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WO1999018225A1 - Expression de la sous-unite de la toxine b du cholera chez les plantes transgeniques et son efficacite dans les vaccins oraux - Google Patents

Expression de la sous-unite de la toxine b du cholera chez les plantes transgeniques et son efficacite dans les vaccins oraux Download PDF

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WO1999018225A1
WO1999018225A1 PCT/US1998/021237 US9821237W WO9918225A1 WO 1999018225 A1 WO1999018225 A1 WO 1999018225A1 US 9821237 W US9821237 W US 9821237W WO 9918225 A1 WO9918225 A1 WO 9918225A1
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ctb
plant
dna construct
protein
subunit
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William H. R. Langridge
Takeshi Arakawa
Daniel Chong
John Lawrence MERRITT
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Loma Linda University
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Loma Linda University
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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
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8257—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits for the production of primary gene products, e.g. pharmaceutical products, interferon
    • C12N15/8258—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits for the production of primary gene products, e.g. pharmaceutical products, interferon for the production of oral vaccines (antigens) or immunoglobulins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/28—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Vibrionaceae (F)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the present invention is related to genetic engineering of plants.
  • the invention is particularly related to the production of transgenic plants that express immunogenic proteins, which are referred to herein as "edible vaccines.”
  • Cholera is a devastating, infectious diarrheal disease that has caused recurrent pandemics throughout the world. Effective prevention of cholera dissemination depends on safe water sources and improved sanitation in countries where cholera is endemic. The poverty in these countries makes the cost of building large-scale water treatment and sanitation systems prohibitive. Thus, the ultimate challenge for cholera prevention lies in establishment of low-cost alternatives, including effective oral vaccines, which provide lasting protection after a single immunization, or convenient and readily available vaccines for frequent admimstration to people living in regions where cholera is endemic. It is widely recognized that oral vaccination is more effective than parenteral vaccination against pathogens that invade through mucosal surfaces. Moreover, oral vaccines are easier and safer to administer.
  • CTX Vibrio cholerae enterotoxin
  • CTB nontoxic B subunit
  • CTX also acts as a strong immunological adjuvant for co- administered antigens (Lycke and Holmgren, 1986; Jackson et al, 1993; Holmgren et al, 1993).
  • the strong adjuvant effect of CTX is due to its ability to interact with many vertebrate cell types by elevating intracellular cAMP levels through activation of adenylate cyclase (Gill and Meren, 1978).
  • CTB is not generally considered to be a strong adjuvant for co- administered antigens, it has been demonstrated to be an effective carrier molecule for induction of mucosal immunity to polypeptides to which it is chemically or genetically conjugated (McKenzie and Halsey, 1984; Czerkinsky et al, 1989; Dertzbaugh and Elson, 1993). In addition, CTB has been shown to function as a carrier of conjugated peptides for induction of immunological tolerance (Sun et al, 1994, 1996).
  • CTB as an immunomodulatory carrier molecule may be due largely to its ability to avidly bind to its natural receptor G M1 -ganglioside, on the surface of mammalian intestinal epithelial cells, including M cells of the gut-associated lymphoid tissues (GALT) (Svennerholm, 1976).
  • CTB subunits can be produced in transgenic food plant tissues for assembly into the pentameric structure, which is essential for high binding affinity for the natural toxin receptor (G M1 -ganglioside). It is also unknown if the subunits can stimulate a significant protective immune response against the biological effects of cholera holotoxin following oral administration.
  • U.S. Patent No. 5,681,571 to Holmgren et al. proposes an immunological tolerance-inducing agent.
  • U.S. Patent No. 5,589,384 to Lipscombe et al. proposes a fusion protein for use in a vaccine that employs a B subunit of an enterotoxin.
  • PCT Publication WO 95/08347 relates efforts to reduce or suppress the immune response of a mammal using transgenic plants.
  • CTB immunomodulatory transmucosal carrier molecules
  • CTB as a carrier for an antigen associated with an autoimmune disease, so that oral tolerance may be achieved so as to prevent or mitigate the disease. Disclosure of the Invention
  • the present invention is directed to producing an enterotoxin peptide covalently linked to a protein sorting signal.
  • a DNA construct encodes a fusion protein comprising a non-toxic subunit of an enterotoxin and a signaling peptide.
  • Representative of the enterotoxin subunit is the B subunit of cholera toxin (the CTB subunit).
  • a preferred signaling peptide is a microsomal retention signal, e.g., ER signal, such as the amino acid sequence Ser-Glu-Lys-Asp-Glu-Leu, which is preferably located at the C- terminus of the fusion protein.
  • a DNA construct of the present invention is operably linked to one or more regulatory sequences, e.g., promoter, enhancer, polyadenylation or termination signal, to effect expression of the coding sequence for the fusion protein. Since the DNA construct is preferably expressed in a plant, the promoter must be functional in the plant.
  • a preferred promoter is selected from cauliflower mosaic virus 35S (CaMV 35S), tomato E8, patatin (such as 1B33), ubiquitin, mannopine synthase PI, mannopine synthase P2, A. tumefaciens gene 5, rice actin 1, B. mori cytoplasmic actin, and tandem repeats thereof.
  • An enhancer such as cucumber mosaic virus ires and tobacco etch virus translation enhancer (TEV) can also be operably linked to a nucleotide sequence of the invention.
  • a DNA construct of the present invention is typically provided with flanking right and left T-DNA border regions of Agrobacterium tumefaciens. These border regions permit transfer of the DNA construct from a suitable cloning vector to a plant cell upon activation of the vir genes of A. tumefaciens.
  • a DNA construct further comprises a nucleotide sequence encoding an antigenic polypeptide, which is positioned between an enterotoxin subunit and a signaling peptide.
  • the antigenic polypeptide is not positioned at the C- terminus of the fusion so as not to interfere with operation of the signal peptide.
  • the enterotoxin subunit is preferably located at the N-terminus of the protein, perhaps to facilitate interaction with ganglioside receptors.
  • the antigenic polypeptide region encoded by the DNA construct is typically a mammalian (such as muc-1), bacterial, viral, or fungal peptide sequence, e.g., a coat protein, which is sufficiently large and properly folded so as to effect an immunogenic response in a host animal.
  • the enterotoxin subunit of the fusion protein can act as a carrier for the antigenic polypeptide, while further providing an adjuvant effect.
  • a further aspect of the invention is a transgenic plant cell, regenerated plant, seed, or progeny of a cell transformed with an instant DNA construct.
  • the DNA construct is integrated into the nuclear genome of the cell; however, the construct can exist as a nuclear episome or extrachromosomal DNA.
  • the cell, seed or plant is of an edible variety, such as tobacco, potato, tomato, banana, soybean, pepper, spinach, carrot, maize, corn, wheat, rye, and rice.
  • a method of transforming a plant cell with a DNA construct of the present invention is also contemplated, which is preferably performed by A. tumefaciens transformation or by microparticle bombardment.
  • the plant cell can be regenerated to an adult plant by conventional techniques, and an immunogenic amount of fusion protein can be expressed in the transgenic plant by cultivating and maintaining it under conditions effective to express the fusion protein.
  • expression can be effected through use of a tissue-specific promoter or one having enhanced effectiveness at a desired stage of differentiation or in the presence of a chemical agent.
  • the CTB coding sequence is operably linked (in- frame) to a nucleotide sequence encoding an ER retention signal (Munro and Pelham, 1987).
