ANTIBODIES AGAINST AQUACULTURE DISEASE-CAUSING AGENTS AND USES THEREOF

FIELD OF THE TECHNOLOGY

[0001] This technology relates to methods and compositions for the control of microorganisms in aquaculture and uses thereof.

BACKGROUND OF THE TECHNOLOGY

[0002] Losses to the aquaculture industry following contamination of livestock with pathogens are a global burden. With a growing global population and no significant increase in the amount of farm land available to agriculture, there is a need to produce larger quantities of food without using more space. Aquaculture is an especially attractive use of this space because the feed conversion ratio for aquaculture organisms is roughly 1:1, whereas the ratio for larger farmed sources of protein is 1:3 or higher ⁽¹⁾ . Losses to the global aquaculture industry due to pathogens is estimated to be around 40%, or $6 billion USD per annum ⁽²⁾ . Traditional treatment of animals with antibiotics is a major contributor to the emergence of multi-drug resistant organisms and is widely recognized as an unsustainable solution to controlling contamination of livestock. There is a need for the development of pathogen-specific molecules that inhibit infection or association of the pathogen with the host, without encouraging resistance.

SUMMARY OF THE TECHNOLOGY

[0003] With reference to the definitions set out below, described herein are polypeptides comprising heavy chain variable region fragments (VHHS) whose intended use includes applications in aquaculture, diagnostics, in vitro assays, feed, therapeutics, substrate identification, nutritional supplementation, bioscientific and medical research, and companion diagnostics. Also described herein are polypeptides comprising VHHS that bind to and decrease the virulence of disease-causing agents in aquaculture. Further to these descriptions, set out below are the uses of polypeptides that comprise VHHS in methods of reducing transmission and severity of disease in host animals, including their use as an ingredient in a product. Further described are the means to produce, characterize, refine and modify VHHS for this purpose.

INCORPORATION BY REFERENCE

[0004] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. BRIEF DESCRIPTION OF THE DRAWINGS

[0005] The novel features of the technology are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present technology will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the technology are utilized, and the accompanying drawings of which:

FIGS. 1A-1B: Panel A shows a schematic of camelid heavy chain only antibodies and their relationship to VHH domains. Panel B illustrates the framework regions (FRs) and complementarity determining regions (CDRs) of the VHH domain.

FIGS. 2A-2M: Show phage ELISA binding data for VHH antibodies of this disclosure.

FIGS. 3A-3B: Shows binding of a selection of recombinantly expressed and purified VHH antibodies to PirA or PirB using a protein pull-down assay.

FIG. 4: Shows the disruption of PirA-PirB complex formation by a selection of recombinantly expressed and purified VHH antibodies using a protein pull-down assay.

FIG. 5: Shows binding of a selection of recombinantly expressed and purified VHH antibodies of this disclosure to PirA and PirB in an ELISA-based assay.

FIG. 6: Shows binding of a selection of recombinantly expressed and purified VHH antibodies of this disclosure to PirB in an ELISA-based assay.

FIG. 7: Shows the stability of a selection of recombinantly expressed and purified VHH antibodies to PirA in shrimp midgut extract fluids.

FIG. 8: Shows the stability of a selection of recombinantly expressed and purified VHH antibodies to PirA or PirB in chicken jejunal extract fluids.

FIG. 9: Shows a timecourse of brine shrimp (Artemia salina) survival in the presence of PirA and PirB.

FIG. 10: Shows the ability of a selection of recombinantly expressed and purified VHH antibodies to protect brine shrimp (Artemia salina) from mortality caused by PirA and PirB. FIG. 11: Shows the impact on PirAB-induced mortality in whiteleg shrimp (Litopenaeus vannamei) of selected VHH antibodies of this disclosure.

FIG. 12: Shows the impact on PirAB-induced mortality in whiteleg shrimp (Litopenaeus vannamei) of selected VHH antibodies of this disclosure under different salinity conditions. FIG. 13: Shows the impact on Vibrio parahaemolyticus -induced mortality in whiteleg shrimp (Litopenaeus vannamei) of selected VHH antibodies of this disclosure. DEFINITIONS

[0006] In describing the present technology, the following terminology is used in accordance with the definitions below. In the following description, certain specific details are set forth in order to provide a thorough understanding of various embodiments. However, one skilled in the art will understand that the embodiments provided may be practiced without these details.

Unless the context requires otherwise, throughout the specification and claims which follow, the word “comprise” and variations thereof, such as, “comprises” and “comprising” are to be construed in an open, inclusive sense, that is, as “including, but not limited to.” As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the content clearly dictates otherwise. It should also be noted that the term “or” is generally employed in its sense including “and/or” unless the content clearly dictates otherwise. Further, headings provided herein are for convenience only and do not interpret the scope or meaning of the claimed embodiments.

Host

[0007] As referred to herein, “host”, “host organism”, “recipient animal”, “host animal” and variations thereof refer to the intended recipient of the product when the product constitutes a feed. In certain embodiments, the host is a crustacean, a shellfish, a shrimp or a prawn.

Shellfish

[0008] As referred to herein, “shellfish” refers to any aquatic exoskeleton-bearing invertebrate. Shellfish can be harvested from the wild or reared. Without limitation, shellfish includes crustaceans, bivalvia, gastropods, cephalopods, octopus, squid, cuttlefish, clams, oysters, mussels, scallops, cockles, whelks, winkles, shrimp, prawns, crawfish, crayfish, lobster, crabs, krill and barnacles.

Aquaculture-specific

[0009] As referred to herein, “aquaculture”, “aquatic” and variations thereof refer to the cultivation or dwelling of organisms, including animals and plants, in water.

Pathogens

[0010] As referred to herein, “pathogen”, “pathogenic”, and variations thereof refer to virulent microorganisms, that can be associated with host organisms, that give rise to a symptom or set of symptoms in that organism that are not present in uninfected host organisms, including the reduction in ability to survive, thrive, reproduce. Without limitation, pathogens encompass parasites, bacteria, viruses, prions, protists, fungi and algae. In certain embodiments, the pathogen is a bacterium belonging to the Vibrio genus. In certain embodiments, the pathogen is the White Spot Syndrome Virus. [0011] “Virulence”, “virulent” and variations thereof refer to a pathogen’s ability to cause symptoms in a host organism. “Virulence factor” refers to nucleic acids, plasmids, genomic islands, genes, peptides, proteins, toxins, lipids, macromolecular machineries or complexes thereof that have a demonstrated or putative role in infection.

[0012] “Disease-causing agent” refers to a microorganism, pathogen or virulence factor with a demonstrated or putative role in infection.

Bacteria

[0013] As referred to herein, “bacteria”, “bacterial” and variations thereof refer, without limitation, to Vibrio species, Aeromonas species, Edwarsiellci species, Streptococcus species, Rickettsia species, or any other bacterial species associated with aquatic organisms or host organisms. In certain embodiments, bacteria may not be virulent in all host organisms it is associated with.

Viruses

[0014] As referred to herein, “virus”, “viral” and variations thereof refer, without limitation, to the White Spot Syndrome Virus, or any other viral species associated with aquatic organisms or host organisms.

Antibodies

[0015] A schematic of camelid heavy chain only antibodies and their relationship to VHH domains and complementarity determining regions (CDRs) is shown in FIG. 1. (Panel A). A camelid heavy chain only antibody consists of two heavy chains linked by a disulphide bridge. Each heavy chain contains two constant immunoglobulin domains (CH2 and CH3) linked through a hinge region to a variable immunoglobulin domain (VHH). (Panel B) are derived from single VHH domains. Each VHH domain contains an amino acid sequence of approximately 1 10- 130 amino acids. The VHH domain consists of the following regions starting at the N-terminus (N): framework region 1 (FR1), complementarity -determining region 1 (CDR1), framework region 2 (FR2), complementarity-determining region 2 (CDR2), framework region 3 (FR3), complementarity-determining region 3 (CDR3), and framework region 4 (FR4). The domain ends at the C-terminus (C). The complementarity-determining regions are highly variable, determine antigen binding by the antibody, and are held together in a scaffold by the framework regions of the VHH domain. The framework regions consist of more conserved amino acid sequences; however, some variability exists in these regions.

[0016] As referred to herein “VHH” refers to an antibody or antibody fragment comprising a single heavy chain variable region which may be derived from natural or synthetic sources. NBXs referred to herein are an example of a VHH. In a certain aspect a VHH may lack a portion of a heavy chain constant region (CH2 or CH3), or an entire heavy chain constant region.

[0017] As referred to herein “heavy chain antibody” refers to an antibody that comprises two heavy chains, and lacking the two light chains normally found in a conventional antibody. The heavy chain antibody may originate from a species of the Camelidae family or Chondrichthyes class. Heavy chain antibodies retain specific binding to an antigen in the absence of any light chain

[0018] As referred to herein “specific binding”, “specifically binds” or variations thereof refer to binding that occurs between an antibody and its target molecule that is mediated by at least one complementarity determining region (CDR) of the antibody’s variable region. Binding that is between the constant region and another molecule, such as Protein A or G, for example, does not constitute specific binding.

