Patent Application
20210269512
The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Feb. 5, 2020, is named 48647_706_301_SL.txt and is 344,000 bytes in size.
This invention relates to methods and compositions for the control of microorganisms associated with necrotic enteritis and uses thereof.
Losses to the agriculture industry following contamination of livestock with pathogens are a global burden. With a growing global population and no significant increase in the amount of farmland available to agriculture, there is a need to produce larger quantities of food without using more space. Traditional treatment of animals with antibiotics is a major contributor to the emergence of multi-drug resistant organisms and is widely recognised 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. Global losses to the poultry industry due to the pathogenic organisms that cause necrotic enteritis has been estimated to be $6 billion(1) USD per annum. The bacterium Clostridium perfringens is the causative agent of necrotic enteritis in poultry in conjunction with a variety of predisposing factors(2).
With reference to the definitions set out below, described herein are polypeptides comprising heavy chain variable region fragments (VHHs) whose intended use includes but is not limited to the following applications in agriculture or an unrelated field: 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 and decrease the virulence of disease-causing agents in agriculture. 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, characterise, 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 from the superorder Galloanserae. In certain embodiments, the host is a poultry animal. In certain embodiments, the poultry animal is a chicken, turkey, duck, quail, pigeon, squab or goose. In certain embodiments, the poultry animal is a chicken.
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 Clostridium genus.
“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 Clostridium species, or any other bacterial species associated with host organisms. In certain embodiments, bacteria may not be virulent in all host organisms it is associated with.
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 lacks 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.
For many years, the agriculture industry has utilized antibiotics to control pathogenic bacteria. These antibiotics also acted as growth promoters. This approach has contributed greatly to the spread of antibiotic resistance amongst pathogenic organisms. To phase out antibiotics for non-medicinal purposes and limit antimicrobial resistance, the use of antibiotics as growth promoters in animal feed has already been banned in Europe (effective from 2006). Widespread protection of farmed animals through vaccination has failed due to the short lifespan of many agriculturally important animals, logistical challenges with vaccination of industrial-sized flocks, and high costs. The withdrawal of prophylactic antibiotics in animal feed and the failure of vaccination to offer widespread protection underpins the need for the development of non-antibiotic products to administer to agricultural animals to prevent infection and promote growth.
Significant pathogens affecting poultry animals include bacteria, such as members of the Clostridium and Salmonella genera, among others, as well as parasites, such as members of the Eimeria genus.
Losses due to Clostridium perfringens, the causative agent of necrotic enteritis are estimated at $6 billion(1) USD per annum. Necrotic enteritis can lead to significant mortality in chicken flocks(3). At subclinical levels, damage to the intestinal mucosa caused by C. perfringens leads to decreased digestion and absorption, reduced weight gain and increased feed conversion ratio(3). Typically, necrotic enteritis occurs after some other predisposing factor causes mucosal damage to the chicken(2) C. perfringens virulence factors associated with necrotic enteritis have been shown to include production of toxins and adherence to collagen(4).
Subclinical infection by Eimeria parasites is one of the most common predisposing factors for necrotic enteritis(2). These parasites can physically damage the epithelial layer and induce mucose generation(5). In addition, Eimeria parasites are also the causative agent of coccidiosis in chickens, a disease that is estimated to cause €10 billion in poultry losses globally(6). Coccidiosis is characterized by reduced weight gain and feed conversion, malabsorption, cell lysis of cells linking, and diarrhea(7).
Changes to the gastrointestinal tract microbiota can also serve to induce necrotic enteritis. For example, early infections early of chicks by Salmonella enterica can result in the development of necrotic enteritis in experimental models, possibly through alteration of the host immune response(8).
Other proposed predisposing factors for the development of necrotic enteritis include immune suppression by viral infections, physical changes to the gut caused by alterations to the diet, and poor animal husbandry(2).