  • the resultant fusion is transferred into potato leaf explants by an Agrobacterium tumefaciens mediated stable transformation method (de Block, 1988).
  • the transgenic potato plants are analyzed for production of CTB retaining native antigenicity, oligomeric structure and G M1 -ganglioside binding capacity.
  • a DNA construct of the invention can further comprise a selectable marker gene, such as one that codes for antibiotic resistance or a visualizable protein.
  • a representative visualizable protein is a luciferase, green fluorescent protein, glucuronosidase or ⁇ - galactosidase.
  • a DNA construct can also comprise a nucleotide sequence encoding a leader sequence at the N-terminus of the fusion protein.
  • An expression vector of the invention preferably also contains an E. coli origin of replication to permit the use of conventional cloning techniques.
  • the expression vector can also comprise an A. tumefaciens origin of replication to permit replication in this host. Accordingly, strains of E. coli and of A. tumefaciens transfected with an expression vector of the invention are contemplated.
  • An A. tumefaciens strain may further comprise a helper Ti plasmid.
  • a central aspect of the present invention is a transgenic plant cell transformed with the DNA construct of the invention, particularly one in which the DNA construct is integrated into the nuclear genome of the cell.
  • the plant cell can be from one of the aforementioned plants.
  • a transgenic plant seed transformed with the DNA construct is contemplated.
  • a transgenic plant such as one regenerated from a transformed cell or progeny from a seed, is contemplated.
  • the transgenic plant is transformed with a DNA construct of the invention.
  • a DNA construct is integrated into the nuclear genomes of each cell of the plant.
  • the transgenic plant is of an edible variety, such as tobacco, potato, tomato, banana, soybean, pepper, spinach, carrot, maize, corn, wheat, rye, and rice.
  • a method of transforming a plant cell with a DNA construct of the invention comprises contacting the plant cell with a strain of A. tumefaciens under conditions effective to transfer and integrate the construct into the nuclear genome of the cell.
  • a transgenic plant can thereby be regenerated from the plant cell.
  • An alternative method, which is preferred for monocots, comprises subjecting the plant cell to microparticle bombardment with solid particles loaded with a DNA construct under conditions effective to transfer and integrate the construct into the nuclear genome of the cell.
  • a method of producing an immunogen in a plant is thereby afforded. The method comprises cultivating a transgenic plant of the invention under conditions effective to express an instant fusion protein.
  • operably linked refers to the respective coding sequence being fused in-frame to a promoter, enhancer, termination sequence, and the like, so that the coding sequence is faithfully transcribed, spliced, and translated, and the other structural features are able to perform their respective functions.
  • a heterologous nucleotide sequence of the present invention can be provided as its wild-type sequence, or as a synthetic sequence, such as a "plant-optimized” sequence.
  • a nucleotide sequence having a high degree of homology to these sequences, so that the encoded amino acid sequence remains substantially unchanged, is contemplated.
  • sequences at least 80%, more preferably 90%, homologous with an aforementioned nucleotide sequence are contemplated.
  • only those epitopes of an expressed antigenic protein essential for generating the desired immune response need be present in the molecule. Accordingly, C- and/or N-terminal fragments, including portions of fusion proteins, presenting the essential epitopes are contemplated within the invention.
  • Preferred heterologous proteins for use with the present invention include reporter molecules, such as firefly luciferase, glucuronosidase (GUS), green fluorescent protein (GFP) compounds, and enhanced versions thereof, particularly for use in optimizing the parameters of this expression system.
  • Preferred antigenic proteins and polypeptides include shigatoxin B (StxB), staphylococcus enterotoxin B (SEB), lethal toxin B (LT-B), Norwalk virus capsid protein (NVCP), and hepatitis B surface antigen (HBsAg).
  • a nucleotide sequence of the invention is preferably operably linked at its 3' end to a plant-functional termination sequence.
  • Preferred termination sequences include nopaline synthase (nos), vegetative storage protein (vsp), and protease inhibitor 2 (pin2) termination sequences.
  • the term "vector”, and the like refers to a nucleic acid construct capable of self-replication.
  • Such a vector includes a plasmid, bacteria transformed with plasmids, phage vectors, cosmids, and bacterial and yeast artificial chromosomes.
  • a vector of the present invention will be a plasmid, whether it is present in vitro, in E. coli, in A. tumefaciens, or as a nuclear episome of a plant. Suitable techniques for assembling the instant structural components into an expression cassette or replicon are described by Maniatis et al. (1982).
  • a strain of bacteria such as E. coli
  • the E. coli can also be mated with A. tumefaciens to introduce the vector therein, where it can reside intact as a shuttle vector.
  • a helper Ti plasmid in the A. tumefaciens can provide the vir genes necessary to transfer the T-DNA directly from the shuttle vector to the plant cell.
  • the vector can undergo homologous recombination with a tumor- inducing (Ti) plasmid and exchange the instant cassette for the T-DNA of the Ti plasmid.
  • Ti tumor- inducing
  • Methods of gene transfer into plants include use of the A. tumefaciens —Ti plasmid system.
  • the tumor-inducing (Ti) plasmids of A. tumefaciens contain a segment of plasmid DNA called transforming DNA (T-DNA), which integrates into the plant host genome.
  • T-DNA transforming DNA
  • a plasmid vector is constructed that replicates in E. coli. This plasmid contains the DNA encoding the protein of interest (an antigenic protein in this invention) and this DNA is flanked by T-DNA border sequences, which define the points at which the DNA integrates into the plant genome.
  • a gene encoding a selectable marker (such as a gene encoding resistance to an antibiotic such as kanamycin) is also inserted between the left border (LB) and right border (RB) sequences.
  • the expression of this gene in transformed plant cells gives a positive selection method to identify those plants or plant cells having an integrated T-DNA region.
  • the plasmid is transferred to Agrobacterium. This can be accomplished via a conjugation mating system, or by direct uptake of plasmid DNA by the Agrobacterium.
  • the Agrobacterium strain utilized must contain a set of inducible virulence (vir) genes, which are essential for T-DNA transfer to plant cells. The A.
  • tumefaciens gene transfer system mentioned above is the etiologic agent of crown gall, a disease of a wide range of dicotyledons and gymnosperms [DeCleene, M. et. al., Bot. Rev. 42, 389 (1976)], that results in the formation of tumors or galls in plant tissue at the site the infection.
  • the Agrobacterium system has been developed to permit routine transformation of a variety of plant tissue [see, e.g., Schell, J. et al,
  • Representative tissues transformed in this manner include tobacco [Barton, K. et al, Cell 32, 1033 (1983)]; tomato [Fillatti, J. et al, Bio/Technology 5, 726 (1987)]; sunflower [Everett, N. et al,. Bio/Technology 5, 1201 (1987)]; cotton [Umbeck, P. et al, Bio/Technology 5, 263 (1987)]; rapeseed [Pua, E. et al, Bio/Technology 5, 815 (1987)]; potato [Facciotti D.
  • Agrobacterium strains and plasmid construction strategies can be used to optimize genetic transformation of plants. For instance, A. tumefaciens may not be the only Agrobacterium strain used. Other Agrobacterium strains such as A. rhizogenes may be more suitable in some applications. A.
  • rhizogenes which incites root hair formation in many dicotyledonous plant species, carries a large extra-chromosomal element called an Ri (root-including) plasmid, which functions in a manner analogous to the Ti plasmid of A. tumefaciens. Transformation using A. rhizogenes has developed analogously to that of A. tumefaciens and has been successfully utilized to transform, for example, alfalfa, [Sukhapinda, K. et al, Plant Mol. Biol. 8, 209 (1987)].