[0019] As referred to herein “antibody fragment” refers to any portion of a conventional or heavy chain antibody that retains a capacity to specifically bind a target antigen and may include a single chain antibody, a variable region fragment of a heavy chain antibody, a nanobody, a polypeptide or an immunoglobulin new antigen receptor (IgNAR).

[0020] As referred to herein an “antibody originates from a species” when any of the CDR regions of the antibody were raised in an animal of said species. Antibodies that are raised in a certain species and then optimized by an in vitro method (e.g., phage display) are considered to have originated from that species.

[0021] As referred to herein “conventional antibody” refers to any full-sized immunoglobulin that comprises two heavy chain molecules and two light chain molecules joined together by a disulfide bond. In certain embodiments, the antibodies, compositions, feeds, products, and methods described herein do not utilize conventional antibodies.

Production System

[0022] As referred to herein, “production system” and variations thereof refer to any system that can be used to produce any physical embodiment of the technology or modified forms of the technology. Without limitation, this includes but is not limited to biological production by any of the following: bacteria, yeast, algae, arthropods, arthropod cells, plants, mammalian cells. Without limitation, biological production can give rise to antibodies that can be intracellular, periplasmic, membrane-associated, secreted, or phage-associated. Without limitation, “production system” and variations thereof also include, without limitation, any synthetic production system. This includes, without limitation, de novo protein synthesis, protein synthesis in the presence of cell extracts, protein synthesis in the presence of purified enzymes, and any other alternative protein synthesis system.

Product

[0023] As referred to herein, “product” refers to any physical embodiment of the technology or modified forms of the technology, wherein the binding of the VHH to any molecule, including itself, defines its use. Without limitation, this includes a feed, a feed additive, a nutritional supplement, a premix, a medicine, a therapeutic, a drug, a diagnostic tool, a component or entirety of an in vitro assay, a component or the entirety of a diagnostic assay (including companion diagnostic assays).

Feed Product

[0024] As referred to herein, “feed product” refers to any physical embodiment of the technology or modified forms of the technology, wherein the binding of the VHH to any molecule, including itself, defines its intended use as a product that is taken up by a host organism. Without limitation, this includes a feed, a pellet, a feed additive, a nutritional supplement, a premix, a medicine, a therapeutic or a drug.

DETAILED DESCRIPTION OF THE TECHNOLOGY

[0025] Descriptions of the technology provided are to be interpreted in conjunction with the definitions and caveats provided herein.

[0026] Some farmed aquatic organisms, such as some crustaceans, lack a true adaptive immune response. Additionally, the administration of therapeutics by injection for small and intensely reared organisms is cumbersome. For these reasons, vaccine-based approaches to protecting farmed aquaculture organisms from pathogenic infection is ineffective. Secondly, the use of antibiotics as growth promoters in animal feed has already been banned in Europe (effective from 2006) in an effort to phase out antibiotics for non-medicinal purposes and limit antimicrobial resistance. Indeed, many bacterial pathogens of aquatic organisms already harbor resistance to common antibiotics. This underpins the need for the development of non-antibiotic products to administer to aquatic organisms to prevent infection and promote growth.

[0027] Significant pathogens affecting farmed aquatic organisms include bacteria, such as members of the Vibrio genus, among others, as well as viruses such as White Spot Syndrome Virus (WSSV). Losses due to Vibrio parahaemolyticus, for example, first emerged in 2009 and have been prevalent ever since ⁽³⁾ . It was not until 2013 that V. parahaemolyticus was shown to be the causative agent of Acute Hepatopancreatic Necrosis Disease (AHPND): a subtype of Early Mortality Syndrome (EMS) that contributes approximately $1 billion USD loss to the shrimp farming industry per annum ⁽⁴ ‘ ⁵⁾ . In 2015 it was demonstrated that the presence of the pVA-1 plasmid and the toxins encoded (PirA and PirB) are directly responsible for AHPND ⁽⁵⁾ . Once infected, organisms are up to 100% moribund within 3 days. V. parahaemolyticus is also a prevalent human pathogen, responsible for gastrointestinal infection and septicemia after exposure to contaminated fish or fisheries ⁽⁶⁾ . In addition to PirA and PirB, V. parahaemolyticus produces several proteinaceous factors that have been demonstrated to facilitate host infection and can be targeted to curb virulence.

[0028] WSSV infection is a longer-standing problem; having been identified in 1992 ⁽⁷⁾ there is still no effective means of controlling viral spread or infection in aquatic organisms. Cumulative losses to the aquaculture industry as a consequence of WSSV are estimated at $15 billion USD ⁽⁸⁾ . Infected organisms are moribund within 3-5 days. The surface of the viral envelope is well characterized and can be targeted to prevent infection.

[0029] Other disease-causing agents affecting farmed aquaculture organisms include bacteria (such as Yersinia spp., Edwarsiella spp., Aeromonas spp., Streptococcus spp. and Rickettsia spp.), viruses (such as White Spot Syndrome Virus (WSSV), Yellowhead virus, tilapia iridovirus, epizootic hematopoietic necrosis virus (EHNV), infectious hematopoietic necrosis virus (IHNV), infectious salmon anemia virus (ISAV), infectious pancreatic necrosis virus (IPNV), infectious hypodermal and hematopoietic necrosis virus (IHHNV), taura syndrome virus (TSV) and white spot bacilloform virus (WSBV), hepatopancreatic parvo-like virus (HPV), reo-like virus, monodon baculovirus (MBV), baculoviral midgut GI and necrosis virus (BMN)), algae, prions, protists, parasites, fungi, peptides, proteins and nucleic acids. To our knowledge, an effective, non-vaccine-based treatment against any of these disease-causing agents has yet to be developed for commercial use.

[0030] Existing methods fail to acknowledge the limited immune development of aquatic organisms affected by the pathogens listed above, and as such rely on the host organism to generate protection against disease-causing agents. This approach is limited by the inadequacies of the host organism’s immune system and therefore does not provide an effective means of protection. This problem is circumvented by introducing exogenous peptides into the host that neutralize the virulence and spread of the disease-causing agent without eliciting the host immune response. Moreover, the methods described herein provide scope for the adaptation and refinement of neutralizing peptides, which provides synthetic functionality beyond what the host is naturally able to produce.

[0031] Antibody heavy chain variable region fragments (VHHS) are small (12-15 kDa) proteins that comprise specific binding regions to antigens. When introduced into an animal, VHHS bind and neutralize the effect of disease-causing agents in situ. Owing to their smaller mass, they are less susceptible than conventional antibodies, such as previously documented IgYs, to cleavage by enzymes found in host organisms, more resilient to temperature and pH changes, more soluble, have low systemic absorption and are easier to recombinantly produce on a large scale, making them more suitable for use in animal therapeutics than conventional antibodies.

Antibodies for preventing or reducing virulence (summary)

[0032] In one aspect, the present technology provides a polypeptide or pluralities thereof comprising a VHH or VHHS that bind disease-causing agents to reduce the severity and transmission of disease between and across species. In certain embodiments, the VHH is supplied to host animals. In certain embodiments, the VHH is an ingredient of a product. Binding to reduce virulence

[0033] In another aspect, the present technology provides a polypeptide or pluralities thereof comprising a VHH or VHHS that bind disease-causing agents, and in doing so, reduce the ability of the disease-causing agent to exert a pathological function or contribute to a disease phenotype. In certain embodiments, binding of the VHH(S) to the disease-causing agent reduces the rate of replication of the disease-causing agent. In certain embodiments, binding of the VHH(S) to the disease-causing agent reduces the ability of the disease-causing agent to bind to its cognate receptor. In certain embodiments, binding of the VHH(S) to the disease-causing agent reduces the ability of the disease-causing agent to interact with another molecule or molecules. In certain embodiments, binding of the VHH(S) to the disease-causing agent reduces the mobility or motility of the disease-causing agent. In certain embodiments, binding of the VHH(S) to the disease-causing agent reduces the ability of the disease-causing agent to reach the site of infection. In certain embodiments, binding of the VHH(S) to the disease-causing agent reduces the ability of the disease-causing agent to cause cell death.

