WO2023205876A1 - Anticorps dirigés contre des agents provoquant une maladie chez des plantes et leurs utilisations - Google Patents
Anticorps dirigés contre des agents provoquant une maladie chez des plantes et leurs utilisations Download PDFInfo
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
- A01N63/10—Animals; Substances produced thereby or obtained therefrom
- A01N63/14—Insects
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
- A01N63/20—Bacteria; Substances produced thereby or obtained therefrom
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
- A01N63/20—Bacteria; Substances produced thereby or obtained therefrom
- A01N63/22—Bacillus
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
- A01N63/30—Microbial fungi; Substances produced thereby or obtained therefrom
- A01N63/32—Yeast
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
- A01N63/50—Isolated enzymes; Isolated proteins
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01P—BIOCIDAL, PEST REPELLANT, PEST ATTRACTANT OR PLANT GROWTH REGULATORY ACTIVITY OF CHEMICAL COMPOUNDS OR PREPARATIONS
- A01P1/00—Disinfectants; Antimicrobial compounds or mixtures thereof
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/12—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria
- C07K16/1203—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-negative bacteria
- C07K16/1228—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-negative bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
- C07K16/1235—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-negative bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia from Salmonella (G)
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/12—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria
- C07K16/1267—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-positive bacteria
- C07K16/1282—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-positive bacteria from Clostridium (G)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
- C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
- C12N15/815—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts for yeasts other than Saccharomyces
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
- C07K2317/22—Immunoglobulins specific features characterized by taxonomic origin from camelids, e.g. camel, llama or dromedary
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
- C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
- C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
Definitions
- the present technology relates to methods and compositions for the control of microorganisms associated with infections in plant xylem and uses thereof.
- Xylella fastidiosa is one of the leading vascular pathogens in economically important perennial plants. This bacterium causes such diseases as Pierce’s Disease in grapes, Almond Leaf Scorch in almonds, and Olive Quick Decline Syndrome in olives (Rapicavoli et al, 2018). Bacteria require an insect vector for transport from diseased to healthy plants (Chatterjee et al, 2008). Diseased plants typically show symptoms of scorched leaves and leaf necrosis at the margins (Rapicavoli et al, 2018). Disease control relies on chemical treatment of insect vectors that propagate Xylella fastidiosa between plants (Kyrkou et al, 2018) or the destruction, through uprooting or burning, of diseased plants (Bucci, 2018).
- VHHS heavy chain variable region fragments
- a polypeptide comprising heavy chain variable region fragments whose intended use includes but is not limited to the following applications in plant health or an unrelated field: diagnostics, in vitro assays, injectables, formulations, prophylactics, therapeutics, substrate identification, nutritional supplementation, bioscientific and medical research, and companion diagnostics.
- polypeptides comprising VHHS that bind and decrease the virulence of disease-causing agents in plants.
- set out below are the uses of polypeptides that comprise VHHS in methods of reducing transmission and severity of disease in host plants, including their use as an ingredient in a product. Further described are the means to produce, characterise, refine, and modify VHHS for this purpose.
- FIG. 1 Shows the structure of the exopolysaccharide (EPS) produced by Xylella fastidiosa (Fastidian Gum) and described in figure 3 of da Silva et al (2001 ).
- EPS exopolysaccharide
- FIG. 2 Shows a schematic of camelid heavy chain only antibodies and their relationship to VHH domains and complementarity determining regions (CDRs).
- FIG. 3 Shows phage ELISA binding data for VHH antibodies of this disclosure.
- FIG. 4 Shows functional assays that can be used to assess the activity of VHH antibodies after passage through plants.
- FIG. 5 Shows that NBX0018 (SEQ ID NO: 115) maintains functional activity after passage through the vascular system of olive, calamansi, fig, orange, almond, and grape plants.
- FIG. 6 Shows that NBX18029 (SEQ ID NO: 118), NBX12006 (SEQ ID NO: 116), NBX0880 (SEQ ID NO: 119), and NBX15016 (SEQ ID NO: 117) maintain binding active after passage through the vascular system of olive plants.
- FIG. 7 Shows that NBX18029 (SEQ ID NO: 118), NBX0880 (SEQ ID NO: 119), and NBX15016 (SEQ ID NO: 117) maintain binding active after passage through the vascular system of grape and almond plants.
- FIG. 8 Shows that NBX18029 (SEQ ID NO: 118), NBX0880 (SEQ ID NO: 119), and NBX15016 (SEQ ID NO: 117) maintain binding activity after passage through the vascular system of orange plants.
- FIG. 9 Shows that NBX18029 (SEQ ID NO: 118), NBX0880 (SEQ ID NO: 119), and NBX15016 (SEQ ID NO: 117) maintain binding activity after passage through the vascular system of fig plants.
- FIG. 10 Shows that NBX18029 (SEQ ID NO: 118), NBX0880 (SEQ ID NO: 119), and NBX15016 (SEQ ID NO: 117) maintain binding activity after passage through the vascular system of calamansi plants.
- FIG. 11 Shows the presence of NBX18029 (SEQ ID NO: 118), NBX0880 (SEQ ID NO: 119), and NBX15016 (SEQ ID NO: 117) in grape and almond plant extracts and the presence of NBX0018 (SEQ ID NO: 115) in almond, grape, and orange plant extracts.
- host refers to the intended recipient of the product when the product constitutes a prophylactic or a therapeutic.
- the host is from the order Vitales.
- the host is from the family Vitaceae.
- the host is from the genus Vitis.
- the host is a grape plant.
- the host is from the order Rosales.
- the host is from the family Rosaceae.
- the host is from the genus Prunus.
- the host is an almond, plum or peach plant.
- the host is from the genus Ficus. In certain embodiments, the host is a fig plant. In certain embodiments, the host is from the order Lamiales. In certain embodiments, the host is from the family Oleaceae. In certain embodiments, the host is from the genus Olea. In certain embodiments, the host is an olive plant. In certain embodiments, the host is from the order Sapindales. In certain embodiments, the host is from the family Rutaceae. In certain embodiments, the host is from the genus Citrus. In certain embodiments, the host is a citrus plant. In certain embodiments, the host is from the order Fabales. In certain embodiments, the host is from the family Fabaceae.
- the host is from the genus Medicago. In certain embodiments, the host is an alfalfa plant. In certain embodiments, the host is from the order Gentianales. In certain embodiments, the host is from the family Apocynaceae. In certain embodiments, the host is from the genus Vinca. In certain embodiments, the host is a periwinkle plant. In certain embodiments, the host is from the order Gentianales. In certain embodiments, the host is from the family Rubiaceae. In certain embodiments, the host is from the genus Coffea. In certain embodiments, the host is a coffee plant. In certain embodiments, the host is from the order Gentianales.