  • Methods of inoculation of the plant tissue vary depending upon the plant species and the Agrobacterium delivery system.
  • a convenient approach is the leaf disc procedure which can be performed with any tissue explant that provides a good source for initiation of whole plant differentiation. The addition of nurse tissue may be desirable under certain conditions.
  • Other procedures such as in vitro transformation of regenerating protoplasts with A. tumefaciens may be followed to obtain transformed plant cells as well.
  • Several so-called "direct" gene transfer procedures have been developed to transform plants and plant tissues without the use of an Agrobacterium intermediate. Plant regeneration from protoplasts is a particularly useful technique [Evans, D. A. et al, Handbook of Plant Cell Culture 1, 124 (1983)].
  • DNA into protoplasts of a plant can be effected by treatment of the protoplasts with an electric pulse in the presence of the appropriate DNA in a process called electroporation.
  • the protoplasts are isolated and suspended in a mannitol solution.
  • Supercoiled or circular plasmid DNA is added.
  • the solution is mixed and subjected to a pulse of about 400 V/cm at room temperature for less than 10 to 100 microseconds. A reversible physical breakdown of the membrane occurs to permit DNA uptake into the protoplasts.
  • DNA viruses have been used as gene vectors in plants.
  • a cauliflower mosaic virus carrying a modified bacterial methotrexate-resistance gene was used to infect a plant.
  • the foreign gene was systematically spread in the plant [Brisson, N. et al, Nature 310, 51 1 (1984)].
  • the advantages of this system are the ease of infection, systematic spread within the plant, and multiple copies of the gene per cell.
  • Liposome fusion has also been shown to be a method for transformation of plant cells.
  • protoplasts are brought together with liposomes carrying the desired gene. As membranes merge, the foreign gene is transferred to the protoplasts
  • Polyethylene glycol (PEG) mediated transformation has been carried out in N tabacum (a dicot) and Lolium multi ⁇ orum (a monocot). It is a chemical procedure of direct gene transfer based on synergistic interaction between Mg 2+ , PEG, and possibly Ca 2+ [Negrutiu, R. et al, Plant Mol. Biol. 8, 363 (1987)].
  • exogenous DNA can be introduced into cells or protoplasts by micro injection. A solution of plasmid DNA is injected directly into the cell with a finely pulled glass needle.
  • a recently developed procedure for direct gene transfer involves bombardment of cells by microprojectiles carrying DNA [Klein, T. M. et al, Nature 327, 70 (1987)].
  • tungsten or gold particles coated with the exogenous DNA are accelerated toward the target cells. At least transient expression has been achieved in onion.
  • This procedure has been utilized to introduce DNA into Black Mexican sweet com cells in suspension culture and maize immature embryos and also into soybean protoplasts [Klein, T. M. et al, Bio/Technology 6, 559 (1988)]. Stably transformed cultures of maize and tobacco have been obtained by microprojectile bombardment.
  • a method of inducing immunity to an enterotoxin in a mammal or avian is thereby provided. Such method entails the animal consuming an immunizing effective amount of an edible part of the transgenic plant. Hence, immunity to cholera is effected when the enterotoxin subunit is the CTB subunit. Antibiotic resistant avians can be particularly advantaged.
  • a method of effecting or boosting immunity of a mammal or avian to a pathogen can be effected, particularly whenever the fusion protein contains an antigenic
  • the antigenic portion is provided by the antigenic polypeptide of the fusion protein.
  • the antigenic polypeptide can assemble as an antigenic complex or particle, and the subunit acts as a carrier for the antigenic polypeptide.
  • transgenic food plants for the production and delivery of oral vaccine against microbial enteropathogens and their toxins is investigated.
  • a cholera toxin (the prototypical diarrhea-inducing enterotoxin) is used as a model system.
  • the oral immunization of CD-I mice by transgenic potato plants transformed with the CTB coding sequence is studied and the levels of serum and mucosal anti-cholera toxin antibodies are determined.
  • the efficacy of an edible vaccine against V. cholerae enterotoxin in mammalian cell culture and in mouse intestine is evaluated.
  • Fig. 1 depicts the structure of the plant expression vector pPCV701FM4- CTB:SEKDEL.
  • the followmg four genes are located within the T-DNA sequence flanked by the right and left border (RB and LB) 25 bp direct repeats required for integration of the T-DNA into plant genomic DNA: (1) 393 bp CTB:SEKDEL coding sequence under control of the mas P2 promoter; (2) the bacterial luciferase AB fusion gene (luxF) under control of the mas PI promoter as a detectable marker; (3) an NPT-II expression cassette for resistance to kanamycin in plants; (4) a ⁇ -lactamase cassette for resistance to ampicillin in E. coli and carbenicillin in A.
  • RB and LB right and left border
  • the g7pA polyadenylation signal is from the A. tumefaciens T L -DNA gene 7; the OcspA polyadenylation signal is from the octopine synthase gene; Pnos is the promoter of the nopaline synthase gene; g4pA is the polyadenylation signal from T L -DNA gene 4; OriT is the origin of transfer derived from pRK2; OriV is the wide host range origin of replication for multiplication of the plasmid in tumefaciens derived from pRK2; and Ori pBR322 is the replication origin of pBR322 for maintenance of the plasmid in E.
  • Fig. 2 shows the luciferase activity in transformed potato leaf tissues. Bacterial luciferase activity was detected in leaves of six kanamycin-resistant potato plants (Nos 1-6) by low-light image analysis after induction on high auxin medium for 48 h (columns A and B in duplicate). Arrows indicate leaves with relatively high light intensities. No luciferase activity was detected in leaves of six untransformed potato plants (Nos 7-12, columns C and D in duplicate). Photon detection period was 5 min.
  • Fig 3 shows the detection of the CTB fusion gene in genomic DNA of transformed potato leaves. Lane 1 is a 1 kb DNA ladder (Gibco Life Technologies).
  • DNA templates used for PCR amplification reaction of the CTB gene were as follows: lane 2, pPCV701FM4-CTB:SEKDEL plasmid DNA (10 ng); lane 3, untransformed potato plant genomic DNA (500 ng); lane 4-6, transformed potato plant genomic DNA (500 ng) from plants No. 1, No. 3, and No. 4.
  • Fig. 4 shows immunoblot detection of plant CTB protein. Auxin-induced leaf callus tissues derived from transgenic potato plant No. 4 were analyzed for the expression of multimeric CTB protein, which dissociated to monomers by heat treatment.
  • Fig. 4A Multimeric CTB. Lane 1, 100 ng bacterial CTB; lane 2, 100 ng bacterial CTB mixed with total protein (100 ⁇ g) from untransformed potato plant leaf callus tissue; lane 3, total protein (100 ⁇ g) from untransformed potato plant leaf callus tissue; lane 4, total protein (100 ⁇ g) from No. 4 transgenic potato plant leaf callus tissue.
  • Fig. 4B Monomeric CTB.
  • Lane 1 100 ng bacterial CTB multimer (M r ⁇ 45 kDa) partially dissociated to monomer (M r ⁇ 12 kDa); lanes 2 (boiled) and 3 (unboiled), total protein (100 ⁇ g) from untransformed potato plant callus tissue; lanes 4 (boiled) and 5 (unboiled), total protein (100 ⁇ g) from No. 4 transgenic potato plant leaf callus tissue. Arrow indicates the band (M r ⁇ 15 kDa) corresponding to leaf callus CTB monomer.