Antibodies derived from llamas

[0034] In a further aspect, the present technology provides a method for the inoculation of Camelid or other species with recombinant virulence factors, the retrieval of mRNA encoding VHH domains from lymphocytes of the inoculated organism, the reverse transcription of mRNA encoding VHH domains to produce cDNA, the cloning of cDNA into a suitable vector and the recombinant expression of the VHH from the vector. In certain embodiments, the camelid can be a dromedary, camel, llama, alpaca, vicuna or guanaco, without limitation. In certain embodiments, the inoculated species can be, without limitation, any organism that can produce single domain antibodies, including cartilaginous fish, such as a member of the Chondrichthyes class of organisms, which includes for example sharks, rays, skates and sawfish. In certain embodiments, the heavy chain antibody comprises a sequence set forth in Table 1. In certain embodiments, the heavy chain antibody comprises an amino acid sequence with at least 80%, 90%, 95%, 97%, 99%, or 100% identity to any sequence disclosed in Table 1. In certain embodiments, the heavy chain antibody possesses a CDR1 set forth in Table 2. In certain embodiments, the heavy chain antibody possesses a CDR2 set forth in Table 2. In certain embodiments, the heavy chain antibody possesses a CDR3 set forth in Table 2.

Antibodies recombinantly expressed

[0035] In another aspect, the present technology provides a method for producing VHH in a suitable producing organism. Suitable producing organisms include, without limitation, bacteria, yeast and algae. In certain embodiments, the producing bacterium is Escherichia coli. In certain embodiments, the producing bacterium is a member of the Bacillus genus. In certain embodiments, the producing bacterium is a probiotic. In certain embodiments, the yeast is Pichia pastoris. In certain embodiments, the yeast is Saccharomyces cerevisiae. In certain embodiments, the algae is a member of the Chlamydomonas or Phaeodactylum genera.

Antibodies added to feed

[0036] In yet another aspect, the present technology provides a polypeptide or pluralities thereof comprising a VHH or VHHS that bind disease-causing agents and are administered to host animals via any suitable route as part of a feed product. In certain embodiments, the animal is selected from the list of host animals described, with that list being representative but not limiting. In certain embodiments, the route of administration to a recipient animal can be, but is not limited to: introduction to the alimentary canal orally or rectally, provided to the exterior surface (for example, as a spray or submersion), provided to the medium in which the animal dwells (including air and water based media), provided by injection, provided intravenously, provided via the respiratory system, provided via diffusion, provided via absorption by the endothelium or epithelium, or provided via a secondary organism such as a yeast, bacterium, algae, bacteriophages, plants and insects. In certain embodiments, the host animal is a shellfish. In certain embodiments, the host animal is shrimp.

Feed product

[0037] In a further aspect, the present technology provides a polypeptide or pluralities thereof comprising a VHH or VHHS that bind disease-causing agents and are administered to host animals in the form of a product. The form of the product is not limited, so long as it retains binding to the disease-causing agent in the desired form. In certain embodiments, the product is feed, pellet, nutritional supplement, premix, therapeutic, medicine, or feed additive, but is not limited to these forms.

Feeding dosage

[0038] In a further aspect, the present technology provides a polypeptide or pluralities thereof comprising a VHH or VHHS that bind disease-causing agents and are administered to host animals as part of a product at any suitable dosage regime. In practice, the suitable dosage is the dosage at which the product offers any degree of protection against a disease-causing agent, and depends on the delivery method, delivery schedule, the environment of the recipient animal, the size of the recipient animal, the age of the recipient animal and the health condition of the recipient animal among other factors. In certain embodiments, VHHS are administered to recipient animals at a concentration in excess of 1 mg/kg of body weight. In certain embodiments, VHHS are administered to recipient animals at a concentration in excess of 5 mg/kg of body weight. In certain embodiments, VHHS are administered to recipient animals at a concentration in excess of 10 mg/kg of body weight. In certain embodiments, VHHS are administered to recipient animals at a concentration in excess of 50 mg/kg of body weight. In certain embodiments, VHHS are administered to recipient animals at a concentration in excess of 100 mg/kg of body weight. In certain embodiments, VHHS are administered to recipient animals at a concentration less than 1 mg/kg of body weight. In certain embodiments, VHHS are administered to recipient animals at a concentration less than 500 mg/kg of body weight. In certain embodiments, VHHS are administered to recipient animals at a concentration less than 100 mg/kg of body weight. In certain embodiments, VHHS are administered to recipient animal at a concentration less than 50 mg/kg of body weight. In certain embodiments, VHHS are administered to recipient animals at a concentration less than 10 mg/kg of body weight.

Feeding frequency

[0039] In a further aspect, the present technology provides a polypeptide or pluralities thereof comprising a VHH or VHHS that bind disease-causing agents and are administered to host animals as part of a product at any suitable dosage frequency. In practice, the suitable dosage frequency is that at which the product offers any protection against a disease-causing agent, and depends on the delivery method, delivery schedule, the environment of the recipient animal, the size of the recipient animal, the age of the recipient animal and the health condition of the recipient animal, among other factors. In certain embodiments, the dosage frequency can be but is not limited to: constantly, at consistent specified frequencies under an hour, hourly, at specified frequencies throughout a 24-hour cycle, daily, at specified frequencies throughout a week, weekly, at specified frequencies throughout a month, monthly, at specified frequencies throughout a year, annually, and at any other specified frequency greater than 1 year.

Feed additives

[0040] In a further aspect, the present technology provides a polypeptide or pluralities thereof comprising a VHH or VHHS that bind disease-causing agents and are administered to host animals as part of a product that also comprises other additives or coatings. In practice, the most suitable coating or additive depends on the method of delivery, the recipient animal, the environment of the recipient, the dietary requirements of the recipient animal, the frequency of delivery, the age of the recipient animal, the size of the recipient animal, the health condition of the recipient animal In certain embodiments, these additives and coatings can include, but are not limited to the following list and mixtures thereof: a vitamin, an antibiotic, a hormone, 1 peptide, a steroid, a probiotic, a bacteriophage, chitin, chitosan, B-l,3-glucan, vegetable extracts, peptone, shrimp meal, krill, algae, B-cyclodextran, alginate, gum, tragacanth, pectin and gelatin. Non-feed uses

[0041] In a further aspect, the present technology provides a polypeptide or pluralities thereof comprising a VHH or VHHS that bind disease-causing agents, and can be used in a non-feed use, such as but not limited to: a diagnostic kit, an ELISA-based assay, a western blot assay, an immunofluorescence assay, or a FRET assay, in its current form and/or as a polypeptide conjugated to another molecule. In certain embodiments, the conjugated molecule is can be but is not limited to: a fluorophore, a chemiluminescent substrate, an antimicrobial peptide, a nucleic acid or a lipid.

Antigens

[0042] In a further aspect, the present technology provides a polypeptide or pluralities thereof comprising a VHH or VHHS that bind disease-causing agents, including toxins, produced by a species of Vibrio. In certain embodiments, the Vibrio species is capable of harbouring the pVA- 1 plasmid. In certain embodiments, the species does not belong to the Vibrio genus but is capable of harbouring disease-causing agents shared by Vibrio species, such as but not limited to the pVA-1 plasmid. In certain embodiments, the Vibrio species refers to both current and reclassified organisms. In certain embodiments, the Vibrio species is V. adaptatus, V. aerogenes, V. aestivus, V. aestuarianus, V. agarivorans, V. albensis, V. alfacsensis, V. alginolyticus, V. anguillarum, V. areninigrae, V. artabrorum, V. atlanticus, V. atypicus, V. azureus, V. brasiliensis, V. bubulus, V. calviensis, V. campbellii, V. casei, V. chagasii, V. cholerae, V. cincinnatiensis , V coralliilyticus, V. crassostreae, V. cyclitrophicus, V. diabolicus, V. diazotrophicus, V. ezurae, V. fluvialis, V. fortis, V. furnissii, V. gallicus, V. gazogenes, V. gigantis, V. halioticoli, V. harveyi, V. hepatarius, V. hippocampi, V. hispanicus, V. ichthyoenteri, V. indicus, V. kanaloae, V. lentus, V. litoralis, V. logei, V. mediterranei, V. metschnikovii, V. mimicus, V. mytili, V. natriegens, V. navarrensis, V. neonatus, V. neptunius, V. nereis, V. nigripulchritudo, V. ordalii, V. orientalis, V. pacinii, V. parahaemolyticus , V. pectenicida, V. penaeicida, V. pomeroyi, V. ponticus, V. proteolyticus, V. rotiferianus, V. ruber, V. rumoiensis, V. salmonicida, V. scophthalmi, V. splendidus, V. superstes, V. tapetis, V. tasmaniensis, V. tubiashii, V. vulnificus, V. wodanis, V. xuii, V. fischer, or V. hollisae.

[0043] In certain embodiments, the VHH or plurality thereof is capable of binding to two or more disease-causing agents, originating from the same or different species. In certain embodiments, the disease-causing agent is a polypeptide with 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100% amino acid sequence identity to PirA (SEQ ID 25). In certain embodiments, the disease-causing agent is a polypeptide with 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100% amino acid sequence identity to PirB (SEQ ID 26). In certain embodiments, the disease-causing agent is an exposed peptide, protein, protein complex, nucleic acid, lipid, or combination thereof, that is associated to the surface of the Vibrio bacterium. In certain embodiments, the disease-causing agent is a pilus, fimbria, flagellum, secretion system or porin. In certain embodiments, the disease-causing agent is the Vibrio bacterium.