Earlier efforts in the field of this invention rely on the host organism to generate protection against disease-causing agents. This approach is often limited by the short lifespan of the host organisms affected by the pathogens listed above, which do allow the host organism's immune system enough time to generate long-lasting immunity. Furthermore, the effectiveness of prior arts is limited by technical challenges associated with widespread vaccination of large flocks of host organisms. These problems are circumvented by introducing exogenous peptides that neutralise the virulence and spread of the disease-causing agent into the host via feed without eliciting the host immune response. Moreover, the methods described herein provide scope for the adaptation and refinement of neutralising 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 neutralise 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 guacano, 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%, or 99% identity to any sequence disclosed in Table 1. In certain embodiments, the heavy chain antibody possess a CDR1 set forth in Table 2. In certain embodiments, the heavy chain antibody possess a CDR2 set forth in Table 2. In certain embodiments, the heavy chain antibody possess 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 alga 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 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 is from the superorder Galloanserae. In certain embodiments, the host is a poultry animal. In certain embodiments, the poultry animal is a chicken, turkey, duck, quail, pigeon, squab or goose. In certain embodiments, the poultry animal is a chicken.
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, an antimicrobial peptide, a steroid, a probiotic, a probiotic, a bacteriophage, chitin, chitosan, B-1,3-glucan, vegetable extracts, peptone, shrimp meal, krill, algae, B-cyclodextran, alginate, gum, tragacanth, pectin, gelatin, an additive spray, a toxin binder, a short chain fatty acid, a medium chain fatty acid, yeast, a yeast extract, sugar, a digestive enzyme, a digestive compound, an essential mineral, an essential salt, or fibre.
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 enzyme-linked immunosorbent assay (ELISA), a western blot assay, an immunofluorescence assay, or a fluorescence resonance energy transfer (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 Clostridium. In certain embodiments, the species does not belong to the Clostridium genus but is capable of harbouring disease-causing agents shared by Clostridium species. In certain embodiments, the Clostridium species refers to both current and reclassified organisms. In certain embodiments, the Clostridium species is Clostridium perfringens.
In certain embodiments, the VHH or plurality thereof is capable of binding to one or more disease-causing agents, originating from the same or different species. In certain embodiments, the disease-causing agent is a polypeptide with 80% or greater amino acid sequence identity to NetB (SEQ ID 207). In certain embodiments, the disease-causing agent is a polypeptide with 80% or greater amino acid sequence identity to Cpa (SEQ ID 208). In certain embodiments, the disease-causing agent is a polypeptide with 80% or greater amino acid sequence identity to Cpb2 (SEQ ID 209). In certain embodiments, the disease-causing agent is a polypeptide with 80% or greater amino acid sequence identity to CnaA (SEQ ID 210). In certain embodiments, the disease-causing agent is a polypeptide with 80% or greater amino acid sequence identity to the collagen-binding domain of CnaA (SEQ ID 211). 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 Clostridium 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 Clostridium bacterium.
In certain embodiments, the disease-causing agent or a derivative thereof can be provided in excess and outcompete the activity of the pathogen expressed disease-causing agent. In certain embodiments, a polypeptide with 80% or greater amino acid sequence identity to CnaA (SEQ ID 210) or the collagen-binding domain of CnaA (SEQ ID 211) can be provided in excess to outcompete the activity of CnaA expressed by the Clostridium perfringens bacterium.
perfringens]
perfringens]
perfringens]
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, 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., 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. 6×His-tagged TEV (“6×His” disclosed as SEQ ID NO: 695) and 6×His-tag (SEQ ID NO: 695) 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 eluate 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 antigens used for llama immunization. Two rounds of panning are performed and antigen-binding clones arising from round 2 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, 6×His-thioredoxin-NBX fusion proteins (“6×His” disclosed as SEQ ID NO: 695) 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. 6×His-tagged TEV and 6×His-tagged thioredoxin (“6×His” disclosed as SEQ ID NO: 695) 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.
Pichia pastoris strain GS115 with constructs for the expression and secretion of 6×His-tagged VHH (“6×His” disclosed as SEQ ID NO: 695) 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 ˜10 mg/ml.
Hepatocellular carcinoma-derived epithelial cells (LMH cells) from Gallus gallus strain Leghorn are adhered to the surface of a tissue-culture treated and gelatin-coated 96-well microtitre plate at 64,000 cells/well overnight at 37° C. and 5% CO2. Recombinantly expressed NetB is preincubated with NBX at a range of concentrations or the buffer in which the NBXs are dissolved (20 mM HEPES pH 7.4, 150 mM NaCl) for 15 minutes at 37° C. and 5% CO2. After 15 minutes the toxin/NBX mixtures are added to triplicate wells of LMH cells. The final concentration of NetB is 5 nM. The final concentrations of NBXs are 1, 3, 9, 27, 81, 243, 729, and 2187 nM. LMH cells with toxin/NBX mixtures are incubated for 5 hours at 37° C. and 5% CO2. Cytotoxicity induced by NetB is measured using the Pierce LDH Cytotoxicity Assay Kit (Thermo Scientific) following the manufacturer's instructions. NetB percent cytotoxicity in the presence of NBX is determined relative to NetB cytotoxicity in the absence of NBX. A non-linear fit of the inhibitor concentration versus response is determined using GraphPad Prism 8 which generates the 50% inhibitory concentration (IC50) which approximates the NBX concentration required to block 50% of the cytotoxicity of 5 nM NetB.