  • Fig. 4C CTB in tuber tissues. Lane 1, 100 ng bacterial CTB (boiled); lane 2, total protein (150 ⁇ g) from No.
  • Fig. 5 illustrates the determination of CTB protein levels in transgenic potato plants using ELISA.
  • the ELISA detection of plant-synthesized CTB using a variety of transgenic plant protein concentrations indicates that CTB protein levels in induced leaf tissues from transgenic potato plant No. 4 reaches a maximum 0.3% of total soluble plant protein.
  • CTB protein levels (% CTB) were plotted against dilutions of plant homogenate. Error bars indicate SE.
  • Fig. 6 compares the G M1 binding ability of plant-derived CTB, bacterial CTB, and bacterial CTX.
  • the ELISA assay was performed by coating microtiter plates with either G M1 -monosialoganglioside, sucrose, galactose, or BSA as CTB or CTX receptor molecules (300 ng/well).
  • G M1 -monosialoganglioside sucrose, galactose, or BSA
  • CTB or CTX receptor molecules 300 ng/well.
  • A a transformed plant tissue homogenate containing approximately 30 ng of recombinant CTB in 10 ⁇ g of total soluble protein was used per well.
  • B and CTX (C) 30 ng of each molecule was mixed with untransformed plant homogenate containing 10 ⁇ g of total soluble protein per well.
  • Relative binding affinity of plant CTB, bacterial CTB, and CTX for receptor molecules was expressed as relative light units (RLU).
  • Fig. 7 A illustrates the CTB plant expression vector pPCV701FM4- CTB:SEKDEL.
  • the T-DNA sequence flanked by right and left borders (RB and LB) contains the / «xF/CTB:SEKDEL expression cassette containing the bi-directional mannopine synthase (mas) PI and P2 promoters (Koncz, C. et al, 1987).
  • the luxF gene is a detectable marker for agrobacteria and plants (Escher, A., et al, 1989; Langridge, W., et al, 1989).
  • the NPT II expression cassette containing the nopaline synthase promoter provides kanamycin resistance in plants
  • the ⁇ -lactamase (Bla) expression cassette provides ampicillin resistance in Escherichia coli and carbenicillin resistance in Agrobacterium tumefaciens.
  • the CTB fusion gene contains the DNA sequence encoding the CTB leader peptide at the 5' end and an ER retention signal (SEKDEL) at the 3' end.
  • the g7pA, g4pA and OcspA sequences are polyadenylation signals from A. tumefaciens T L -DNA gene 7, gene 4, and the octopine synthase gene, respectively.
  • Ori pBR is the origin of replication from plasmid pBR322.
  • Fig. 7B shows the immunoblot detection of CTB protein in a transgenic potato plant. Microtuber homogenate from a transformed potato plant (lane 3; 100 ⁇ g total soluble protein/lane) revealed the chimeric CTB pentamer ( ⁇ 50 kDa), which was slightly larger than the bacterial CTB pentamer ( ⁇ 45 kDa), (lane 1; 100 ng). Untransformed potato tuber proteins did not react with anti-CT antibody (lane 2; 100 ⁇ g total soluble protein/lane).
  • Fig. 7C shows the effects of boiling tuber tissues on CTB pentamer dissociation.
  • the amount of CTB in the tuber homogenate prior to boiling (time 0) was considered to be 100%.
  • the increase in CTB amount detected after 0.5 min boiling may indicate enhanced extraction of plant soluble protein due to tissue softening.
  • Transgenic potato tissues became soft after 3 min boiling which conesponded to CTB levels of 50%. Error bars indicate standard errors of the mean for four separate measurements.
  • Fig. 8A illustrates anti-CTB antibody endpoint titer determination.
  • Baseline RLU background signal from the enzyme-substrate reaction alone.
  • R 2 regression coefficient. The thin line is an extrapolation of the sample value to the baseline for determination of the E titer.
  • Fig. 8B shows serum anti-CTB antibody titers.
  • the E titers of three isotypes (Bl, IgG; B2, IgA; B3, IgM) of serum anti-CTB antibody are expressed for days 35 (___l)and 70 ( ⁇ ).
  • Bacterial CTB is the serum sample obtained from mice orally immvmized with 30 ⁇ g of bacterial CTB.
  • Potato (lg) and (3g) are serum samples obtained from mice orally immunized with 1 g and 3 g of transgenic potato tissues, respectively.
  • Negative control serum sample derived from mice orally immunized with 1 g of untransformed potato tissues. Error bars were determined based on the fluctuations of RLU baseline values which affect the E titer.
  • Fig. 8C shows mucosal anti-CTB antibody titers. The E titers of mucosal
  • Fig. 9 depicts an ileal loop ligation assay and the effect of oral immunization with bacterial or plant CTB on the small intestine.
  • Fig. 9A shows ileal loops excised from un- immunized mice.
  • Fig. 9B shows the same from an orally immunized mouse. The middleand right loops were challenged with CT (125 ng/loop). The left loop was injected with physiological saline.
  • Fig. 9C shows the intestinal fluid accumulation per unit length of ligated ileal loops from un-immunized and immunized mice. The reduction in fluid secretion into the ileal loop in the immunized mice in comparison with un-immunized mice (*) is expressed as % protection above each bar. A significant difference in reduction of fluid accumulation in mice orally immunized with plant or bacterial CTB was observed in comparison with mice fed untransformed potato tissues (p ⁇ 0.05).
  • Fig. 10 depicts a G M] -ELISA assay of CT binding to G M ,-ganglioside.
  • the G M1 - ELISA method described in the section on materials and methods was used to elucidate the protective mechanism of anti-CTB antibody. Approximately 35% less RLU signal was detected from G M1 -ganglioside-coated microtiter wells [GM1(+)] when CT was incubated with immune serum prior to assay than when CT was incubated with non- immune serum. In the absence of G M1 -ganglioside [GM1(-)], CT incubated with either immune or non-immune serum resulted in similar RLU levels.
  • Example 1 Construction of plant expression vector pPCV701FM4-CTB:SEKDEL.
  • the plant expression vector pPCV701FM4 was derived from plasmid pPCV701 by addition of multiple cloning sites immediately downstream from the mannopine synthase (mas) P2 promoter.
  • the vector was digested with Xbal and S ⁇ cl restriction endonucleases within the multiple clomng site to insert a gene encoding the cholera toxin B subunit from plasmid pRT42 containing the ctxAB operon (provided by Dr. J. Mekalanos, Harvard Medical School).
  • the 3' primer was designed to contain a nucleotide sequence encoding a hexapeptide ER retention signal (SEKDEL) in frame with the CTB open reading frame.
  • SEKDEL hexapeptide ER retention signal
  • the reaction mixture was used to transform Escherichia coli strain HB101 by electroporation (Gene Pulser, Bio-Rad, Inc. Hercules, CA) at a setting of 250 ⁇ FD, 200 ⁇ , and 2,500 V. Ampicillin resistant colonies were isolated after overnight culture at 37 °C.
  • the plasmid was isolated from individual colonies of transformants and subjected to DNA sequence analysis with the forward primer (5'-ACCAATACATTACACTAGCATCTG-3 ' ) specific for the mas P2 promoter and the reverse primer (5'-GACTGAGTGCGATATTATGTGTAATAC-3 ' ) specific for the gene 7 poly(A) signal in a model 373 A DNA Sequencer (Applied Biosystems, Inc.).