[0044] In a further aspect, the present technology provides a polypeptide or pluralities thereof comprising a VHH or VHHS that bind disease-causing agents produced by White Spot Syndrome Virus. In certain embodiments, the disease-causing agent is a polypeptide with 60%, 70% 80%, 90%, 95%, 98%, 99%, or 100% amino acid sequence identity VP24 (SEQ ID 27). In certain embodiments, the disease-causing agent is a polypeptide with 60%, 70% 80%, 90%, 95%, 98%, 99%, or 100% amino acid sequence identity to VP28 (SEQ ID 28). In certain embodiments, the disease-causing agent is viral protein associated with or hypothesised to be associated with the envelope of the White Spot Syndrome Virus. In certain embodiments, the disease-causing agent is the White Spot Syndrome Virus.

SEQ ID 25:

PirA

>tr|A0A085YLC0|A0A085YLC0_VIBPH JHE-like toxin PirA-like OS=Vibrio parahaemolyticus OX=670 GN=vpl9 PE=4 SV=1 MSNNIKHETDYSHDWTVEPNGGVTEVDSKHTPIIPEVGRSVDIENTGRGELTIQYQWGA PFMAGGWKVAKSHVVQRDETYHLQRPDNAFYHQRIVVINNGASRGFCTIYYH

SEQ ID 26: PirB

>tr|A0A085YLCl|A0A085YLCl_VIBPH JHE-like toxin PirB-like OS=Vibrio parahaemolyticus OX=670 GN=BTO19_25780 PE=4 SV=1

MTNEYVVTMSSLTEFNPNNARKSYLFDNYEVDPNYAFKAMVSFGLSNIPYAGGFLST L

WNIFWPNTPNEPDIENIWEQLRDRIQDLVDESIIDAINGILDSKIKETRDKIQDINE TIENFG YAAAKDDYIGLVTHYLIGLEENFKRELDGDEWLGYAILPLLATTVSLQITYMACGLDY KDEFGFTDSDVHKLTRNIDKLYDDVSSYITELAAWADNDSYNNANQDNVYDEVMGAR SWCTVHGFEHMLIWQKIKELKKVDVFVHSNLISYSPAVGFPSGNFNYIATGTEDEIPQPL KPNMFGERRNRIVKIESWNSIEIHYYNRVGRLKLTYENGEVVELGKAHKYDEHYQSIEL

NGAYIKYVDVIANGPEAIDRIVFHFSDDRTFVVGENSGKPSVRLQLEGHFICGMLAD QE GSDKVAAFSVAYELFHPDEFGTEK

SEP ID 27:

VP24

>tr|Q9E7K6|Q9E7K6_WSSV Major structural protein VP24 OS=White spot syndrome virus OX=92652 GN=VP24 PE=4 SV=1

MHMWGVYAAILAGLTLILVVISIVVTNIELNKKLDKKDKDAYPVESEIINLTINGVA RGN

HFNFVNGTLQTRNYGKVYVAGQGTSDSELVKKKGDIILTSLLGDGDHTLNVNKAESK E LELYARVYNNTKRDITVDSVSLSPGLNATGREFSANKFVLYFKPTVLKKNRINTLVFGA TFDEDIDDTNRHYLLSMRFSPGNDLFKVGEK

SEP ID 28:

VP28

>tr|A6ZI33|A6ZI33_WSSV Coat protein OS=White spot syndrome virus OX=92652 GN=VP28 PE=4 SV=1

MDLSFTLSVVSAILAITAVIAVFIVIFRYHNTVTKTIETHTGNIETNMDENLRIPVT AEVGS GYFKMTDVSFDSDTLGKIKIRNGKSDAQMKEEDADLVITPVEGRALEVTVGQNLTFEG TFKMWNNTSRKINITGMQMVPKINPSKAFVGSSNTSSFTPVSIDEDEVGTFVCGTTFGAP IAATAGGNLFDMYVHVTYSGTETE

SEP ID 1657;

WSSV067

>AAL88935.1 WSSV067 [Shrimp white spot syndrome virus]

MWLMTSCPIYVLVGCNTSLYKAIEGVKTKHTMVLTLSCTTRRVASSKGNFSKEDAVL G

NQFPILKKSNNLSIARPPSIESFSASVEKIFREWNESGGEKIFDISQNEEEWMDIIS LVESVY EPVFSKSLKPDKLADKTCLTAAAFAALASAVDEKLTILSGSDGSVLQRTTKVMKKDPK KIAESLLNNEKWTSILLDRLKTAKKLLSRRGALKSAERVEVLHRLNKLKEAPLPHHPSLF DNFSGGKTSAVSAGTVIASDMHFKLVEHIFKVSFRKWGPCGDKTESGEEEDEEEEEEEK

KHSISRFVLQFMNGHNGQHYHRPESASVYFCDYYDYLAYRNLPNEYKLSSMHPGTFN MEDLPFRPFAVPSTYKTELEYKRFVQSTNLPQLSFDYGEFLCYCIFGADWYKHLGDVVD SLENSSMISFDSQTLSGVYKNTANYKRLGKKRNGIADLAVRSMAEFIRTEAHKALTAEE MEEEEEEEEAEEEAMDQEPAEVDFLSVPHLRRKIRQAVSVLNNFVENDLSILVSNFKNV

LTDDTVSGTDTDNFGSSGEFEALSSHLFLSRILDEVHILRNTDIQRTLFSTHVSLSD KSPPS RVRGSNVNFNNNAGNISSLQTYGGIEELPENVLVGLSGGFEDTDMYSGEDVVVVWDGC DGGKVLSVTFNCGDNFIQLHEKTAETFKDDTDLVERIRDVLQTASKTGNLNKKAYSRK NIYAVLRENGIERPGDDFTEKGIALKDKTNOPPPPARSAKITVEGVKGFFSGFRDILETR A

LTTYSAETFRDLGQGIVKETEGLTAATVAETSFSEGLAESLRSDANLGLEFSEDAKT VVF

KNDTSRSLLEETRALRANNTSFSSFARDMGVQVSADLDAEFAAEMRETYPDAALEQN L

KDLDKFEETIPESOVKKLKKIDSYLTENPERAGKEINDTELSKATDSVLGKKLGNAV TV

LMNNFGKVTIVVGASVVAGFLGPAAVALVHASRGAHLNVVDHTSPKGVISYKIVDFS C

ADRNTGWAKPTKHPFREEIDHVIALDASFLTENGAYVFPEDGGPKSKYKAYAPICGT KD

AAQGECGSWATFDDPHSVLPWVASMKDLPKGQSLSCDKGMSTLKAVSSVLLSIGKDV AEAIFEVAEDAVVGLASKAISAVINNPLFIFGVPLGFGIAATRLNPSNWKTGLIVFSILL V VILIVRFFAGSGPLTLNWFGAKNSAKRKQTEQFEDGGGNRSKIVLAEKDNANSKLQSRR NETGPMRLEELPGHEDLRPVFFPATTNYSKSAKILGYKSKPFNDFYTKIINTDIIKMDR

SEP ID 1658:

VP53A (Underlined portion of WSSV067 in SEQ ID 1657. See Chen et af^ for more details.) >VP53A

ITVEGVKGFFSGFRDILETRALTTYSAETFRDLGQGIVKETEGLTAATVAETSFSEG LAES LRSDANLGLEFSEDAKTVVFKNDTSRSLLEETRALRANNTSFSSFARDMGVQVSADLD AEFAAEMRETYPDAALEQNLKDLDKFEETIPESQVKKLKKIDSYLTENPERAGKEINDT ELSKATDSVLGKKLGNAVTVLM

SEQ ID 1659:

WSV256

>NP_477778.1 wsv256 [Shrimp white spot syndrome virus]

MIFYTMQPFLGFLVFSVLIVIVMTVLAVYTAPQIKKSKKRKIEDENEEEPVKTLEDF VKG

RLLNAVKEKPAEYFELLISADTEAALKTAEETALRDFVIENDSVEIDVEEVLEEKPR EYV

FKLAGATSETLTNTIIAEVQKKAALITEEDITIKMLKQFRAANKDNKDGEATPEEKE DFT NNSDLVGLYLNEVVEKTTNIVINKIFPHEMVFERCAILIEDFDTGVVTDOAIQIPSNKYK I RLVEGDEPEVFPGDCLDLAVSVDKINHVLKISAKNGCENNCFVIIPRFSPVGSVSSMILG S TDOVKPKTFLFLANKNDSTHFOFTMDKQHSVGCELDMLIFSERNLRNLPDSKPRPLSDA DILASYGKRLGTGVFTTENLVDD

SEP ID 1660:

VP51B (Underlined portion of WSV256 in SEQ ID 1658.) EMVFERCAILIEDFDTGVVTDQAIQIPSNKYKIRLVEGDEPEVFPGDCLDLAVSVDKINH VLKISAKNGCENNCFVIIPRFSPVGSVSSMILGSTDQVKPKTFLFLANKNDSTHFQFTMD KQHSVGCELDMLIFSERNLRNLPDSKPRPLSDADILASYGKRLGTGVFTTENLVDD

EXAMPLES

[0045] The following illustrative examples are representative of the embodiments of the applications, systems and methods described herein and are not meant to be limiting in any way. [0046] While preferred embodiments of the present technology are shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the technology. It should be understood that various alternatives to the embodiments of the technology described herein may be employed in practicing the technology.