- the host is from the family Apocynaceae. In certain embodiments, the host is from the genus Nerium. In certain embodiments, the host is an oleander plant. In certain embodiments, the host is from the order Fagales. In certain embodiments, the host is from the family Juglandaceae. In certain embodiments, the host is from the genus Carya. In certain embodiments, the host is a pecan plant. In certain embodiments, the host is from the order Ericales. In certain embodiments, the host is from the family Ericaceae. In certain embodiments, the host is from the genus Vaccinium. In certain embodiments, the host is a berry-producing plant.
- the host is from the order Hemiptera. In certain embodiments, the host is from the family Cicadellidae. In certain embodiments, the host is a leafhopper. In certain embodiments, the host is a sharpshooter. In certain embodiments, the host is from the order Hemiptera. In certain embodiments, the host is from the family Aphrophoridae. In certain embodiments, the host is a spittlebug.
- pathogen refers 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, or reproduce.
- pathogens encompass parasites, bacteria, viruses, prions, protists, fungi, and algae.
- the pathogen is a bacterium belonging to the order Xanthomonadales.
- the pathogen is a bacterium belonging to the family Xanthomonadaceae.
- the pathogen is a bacterium belonging to the genus Xylella. In certain embodiments, the pathogen is Xylella fastidiosa. In certain embodiments, the pathogen is Xylella fastidiosa subspecies fastidiosa. In certain embodiments, the pathogen is Xylella fastidiosa subspecies multiplex. In certain embodiments, the pathogen is Xylella fastidiosa subspecies pauca. [0021] “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.
- bacteria refers, without limitation, to bacteria of the Xylella genus, or any other bacterial species associated with host organisms. In certain embodiments, a bacterium may not be virulent in all host organisms it is associated with.
- exopolysaccharide As referred to herein, “exopolysaccharide”, “EPS”, “fastidian gum”, “gum” and variations thereof refer, without limitation, to the sugar-containing, high molecular weight polymers synthesized by Xylella fastidosa, or any other bacteria.
- FIG. 2 A schematic of camelid heavy chain only antibodies and their relationship to VHH domains and complementarity determining regions (CDRs) is shown in FIG. 2.
- 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).
- VHH variable immunoglobulin domain
- Each VHH domain contains an amino acid sequence of approximately 110-130 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).
- N N-terminus
- 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.
- 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.
- a VHH may lack a portion of a heavy chain constant region (CH2 or CH3), or an entire heavy chain constant region.
- 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.
- binding 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.
- CDR complementarity determining region
- 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).
- IgNAR immunoglobulin new antigen receptor
- 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 are considered to have originated from that species.
- 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.
- the antibodies, compositions, therapeutic formulations, prophylactic formulations, products, and methods described herein do not utilize conventional antibodies.
- 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.
- 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 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 formulation, a formulation additive, a nutritional supplement, a premix, a medicine, a prophylactic, a spray, a therapeutic, an injectable, 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), a component of a delivery system, or an immunomodulatory molecule
- formulation 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 formulation, a formulation additive, a nutritional supplement, a premix, a medicine, a prophylactic, a therapeutic, a spray, an injectable, or a drug.
- vascular pathogens include bacteria, such as members of the Xylella genus and fungi. From this group, one organism of particular interest is the bacterium Xylella fastidiosa which causes Pierce’s Disease in grapes, Almond Leaf Scorch in almonds, and Olive Quick Decline Syndrome in olives (Rapicavoli et al, 2018). Together, these diseases cause annual losses in the 100s of millions of dollars to producers.
- Xylella fastidiosa Other plant diseases caused by Xylella fastidiosa include citrus variegated chlorosis, alfalfa dwarf, periwinkle wilt, peach phony disease and leaf scorch in olives, plums, coffee, oleander, pecan, and vaccinium berry plants.
- Xylella fastidiosa The lifecycle of Xylella fastidiosa within the plant is a complex one (Bucci, 2018). Initially, after Xylella fastidiosa enters a plant xylem, planktonic cells utilize the xylem to spread within the plant. Next the bacteria adhere to host cells and form bacterial cell aggregates known as biofilm. As bacterial numbers increase, they begin producing enzymes that degrade host cell walls and expand pores between xylem vessels, further allowing the bacteria to spread through the plant. Diseased plants exhibit such symptoms as leaf scorching, necrosis at leaf margins, and berry desiccation (Rapicavoli et al, 2018). To spread between plants, Xylella fastidiosa requires an insect vector.
- Xylem sap- feeding insects such as leafhoppers and sharpshooters, ingest bacteria from the xylem of an infected plant and then directly inoculate the xylem of healthy plants with the bacteria (Chatterjee et al, 2008).
- Xylella fastidiosa proteins that have been implicated in nutrient acquisition or host cell wall degradation include ChiA (Labroussaa et al, 2017), LesA (Nascimento et al, 2016), and PglA (Roper et al, 2007).
- the type 1 fimbriae (Li et al, 2007; Feil et al, 2007) and the autotransporter XatA (Matsumoto et al, 2012) of Xylella fastidiosa are both involved in virulence through roles in cell-cell aggregation and biofilm formation.
- exopolysaccharide (EPS) produced by Xylella fastidiosa contributes to virulence by facilitating bacterial adhesion to surfaces and biofilm formation (Killiny et al, 2013).
- EPS exopolysaccharide
- TolC of Xylella fastidiosa is likely playing a role in virulence through both the secretion of enzymes necessary for virulence and the removal of toxic phytochemicals from the bacteria (Reddy etal, 2007). Genetic disruption of any of the systems described above gives rise to Xylella fastidiosa isolates with significantly decreased capacity to cause disease in plants.
- Some of these virulence factors include ChiA (Labroussaa et al, 2017), PglA (Killiny and Almeida, 2014), EPS (Killiny et al, 2013), also contribute to vector transmission of Xylella fastidiosa between plants.
- VHHS antibody heavy chain variable region fragments
- IgYs IgYs
- HHS introduced into plants can be used to deliver other molecules for specific pathogen elimination.
- VHHS may also modulate plant immunity.
- Antibodies for preventing or reducing virulence (summary)
- 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.
- the VHH is supplied to host plants.
- the VHH is an ingredient of a product.
- 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.
- binding of the VHH(S) to the disease-causing agent reduces the rate of replication of the disease-causing agent.
- binding of the VHH(S) to the disease-causing agent reduces the ability of the disease-causing agent to bind to its cognate receptor.
- binding of the VHH(S) to the disease-causing agent reduces the ability of the disease-causing agent to degrade components of the host organism.
- 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 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.
- the present technology provides a polypeptide or pluralities thereof comprising a VHH or VHHS that bind disease-causing agents and can be bound to another molecule.