  • the shuttle vector was transferred into A. tumefaciens recipient strain GV3101 pMP90RK by the same electroporation conditions described for E. coli transformation.
  • the bacteria were grown at 29 °C on YEB solid medium (beef extract 5.0 g/L, Bacto yeast extract 1.0 g/L, Bacto peptone 1.0 g/L, sucrose 5.0 g/L, MgSO 4 *7H 2 O 0.1 g/L) containing the antibiotics carbenicillin (100 ⁇ g /mL), rifampicin (100 ⁇ g /mL), kanamycin (25 ⁇ g/mL), and gentamycin (25 ⁇ g /mL).
  • the plasmid was isolated from an A. tumefaciens transformant and transferred back into E. coli HB101, by electroporation to confirm by restriction endonuclease analysis, that no significant deletion had occurred in the vector. Structural confirmation of the plasmid was required because recombination events within the rec + A. tumefaciens strain could alter the T-DNA sequence. Transfer of the plasmid from A. tumefaciens back to the E. coli host was necessary because significant amounts of plasmid are difficult to isolate directly from tumefaciens. Agrobacteria carrying the plant expression vector were grown on YEB solid medium containing all antibiotics for 48 h at 29 °C and directly used for transformation of sterile potato leaf explants.
  • Sterile potato plants Solanum tuber osum cv. Bintje were grown in Magenta boxes (Sigma) or Mason jars on solid Murashige and Skoog (MS) complete organic medium (JRH Biosciences No. 56-750-015) containing 3.0% sucrose and 0.2% gelrite (a clear Pseudomonas polysaccharide solid support medium).
  • Leaf explants were excised from the young plants and laterally bisected in a 9 cm diameter culture dish containing an overnight culture of A. tumefaciens suspension (2-5 xlO cells/mL) harboring pPCV701FM4-CTB: SEKDEL. Acetosyringone (370 ⁇ M) was added to the bacterial suspension to facilitate transformation.
  • the explants were incubated in the bacterial suspension for 5 min, blotted on sterile filter paper, and transferred to MS solid medium, pH 5.7, containing the plant hormones auxin (0.1 ⁇ g/mL naphthalene acetic acid (NAA)) and cytokinin (1.0 ⁇ g/mL trans-zeatin).
  • the leaf explants were incubated for 48-72 h at room temperature on MS solid medium to permit T-DNA transfer into the plant genome.
  • the leaf explants were transferred to MS solid medium containing the antibiotics kanamycin (100 ⁇ g mL) and claforan (400 ⁇ g/mL), for selection of transformed plant cells and for counterselection against continued Agrobacterium growth, respectively.
  • Transformed plant cells formed calli on the selective medium after continuous incubation for 2-3 weeks at 25 °C in a light room under cool white fluorescent tubes on a 12-h photoperiod regime.
  • transformed calli grew to 5-10 mm in diameter
  • the leaf tissue was transferred to MS medium containing 1.0 ⁇ g/mL trans-zeatin, 50 ⁇ g/mL kanamycin and 400 ⁇ g/mL claforan for shoot induction.
  • regenerated shoots were excised at the base from the calli and transferred to MS solid medium without plant hormones or antibiotics to stimulate root formation. Plantlets were obtained after about 6 weeks further growth under sterile conditions in Mason jars.
  • the plantlets were grown into mature plants (4-6 weeks) in potting soil in the greenhouse under a 12-h photoperiod. Activity of the luciferase reporter gene was detected in leaf tissues of the putative transformed plants by low-light image analysis with a Hamamatsu Argus-100 intensified camera system (Hamamatsu Photonics, K.K., Japan).
  • Example 3 Detection of luciferase activity in transformed A. tumefaciens and transgenic plants.
  • bacterial luciferase gene expression under control of the mas PI promoter was monitored by low- light image analysis (Langridge et al, 1991).
  • the volatile substrate N-decyl aldehyde (Sigma D-7384) was applied to a 9 cm diameter glass culture plate lid by swabbing the plate with substrate-saturated cotton.
  • a culture plate containing bacterial colonies grown for 24-48 h on YEB solid medium was covered with the substrate coated glass lid, and the culture plate was transferred into the photon counting chamber of the Argus- 100 intensified camera system for photon counting for a period of 1-5 min.
  • Bacterial luciferase bioluminescence was also used to detect insertion of the T- DNA into the plant genome and to estimate the level of mas P2 promoter driven expression of the CTB gene by the level of mas PI driven expression of the luxF gene.
  • Leaves excised from putative transformants were wounded by cutting perpendicular to the central vein with a sterile scalpel blade followed by incubation of the wounded leaf tissue on MS solid medium containing NAA (5.0 mg/L) and 2,4-dichlorophenoxy acetic acid (2,4-D) (6.0 mg/L) for 48 h. Wounding and subsequent incubation on high auxin medium for several days is necessary to detect the maximum amount of gene expression from the mas promoters in potato plant tissues. Light emission from the wounded leaf tissues was detected as described for agrobacteria. Approximately 5-30 min exposure was required to obtain an coherent photon emission image.
  • Genomic DNA was isolated from transformed potato leaf tissues as described by Doyle and Doyle (1992) with the following modification: biological grinding spheres (Boehringer Mannheim) were used instead of a mortar and pestle for grinding the plant tissues. Presence of the CTB gene was determined by PCR analysis using the oligonucleotide forward and reverse primers specific for the pPCV701FM4 vector.
  • Transformed plant genomic DNA 500 ng was used as a template to detect the CTB gene under the following PCR conditions: 94 °C for 45 s, 55 °C for 60 s, and 72 °C for 60 s for a total of 30 cycles. PCR samples were separated by electrophoresis on a 1% agarose gel and stained with ethidium bromide. Example 5. Immunoblot detection of CTB protein in transformed potato tissues.
  • Transgenic potato tissues were evaluated for the presence of CTB protein by immunoblot analysis using a Bio-Rad Immun-Lite Assay Kit (Bio-Rad 170-6471). Callus tissues were derived from leaf or tuber tissues incubated for 5-7 weeks on MS solid medium containing NAA (5.0 mg/L) and 2,4-D (6.0 mg/L).
  • Tissues ( ⁇ 1 g fresh weight) were homogenized by grinding in a mortar and pestle on ice in 1.0 mL of extraction buffer (200 mM Tris-Cl, pH 8.0, 100 mM NaCl, 400 mM sucrose, 10 mM EDTA, 14 mM 2-mercaptoethanol, 1 mM phenylmethylsulfonyl fluoride, 0.05% Tween- 20).
  • the tissue homogenate was centrifuged twice at 17,000 x g in a Beckman GS-15R centrifuge for 15 min at 4 °C to remove insoluble cell debris.
  • the separated protein bands were transferred from the gel to Immun-Lite membranes by electroblotting on a semi-dry blotter (Labconco) for 60-90 min at 15 V and 100 mA.
  • Nonspecific antibody reactions were blocked by incubation of the membrane in 25 mL of 5% non-fat dry milk in TBS buffer (20 mM Tris, pH 7.5 and 500 mM NaCl) for 1 h on a rotary shaker (40 rpm), followed by washing in TBS buffer for 5 min with gentle agitation.