Production of antigens

[0047] Recombinant antigens can be purified from an E. coli expression system. For example, the gene for an antigen can be expressed at 18°C in E. coli BL21 (DE3) cells grown overnight in autoinducing media (Formedium). Cells are then lysed by sonication in buffer A (250 mM NaCl, 50 mM CaCh, 20 mM Imidazole and 10 mM HEPES, pH 7.4) with 12.5 pg/ml DNase I, and IX Protease inhibitor cocktail (Bioshop). The lysate is cleared by centrifugation at 22000 x g for 30 minutes at 4°C, applied to a 5 ml HisTrap HP column (GE Healthcare) pre-equilibrated with buffer A, washed with ten column volumes of buffer A and eluted with a gradient of 0% to 60% (vol/vol) buffer B (250 mM NaCl, 50 mM CaCh, 500 mM imidazole andlO mM HEPES, pH 7.4). The protein is then dialyzed overnight in the presence of TEV against buffer C (250 mM NaCl, 10 mM HEPES, pH 7.4 and 5 mM [3-mercaptoethanol) at 4°C. The dialyzed protein is applied to a HisTrap HP column (GE Biosciences) pre-equilibrated with buffer C. 6xHis-tagged TEV and 6xHis-tag are bound to the column and the antigen is collected in the flowthrough. The sample is dialyzed overnight against buffer D (5 mM NaCl and 10 mM Tris pH 8.8) and then applied to a 5 ml HiTrap Q HP column (GE Healthcare). The protein is eluted with a gradient of 0% to 50% (vol/vol) buffer E (1.0 M NaCl and 10 mM Tris pH 8.8). Lastly, the elution is loaded onto a Superdex 75 Increase 10/300 GL gel filtration column (GE Healthcare) using buffer F (400 mM NaCl and 20 mM HEPES pH 7.4). The protein sample is then concentrated to 1 mg/mL using Amicon concentrators with appropriate molecular weight cut-off (MWCO; Millipore). The purified protein is stored at -80°C.

Production of NBXs and panning

Llama immunization

[0048] A single llama is immunized with purified disease-causing agents, such as the antigens listed, which may be accompanied by adjuvants. The llama immunization is performed using 100 pg of each antigen that are pooled and injected for a total of four injections. At the time of injection, the antigens are thawed, and the volume increased to 1 ml with PBS. The 1 ml antigen-PBS mixture is then mixed with 1 ml of Complete Freund’s adjuvant (CFA) or Incomplete Freund’s adjuvant (IF A) for a total of 2 ml. A total of 2 ml is immunized per injection. Whole llama blood and sera are then collected from the immunized animal on days 0, 28, 49, 70. Sera from days 28, 49 and 70 are then fractionated to separate VHH from conventional antibodies. ELISA can be used to measure reactivity against target antigens in polyclonal and VnH-enriched fractions. Lymphocytes are collected from sera taken at days 28, 49, and 70.

Panning

[0049] RNA isolated from purified llama lymphocytes is used to generate cDNA for cloning into phagemids. The resulting phagemids are used to transform E. coli TG-1 cells to generate a library of expressed VHH genes. The phagemid library size can be -2.5 x 10 ⁷ total transformants and the estimated number of phagemid containing VHH inserts can be estimated to be -100%. High affinity antibodies are then selected by panning against the Vibrio or WSSV antigens used for llama immunization. At least two rounds of panning are performed and antigen-binding clones arising from rounds 2 or later are identified using phage ELISA. Antigen-binding clones are sequenced, grouped according to their CDR regions, and prioritized for soluble expression in E. coli and antibody purification.

[0050] Figure 2 shows the Phage ELISA results for all antibodies of this disclosure. Black bars show binding to wells coated with the antigen specified in Tables 1 and 2 dissolved in phosphate-buffered saline (PBS). Grey bars are negative controls that show binding to wells coated with PBS only. In all cases binding to the antigen target is at least 50% above binding to the PBS-coated wells. Panel A shows the results for NBX0401 to NBX0406. Panel B shows the results forNBX0601 to NBX0630. Panel C shows the results for NBX0631 to NBX0637, NBX0813 to NBX0825, NBX0845, NBX0846, and NBX0849. Panel D shows the results for NBX0638 to NBX0650, and NBX0826 to NBX0844. Panel E shows the results for NBX0850 to NBX0865, and NBX09001 to NBX09011. Panel F shows the results for NBX0722 to NBX0725, NBX0730, NBX0738, NBX0739, NBX0745, and NBX0746. Panel G shows the results for NBX0651 to NBX0654, NBX0754, NBX0759 to NBX0763, NBX0770 to NBX0772, NBX0774 to NBX0789, NBX08109 to NBX08115, NBX08310 to NBX08137, and NBX08146 to NBX08148. Panel H shows the results for NBX09012, NBX09015 to NBX09019, NBX10101 to NBX10106, NBX12001 to NBX12003, NBX12020 to NBX12022, NBX12049 to NBX12051, NBX12083 to NBX12086, and NBX15067 to NBX15094. Panel I shows the results for NBX16048 to NBX16053, NBX16055 to NBX16072, NBX19024 to NBX19026, NBX19028, NBX19035, NBX19036, NBX19039 to NBX19042, and NBX19044 to NBX19059. Panel J shows the results for NBX19060, NBX19061, NBX19063, NBX19064, NBX19066 to NBX19070, NBX21017 to NBX21022, NBX21036, NBX21037, NBX21060 to NBX21067, NBX21087 to NBX21090, NBX22001 to NBX22018, and NBX22047 to NBX22049. Panel K shows the results for NBX22050 to NBX22054, NBX22071 to NBX22082, NBX22113 to NBX22118, and NBX23005 to NBX23024. Panel L shows the results for NBX23025 to NBX23037, NBX23042 to NBX23053, and NBX26001 to NBX26017. Panel M shows the results forNBX26028, NBX26020, and NBX27001 to NBX27013.

Purification of VuHs from E. coli

[0051] TEV protease-cleavable, 6xHis-thioredoxin-NBX fusion proteins are expressed in the cytoplasm of E. coli grown in autoinducing media (Formedium) for 24 hours at 30°C. Bacteria are collected by centrifugation, resuspended in buffer A (10 mM HEPES, pH 7.5, 250 mM NaCl, 20 mM Imidazole) and lysed using sonication. Insoluble material is removed by centrifugation and the remaining soluble fraction is applied to a HisTrap column (GE Biosciences) pre-equilibrated with buffer A. The protein is eluted from the column using an FPLC with a linear gradient between buffer A and buffer B (10 mM HEPES, pH 7.5, 500 mM NaCl, 500 mM Imidazole). The eluted protein is dialyzed overnight in the presence of TEV protease to buffer C (10 mM HEPES, pH 7.5, 500 mM NaCl). The dialyzed protein is applied to a HisTrap column (GE Biosciences) pre-equilibrated with buffer C. 6xHis-tagged TEV and 6xHis-tagged thioredoxin are bound to the column and highly purified NBX is collected in the flowthrough. NBX proteins are dialyzed overnight to PBS and concentrated to ~10 mg/ml.

Purification of VuHs from P. vastoris

[0052] Pichia pastoris strain GS115 with constructs for the expression and secretion of 6xHis- tagged VHH are grown for 5 days at 30oC with daily induction of 0.5% (vol/vol) methanol. Yeast cells are removed by centrifugation and the NBX-containing supernatant is spiked with 10 mM imidazole. The supernatant is applied to a HisTrap column (GE Biosciences) preequilibrated with buffer A (10 mM HEPES, pH 7.5, 500 mM NaCl). The protein is eluted from the column using an FPLC with a linear gradient between buffer A and buffer B (10 mM HEPES, pH 7.5, 500 mM NaCl, 500 mM Imidazole). NBX proteins are dialyzed overnight to PBS and concentrated to ~1.5 mg/ml.

NBX Pull-downs

[0053] Approximately 0.1 mg of polyhistidine-tagged PirA or PirB is incubated with NBX at a 1:5 molar ratio in 200 pl of binding buffer (10 mM phosphate buffer pH7.4 and 500 mM NaCl) for 30 minutes at room temperature, and then applied onto a column containing Ni-NTA (nickelnitrilotriacetic acid) resin pre-equilibrated with the binding buffer. Protein mixture and the resin are incubated for 30 minutes before the resin is washed with the binding buffer and then with the binding buffer plus 20 mM Imidazole. Bound proteins are eluted with 100 pl of 1 M imidazole, pH 7.4. The presence or absence of NBX in the various fractions is analyzed on an SDS-PAGE gel. A protein solution containing only the NBX is also applied to a separate column to assess non-specific binding of the NBX to the resin.