- the VHH or VHHS deliver the other molecule to a specific pathogen.
- the other molecule can eliminate the specific pathogen targeted by the VHH or VHHS.
- 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.
- the camelid can be a dromedary, camel, llama, alpaca, vicuna, or guanaco, without limitation.
- 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.
- the heavy chain antibody comprises a sequence set forth in Table 1 .
- 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.
- the heavy chain antibody possesses a CDR1 set forth in Table 2.
- the heavy chain antibody possesses a CDR2 set forth in Table 2.
- the heavy chain antibody possesses a CDR3 set forth in Table 2.
- the present technology provides a method for producing VHH in a suitable producing organism.
- suitable producing organisms include, without limitation, bacteria, yeast, and algae.
- the producing bacterium is Escherichia coli.
- the producing bacterium is a member of the Bacillus genus.
- the producing bacterium is a probiotic.
- the yeast is Pichia pastoris.
- the yeast is Saccharomyces cerevisiae.
- the yeast is Yarrowia lipolytica.
- the alga is a member of the Chlamydomonas or Phaeodactylum genera.
- the present technology provides a polypeptide or pluralities thereof comprising a VHH or HHS that bind disease-causing agents and are administered to host plants via any suitable route as a therapeutic.
- the plant is selected from the list of host plant described, with that list being representative but not limiting.
- the route of administration to a recipient plant can be, but is not limited to: inclusion in any part of the plant, introduction to the xylem, introduction to the phloem, provided to the exterior surface (for example, as a spray or submersion), provided to the medium in which the plant dwells, provided by injection, provided via diffusion, provided via absorption by roots, provided genetically as a genetically-modified plant, or provided via a secondary organism such as a yeast, bacterium, algae, bacteriophages, plants and insects.
- the route of administration to a recipient insect can be but is not limited to: inclusion in any part of the insect, provided by insect feed, provided by insect traps, or provided via a secondary organism such as a yeast, bacterium, algae, bacteriophages, plants and insects.
- the VHH or VHHS deliver a secondary molecule to the pathogen.
- the VHH or VHHS modulate the immune system of the host.
- the host is from the order Vitales.
- the host is from the family Vitaceae.
- the host is from the genus Vitis.
- the host is a grape plant.
- the host is from the order Rosales.
- the host is from the family Rosaceae. In certain embodiments, the host is from the genus Prunus. In certain embodiments, the host is an almond, plum or peach plant. In certain embodiments, the host is from the genus Ficus. In certain embodiments, the host is a fig plant. In certain embodiments, the host is from the order Lamiales. In certain embodiments, the host is from the family Oleaceae. In certain embodiments, the host is from the genus Olea. In certain embodiments, the host is an olive plant. In certain embodiments, the host is from the order Sapindales. In certain embodiments, the host is from the family Rutaceae. In certain embodiments, the host is from the genus Citrus.
- the host is a citrus plant. In certain embodiments, the host is from the order Fabales. In certain embodiments, the host is from the family Fabaceae. In certain embodiments, the host is from the genus Medicago. In certain embodiments, the host is an alfalfa plant. In certain embodiments, the host is from the order Gentianales. In certain embodiments, the host is from the family Apocynaceae. In certain embodiments, the host is from the genus Vinca. In certain embodiments, the host is a periwinkle plant. In certain embodiments, the host is from the order Gentianales. In certain embodiments, the host is from the family Rubiaceae.
- the host is from the genus Coffea. In certain embodiments, the host is a coffee plant. In certain embodiments, the host is from the order Gentianales. In certain embodiments, the host is from the family Apocynaceae. In certain embodiments, the host is from the genus Nerium. In certain embodiments, the host is an oleander plant. In certain embodiments, the host is from the order Fagales. In certain embodiments, the host is from the family Juglandaceae. In certain embodiments, the host is from the genus Carya. In certain embodiments, the host is a pecan plant. In certain embodiments, the host is from the order Ericales. In certain embodiments, the host is from the family Ericaceae.
- the host is from the genus Vaccinium. In certain embodiments, the host is a berry-producing plant. In certain embodiments, the host is from the order Hemiptera. In certain embodiments, the host is from the family Cicadellidae. In certain embodiments, the host is a leafhopper. In certain embodiments, the host is a sharpshooter. In certain embodiments, the host is from the order Hemiptera. In certain embodiments, the host is from the family Aphrophoridae. In certain embodiments, the host is a spittlebug.
- the present technology provides a polypeptide or pluralities thereof comprising a VHH or VHHS that bind disease-causing agents to host plants as part of a product at any suitable dosage regime.
- the suitable dosage is the dosage at which the product offers any degree of protection against a diseasecausing agent, and depends on the delivery method, delivery schedule, the environment of the recipient plant, the size of the recipient plant, the age of the recipient plant and the health condition of the recipient plant among other factors.
- VHHS are administered to recipient plants at a concentration in excess of 1 mg/kg of plant weight. In certain embodiments, VHHS are administered to recipient plants at a concentration in excess of 5 mg/kg of plant weight.
- VHHS are administered to recipient plants at a concentration in excess of 10 mg/kg of plant weight. In certain embodiments, VHHS are administered to recipient plants at a concentration in excess of 50 mg/kg of plant weight. In certain embodiments, VHHS are administered to recipient plants at a concentration in excess of 100 mg/kg of plant weight. In certain embodiments, VHHS are administered to recipient plants at a concentration less than 1 mg/kg of plant weight. In certain embodiments, VHHS are administered to recipient plants at a concentration less than 500 mg/kg of plant weight. In certain embodiments, VHHS are administered to recipient plants at a concentration less than 100 mg/kg of plant weight. In certain embodiments, VHHS are administered to recipient plants at a concentration less than 50 mg/kg of plant weight.
- VHHS are administered to recipient plants at a concentration less than 10 mg/kg of plant weight. In certain embodiments, VHHS are administered to fields at a concentration in excess of 1 kg/ha. In certain embodiments, VHHS are administered to fields at a concentration in excess of 5 kg/ha. In certain embodiments, VHHS are administered to fields at a concentration in excess of 10 kg/ha. In certain embodiments, VHHS are administered to fields at a concentration in excess of 50 kg/ha. In certain embodiments, VHHS are administered to fields at a concentration in excess of 100 kg/ha. In certain embodiments, VHHS are administered to fields at a concentration less than 1 kg/ha.
- the present technology provides a polypeptide or pluralities thereof comprising a VHH or VHHS that bind disease-causing agents and are administered to host plants as part of a product at any suitable dosage frequency.
- 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 plant, the size of the recipient plant, the age of the recipient plant and the health condition of the recipient plant, among other factors.
- 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.
