYRVVSAFPIQHQDWTGGKEFKCKVNNKGFSAPIVRIIS

RSKGPAREPQVYVFDPPKEEFSKSTFSVTCMVTGFYPEDVAVEWQRNRQTESEDKYRTTPPQ LDTDRSYFLY SKLRVDRN SW QEGDTYTCVVMHEALHNHYMQKSTSKS AGAGDPGENMGD

YIRLATSERATLQSELNQIKNWLIFSLGKRVGKKAFFTNGKKMPFNEVKTLCAQFQGRVATP

MNAEENRALKDLVTEEAFLGITDQETEGKFVDLTGKGVTYQNWNDGEPNNASPGEHCVTLL

SDGTWNDIACSASFLTVCEFSL

[00224] SEQ ID NO: 4 (Fig. 4: BtFcMBL-4)

AKTVDKAVGVSIDCSKCHNQPCVREPSVFIFPPKPKDTLMITGTPEVTCVVVNVGHDNPEVQ

FSWFVDDVEVHTARSKPREEQFDSTYRVVSALPIQHQDWTGGKEFKCKVNNKGLSAPIVRIIS

RSKGPAREPQVYVLDPPKEELSKSTLSVTCMVTGFYPEDVAVEWQRNRQTESEDKYRTTPPQ

LDTDRSYFLY SKLRVDRN SW QEGDTYTCVVMHEALHNHYMQKSTSKS AGAGDPGENMGD

YIRLATSERATLQSELNQIKNWLIFSLGKRVGKKAFFTNGKKMPFNEVKTLCAQFQGRVATP

MNAEENRALKDLVTEEAFLGITDQETEGKFVDLTGKGVTYQNWNDGEPNNASPGEHCVTLL

SDGTWNDIACSASFLTVCEFSL

[00225] SEQ ID NO: 5 (Fig. 5: BtFcMBL-5)

AKTTIPPGKPTTQESEVEKTPCQCSKCPEPLGGLSVFIFPPKPKDTLTISGTPEVTCVVVDVGQD

DPEVQFSWFVDDVEVHTARTKPREEQFNSTYRVVSALRIQHQDWLQGKEFKCKVNNKGLPA

PIVRTISRTKGQAREPQVYVLAPPREELSKSTLSLTCLITGFYPEEIDVEWQRNGQPESEDKYH

TTAPQLDADGSYFLYSRLRVNKSSWQEGDHYTCAVMHEALRNHYKEKSISRSPGAGDPGEN

MGDYIRLATSERATLQSELNQIKNWLIFSLGKRVGKKAFFTNGKKMPFNEVKTLCAQFQGRV

ATPMNAEENRALKDLVTEEAFLGITDQETEGKFVDLTGKGVTYQNWNDGEPNNASPGEHCV

TLLSDGTWNDIAC S A SFLTV CEF SL

[00226] SEQ ID NO: 6 (Fig. 6: BtFcMBL-6)

AKTTIPPGKPTTQESEVEKTPCQCSKCPEPLGGLSVFIFPPKPKDTLTISGTPEVTCVVVDVGQD

DPEVQFSWFVDDVEVHTARTKPREEQFDSTYRVVSALRIQHQDWLQGKEFKCKVNNKGLPA

PIVRTISRTKGQAREPQVYVLAPPREELSKSTLSLTCLITGFYPEEIDVEWQRNGQPESEDKYH

TTAPQLDADGSYFLYSRLRVNKSSWQEGDHYTCAVMHEALRNHYKEKSISRSPGAGDPGEN

MGDYIRLATSERATLQSELNQIKNWLIFSLGKRVGKKAFFTNGKKMPFNEVKTLCAQFQGRV

ATPMNAEENRALKDLVTEEAFLGITDQETEGKFVDLTGKGVTYQNWNDGEPNNASPGEHCV

TLLSDGTWNDIAC S A SFLTV CEF SL

[00227] SEQ ID NO: 7 (Bovine IgGl Fc fragment)

VDKAVDPRCKTTCDCCPPPELPGGPSVFIFPPKPKDTLTISGTPEVTCVVVDVGHDDPEVKFS WFVDDVEVNTATTKPREEQFN STYRVV S ALRIQHQDWTGGKEFKCKVHNEGLPAPIVRTISR TKGPAREPQVYVLAPPQEELSKSTVSLTCMVTSFYPDYIAVEWQRNGQPESEDKYGTTPPQL DADGSYFLY SRLRVDRN SW QEGDTYTCVVMHEALHNHYTQKSTSKS AGK

[00228] SEQ ID NO: 8 (Bovine IgG2 Fc Fragment)