  • the membrane was incubated overnight at room temperature with gentle agitation in 30 mL of a 1:5,000 dilution of rabbit anti-cholera antiserum (Sigma C-3062) in antibody dilution buffer (TBST (TBS with 0.05% Tween-20) containing 1% non-fat dry milk) followed by washing three times in TBST buffer.
  • TST rabbit anti-cholera antiserum
  • the membrane was incubated for 1 h at room temperature with gentle agitation in 30 mL of a 1 : 10,000 dilution of mouse anti -rabbit IgG conjugated with alkaline phosphatase (Sigma A-2556) in antibody dilution buffer.
  • the membrane was washed three times in TBST buffer as before and once with TBS buffer, followed by incubation in 20 mL of lx substrate buffer containing 36 ⁇ L of chemiluminescent substrate CSPDJ for 5 min at room temperature with gentle agitation.
  • the membrane was placed inside a household seal-a-meal bag after removing excess substrate buffer and placed in a photocassette on Kodak X-OMAT film (No. 1651454).
  • the membrane was also used to image chemiluminescent light intensity in both the numerical and graphic form by the Argus- 100 video image analysis.
  • the film was subjected to 1-15 min exposure at room temperature in the dark for optimal image development.
  • the exposed film was developed in a Kodak M35 A X-OMAT Processor.
  • Example 6 Quantitation of CTB protein level in transgenic potato tissues.
  • CTB protein levels in transgenic potato plants were determmed by quantitative chemiluminescent ELISA assays.
  • a 96-well microtiter plate (MicroliteTM 2, Dynatech Laboratories), loaded with 100 ⁇ L/well of selected concentrations of total soluble potato protein in bicarbonate buffer, pH 9.6 (15 mM NajCOj, 35mM NaHCO 3 ) was incubated overnight at 4 °C. The plate was washed three times in PBST (phosphate buffered saline (PBS) containing 0.05% Tween-20).
  • PBST phosphate buffered saline
  • the backgroimd was blocked by incubation in 1% bovine serum albumin (BSA) in PBS (300 ⁇ L/well) at 37 °C for 2 h followed by washing three times with PBST.
  • BSA bovine serum albumin
  • the plate was incubated in a 1 :8,000 dilution of rabbit anti- cholera toxin antibody (Sigma C-3062), (100 ⁇ L/well) for 2 h at 37 °C, followed by washing the wells three times with PBST.
  • the plate was incubated with a 1 : 80,000 dilution of anti-rabbit IgG conjugated with alkaline phosphatase (Sigma A-2556), (100 ⁇ g/well) for 2 h at 37 °C and washed three times with PBST.
  • the plate was finally incubated with 100 ⁇ L/well of Lumi-Phos® Plus (Lumigen, Inc. P-701) for 20 min at 37 °C to maximize the reaction rate.
  • the plate was cooled to room temperature before the enzyme-substrate reaction was measured in a MicroliteTM ML3000 Microtiter® Plate Luminometer (Dynatech Laboratories), operated according to the manufacturer ' s instructions.
  • chemiluminescent light intensities of the enzyme-substrate reaction from bacterial and plant CTB protem bands blotted on the Immun-Lite membranes after SDS-PAGE were quantified by the Argus- 100 Data Analysis Program (Hamamatsu Photonics, K.K.). The numeric value of the total light intensity of the area defined by a rectangular window surrounding the plant CTB protein band was compared with the bacterial CTB band, and the amount of plant CTB was estimated based on the known amount of bacterial CTB.
  • Example 7 CTB-G M , binding assay.
  • a G M1 -ELISA assay was performed to determine the affinity of plant derived CTB for G M1 -ganglioside.
  • the microtiter plate was coated with monosialoganglioside-G M1 (Sigma G-7641) by incubating the plate with 100 ⁇ L/well of G M1 (3.0 ⁇ g/mL) in bicarbonate buffer, pH 9.6 (15 mM Na 2 CO 3 , 35mM NaHCO 3 ) at 4 °C overnight.
  • the wells were coated with 100 ⁇ L/well of BSA, sucrose, or galactose (3.0 ⁇ g/mL each) as controls.
  • the plates were incubated with transformed plant total soluble protein, bacterial CTB (Sigma C-9903), or CTX (Sigma C-8052) in PBS (100 ⁇ L/well) overnight at 4 °C. The remainder of the procedure was identical to the ELISA described above.
  • Example 8 Construction of transgenic potato plants producing CTB: SEKDEL fusion peptide.
  • the CTB coding sequence including its putative leader peptide (which is absent in mature cholera toxin B subunit in V. cholerae), was PCR amplified from the ctxAB operon in the plasmid pRT42 (provided by Dr. J. Mekalanos).
  • the 3' PCR primer contained an oligonucleotide sequence encoding a hexapeptide endoplasmic reticulum (ER) retention signal (SEKDEL) in frame with the CTB gene.
  • SEKDEL hexapeptide endoplasmic reticulum
  • the amplified DNA fragment containing the CTB: SEKDEL fusion gene was cloned into plant transformation vector pPCV701FM4 (Koncz, C, et al, 1987).
  • the resultant plasmid was introduced into Agrobacterium tumefaciens strain GV3101 pMP90RK by electroporation (Koncz, C, et al, 1987). Potato plants (Solanum tuberosum cv. Bintje) were transformed with agrobacteria harboring pPCV701FM4-CTB: SEKDEL as previously described above.
  • Example 9 Immunization Antigens. Purified cholera holotoxin and its B subunit were purchased from Sigma Chemical Co. (St. Louis, MO). Immunization was performed with CTB. Cholera holotoxin was used in mouse ileal loop ligation and in vitro toxin neutralization assay experiments.
  • Potato tuber and leaf callus tissues producing approximately 0.3% of total soluble protein as CTB pentamers were used for oral immunization of CD-I mice.
  • ATCC Manassas, VA
  • DMEM Modified Eagle's Medium
  • Example 11 Oral immunization with CTB.
  • mice were fed ad libitum transgenic (microtuber or leaf callus) potato tissues containing approximately 30 ⁇ g CTB/g fresh weight, previously determined by CL- ELISA and immunoblot experiments with transformed plant tissue homogenates as described above. Mice were fed four times on days 0, 6, 17, 24 with a final booster dose on day 65. A group of 10 mice were fed with 1 g of transgenic potato tissues, and a group of 8 mice were fed with 3 g of transgenic potato tissues.
  • Example 12 Serum and fecal sample preparation.
  • mice were bled on days 35 and 70 of the experiment.
  • Fecal pellets were collected on days 25, 28, 31, 38, 45, 65, and 70 of the experiment to determine the presence of mucosal antibodies (IgA and IgG) secreted in response to CTB ingestion.
  • Fecal antibodies were detected according to the co- proantibody isolation method described by de Vos and Dick. Example 13. Chemiluminescent ELISA.
  • Anti-mouse antibody [1 :20,000 dilution of anti-mouse IgG (Sigma A-3688), 1:20,000 IgA (Sigma A-4937), or 1 :30,000 IgM (Sigma A-9688)] diluted in PBS containing 0.5% BSA was added (100 ⁇ L/well), and incubated for 2 h at 37 °C. The wells were washed and incubated with the chemiluminescent substrate, Lumi-Phos® Plus, (100 ⁇ L/well) (Lumigen Inc., Southfield, MI.) for 20 min at 37 °C. The plates were read in a MicroliteTM ML3000 Microtiter® Plate Luminometer (Dynatech Laboratories).