[0054] Figure 3A shows representative results for four unique NBXs. For each of the four antibodies, the lanes are as follows. (1) Starting material of PirA(*) and NBX( ⁺ ) mixture prior to application to Ni-NTA resin. (2) Flow-through of PirA and NBX through the Ni-NTA resin. (3) Final wash of the Ni-NTA resin prior to protein elution. (4) Elution of PirA and NBX from the Ni-NTA resin. (5) Elution from Ni-NTA resin to which only NBX was applied. (6) Final wash of Ni-NTA resin to which only NBX was applied. (7) NBX( ⁺ ) only mixture prior to application to Ni-NTA resin. NBXs that can successfully be pulled down by PirA are those that appear in the lane 4 elution but not in the lane 5 elution. For each gel a ladder of proteins of known sizes in kilodaltons (kDa) are shown for reference.

[0055] Figure 3B shows representative results for five unique NBXs. For each of the five antibodies, the lanes are as follows. (1) Starting material of PirA(*) or PirB(**) and NBX( ⁺ ) mixture prior to application to Ni-NTA resin. (2) Flow-through of PirA or PirB and NBX through the Ni-NTA resin. (3) Final wash of the Ni-NTA resin prior to protein elution. (4) Elution of PirA or PirB and NBX from the Ni-NTA resin. (5) Elution from Ni-NTA resin to which only NBX was applied. (6) Final wash of Ni-NTA resin to which only NBX was applied. (7) Flow-through of NBX only mixture through the Ni-NTA resin. (8) NBX( ⁺ ) only mixture prior to application to Ni-NTA resin. NBXs that can successfully be pulled down by PirA or PirB are those that appear in the lane 4 elution but not in the lane 5 elution.

[0056] Table 3 indicates, for all NBXs tested, whether the NBX can be pulled-down by the either PirA or PirB after application to Ni-NTA resin. Disruption of PirA-PirB complex by NBXs

[0057] PirA and PirB are known to form a protein-protein complex ⁽¹⁰⁾ . The NBX pulldown described in Example 3 above was adapted to identify those NBXs that block the formation of the PirA/PirB complex. For NBXs that bind PirA, polyhistidine-tagged PirA, NBX, and untagged PirB are mixed at a molar ratio of 1:5:2 in 200 pl of binding buffer (10 mM phosphate buffer pH7.4 and 500 mM NaCl). For NBXs that bind PirB, polyhistidine-tagged PirB, NBX, and untagged PirA are mixed at a molar ratio of 1:5:2 in 200 pl of binding buffer (10 mM phosphate buffer pH7.4 and 500 mM NaCl). After a 30 minute incubation at room temperature, the mixture is applied onto a column containing Ni-NTA (nickel-nitrilotriacetic acid) resin preequilibrated with the binding buffer. The protein mixture and resin are incubated for 30 minutes before the resin is washed with the binding buffer and then with the binding buffer plus 20 mM Imidazole. Bound proteins are eluted with 100 pl of 1 M imidazole, pH 7.4. The presence or absence of all three proteins in the various fractions is analyzed on an SDS-PAGE gel. A protein solution containing only PirA and PirB is also applied to a separate column to confirm the expected interaction between PirA and PirB in the absence of the NBX. A protein solution lacking the polyhistidine-tagged protein protein is also applied to a separate column to assess non-specific binding of all untagged proteins to the resin.

[0058] Figure 4 shows representative results for two unique NBXs and control samples. For each gel a ladder of proteins of known sizes in kilodaltons (kDa) are shown for reference. [0059] In the left most panel, the lanes are as follows. (1) Starting material of polyhistidine- tagged PirA(*) and untagged PirB(**) mixture prior to application to Ni-NTA resin. (2) Flowthrough of polyhistidine-tagged PirA and untagged PirB. (3) Final wash of the Ni-NTA resin prior to protein elution. (4) Elution of polyhistidine-tagged PirA and untagged PirB from the Ni- NTA resin. (5) Elution from Ni-NTA resin to which only untagged PirB was applied. (6) Final wash of Ni-NTA resin to which only untagged PirB was applied. (7) Untagged PirB(**) only mixture prior to application to Ni-NTA resin. The results of the left most panel indicate that untagged PirB can be pulled down by polyhistidine-tagged PirA (elutes in lane 4) but does not interact non-specifically with the resin (does not elute in lane 5).

[0060] In the middle panel, the lanes are as follows. (1) Starting material of polyhistidine-tagged PirA(*), NBX0820( ⁺ ), and untagged PirB(**). (2) Flow-through of polyhistidine-tagged PirA, NBX0820, and untagged PirB mixture. (3) Final wash of the Ni-NTA resin prior to protein elution. (4) Elution of polyhistidine-tagged PirA and NBX0820, but not untagged PirB from the Ni-NTA resin. (5) Elution from Ni-NTA resin to which only untagged PirB and NBX0820 were applied. (6) Final wash of Ni-NTA resin to which only untagged PirB and NBX0820 were applied. (7) Untagged PirB(**) and NBX0820( ⁺ ) mixture prior to application to Ni-NTA resin. The results of the middle panel demonstrate that in the presence of NBX0820, untagged PirB fails to bind polyhistidine-tagged PirA (no untagged PirB elution in lane 4). Therefore, NBX0820 inhibits PirA-PirB complex formation.

[0061] In the right most panel, the lanes are as follows. (1) Starting material of polyhistidine- tagged PirA(*) and untagged PirB(**) mixture prior to application to Ni-NTA resin. (2) Flow- through of polyhistidine-tagged PirA and untagged PirB. (3) Final wash of the Ni-NTA resin prior to protein elution. (4) Elution of polyhistidine-tagged PirA and untagged PirB from the Ni- NTA resin. (5) Starting material of polyhistidine-tagged PirA(*), NBX15077( ^# ), and untagged PirB(**). (6) Flow-through of polyhistidine-tagged PirA, NBX15077, and untagged PirB mixture. (7) Final wash of the Ni-NTA resin prior to protein elution. (8) Elution of polyhistidine-tagged PirA and NBX15077, but not untagged PirB from the Ni-NTA resin. The results of the right most panel demonstrate that in the presence of NBX15077, untagged PirB fails to bind polyhistidine-tagged PirA (no untagged PirB elution in lane 8). Therefore, NBX15077 inhibits PirA-PirB complex formation.

[0062] Table 4 indicates, for all NBXs tested, whether the NBX can disrupt PirA-PirB complex formation.

Affinity of NBXs for antigens as measured in an ELISA-based assay

[0063] A 96-well plate was coated overnight at 4°C with 100 pL of PirA (10 pg/ml), PirB (20 pg/ml), VP24 (10 pg/ml), VP28 (10 pg/ml), VP51B (2.5 pg/ml), or VP53A (5 pg/ml) in PBS. After the overnight incubation, antigen solutions were removed, wells were washed three times with 300 pL of PBS and blocked with 200 pL of 5% skim milk powder in PBS + 0.05% Tween- 20 (PBST) for two hours at room temperature. Blocking solution was removed and wells were washed one time with 300 pL of PBST. NBXs were diluted in blocking solution, and 100 pL was added to wells. The final NBX concentrations ranged from 4 pM to 40 pM. Plates were incubated with NBX solutions for one hour at room temperature. NBX solutions were removed, wells were washed three times with 300 pL of PBST, and 100 pL of HRP-conjugated rabbit IgG anti-VnH cocktail (GenScript) diluted 1:8000 in blocking solution was added to wells and incubated for one hour at room temperature. Anti-VnH antibody solution was removed, wells were washed three times with 300 pL of PBST, and 100 pL of 3,3’,5,5’-tetramethylbenzidine (TMB) substrate (Abeam) was added to wells and incubated for 30 minutes at room temperature. Reactions were stopped with the addition 50 pL of 1 M HC1 and absorbance at 450 nm was measured.

[0064] Figure 5 shows the binding of four representative NBXs to both PirA (left panel) and PirB (right panel). The data shown are for NBX0820 (blue, circles), NBX15077 (red, square), NBX23022 (green, triangle), and NBX23032 (purple, inverted triangle). As expected NBX0820 and NBX15077 bound to PirA but not PirB, while NBX23022 and NBX23032 bound to PirB but not PirA. For all four NBXs, binding is detected at sub-nanomolar concentrations, with midpoints of saturation of 1.7 nM for NBX0820 to PirA, 1.4 nM for NBX15077 to PirA, 0.5 nM for NBX23022 to PirB, and 1.1 nM for NBX23032 to PirB. These results indicate that the representative NBXs have high affinity for their expected antigens.