VDKA V GV SIDC SKCHN QPCVREP S VFIFPPKPKDTLMITGTPEVTC VVVNV GHDNPEV QF SW FVDDVEVHTARSKPREEQFNSTYRVVSALPIQHQDWTGGKEFKCKVNNKGLSAPIVRIISRSK GPAREPQVYVLDPPKEELSKSTLSVTCMVTGFYPEDVAVEWQRNRQTESEDKYRTTPPQLDT

DRSYFLYSKLRVDRNSWQEGDTYTCVVMHEALHNHYMQKSTSKSAGK

[00229] SEQ ID NO: 9 (Bovine IgG3 Fc Fragment):

KTTIPPGKPTTQESEVEKTPCQCSKCPEPLGGLSVFIFPPKPKDTLTISGTPEVTCVVVDVGQDD PEVQFSWFVDDVEVHTARTKPREEQFNSTYRVVSALRIQHQDWLQGKEFKCKVNNKGLPAP IVRTISRTKGQAREPQVYVLAPPREELSKSTLSLTCLITGFYPEEIDVEWQRNGQPESEDKYHT TAPQLDADGSYFLYSRLRVNKSSWQEGDFIYTCAVMHEALRNFIYKEKSISRSPGK

[00230] SEQ ID NO: 10 (Bovine IgGl Fc Fragment 1)

AKTVDKAVDPRSKTTCDCCPPPELPGGPSVFIFPPKPKDTLTISGTPEVTCVVVDVGHDDPEV

KFSWFVDDVEVNTATTKPREEQFDSTYRVVSALRIQHQDWTGGKEFKCKVFINEGLPAPIVR

TISRTKGPAREPQVYVLAPPQEELSKSTVSLTCMVTSFYPDYIAVEWQRNGQPESEDKYGTTP

PQLDADGSYFLYSRLRVDRNSWQEGDTYTCVVMHEALFINFIYTQKSTSKSAGK

[00231] SEQ ID NO: 11 (Bovine IgG2 Fc Fragment 1 (Fc2NK))

AKTVDKAVGVSIDCSKCFINQPCVREPSVFIFPPKPKDTLMITGTPEVTCVVVNVGHDNPEVQ

FSWFVDDVEVHTARSKPREEQFNSTYRVVSALPIQHQDWTGGKEFKCKVNNKGLSAPIVRIIS

RSKGPAREPQVYVLDPPKEELSKSTLSVTCMVTGFYPEDVAVEWQRNRQTESEDKYRTTPPQ

LDTDRSYFLYSKLRVDRNSWQEGDTYTCVVMHEALFINFIYMQKSTSKSAGK

[00232] SEQ ID NO: 12 (Bovine IgG2 Fc Fragment 2 (Fc2DFlag))

AKTAKTVDKAVGVSIDCSKCFINQPCVREPSVFIFPPKPKDTLMITGTPEVTCVVVNVGHDNP

EVQFSWFVDDVEVHTARSKPREEQFDSTYRVVSALPIQHQDWTGGKEFKCKVNNKGLSAPI

VRIISRSKGPAREPQVYVLDPPKEELSKSTLSVTCMVTGFYPEDVAVEWQRNRQTESEDKYR

TTPPQLDTDRSYFLYSKLRVDRNSWQEGDTYTCVVMHEALFINFIYMQKSTSKSAGADYKDH

DGDYKDHDIDYKDDDDK

[00233] SEQ ID NO: 13 (Bovine IgG2 Fc Fragment 3 (Fc2D))

AKTVDKAVGVSIDCSKCFINQPCVREPSVFIFPPKPKDTLMITGTPEVTCVVVNVGHDNPEVQ

FSWFVDDVEVHTARSKPREEQFDSTYRVVSALPIQHQDWTGGKEFKCKVNNKGLSAPIVRIIS

RSKGPAREPQVYVLDPPKEELSKSTLSVTCMVTGFYPEDVAVEWQRNRQTESEDKYRTTPPQ

LDTDRSYFLYSKLRVDRNSWQEGDTYTCVVMHEALFINFIYMQKSTSKSAGA

[00234] SEQ ID NO: 14 (Bovine IgG2 Fc Fragment 4 (Fc2short))

AKTVFIFPPKPKDTLMITGTPEVTCVVVNVGHDNPEVQFSWFVDDVEVHTARSKPREEQFNS

TYRVVSALPIQHQDWTGGKEFKCKVNNKGLSAPIVRIISRSKGPAREPQVYVLDPPKEELSKS

TLSVTCMVTGFYPEDVAVEWQRNRQTESEDKYRTTPPQLDTDRSYFLYSKLRVDRNSWQEG

DAYTCVVMHEALFINFIYMQKSTSKSAGK

[00235] SEQ ID NO: 15 (Bovine IgG2 Fc Fragment 5 (Fc2deltaCys))