Table 3 indicates, for all NBXs tested, whether the NBX can neutralize the activity of NetB against LMH cells with an IC50-value less than 1 μM and/or less than 50 nM.
In a 96-well microtiter plate, 2 μg of collagen is incubated in 100 μl of PBS per well overnight at 4° C. The plate is washed with 200 μl of PBS and then blocked with 200 μl of 5% skim milk in PBS for 2 hours at 37° C. During the blocking step, 200 nM or 2 μM of individual NBXs are mixed with or without 100 nM of 6×-Histidine (SEQ ID NO: 695) and Maltose-binding-protein (MBP) tagged CnaA in PBS for 30 minutes at 37° C. The plate is washed with 200 μl of PBS three times, and 100 μl of NBXs or NBX/MBP-CnaA mixture is added to each well for a 2-hour incubation at 37° C. After washing with 200 μl of PBS three times, 100 μl of 0.125 μg/ml of anti-His conjugated with HRP is added to each well and incubated for 1 hour at room temperature. The plate is then washed with 200 μl of PBS three times, and 100 μl of TMB substrate is added to each well and allowed to develop for 30 minutes. To stop the reaction, 50 μl of 1 M HCl is added to each well. Absorbance of the plate at 450 nm is read to quantify binding. To quantify the reduction of CnaA binding to collagen in the presence of NBX, a percent reduction is calculated relative to the binding of CnaA in the absence of NBX (100% binding).
Table 4 indicates, for all NBXs tested, whether the NBX can reduce binding of CnaA to collagen by more than 50% when the NBX is supplied at 2 μM and/or at 200 nM.
Cpa is mixed with NBX or PBS to achieve a final concentration of 100 nM (Cpa) and 1 uM (NBX) in a total store-bought, free-range eggs by separation from the white. The yolk is punctured carefully then 5 ml is removed and mixed thoroughly with 45 ml PBS to create a 10% solution. The solution is centrifuged at 500 g to remove large aggregates and then passed through a 0.45 um GD/X syringe filter. 60 ul of the filtered yolk solution is added to the Cpa or Cpa/NBX wells to achieve a final concentration of 5% v/v egg yolk. The plate is incubated for 1 hr at 37° C. after which the optical density of the plate is measured at 620 nm. NBX neutralization of Cpa lecithinase activity is determined relative to Cpa lecithinase activity in the absence of NBX (100%).
Table 5 indicates, for all NBXs tested, whether the NBX can reduce Cpa lecithinase activity by more than 40% when the NBX is supplied at 1 μM.
In a 96-well microtiter plate, 2 μg of collagen is incubated in 100 μl of PBS per well overnight at 4° C. The plate is washed with 200 μl of PBS and then blocked with 200 μl of 5% skim milk in PBS for 2 hours at 37° C. During the blocking step, 100 nM of 6×-Histidine (SEQ ID NO: 695) and Maltose-binding-protein (MBP) tagged CnaA is mixed with between 0 and 2000 nM untagged CnaA in PBS for 30 minutes at 37° C. The plate is washed with 200 μl of PBS three times, and 100 μl of MBP-CnaA or MBP-CnaA/untagged CnaA mixture is added to each well for a 2-hour incubation at 37° C. After washing with 200 μl of PBS three times, 100 μl of 0.125 μg/ml of anti-His conjugated with HRP is added to each well and incubated for 1 hour at room temperature. The plate is then washed with 200 μl of PBS three times, and 100 μl of TMB substrate is added to each well and allowed to develop for 30 minutes. To stop the reaction, 50 μl of 1 M HCl is added to each well. Absorbance of the plate at 450 nm is read to quantify binding.
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. 62/694,164, filed Jul. 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.
| Number | Date | Country | |
|---|---|---|---|
| 62694164 | Jul 2018 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/IB2019/001198 | Jul 2019 | US |
| Child | 17141052 | US |
