Patent Application
20220119506
This invention relates to methods and compositions for the control of microorganisms in aquaculture and uses thereof.
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.
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.
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.
The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:
In describing the present invention, 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.
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.
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.
As referred to herein, “aquaculture”, “aquatic” and variations thereof refer to the cultivation or dwelling of organisms, including animals and plants, in water.
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.
“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.
“Disease-causing agent” refers to a microorganism, pathogen or virulence factor with a demonstrated or putative role in infection.
As referred to herein, “bacteria”, “bacterial” and variations thereof refer, without limitation, to Vibrio species, Aeromonas species, Edwarsiella 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.
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.
A schematic of camelid heavy chain only antibodies and their relationship to VHH domains and complementarity determining regions (CDRs) is shown in
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.
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
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.
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).
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.
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.
As referred to herein, “production system” and variations thereof refer to any system that can be used to produce any physical embodiment of the invention or modified forms of the invention. 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.
As referred to herein, “product” refers to any physical embodiment of the invention or modified forms of the invention, 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).
As referred to herein, “feed product” refers to any physical embodiment of the invention or modified forms of the invention, 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.
Descriptions of the invention provided are to be interpreted in conjunction with the definitions and caveats provided herein.
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.
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.
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.
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.
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.
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.
In one aspect, the present invention 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.
In another aspect, the present invention 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
In a further aspect, the present invention 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.
In another aspect, the present invention 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.
In yet another aspect, the present invention 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.
In a further aspect, the present invention 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.
In a further aspect, the present invention 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.
In a further aspect, the present invention 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.
In a further aspect, the present invention 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-1,3-glucan, vegetable extracts, peptone, shrimp meal, krill, algae, B-cyclodextran, alginate, gum, tragacanth, pectin and gelatin.
In a further aspect, the present invention 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.
In a further aspect, the present invention 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. nignpulchritudo, 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.
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.
In a further aspect, the present invention 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.
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.
While preferred embodiments of the present invention 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 invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention.
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 CaCl2, 20 mM Imidazole and 10 mM HEPES, pH 7.4) with 12.5 μg/ml DNase I, and 1× Protease inhibitor cocktail (Bioshop). The lysate is cleared by centrifugation at 22000×g for 30 minutes at 4° C., and is then 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 CaCl2, 500 mM imidazole and 10 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 β-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.
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 μg 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 (IFA) 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 VHH-enriched fractions. Lymphocytes are collected from sera taken at days 28, 49, and 70.
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 ˜2.5×107 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.
Purification of VHHs from E. coli
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 VHHs from P. pastoris
Pichia pastoris strain GS115 with constructs for the expression and secretion of 6xHis-tagged VHH are grown for 5 days at 30° C. 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) pre-equilibrated 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.
Approximately 0.1 mg of antigen is incubated with NBX at a 1:5 molar ratio in 200 μl 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 (nickel-nitrilotriacetic 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 μl 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.
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 8×0.2 mL strip tubes on ice. Set up two reactions in volumes of 10 μlon ice. The first reaction contains no shrimp midgut extract and consists of 5 μg NBX in 3.2 μL PBS and 4.8 μL of 150 mM NaCl. The second reaction contains shrimp midgut extract and is generated using the following ratios: 2.4 μL shrimp midgut extract, 5 μg NBX in 0.8 μL PBS, and 4.8 μL of 150 mM NaCl. The tubes are incubated on ice for 5 minutes (corresponds to time=0 minutes in
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.
The following references are incorporated by reference in their entirety.
While preferred embodiments of the present invention 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 invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.
This application claims the benefit of U.S. Provisional Application No. 62/680,736, filed Jun. 5, 2018, which application is incorporated herein by reference. Priority is claimed pursuant to 35 U.S.C. § 119. The above noted patent application is incorporated by reference as if set forth fully herein.
| Filing Document | Filing Date | Country | Kind |
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| PCT/IB2019/000687 | 6/4/2019 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 62680736 | Jun 2018 | US |