AKTSKCFINQPCVREPSVFIFPPKPKDTLMITGTPEVTCVVVNVGHDNPEVQFSWFVDDVEVH

TARSKPREEQFDSTYRVVSALPIQHQDWTGGKEFKCKVNNKGLSAPIVRIISRSKGPAREPQV YVLDPPKEELSKSTLSVTCMVTGFYPEDVAVEWQRNRQTESEDKYRTTPPQLDTDRSYFLYS

KLRVDRNSWQEGDTYTCVVMHEALHNHYMQKSTSKSAGALAICEFPA

[00236] SEQ ID NO: 16 (Bovine MBL, liver A,GenBank: AAI09675.1)

[00237] MFLFSSLPVL LCLVTVSFSN TKAIEDAQKT CPVVACAIPV TNGTPGRDGR DGPKGEKGEPGQGLRGS QGP PGKMGPPGNI GNPGLPGPRG YKGDRGDSSV AEAKLASLER QIRDLRSELDHVKKLQTFSL GKKSGKKLYV TNREKMPFSS VKALCTALGA TVATPKNAEE NKAIQDMASD TAFLGITDEV TEGQFMYVTG GRLGY SNWKK NEPNNYGSGE DCVSLLPDGL WNDISCSSSFLAICEFPA

[00238] SEQ ID NO: 17 (Bovine MBL protein C isoform XI, NCBI Reference Sequence:

XP_005225409.1)

[00239] MSLFTSLPFL LLTAVTASCA DTETENCENI RKTCPVIACG PPGINGIPGK DGRDGAKGEK GEPGQGLRGS QGPPGKMGPQ GTPGIPGIPG PIGQKGDPGE NMGDYIRLAT SERATLQSEL NQIKNWLIFS LGKRV GKKAF FTNGKKMPFN EVKTLCAQFQ GRVATPMNAE ENRALKDLVT EEAFLGITDQ ETEGKFVDLT GKGVTY QNWN DGEPNNASPG EHCVTLLSDG TWNDIACSAS FLTVCEFSL

Claims

CLAIMS What is claimed herein is:

1. A composition comprising:

an engineered fusion polypeptide comprising:

c. an antibody fragment comprising the C2 and/ or C3 domains of a bovine

immunoglobulin G (IgG); and

d. at least one bovine mannose binding lectin (MBL) domain.

2. The composition of claim 1, wherein the fusion polypeptide comprises at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten or more MBL domains.

3. The composition of claim 1, wherein the MBL domain binds to a microorganism or a

microorganism -derived biomolecule.

4. The composition of claim 3, wherein the microorganism is a virus, a fungus, a bacterium, a parasite, or a yeast.

5. The composition of any of claims 1-4, wherein the fusion polypeptide further comprises a polypeptide linker domain between the antibody fragment and the MBL domain.

6. The composition of any of claims 1-5, wherein the bovine antibody fragment is linked to the N-terminus or C-terminus of the bovine MBL domain.

7. The composition of any of claims 1-6, wherein the fusion polypeptide comprises the sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6.

8. The composition of any of claims 1-7, wherein the bovine antibody fragment is derived from Bos taurus.

9. The composition of any of claims 1-8, wherein the bovine antibody fragment is derived from bovine IgGl, IgG2, or IgG3.

10. The composition of any of claims 1-9, wherein the bovine antibody fragment is a polypeptide comprising the sequence of SEQ ID NO: 7, or SEQ ID NO: 8, or SEQ ID NO: 9, or SEQ ID NO: 10 or SEQ ID NO: 11, or SEQ ID NO: 12, or SEQ ID NO: 13, or SEQ ID NO: 14, or SEQ ID NO: 15.

11. The composition of any of claims 1-10, wherein one or more amino acids within the fusion polypeptide is substituted relative to a wild-type bovine sequence to prevent glycosylation.

12. The composition of claim 11, wherein the one or more substituted amino acids is substituted with an aspartate.

13. The composition of any of claims 1-12, wherein two or more amino acids within the fusion polypeptide are substituted relative to a wild-type bovine sequence to stimulate glycosylation.

14. The composition of any of claims 1-13, wherein the first amino acid of the fusion polypeptide is substituted with an aspartate and the terminal lysine of the fusion polypeptide is substituted with an alanine.

15. The composition of any of claims 1-14, further comprising at least one microorganism or microorganism -derived biomolecule bound to the fusion polypeptide.