- the present technology provides a polypeptide or pluralities thereof comprising a VHH or VHHS that bind disease-causing agents and are administered to host plants via any suitable route as part of a prophylactic or therapeutic product.
- the plant is selected from the list of host plants described, with that list being representative but not limiting.
- the route of administration to a recipient plant can be, but is not limited to: inclusion in any part of the plant, introduction to the xylem, introduction to the phloem, provided to the exterior surface (for example, as a spray or submersion), provided to the medium in which the plant dwells, provided by injection, provided via diffusion, provided via absorption by roots, provided genetically as a genetically-modified plant, or provided via a secondary organism such as a yeast, bacterium, algae, bacteriophages, plants and insects.
- the route of administration to a recipient insect can be, but is not limited to: inclusion in any part of the insect, provided by insect feed, provided by insect traps, or provided via a secondary organism such as a yeast, bacterium, algae, bacteriophages, plants and insects.
- the host is from the order Vitales.
- the host is from the family Vitaceae.
- the host is from the genus Vitis.
- the host is a grape plant.
- the host is from the order Rosales.
- the host is from the family Rosaceae.
- the host is from the genus Prunus.
- the host is an almond, plum or peach plant. In certain embodiments, the host is from the genus Ficus. In certain embodiments, the host is a fig plant. In certain embodiments, the host is from the order Lamiales. In certain embodiments, the host is from the family Oleaceae. In certain embodiments, the host is from the genus Olea. In certain embodiments, the host is an olive plant. In certain embodiments, the host is from the order Sapindales. In certain embodiments, the host is from the family Rutaceae. In certain embodiments, the host is from the genus Citrus. In certain embodiments, the host is a citrus plant. In certain embodiments, the host is from the order Fabales.
- the host is from the family Fabaceae. In certain embodiments, the host is from the genus Medicago. In certain embodiments, the host is an alfalfa plant. In certain embodiments, the host is from the order Gentianales. In certain embodiments, the host is from the family Apocynaceae. In certain embodiments, the host is from the genus Vinca. In certain embodiments, the host is a periwinkle plant. In certain embodiments, the host is from the order Gentianales. In certain embodiments, the host is from the family Rubiaceae. In certain embodiments, the host is from the genus Coffea. In certain embodiments, the host is a coffee plant.
- the host is from the order Gentianales. In certain embodiments, the host is from the family Apocynaceae. In certain embodiments, the host is from the genus Nerium. In certain embodiments, the host is an oleander plant. In certain embodiments, the host is from the order Fagales. In certain embodiments, the host is from the family Juglandaceae. In certain embodiments, the host is from the genus Carya. In certain embodiments, the host is a pecan plant In certain embodiments, the host is from the order Ericales. In certain embodiments, the host is from the family Ericaceae. In certain embodiments, the host is from the genus Vaccinium. In certain embodiments, the host is a berry-producing plant.
- the host is from the order Hemiptera. In certain embodiments, the host is from the family Cicadellidae. In certain embodiments, the host is a leafhopper. In certain embodiments, the host is a sharpshooter. In certain embodiments, the host is from the order Hemiptera. In certain embodiments, the host is from the family Aphrophoridae. In certain embodiments, the host is a spittlebug.
- the present technology provides a polypeptide or pluralities thereof comprising a VHH or HHS that bind disease-causing agents and are administered to host plants in the form of a product.
- the form of the product is not limited.
- the product is a formulation, nutritional supplement, premix, prophylactic, therapeutic, spray, injectable, medicine, formulation additive, or feed but is not limited to these forms.
- the present technology provides a polypeptide or pluralities thereof comprising a VHH or HHS that bind disease-causing agents and are administered to host plants as part of a product that also comprises other additives or coatings.
- the most suitable coating or additive depends on the method of delivery, the recipient plant, the environment of the recipient, the nutritional requirements of the recipient plant, the frequency of delivery, the age of the recipient plant, the size of the recipient plant, or the health condition of the recipient plant.
- these additives and coatings can include but are not limited to the following list and mixtures thereof: a vitamin, an amino acid, a dye, an antibiotic, an antiviral, a hormone, an antimicrobial peptide, a steroid, a prebiotic, a probiotic, a bacteriophage, chitin, chitosan, B-1 ,3-glucan, vegetable extracts, peptone, an additive spray, a toxin binder, a yeast extract, a plant extract, sugar, a detoxifying enzyme, an enzyme inhibitor, an essential mineral, carnitine, glucosamine, an essential salt, a preservative, a stabilizer, a pesticide, a herbicide, or water.
- 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-prophylactic or non-therapeutic use, such as but not limited to: a diagnostic kit, an enzyme-linked immunoabsorbent 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.
- the conjugated molecule can be but is not limited to: a fluorophore, a chemiluminescent substrate, an antimicrobial peptide, a nucleic acid, or a lipid.
- the present technology provides a polypeptide or pluralities thereof comprising a VHH orVnHs that bind disease-causing agents, produced by a bacterium of the order Xanthomonadales.
- the Xanthomonadales bacterium refers to both current and reclassified bacteria.
- the pathogen is a bacterium belonging to the Xanthomonadaceae family.
- the pathogen is a bacterium belonging to the genus Xylella.
- the pathogen is Xylella fastidiosa.
- the VHH or plurality thereof is capable of binding to one or more disease-causing agents, originating from the same or different bacteria.
- the disease-causing agent is a polypeptide with 80% or greater amino acid sequence identity to Xylella fastidiosa protein ChiA (SEQ ID NO: 109).
- the disease-causing agent is a polypeptide with 80% or greater amino acid sequence identity to Xylella fastidiosa protein LesA (SEQ ID NO: 110).
- the disease-causing agent is a polypeptide with 80% or greater amino acid sequence identity to Xylella fastidiosa protein PglA (SEQ ID NO: 111 ).
- the disease-causing agent is a polypeptide with 80% or greater amino acid sequence identity to Xylella fastidiosa protein FimF (SEQ ID NO: 112). In certain embodiments, the disease-causing agent is a polypeptide with 80% or greater amino acid sequence identity to Xylella fastidiosa protein XatA (SEQ ID NO: 113). In certain embodiments, the disease-causing agent is a polypeptide with 80% or greater amino acid sequence identity to Xylella fastidiosa protein TolC (SEQ ID NO: 114). In certain embodiments, the disease-causing agent is the exopolysaccharide produced by Xylella fastidiosa (FIG. 1 ).
- the preferred antigens are ChiA (SEQ ID NO: 109), LesA (SEQ ID NO: 10), and PglA (SEQ ID NO: 111 ) as their recombinant expression and purification is anticipated to be simpler than other antigens.
- the disease-causing agent is a molecule secreted by Xylella fastidiosa.
- the disease-causing agent is a surface associated molecule of Xylella fastidiosa.