[0065] Figure 6 shows the binding of two additional NBXs to PirB. The data shown are for NBX23021 (left panel) and NBX23036 (right panel). For both NBXs, binding is detected at sub- nanomolar concentrations, with midpoints of saturation of 3.7 nM for NBX23021 to PirB and 0.4 nM for NBX23026 to PirB. These results indicate that the representative NBXs have high affinity for their expected antigens. [0066] Table 5 indicates, for all NBXs tested, whether the NBX can bind its expected antigen with a midpoint of saturation below 100 nM in the ELISA-based binding assay.

Protein stability in intestinal tract fluids

[0067] To use NBXs as an oral application in animals, high proteolytic stability in the intestinal tract is required. Depending on extract availability the NBXs of this disclosure were tested in either a shrimp midgut extract or a chicken jejunal extract as a surrogate when the shrimp extract was not available.

[0068] Thaw frozen shrimp midgut extract and NBX at room temperature, and immediately place on ice. Spin shrimp midgut extract and protein at 10,000 RCF for 1 minute to pellet and remove any precipitation. Prechill PBS and saline on ice. Label and prechill 8 x 0.2 mL strip tubes on ice. Set up two reactions in volumes of 10 pl on ice. The first reaction contains no shrimp midgut extract and consists of 5 pg NBX in 3.2 pL PBS and 4.8 pL of 150 mM NaCl. The second reaction contains shrimp midgut extract and is generated using the following ratios: 2.4 pL shrimp midgut extract, 5 pg NBX in 0.8 pL PBS, and 4.8 pL of 150 mM NaCl. The tubes are incubated on ice for 5 minutes (corresponds to time = 0 minutes in Figure 1) followed by 26° C for up to 24 hours. The final incubation temperature (26°C) is the internal temperature of a shrimp. After incubation, add 8 pL of preheated 2X SDS sample buffer to stop the reaction. Boil at 95-100°C for 5 minutes. The stability of each NBXs is assessed by the presence or absence of the NBX on an 18% SDS-PAGE gel. Alternatively, the shrimp midgut extract is replaced with a chicken jejunal extract. For such experiments, the final incubation temperature is increased to 42°C.

[0069] Figure 7 shows representative results for four unique NBXs tested in shrimp midgut extracts. For each of the four antibodies shown SDS-PAGE gels are arranged from left to right as follows. A ladder of proteins of known sizes in kilodaltons (kDa) are shown for reference. The next two lanes show the NBX at the beginning and end of the experiment in the absence of shrimp midgut extract. These lanes show that the NBX is not degraded over time in the absence of shrimp midgut extract. The subsequent lane shows the appearance of the shrimp midgut extract at the start of the experiment without NBX added. This lane allows for the visualization of naturally occurring proteins in the extract. The subsequent 7-9 lanes show the time course of NBX stability in the shrimp midgut extract. These lanes allow for the visualization of the relative stability of the NBX. The longer the full-sized NBX can be visualized on the gel the more stable it is. The final lane shows the shrimp midgut extract in the absence of NBX at the endpoint of the assay. Times indicated are in minutes, unless they are denoted with an “h”, in which case they are in hours. 151 NBXs of this disclosure have been tested for stability in the shrimp midgut extract. NBX0625 is in the top 6% of the most stable NBXs in this extract. NBX0820 is in the top 22% of the most stable NBXs in this extract. NBX0821 is in the top 13% of the most stable NBXs in this extract. NBX0845 is within the top 33% of the most stable NBXs in this extract.

[0070] Table 6 indicates, for all NBXs tested, whether or not the NBX is in the top 33% of the most stable NBXs in the shrimp midgut extract.

[0071] Table 7 indicates all NBXs that are in the top 10% of the most stable NBXs in the shrimp midgut extract.

[0072] Figure 8 shows representative results for six unique NBXs tested in chicken jejunal extracts. For each of the six antibodies shown SDS-PAGE gels are arranged from left to right as follows. A ladder of proteins of known sizes in kilodaltons (kDa) are shown for reference. The next two lanes show the NBX at the beginning and end of the experiment in the absence of chicken jejunal extract. These lanes show that the NBX is not degraded over time in the absence of chicken jejunal extract. The subsequent lane shows the appearance of the chicken jejunal extract at the start of the experiment without NBX added. This lane allows for the visualization of naturally occurring proteins in the extract. The subsequent 7-9 lanes show the time course of NBX stability in the chicken jejunal extract. These lanes allow for the visualization of the relative stability of the NBX. The longer the full-sized NBX can be visualized on the gel the more stable it is. The final lane shows the chicken jejunal extract in the absence of NBX at the endpoint of the assay. Times indicated are in hours. 260 NBXs of this disclosure have been tested for stability in the chicken jejunal extract. NBX0820 is in the top 66% of the most stable NBXs in this extract. NBX15077 is in the top 3% of the most stable NBXs in this extract. NBX23021 is in the top 28% of the most stable NBXs in this extract. NBX23022, NBX23026, and NBX23032 are all within the top 10% of the most stable NBXs in this extract.

[0073] Table 8 indicates, for all NBXs tested, whether or not the NBX is in the top 33% of the most stable NBXs in the chicken jejunal extract.

[0074] Table 9 indicates all NBXs that are in the top 10% of the most stable NBXs in the chicken j ej unal extract.

NBX Protection of brine shrimp from PirA/PirB toxin

[0075] Brine shrimp (Artemia salina) were used as a surrogate for whiteleg shrimp to assess the ability of NBXs to neutralize the PirA/PirB toxin and reduce mortality. Artemia salina cysts were purchased from Aquarium Direct (Saint-Charles-Borromee, QC, Canada) and rehydrated in 1.7 L of artificial seawater (pH 8.4-8.6; Alkalinity 3.2-3.8 mEq/L) prepared following manufacturer’s instruction (Salinity™; Aquavitro®; Madison, GA, USA) in an Artemia hatchery blender (ZH-2000, Ziss Artemia Blender 2.0 L; 18cm* 14cm* 13cm). After 48 hours incubation between 26-28°C under constant aeration with a constant light source, the growth stage of Artemia was confirmed under a microscope. Only those that reached instar II larvae were used in the challenge test.

[0076] About 10 to 12 stage II Artemia were allocated into 0.5-mL artificial seawater per well in 24-well plates and acclimatized for 1 hour at 26-28°C. Diluted protein treatment solutions (0.5 mL) were added to each well to achieve final concentrations of 3 pM PirA, 3 pM PirB, and 15 pM nanobody in 10% PBS. The 24-well plates were placed on a shaking platform (90 rpm) for 5 minutes to ensure homogenous mixture of treatment solution with seawater. The plates were incubated in a non-shaking incubator between 26-28°C with a constant light source and mortality was measured up to 48 hours post challenge. Each treatment group had 12 replicates wells. A negative control group that only received 10% PBS and a positive control group that received PirA and PirB but no nanobody were included in each experiment.

[0077] Figure 9 shows that there was little brine shrimp mortality after 48 hours in the presence of seawater (blue, circles), a PBS buffer control (green, triangle), or an off target NBX that does not bind either PirA or PirB (orange, diamond). Significant brine shrimp mortality in the presence of 3 mM PirAB (red, square) occured after 24 hours of incubation. The mortality seen in the presence of PirAB was unaltered when the off target NBX was added (black, inverted triangle). This last result indicates that there are no non-specific interactions between PirAB and NBXs that impact the assay. Based on these results, in subsequent experiments testing PirA or PirB binding NBXs, brine shrimp mortality was measured at 42 hours.

[0078] Figure 10 shows representative results for six NBXs tested for the ability to protect brine shrimp from PirA and PirB. For each NBX, the results shown are from three to four experiments. The data was collected after 42 hours of treatment. Bars in the graphs represent the means and error bars represent the standard deviations. All six NBXs (NBX0820, NBX15077, NBX23021, NBX23022, NBX23026, and NBX23032) provided protection to the brine shrimp and increased their survival in the presence of PirA and PirB.

[0079] Table 8 indicates, for all NBXs tested, whether the NBX reduced brine shrimp mortality caused by PirAB by at least 30%. NBX Protection of whiteleg shrimp from PirA/PirB toxin

[0080] Post-larva whiteleg shrimp (PL10, 10 mm +/- 1 mm in length, Litopenaeus vannamei) were used for assessing the efficacy of NBXs in neutralizing the PirA/PirB toxin and reducing mortality. One PL10 is put into 0.5-ml seawater (20 ppt of salinity, pH-7.8-8.2, 120-160 ppm of alkalinity) per well in 24-well plates and allowed to acclimatize for 1 to 2 hours. During acclimatization, PirA, PirB, NBXs, and seawater were mixed as a 2X treatment solution and allowed to equilibrate for 30 minutes. After the PL10 acclimatization period, 0.5 mL of 2X treatment solutions were added to each well to start the test. The final NBX concentration is either 4 or 8 pM. The final PirA and PirB concentrations vary by batch and was defined as a dose that gives a slowly increasing mortality curve over a 24-hour period and reaches 80-100% mortality by 24 hours. A typical PirA and PirB concentration used was 125 nM. Each treatment group had a total of 12 PL 10s. Shrimp death was monitored every 2 hours for 24 hours. For each test, a negative control that contained only seawater and a positive control that contained PirA and PirB but no NBX were included as additional groups.