  • Serum and fecal endpoint titers were determined as described elsewhere (Jackson, R., et al, 1996). Briefly, serial dilutions of serum or fecal extracts from immunized or unimmunized mice were transferred in duplicate into microtiter plates for CL-ELISA. Background RLU from the fecal or serum sample of unimmunized mice was subtracted from the RLU of samples from immunized mice. The resultant RLU was plotted on a log 10 scale against two-fold dilutions (log 2 ) of the samples. The graphic data were extrapolated for three groups of immunized mice to the level of 0.5 RLU which was the nonspecific background signal generated from the enzyme-substrate reaction alone in this assay system.
  • Example 15 Vero-cell based CT neutralization assay.
  • Vero cells were grown at 37 °C in DMEM supplemented with 5% FCS in a 5% CO 2 atmosphere.
  • concentrations of CT required for a cytotoxic response were determined initially by adding serial dilutions of CT to Vero cell monolayers (Stavric, S., et al, 1978).
  • the changes in cellular morphology were scored as 0, 1, 2, 3, 4, or 5, corresponding roughly to 0, ⁇ 25, 25-50, 50-75, 75-90, or > 90% cells affected.
  • a final concentration of 25-30 ng CT/mL was found to give scores above 3 and was used for toxin neutralization assays.
  • the relative efficacy of toxin neutralization of antisera was assayed by incubating 100 ⁇ L of two-fold dilutions of pooled serum in PBS prepared from immunized or unimmunized mice with 25 ng CT for 1 h at 35 °C.
  • the growth medium Prior to addition of the toxin-antiserum mixture to confluent monolayers, the growth medium was replaced with 1 mL of fresh medium containing 5% FCS, followed by 1 h incubation at 37 °C in a 5% CO 2 atmosphere.
  • the toxin-antiserum mixture was added to the cell monolayers and cytotoxic responses were observed after 20 h incubation at 37 °C.
  • Example 16 Mouse ileal loop ligation assay.
  • a mouse ileal loop ligation experiment was conducted essentially as described elsewhere (Punyashthiti and Finkelstein, 1971) on day 70 of the CTB oral immunization experiment. Briefly, animals were starved for 48 h and three equal loops 2 to 3 cm in length were ligated. Cholera toxin (125 ng in 30 ⁇ l physiological saline) was injected into each of the two loops, and the third loop was injected with saline only. After 24 h incubation the three consecutive ileal loops excised and were punctured to measure the fluid volume and the length of the empty loops. Student's t-test was used to determine the significance of fluid reduction between control mice and immunized mice.
  • Example 17 Antiserum-mediated inhibition of CT binding to G M1 -ganglioside by G m - ELISA.
  • a 96-well microtiter plate was coated with 100 ⁇ L/well of G M1 -ganglioside (3.0 ⁇ g/mL) (Sigma G-7641) in bicarbonate buffer, pH 9.6 and incubated at 4 °C overnight.
  • G M1 -ganglioside 3.0 ⁇ g/mL
  • B-7641 bicarbonate buffer, pH 9.6
  • Pooled serum (100 ⁇ L) from mice fed 1 g of transgenic potato tissues, or from mice fed 1 g of untransformed potato tissues was mixed with 2.5 ng of CT and incubated at 35 °C for 1 h.
  • the serum-CT mixture was loaded into the G M1 -ganglioside-coated or uncoated wells and the plate was incubated at 4 °C overnight.
  • Plant expression vector harboring CTB: SEKDEL fusion gene The CTB: SEKDEL fusion gene was inserted into the plant expression vector pPCV701FM4 resulting in pPCV701FM4-CTB: SEKDEL (Fig. 1).
  • Plant expression vector pPCV701FM4 harbors a 430 bp DNA fragment containing the A. tumefaciens bidirectional mannopine synthase (mas PI, P2) promoters (Koncz et al, 1987). The PI promoter is fused to the bacterial luciferase reporter gene (luxF) (Escher et al, 1989), and the P2 promoter is linked to the CTB:SEKDEL fusion gene.
  • luxF bacterial luciferase reporter gene
  • the plant expression vector contains the ⁇ -lactamase gene, which confers ampicillin resistance in E. coli and carbenicillin resistance in A. tumefaciens.
  • the neomycin phosphotransferase II gene (NPT-II) linked to the nopaline synthase (NOS) promoter provides selection for transformed plant cells.
  • the oligonucleotide sequence surrounding the translation initiation codon of the CTB gene was changed to a preferred nucleotide context for translation in eukaryotic cells (Kozak, 1981), and a putative Shine-Dalgarno sequence (AGGA) present in the ctxAB operon in plasmid pPT42 was also removed.
  • the DNA fragment encoding the 2 l-amino acid leader peptide of the CTB protein was retained to direct the newly synthesized CTB protein into the lumen of the ER.
  • An oligonucleotide sequence encoding the ER retention signal (SEKDEL) and using codon usage favored in potato was inserted at the 3 ' end of the coding sequence of the CTB gene to sequester CTB protein within the lumen of the ER.
  • SEKDEL fusion gene was inserted into the multiple cloning site immediately downstream of the mas P2 promoter.
  • Potato plant transformation efficiencies were found to vary considerably between experiments, suggesting the possibility that differences in aspects of the transformation method such as physiological state of leaf explants and the growth state of the agrobacteria used for transformation may result in substantial differences in transformation efficiency.
  • the number of kanamycin-resistant plants regenerated per transformation experiment varied dramatically ranging from 0 to 200 per 50 leaf explants transformed. Therefore, the presence of a convenient detectable marker gene, such as luciferase, in addition to selectable marker gene significantly enhances the process of screening large numbers of antibiotic resistant putative transformants.
  • plant-synthesized CTB did not show the presence of A subunit (Fig. 4A, lane 4). Homogenates from untransformed plants did not interfere with the antigenicity of exogenously added cholera toxin B subunit (Fig. 4A, lane 2). Anti- cholera toxin antibodies did not show a significant cross reaction with potato plant proteins (Fig. 4A, lane 3). Plant-produced CTB protein dissociated into monomers with molecular weight of approximately 15 kDa when the homogenate was boiled for 3 min prior to SDS-PAGE (Fig. 4B, lane 4).
  • Presence of the SEKDEL hexapeptide at the C-terminus of proteins has been shown to significantly increase protein accumulation within plant tissues such as potato leaves and tubers, tobacco and alfalfa leaves as well as in COS cells (Munro and Pelham, 1987; Wandelt et al, 1992; Haq et al, 1995), thereby facilitating protein subunit oligomerization.
  • Immunoblot analysis of plant-synthesized CTB showed that the monomers were significantly less immunoreactive in comparison to the multimeric form (Fig. 4B, compare lanes 4 and 5).
  • bacterial CTB was found to be substantially less reactive when dissociated by heat.
  • Transgenic potato microtuber tissues were analyzed for the presence of multimeric CTB proteins (Fig. 4C). Following auxin induction, homogenates prepared from microtuber tissues revealed biochemical characteristics of multimeric CTB protein identical to that found in leaf callus tissues, confirming auxin-induced CTB gene expression in all tissues of transformed potato plants.
  • the amount of plant CTB protein was measured by comparison of the relative light units (RLU) from a known amount of bacterial CTB protein-antibody complex with that emitted from a known amount of transformed plant soluble protein.