- the disease-causing agent is a molecule in the outer membrane of Xylella fastidiosa.
- Recombinant antigens can be purified from an E. coli expression system.
- a 6xHis-tagged ChiA was expressed at 30°C in E. coli BL21 (DE3) cells grown overnight in LB media with 0.5 mM IPTG induction at an optical density at 600 nM of 0.8. Cells were then lysed by sonication in buffer A (250 mM NaCI, 50 mM CaCL, 20 mM Imidazole and 10 mM HEPES, pH 7.5) with 12.5 pg/ml DNase I, and 1 mM PMSF.
- buffer A 250 mM NaCI, 50 mM CaCL, 20 mM Imidazole and 10 mM HEPES, pH 7.5
- the lysate was 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 NaCI, 500 mM Imidazole and 10 mM HEPES, pH 7.5). The protein was then dialyzed overnight into buffer C (10 mM Tris, pH 8.8, 5 mM NaCI) at 4°C. The dialyzed protein was applied to a HiTrap HP ion exchange column (GE Biosciences) preequilibrated with buffer C.
- buffer C 10 mM Tris, pH 8.8, 5 mM NaCI
- the protein was eluted with a gradient of 0% to 50% (vol/vol) buffer D (10 mM Tris, pH 8.8, 1 .0 M NaCI). Lastly, the eluate was loaded onto a Sephacryl S100 gel filtration column (GE Healthcare) using PBS buffer. The protein sample was then concentrated to 1 mg/mL using Am icon concentrators with appropriate molecular weight cut-off (MWCO; Millipore). The purified protein was stored at -80°C.
- a single llama was immunized with purified disease-causing agents, such as the antigens listed, which may be accompanied by adjuvants.
- the llama immunization was performed using 100 pg of each antigen that were pooled and injected. At the time of injection, the antigens were thawed, and the volume increased to 1 ml with PBS. The 1 ml antigen-PBS mixture was 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 was immunized per injection.
- CFA Complete Freund’s adjuvant
- IFA Incomplete Freund’s adjuvant
- the immunization was done five times (days 0, 21 , 42, 63, and 70).
- Whole llama blood and sera were collected from the immunized animal on days 0, 28, 49, 84.
- Sera from days 28, 49 and 84 were fractionated to separate VHH from conventional antibodies.
- ELISA were be used to measure reactivity against target antigens in polyclonal and VHH-enriched fractions.
- Lymphocytes were collected from sera taken at days 28, 49, and 84.
- RNA isolated from purified llama lymphocytes was used to generate cDNA for cloning into phagemids.
- the resulting phagemids were used to transform E. coh TG- 1 cells to generate a library of expressed VHH genes.
- the phagemid library size was ⁇ 2.5 x 10 7 total transformants and the estimated number of phagemid containing VHH inserts was >90%.
- High affinity antibodies were then selected by panning against the antigens used for llama immunization. Three rounds of panning were performed and antigen-binding clones arising from round 3 were identified using phage ELISA. Antigenbinding clones were sequenced, grouped according to their CDR sequences, and prioritized for soluble expression in E. coh and antibody purification.
- FIG. 3 shows the phage ELISA results for 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 four times above binding to the PBS-coated wells.
- TEV protease-cleavable, 6xHis-thioredoxin-NBX fusion proteins were expressed in the cytoplasm of E. coh grown in autoinducing media (Formedium) for 24 hours at 30°C. Bacteria were collected by centrifugation, resuspended in buffer A (10 mM HEPES, pH 7.5, 250 mM NaCI, 20 mM Imidazole) and lysed using sonication. Insoluble material was removed by centrifugation and the remaining soluble fraction was 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, 250 mM NaCI, 500 mM Imidazole).
- the eluted protein was dialyzed overnight in the presence of TEV protease to buffer C (10 mM HEPES, pH 7.5, 250 mM NaCI).
- the dialyzed protein was applied to a HisTrap column (GE Biosciences) pre-equilibrated with buffer C. 6xHis-tagged TEV and 6xHis-tagged thioredoxin were bound to the column and highly purified NBX was collected in the flow through.
- NBX proteins were further purified using ion-exchange chromatography.
- NBX 1 proteins were dialyzed overnight to 20 mM HEPES, pH7.4, 150 mM NaCI and concentrated to ⁇ 10 mg/ml.
- Pichia pastoris strain GS115 with constructs for the expression and secretion of 6xHis-tagged VHH were grown for 5 days at 30°C with daily induction of 0.5% (vol/vol) methanol.
- Yeast cells were removed by centrifugation and the NBX- containing supernatant was spiked with 10 mM imidazole. The supernatant was applied to a HisTrap column (GE Biosciences) pre-equilibrated with buffer A (10 mM HEPES, pH 7.5, 250 mM NaCI).
- the protein was eluted from the column using an FPLC with a linear gradient between buffer A and buffer B (10 mM HEPES, pH 7.5, 250 mM NaCI, 500 mM Imidazole).
- buffer A and buffer B 10 mM HEPES, pH 7.5, 250 mM NaCI, 500 mM Imidazole.
- NBX proteins were further purified using ion-exchange chromatography. NBX proteins were dialyzed overnight to 20 mM HEPES, pH7.4, 150 mM NaCI and concentrated to ⁇ 10 mg/ml.
- NBXs can be introduced into the vascular system of a plant and flow to other portions of the plant, where they can be recovered and their activity is maintained.
- NBXs selected for use in these experiments bind to epitopes of antigens of Salmonella enterica serovar Typhimurium.
- Salmonella bacteria can enter plants through roots, stomata, hydathodes, or at points of plant damage and colonize plant tissues, including edible tissues (Karmakar et al, 2018). This allows Salmonella to cause food- borne infections in humans. In the presence of plant pathogenic bacteria, Salmonella may benefit and increase in numbers within plant tissues (George et al, 2018). As such, existing NBXs targeting Salmonella antigens serve as a good surrogate for the study of the flow of NBXs through plant tissues.
- NBX0018 SEQ ID NO: 115
- NBX12006 SEQ ID NO: 116
- NBX18029 SEQ ID NO: 118
- Salmonella ente ca serovar Typhimurium StdD SEQ ID NO: 121
- NBX15016 SEQ ID NO: 117
- NBX0880 SEQ ID NO: 119
- Clostridium perfringens NetB SEQ ID NO: 123
- Salmonella enterica serovar Typhimurium FliC (SEQ ID NO: 120)
- Salmonella enterica serovar Typhimurium StdD (SEQ ID NO: 121 )
- Salmonella enterica serovar Typhimurium SipD (SEQ ID NO: 122)
- NBXs can block the function of the virulence factor they bind in vitro. This can allow for the assessment of NBX function after collection from a plant and is in addition to measuring the binding of each NBX to its target antigen.