[0081] Figure 11 shows the impact of selected NBXs on whiteleg shrimp mortality induced by PirAB. The data for whiteleg shrimp mortality in the presence of each NBX over 24 hours are shown in individual panels. Each panel shows the data from untreated (green, triangle) and PirAB treated (black, inverted triangles) from experiments which also contained the particular NBX. A third line (red, squares) shows the mortality of whiteleg shrimp treated with PirAB and the NBX. Significant variation is seen in the effectiveness of the NBXs at protecting whiteleg shrimp from PirAB. Some NBXs, such as NBX15077 and NBX 23022, provide complete protection to the shrimp and reduce mortality to levels seen in the untreated shrimp. Some NBXs, such as NBX0820, NBX23021, NBX23026, NBX23032, and NBX23043 provide partial protection, with mortality reduced but not to the levels seen in untreated shrimp. Finally, some NBXs, such as NBX15081, NBX19055, and NBX23010 do not provide significant protection to whiteleg shrimp. Data shown is from representative NBXs that did not provide significant protection in our experimental model. Data shown in each graph are the means and standard error of the means from three to five experiments, except for NBX0820, for which there is only one experiment.

[0082] Since shrimp aquaculture occurs in range of salinities, selected NBXs were tested for the ability to protect whiteleg shrimp under different salinities (3, 10, 20, and 30 ppt) using the protocol described above with 8 pM of NBXs.

[0083] Figure 12 shows the impact of two NBXs, NBX15077 and NBX23022 on whiteleg shrimp mortality in the presence of PirAB at a range of salinities. There is very little death in untreated whiteleg shrimp (green, triangles) at any salinity. The mortality of whiteleg shrimp in the presence of PirAB (black, inverted triangle) increases as salinity increases. Treatment with NBX15077 (red, square) reduces mortality to levels comparable to untreated at all salinities tested. Treatment with NBX23033 (blue, circle) reduces mortality to levels comparable to untreated at 3, 10, and 20 ppt and significantly delays mortality at 30 ppt.

NBX Protection of whiteleg shrimp from Vibrio parahaemolyticus

[0084] Post-larva whiteleg shrimp (PL10, 10 mm +/- 1 mm in length, Litopenaeus vannamei) were used for assessing the efficacy of NBXs in neutralizing Vibrio parahaemolyticus strain LA37 and reducing mortality. One PL10 is put into 0.5-ml seawater (20 ppt of salinity, pH-7.8- 8.2, 120-160 ppm of alkalinity) per well in 24-well plates and allowed to acclimatize for 1 to 2 hours. During acclimatization, Vibrio parahaemolyticus, NBXs, and seawater were mixed as a 2X treatment solution and allowed to equilibrate for 30 minutes. After the PL10 acclimatization period, 0.5 mL of 2X treatment solutions were added to each well to start the test. The final NBX concentration is either 4 or 8 pM. The final Vibrio parahaemolyticus dose was 2 x 10 ⁶ colony forming units/well. Each treatment group had a total of 12 PLIOs. Shrimp death was monitored every 2 hours for 24 hours. For each test, a negative control that contained only seawater and a positive control that contained Vibrio parahaemolyticus but no NBX were included as additional groups.

[0085] Figure 13 shows the impact of two NBXs, NBX15077 and NBX23022 on whiteleg shrimp mortality in the presence of Vibrio parahaemolyticus . The data for whiteleg shrimp mortality in the presence of each NBX over 24 hours are shown in individual panels. Each panel shows the data from untreated (green, triangle) and Vibrio parahaemolyticus treated (black, inverted triangles) from experiments which also contained the particular NBX. A third line (red, squares) shows the mortality of whiteleg shrimp treated with Vibrio parahaemolyticus and the NBX. Compared to the results presented above, when disease is induced by purified PirAB toxin, the protection offered by these NBXs is decreased. We have observed that the concentration of NBXs in this experimental setup decreases over time. When challenging whiteleg shrimp with the PirAB toxin and NBXs together, the PirAB toxin dose is highest at the beginning and likely also decreases over time. In contrast, when challenging with Vibrio parahaemolyticus, the PirAB must be first produced by the bacterium and likely increases over time in this assay setup. As such a later application of the NBX is predicted to be potentially more neutralizing in this experimental setup. In upcoming experiments, the exact same challenges will be conducted, except the addition of the NBX will be delayed until approximately six hours after the whiteleg shrimp are exposed to the Vibrio parahaemolyticus. [0086] All publications, patent applications, issued patents, and other documents referred to in this specification are herein incorporated by reference as if each individual publication, patent application, issued patent, or other document is specifically and individually indicated to be incorporated by reference in its entirety. Definitions that are contained in text incorporated by reference are excluded to the extent that they contradict definitions in this disclosure.

[0087] The following references are incorporated by reference in their entirety.

1. Kierath, D. (2015, March). The growth of global aquaculture - Fishy business. Retrieved from deloitte.com/au/en/pages/consumer-business/articles/the-grow th-of-aqua-culture-fishy- business

2. Stentiford, G. D., Sritunyalucksana, K., Flegel, T. W., Williams, B. A. P., Withyachumnamkul, B., Itathitphaisam, O., Bass, D. (2017). New paradigms to help solve the global aquaculture disease crisis. PLos Pathogens, 13(2), pp. 1-6

3. Lee, C. T., Chen, T. I., Yang, Y. T., Ko, T. P., Huang, Y. T., Huang., J. Y., Huang, M. F., Lin, S. J., Chen, C. Y., Lin, S. S., Lightner, D. V., Wang, H. C., Wang, A. H. J., Wang, H. C., Hor, L. I., Lo, C. F. (2015) The opportunistic marine pathogen Vibrio parahaemolyticus becomes virulent by acquiring a plasmid that expresses a deadly toxin. PNAS, 112(34), pp. 10789-10803.

4. FAO Fisheries and Aquaculture (2013) Report of the FAO/MARD Technical Workshop on Early Mortality Syndrome (EMS) or Acute Hepatopancreatic Necrosis Syndrome (AHPNS) of Cultured Shrimp (under TCP/VIE/3304). rep. no. 1053. Retrieved from www.fao.org/docrep/018/i3422e/i3422e.pdf

5. Tran, L., Numan, L., Redman, R. M., Mohney, L. L., Pantoja, C. R., Fitzsimmons, K., Lightner, D. V. (2013) Determination of the infectious nature of the agent of acute hepatopancreatic necrosis syndrome affecting penaeid shrimp. Diseases of Aquatic Organisms, 105(1), pp. 45-55.

6. Ahmed, H. A., El Bayomi, R. M., Hussein, M. A., Khedr, M. H. E., Abo Ramela, E. M., El- Ashram, A. M. M. (2018) Molecular characterization, antibiotic resistance pattern and biofilm formation of Vibrio parahaemolyticus and V. cholerae isolated from crustaceans and humans. International Journal of Food Microbiology, 274, pp. 31-37.

7. Flegel, T. (2012) Historic emergence, impact and current status of shrimp pathogens in Asia, Journal of Invertebrate Pathology, 110(2), pp. 166-173.

8. Lightner, D. V., Redman, R. M., Pantoja, C. R., Tang, K. F. J., Noble, B. L., Schofield, P., Mohney, L. L., Nunan, L. M., Navarro, S. A. (2012) Historic emergence, impact and current status of shrimp pathogens in the Americas, Journal of Invertebrate Pathology, 110(2), pp. 174- 183.

9. Chen, L.-L., Lu, L.-C., Wu, W.-J., Lo, C.-F., Huang, W.-P. (2007) White spot syndrome virus envelope protein VP53A interacts with Penaeus monodon chitin-binding protein (PmCBP), Diseases of Aquatic Organisms, 74(3), pp. 171-178.

10. Lin, S.-J., Chen, Y.-F., Hsu, K.-C., Chen, Y.-L., Ko, T.-P., Lo, C.-F., Wang, H.-C., Wang, H.-C. (2019) Structural insights to the heterotetrameric interaction between the Vibrio parahaemolyticus PirA^ and PirB' ⁷ ' toxins and activation of the Cry -like pore-forming domain, Toxins, 11, Article 233.

[0088] While preferred embodiments of the present technology have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the technology. It should be understood that various alternatives to the embodiments of the technology described herein may be employed in practicing the technology. It is intended that the following claims define the scope of the technology and that methods and structures within the scope of these claims and their equivalents be covered thereby.