16. The composition of claim 15, wherein the microorganism is a bovine pathogen.

17. The composition of claim 16, wherein the bovine pathogen is Clostridium tetani,, Clostridium novyi, Clostridium perfringens, Clostridium perfringens, Clostridium perfringens,

Clostridium septicum, Clostridium chauvoei, Clostridium sordellii, Clostridium hemolyticum, Steptococcus uberis, Staphylococcus aureus, Escherichia coli, Streptococcus agalactiae, Streptococcus dysglalactiae, Coorynebacterium bovis, Mycoplasma bovis, Mannheimia haemolytica, Pasteurella multocida, Histophilus somni, Mycobacterium tuberculosis, bovine viral diarrhea virus, bovine respiratory syncyntial virus, rabies virus, bovine Herpes virus 1, Bovine Herpes virus 4, bovine rhinovirus, bovine enterovirus, bovine coronavirus, bovine reovirus, parainfluenza- 3 virus, or a round worm.

18. The composition of any of claims 1-17, wherein the composition is multivalent.

19. The composition of claim 18, wherein the composition is multivalent and comprises multiple microorganisms or microorganism-derived biomolecules bound to the multivalent fusion polypeptide.

20. The composition of claim 19, wherein the multiple microorganisms or microorganism-derived biomolecules are or are derived from different strains of the same species.

21. The composition of any of claims 1-20, wherein the fusion polypeptide is attached to a solid substrate.

22. The composition of claim 21, wherein the solid substrate is selected from a group consisting of: a magnetic microbead, a paramagnetic microbead, a microporous membrane, a hollow fiber, any other fluid filtration membrane, flow device, microtiter plate, cell culture plate, glass beads, latex beads, a living cell, an extracellular matrix of a biological tissue or organ, and a phagocyte.

23. A pharmaceutical or vaccine composition comprising the composition of any of claims 1-22 and at least one of a pharmaceutically acceptable carrier and an adjuvant.

24. A method of treating or preventing an infectious disease in a subject, the method comprising administering the composition of any of claims 1-23.

25. The method of claim 24, wherein the subject is a cow.

26. The method of any of claims 24-25, wherein the infectious disease is selected from the group consisting of: mastitis, ephemeral fever, actinomycosis, bovine respiratory disease, bovine respiratory syncytial virus, infectious bovine rhinotracheitis, balanoposthitis, influenza, tuberculosis, Clostridium infection, and pneumonia.

27. A method of stimulating an immune response in a subject or vaccinating a subject, the method comprising administering the composition of any of claims 1-23 to the subject.

28. The method of claim 27, wherein the subject is a cow.

29. The method of any of claims 27-28, wherein the immune response or vaccination is specific to a disease selected from the group consisting of: mastitis, ephemeral fever, actinomycosis, bovine respiratory disease, bovine respiratory syncytial virus, infectious bovine

rhinotracheitis, balanoposthitis, influenza, tuberculosis, Clostridium infection, and pneumonia.

30. A method of preparing a vaccine for a bovine subject, the method comprising contacting the composition of any of claims 1-23 with a microorganism or at least one microorganism- derived biomolecule, whereby the microorganism and/or biomolecule are bound by the composition.

31. The method of claim 30, further comprising purifying the microorganism and/or biomolecule from the composition.

32. The method of any of claims 30-31, wherein the microorganism is a bovine pathogen.

33. The method of claim 32, wherein the bovine pathogen is Clostridium tetani,, Clostridium novyi, Clostridium perfringens, Clostridium perfringens, Clostridium perfringens,

Clostridium septicum, Clostridium chauvoei, Clostridium sordellii, Clostridium hemolyticum, Steptococcus uberis, Staphylococcus aureus, Escherichia coli, Streptococcus agalactiae, Streptococcus dysglalactiae, Coorynebacterium bovis, Mycoplasma bovis, Mannheimia haemolytica, Pasteurella multocida, Histophilus somni, Mycobacterium tuberculosis, bovine viral diarrhea virus, bovine respiratory syncyntial virus, rabies virus, bovine Herpes virus 1, Bovine Herpes virus 4, bovine rhinovirus, bovine enterovirus, bovine coronavirus, bovine reovirus, parainfluenza- 3 virus, or a round worm.

34. The method of any of claims 30-33, wherein the vaccine is specific to a disease selected from the group consisting of: mastitis, ephemeral fever, actinomycosis, bovine respiratory disease, bovine respiratory syncytial virus, infectious bovine rhinotracheitis, balanoposthitis, influenza, tuberculosis, Clostridium infection, and pneumonia.