- NBX0018 reduces the motility of Salmonella enterica serovar Typhimurium strain SL1344 (FIG. 4A) in an experiment conducted as follows. Overnight cultures of Salmonella enterica serovar Typhimurium strain SL1344 and a non-motile Dflif mutant were used to inoculate subcultures which were grown at 37°C in a shaking incubator until logarithmic growth was reached. Five pl of log phase bacteria were mixed with 10 pl of NBX0018 or PBS. Mixtures were incubated at room temperature for 1 hour and observed at 400x magnification with an Olympus IX70 inverted microscope with an Olympus DP80 camera.
- Salmonella motility was tracked and analyzed using Velocity image analysis software.
- the non-motile Dflif mutant shows low overall motility, while the wild-type bacterium has high overall motility in the presence of PBS.
- NBX0018 reduces the motility of the wild-type strain in a dose-dependent fashion.
- Each point on the graph indicates the average speed of a tracked bacterium, with hundreds of bacteria tracked for each mixture. The lines indicate the average movement speed of each mixture.
- NBX15016 reduces the invasiveness of Salmonella enterica serovar Typhimurium into tissue-cultured HeLa cells (FIG. 4B) in an experiment conducted as follows. Overnight cultures of Salmonella enterica serovar Typhimurium strain SL1344 and a non-invasive DsipD mutant were used to inoculate subcultures which were grown at 37°C in a shaking incubator until logarithmic growth was reached. Bacterial cultures were incubated for 30 minutes with NBX15016 or PBS and then the mixtures were applied to HeLa cells for one hour. Non-invaded bacterial cells were removed through washing and treatment with gentamicin.
- NBX0880 prevents the NetB toxin (SEQ ID NO: 123) from killing tissue-cultured LMH cells (FIG. 4C) in an experiment conducted as follows. Purified NetB protein was mixed with NBX0880 at a range of concentrations for 30 minutes and then the mixtures were applied to LMH cells for five hours. After five hours, lysis of the LMH cells was assessed using a commercial LDH cytotoxicity kit (ProMega). Lysis by NetB in the presence of NBX0880 was scaled relative to the amount of NetB- induced lysis in the absence of NBX0880. As indicated in the figure, NBX0880 reduces NetB toxicity in a dose-dependent fashion.
- NBX0018 SEQ ID NO: 115
- PBS olive plant
- the NBX solution was injected at a rate of 0.4 mL/hour for 5.5 hours.
- water droplets from leaves were collected (referred to as leaf sweat below).
- the leaf sweat was applied to a Salmonella enterica motility assay as follows. Overnight cultures of Salmonella enterica serovar Typhimurium strain SL1344 and a non-motile Dflif mutant were used to inoculate subcultures which were grown at 37°C in a shaking incubator until logarithmic growth was reached.
- FIG. 5B Similar results (FIG. 5B) were obtained when NBX0018 was injected into calamansi (Citrus x microcarpa), fig (Ficus carica), and orange (Citrus x sinensis) plants and collected from leaf water droplets. Similar results (FIG. 5B) were also obtained when NBX0018 was injected into the stems of almond (Prunus amygdalus) and grape (Vitis vinifera) plants and material was collected from the opposite end of the stem.
- leaf sweat After injection, water droplets from leaves were collected (referred to as leaf sweat below) and leaves were collected and ground in an extraction buffer (20 mM Tris-HCI pH7.4, 150 mM NaCI, 2.6 mM KOI, 2% PVP40, 0.05% Tween- 20) at a ratio of 1 g of leaves to 5 ml of buffer (referred to as leaf extract below).
- the leaf extracts were clarified by centrifugation and the supernatants were used in the ELISA as follows.
- ELISA plate was coated with 100 ml of 1 -10 mg/ml antigens overnight, and then blocked with 5% skim milk in PBS+0.05% Tween-20 (PBST).
- each leaf sweat sample, leaf extract sample or standard curve sample was added to ELISA plate and detected with 1 :5000 dilution of an anti-VHH-HRP antibody (GenScript, A02014-200).
- 100 ml of TMB substrate (Abeam, ab171523) was added to the plate and incubated for 30 minutes, followed by the addition of 50 ml of 1 M HCI to stop the reaction. Absorbance at 450 nm was measured.
- FIG. 6 Data for this example are shown in FIG. 6. Standard curves using known concentrations of NBX protein purified from E. coli with wells coated with the known antigens were produced. The standard curves are shown in panel A, which shows the binding of NBX18029 (SEQ ID NO: 118) to StdD (SEQ ID NO: 121 ), panel C, which shows the binding of NBX12006 (SEQ ID NO: 116) to StdD (SEQ ID NO: 121 ), panel E, which shows the binding of NBX0880 (SEQ ID NO: 119) to NetB (SEQ ID NO: 123), and panel F, which shows the binding of NBX15016 (SEQ ID NO: 117) to SipD (SEQ ID NO: 122).
- Panel B shows that after injection of NBX18029 (SEQ ID NO: 118) into an olive plant and collection of leaf sweat and leaf extract, NBX18029 (SEQ ID NO: 118) binds to wells (+Ag) coated with StdD (SEQ ID NO: 121 ) but not to uncoated wells (-Ag).
- Panel D shows that after injection of NBX12006 (SEQ ID NO: 116) into an olive plant and collection of leaf sweat and leaf extract, NBX12006 (SEQ ID NO: 116) binds to wells (+Ag) coated with StdD (SEQ ID NO: 121 ) but not to uncoated wells (-Ag).
- Panel F shows that after injection of NBX0880 (SEQ ID NO: 119) into an olive plant and collection of leaf sweat and leaf extract, NBX0880 (SEQ ID NO: 119) binds to wells (+Ag) coated with NetB (SEQ ID NO: 123) but not to uncoated wells (-Ag).
- Panel G shows that after injection of NBX15016 (SEQ ID NO: 117) into an olive plant and collection of leaf sweat and leaf extract, NBX15016 (SEQ ID NO: 117) binds to wells (+Ag) coated with SipD (SEQ ID NO: 122) but not to uncoated wells (-Ag).
- ELISA plate was coated with 100 ml of 1-10 mg/ml antigens overnight, and then blocked with 5% skim milk in PBS+0.05% Tween-20 (PBST). 100 ml of each plant sample or standard curve sample was added to ELISA plate and detected with 1 :5000 dilution of an anti-VHH-HRP antibody (GenScript, A02014-200). 100 ml of TMB substrate (Abeam, ab171523) was added to the plate and incubated for 30 minutes, followed by the addition of 50 ml of 1 M HCI to stop the reaction. Absorbance at 450 nm was measured.
- FIG. 7 Data for this example are shown in FIG. 7. Standard curves using known concentrations of NBX protein purified from E. coli with wells coated with the known antigens were produced. The standard curves are shown in panel A, which shows the binding of NBX18029 (SEQ ID NO: 118) to StdD (SEQ ID NO: 121 ), panel C, which shows the binding of NBX0880 (SEQ ID NO: 119) to NetB (SEQ ID NO: 123), and panel E, which shows the binding of NBX15016 (SEQ ID NO: 117) to SipD (SEQ ID NO: 122).
- Panel B shows that after injection of the NBX mixture to both almond (Prunus amygdalus) and grape (Vitis vinifera) stems and collection of material from the opposite end of the stems, NBX18029 (SEQ ID NO: 118) binds to wells (+Ag) coated with StdD (SEQ ID NO: 121 ) but not to uncoated wells (-Ag).
- Panel D shows that after injection of the NBX mixture to both almonds and grape stems and collection of material from the opposite end of the stems, NBX0880 (SEQ ID NO: 119) binds to wells (+Ag) coated with NetB (SEQ ID NO: 123) but not to uncoated wells (-Ag).
- Panel F shows that after injection of the NBX mixture to both almonds and grape stems and collection of material from the opposite end of the stems, NBX15016 (SEQ ID NO: 117) binds to wells (+Ag) coated with SipD (SEQ ID NO: 122) but not to uncoated wells (-Ag).
- NBX15016 SEQ ID NO: 117
- SipD SipD
- -Ag uncoated wells
- leaf sweat After injection, water droplets from leaves were collected (referred to as leaf sweat below) and leaves were collected and ground in an extraction buffer (20 mM Tris-HCI pH7.4, 150 mM NaCI, 2.6 mM KCI, 2% PVP40, 0.05% Tween-20) at a ratio of 1 g of leaves to 5 ml of buffer (referred to as leaf extract below).
- the leaf extracts were clarified by centrifugation and the supernatants were used in the ELISA as follows.
- ELISA plate was coated with 100 ml of 1 -10 mg/ml antigens overnight, and then blocked with 5% skim milk in PBS+0.05% Tween-20 (PBST).
- each leaf sweat sample, leaf extract sample or standard curve sample was added to ELISA plate and detected with 1 .5000 dilution of an anti-VHH-HRP antibody (GenScript, A02014-200).
- 100 ml of TMB substrate (Abeam, ab171523) was added to the plate and incubated for 30 minutes, followed by the addition of 50 ml of 1 M HCI to stop the reaction. Absorbance at 450 nm was measured.
- FIG. 8 Data for this example are shown in FIG. 8. Standard curves using known concentrations of NBX protein purified from E. coli with wells coated with the known antigens were produced. The standard curves are shown in panel A, which shows the binding of NBX18029 (SEQ ID NO: 118) to StdD (SEQ ID NO: 121 ), panel C, which shows the binding of NBX0880 (SEQ ID NO: 119) to NetB (SEQ ID NO: 123), and panel E, which shows the binding of NBX15016 (SEQ ID NO: 117) to SipD (SEQ ID NO: 122).
- Panel B shows that after injection of NBX18029 (SEQ ID NO: 118) into an orange plant and collection of leaf sweat and leaf extract, NBX18029 (SEQ ID NO: 118) binds to wells (+Ag) coated with StdD (SEQ ID NO: 121 ) but not to uncoated wells (-Ag).
- Panel D shows that after injection of NBX0880 (SEQ ID NO: 119) into an orange plant and collection of leaf sweat and leaf extract, NBX0880 (SEQ ID NO: 119) binds to wells (+Ag) coated with NetB (SEQ ID NO: 123) but not to uncoated wells (-Ag).
- Panel F shows that after injection of NBX15016 (SEQ ID NO: 117) into an orange plant and collection of leaf sweat and leaf extract, NBX15016 (SEQ ID NO: 117) binds to wells (+Ag) coated with SipD (SEQ ID NO: 122) but not to uncoated wells (-Ag).
- NBX15016 SEQ ID NO: 117
- leaf sweat After injection, water droplets from leaves were collected (referred to as leaf sweat below) and leaves were collected and ground in an extraction buffer (20 mM Tris-HCI pH7.4, 150 mM NaCI, 2.6 mM KOI, 2% PVP40, 0.05% Tween-20) at a ratio of 1 g of leaves to 5 ml of buffer (referred to as leaf extract below).
- the leaf extracts were clarified by centrifugation and the supernatants were used in the ELISA as follows.
- ELISA plate was coated with 100 ml of 1-10 mg/ml antigens overnight, and then blocked with 5% skim milk in PBS+0.05% Tween-20 (PBST).
- each leaf sweat sample, leaf extract sample or standard curve sample was added to ELISA plate and detected with 1 :5000 dilution of an anti- VHH-HRP antibody (GenScript, A02014-200).
- 100 ml of TMB substrate (Abeam, ab171523) was added to the plate and incubated for 30 minutes, followed by the addition of 50 ml of 1 M HCI to stop the reaction. Absorbance at 450 nm was measured.
- FIG. 9 Data for this example are shown in FIG. 9. Standard curves using known concentrations of NBX protein purified from E. coli with wells coated with the known antigens were produced. The standard curves are shown in panel A, which shows the binding of NBX18029 (SEQ ID NO: 118) to StdD (SEQ ID NO: 121 ), panel C, which shows the binding of NBX0880 (SEQ ID NO: 119) to NetB (SEQ ID NO: 123), and panel E, which shows the binding of NBX15016 (SEQ ID NO: 117) to SipD (SEQ ID NO: 122).
- Panel B shows that after injection of NBX18029 (SEQ ID NO: 118) into a fig plant and collection of leaf sweat and leaf extract, NBX18029 (SEQ ID NO: 118) binds to wells (+Ag) coated with StdD (SEQ ID NO: 121 ) but not to uncoated wells (-Ag).
- Panel D shows that after injection of NBX0880 (SEQ ID NO: 119) into a fig plant and collection of leaf sweat and leaf extract, NBX0880 (SEQ ID NO: 119) binds to wells (+Ag) coated with NetB (SEQ ID NO: 123) but not to uncoated wells (-Ag).
- Panel F shows that after injection of NBX15016 (SEQ ID NO: 117) into a fig plant and collection of leaf sweat and leaf extract, NBX15016 (SEQ ID NO: 117) binds to wells (+Ag) coated with SipD (SEQ ID NO: 122) but not to uncoated wells (-Ag).
- NBX15016 SEQ ID NO: 117
- NBX movement through plants a mixture of 30 mM NBX0880 (SEQ ID NO: 119), 30 mM NBX15016 (SEQ ID NO: 117), and 30 mM NBX18029 (SEQ ID NO: 118) diluted in PBS was injected into a calamansi plant (Citrus x microcarpa). The NBX solution was injected at a rate of 0.4 mL/hour for 6 hours.
- leaf sweat After injection, water droplets from leaves were collected (referred to as leaf sweat below) and leaves were collected and ground in an extraction buffer (20 mM Tris-HCI pH7.4, 150 mM NaCI, 2.6 mM KCI, 2% PVP40, 0.05% Tween-20) at a ratio of 1 g of leaves to 5 ml of buffer (referred to as leaf extract below).
- the leaf extracts were clarified by centrifugation and the supernatants were used in the ELISA as follows.
- ELISA plate was coated with 100 ml of 1 -10 mg/ml antigens overnight, and then blocked with 5% skim milk in PBS+0.05% Tween-20 (PBST).
- each leaf sweat sample, leaf extract sample or standard curve sample was added to ELISA plate and detected with 1 :5000 dilution of an anti-VHH-HRP antibody (GenScript, A02014-200).
- 100 ml of TMB substrate (Abeam, ab171523) was added to the plate and incubated for 30 minutes, followed by the addition of 50 ml of 1 M HCI to stop the reaction. Absorbance at 450 nm was measured.
- FIG. 10 Data for this example are shown in FIG. 10. Standard curves using known concentrations of NBX protein purified from E. coli with wells coated with the known antigens were produced. The standard curves are shown in panel A, NBX0880 (SEQ ID NO: 119) to NetB (SEQ ID NO: 123), panel C, which shows the binding of NBX15016 (SEQ ID NO: 117) to SipD (SEQ ID NO: 122), and panel E, which shows the binding of NBX18029 (SEQ ID NO: 118) to StdD (SEQ ID NO: 121 ).
- Panel B shows that after injection of NBX0880 (SEQ ID NO: 119) into a calamansi plant and collection of leaf sweat and leaf extract, NBX0880 (SEQ ID NO: 119) binds to wells (+Ag) coated with NetB (SEQ ID NO: 123) but not to uncoated wells (-Ag).
- Panel D shows that after injection of NBX15016 (SEQ ID NO: 117) into a calamansi plant and collection of leaf sweat and leaf extract, NBX15016 (SEQ ID NO: 117) binds to wells (+Ag) coated with SipD (SEQ ID NO: 122) but not to uncoated wells (-Ag).
- Panel F shows that after injection of NBX18029 (SEQ ID NO: 118) into a calamansi plant and collection of leaf sweat and leaf extract, NBX18029 (SEQ ID NO: 118) binds to wells (+Ag) coated with StdD (SEQ ID NO: 121 ) but not to uncoated wells (-Ag).
- NBX18029 SEQ ID NO: 118
- FIG. 11 Panel A demonstrates the transfer of a mixture of NBX18029 (SEQ ID NO: 118), NBX0880 (SEQ ID NO: 119), and NBX15016 (SEQ ID NO: 117) through the stems of almond (Prunus amygdalus) and grape (Vitis vinifera) plants.
- Lane 1 NBX mixture diluted in PBS prior to injection.
- Lane 2 Stem extract of grape plant after injection with PBS.
- Lane 3 Stem extract of grape plant after injection with NBX mixture.
- Lane 4 Stem extract of almond plant after injection with PBS.
- Lane 5 Stem extract of almond plant after injection with NBX mixture.
- Lane 6 Molecular weight marker.
- FIG. 11 Panel B demonstrates the transfer of NBX0018 (SEQ ID NO: 115) through the stems of almond (Prunus amygdalus) and grape (Vitis vinifera) plants and to the leaves of an orange (Citrus x sinensis) plant.
- Lane 1 Leaf sweat of orange plant after injection of NBX0018.
- Lane 2 Leaf sweat of orange plant after injection of PBS.
- Lane 3 Stem extract of almond plant after injection with NBX0018.
- Lane 4 Stem extract of almond plant after injection with PBS.
- Lane 5 Stem extract of grape plant after injection with NBX0018.
- Lane 6 Stem extract of grape plant after injection with PBS.
- Lane 7 NBX0018 solution prior to injection.
- Lane 8 Molecular weight marker.
- the exopolysaccharide of Xylella fastidiosa is essential for biofilm formation, plant virulence, and vector transmission.
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Abstract
La présente technologie concerne de manière générale des procédés et des anticorps utiles pour réduire, éliminer ou prévenir une infection par une population bactérienne chez une plante.
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WO2020234642A1 (fr) * | 2019-05-20 | 2020-11-26 | Novobind Livestock Therapeutics Inc. | Anticorps contre des agents pathogènes chez les volailles et leurs utilisations |
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WO2020234642A1 (fr) * | 2019-05-20 | 2020-11-26 | Novobind Livestock Therapeutics Inc. | Anticorps contre des agents pathogènes chez les volailles et leurs utilisations |
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AZIZI ARMAGHAN, ARORA ARINDER, MARKIV ANATOLIY, LAMPE DAVID J., MILLER THOMAS A., KANG ANGRAY S.: "Ribosome Display of Combinatorial Antibody Libraries Derived from Mice Immunized with Heat-Killed Xylella fastidiosa and the Selection of MopB-Specific Single-Chain Antibodies", APPLIED AND ENVIRONMENTAL MICROBIOLOGY, AMERICAN SOCIETY FOR MICROBIOLOGY, US, vol. 78, no. 8, 15 April 2012 (2012-04-15), US , pages 2638 - 2647, XP093106313, ISSN: 0099-2240, DOI: 10.1128/AEM.07807-11 * |
KILLINY N., MARTINEZ R. HERNANDEZ, DUMENYO C. KORSI, COOKSEY D. A., ALMEIDA R. P. P.: "The Exopolysaccharide of Xylella fastidiosa Is Essential for Biofilm Formation, Plant Virulence, and Vector Transmission", MOLECULAR PLANT-MICROBE INTERACTIONS, AMERICAN PHYTOPATHOLOGICAL SOCIETY, US, vol. 26, no. 9, 1 September 2013 (2013-09-01), US , pages 1044 - 1053, XP093106318, ISSN: 0894-0282, DOI: 10.1094/MPMI-09-12-0211-R * |
LABROUSSAA FABIEN, IONESCU MICHAEL, ZEILINGER ADAM R, LINDOW STEVEN E, ALMEIDA RODRIGO P. P: "A chitinase is required for Xylella fastidiosa colonization of its insect and plant hosts", MICROBIOLOGY, SOCIETY FOR GENERAL MICROBIOLOGY, READING, vol. 163, no. 4, 1 April 2017 (2017-04-01), Reading , pages 502 - 509, XP093106316, ISSN: 1350-0872, DOI: 10.1099/mic.0.000438 * |
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