  • the amount of CTB detected was expressed as a percentage of total soluble plant protein (% CTB) in the sample (Fig. 5).
  • % CTB total soluble plant protein
  • Optimal concentrations of soluble protein loaded in the wells of the microtiter plate yielded CTB protein levels reaching 0.3% of total soluble protein in auxin-induced potato tissues (1.5 ng of plant CTB protein detected in 0.5 ⁇ g of total plant soluble protein). When the concentration of total protein deviated from optimal levels, the amount of CTB protein detected decreased.
  • This result may be due to the binding characteristics of the microtiter plate wells to CTB protein in a mixture of total plant proteins. With increasing plant protein levels, increasing amounts of CTB protein may be unable to bind to the wells and is eventually lost through washing. Alternatively, the sensitivity of CTB detection may decrease with lower plant protein amount, eventually reaching undetectable levels.
  • chemiluminescent intensities of bacterial and plant CTB protein bands blotted on Immun-Lite membranes after SDS-PAGE were measured by the Argus- 100 low- light imager Data Analysis Program.
  • the number of photons emitted from either bacterial CTB (Fig. 4B, lane 1) or plant CTB (Fig. 4B, lane 5) protein bands was quantified, and their values compared to provide a semi-quantitative estimate of the amount of plant synthesized-CTB protein. Based on the amount of light emission detected from a known amount of bacterial CTB protein (100 ng), the amount of plant CTB protein was calculated to be approximately 350 ng.
  • the percentage of CTB protein in the plant was calculated based on the amount of soluble plant protein (100 ⁇ g) used in the assay. Based on this method, the percentage of plant CTB protein was found to be approximately 0.35% of total soluble plant protein, a value in close agreement with measurements made by the chemiluminescent ELISA method.
  • Tryptophan at this location is essential for both pentamerization of the B monomer as well as binding of the pentamer to the oligosaccharide moiety of G M1 (de Wolf et al, 1981a,b). Boiling the plant and bacterial CTB prior to G MI -ELISA abolished anti-CTB antibody detection (result not shown). This result could be explained by the fact that monomeric CTB is unable to bind to G M1 - ganglioside, and/or that monomers are weaker in antigenicity in comparison with oligomeric CTB.
  • mas promoter system for expression of multiple genes
  • Auxin induction of the mas PI and P2 promoters results in expression of foreign proteins in plants at levels equivalent to or greater than strong constitutive promoters such as the cauliflower mosaic virus (CaMV) 35 S promoter (Mason et al, 1992; Haq et al, 1995; Mason et al, 1996).
  • CaMV cauliflower mosaic virus
  • Auxin induction provides a substantial contribution to CTB gene expression. Approximately 100-fold lower amounts of CTB protein and luciferase activity were detected in leaf and tuber tissues without induction by this plant hormone (results not shown).
  • auxin induction While in potato transformation experiments, exogenous auxin addition was required to stimulate CTB gene expression from the mas promoter in leaf and microtuber tissues, food plants like tomato which make large amounts of auxin during fruit ripening may not require auxin induction for maximum gene induction.
  • the combination of a convenient method for screening large numbers of transformants via the bacterial luciferase reporter gene and the hormone-inducible mas promoter system provides the potential for rapid screening of large numbers of transgenic plants to select those with the highest transgene expression levels.
  • the luciferase reporter gene was not essential for identification of transformants as only six kanamycin-resistant plants were regenerated and all of them were observed to contain the CTB gene sequence.
  • presence of the luciferase gene on the mas PI promoter can be extremely useful for selection of transformed plants.
  • the results of our experiment indicate that the mas dual promoter system can serve as a model system for simultaneous expression of two desired gene products in food plant tissues.
  • the mas dual promoter system provides a distinct advantage when two proteins are expected to be produced in the plant, for example, when two recombinant protein antigens are desired for construction of a vaccine against multiple pathogens. Future perspectives for food plant-derived recombinant CTB
  • oligomeric CTB protein in edible plants may induce mucosal and systemic anti-cholera toxin antibodies in mammals at levels sufficient to provide protective immunity against cholera toxin challenge upon feeding transgenic plant tissues.
  • LT-B enterotoxigenic Escherichia coli heat- labile enterotoxin B subunit
  • Transgenic potato plants producing pentameric CTB The oligonucleotide sequence encoding the endoplasmic reticulum (ER) retention signal (SEKDEL) was fused to the 3' end of the CTB gene and cloned into the plant expression vector pPCV701FM4 (Fig. 7A). Following Agrobacterium-mediated potato leaf transformation, the CTB fusion gene was expressed in both microtuber and leaf tissues up to 0.3% of total soluble protein. In addition, the chimeric protein monomers assembled into pentamers which exhibited native antigenicity with a small molecular weight increase in comparison with the bacterial CTB pentamer (45 kDa vs. 50 kDa).
  • Potato tissues were orally administered to mice four times at weekly intervals for a month with a final booster feeding.
  • Systemic and mucosal CTB-specific antibody titers were determined in both serum and feces collected from immunized mice by the class- specific chemiluminescent ELISA (CL-ELISA) method and expressed as endpoint (E) titers (Jackson, R. et al, 1996) Essentially, the E titers for the three antibody isotypes (IgM, IgG, and IgA) were determined in serum and fecal samples as shown in Fig. 8A.
  • IgM, IgG, and IgA endpoint
  • the E titers of the three serum anti-CTB antibody isotypes (IgG, IgA and IgM) were determined and the results expressed for days 35 and 70 (Fig. 8B, lanes 1-3).
  • Anti- CTB titers for IgG and IgA slightly increased from day 35 to day 70, while the IgM titer decreased.
  • the E titers of fecal (intestinal) anti-CTB antibody isotypes (IgA and IgG) were determined in a similar fashion, and the results expressed for each day of sample collection (Fig. 8C).
  • Both fecal IgA and IgG titers reached the highest level around day 28, four days after the 4th feeding, and gradually decreased over the next 40 days until the booster feeding on day 65.
  • fecal IgA and IgG titers increased after the booster dose.
  • mice fed 3 g of transformed potato a single booster feeding increased the IgA titer to approximately the highest titer observed on day 28.
  • fecal IgA titers were higher than IgG titers for three groups of immunized mice for each day of fecal sample collection. Mice immunized with 30 ⁇ g of bacterial CTB and 3 g of transformed potato tissues showed similar mucosal IgA antibody titers on day 70 of the ileal loop ligation experiment.
  • Vero cell morphology Normal and affected Vero cell morphology was detected by light microscopy. Vero cells affected by cholera toxin appeared refractile, thick-walled, and possessed filamentous tendrils.
  • the neutralization titer was defined as the highest serum dilution providing complete neutralization of CT cytotoxicity. Serum derived from mice fed lg of untransformed potato tissues showed no protection against CT-induced cytotoxicity, and addition of immune serum alone to the cell monolayers did not adversely affect cell morphology, indicating that mouse serum contains no factors which abrogate the toxin effect, nor factors which alter Vero cell morphology.
  • mice orally immunized four times followed with a final booster of 30 ⁇ g bacterial CTB showed a titer of 1:32 for complete protection, in comparison with a titer of 1 :8 for mice fed with 3 g of transformed potato tissues and a titer of 1 :2 for mice fed with lg of transformed potato tissues (Table 1).
  • cholera toxin neutralization titers are expressed as the highest dilutions of 100 ⁇ L of pooled immune serum which conferred complete protection against cholera toxin (25 ng/mL final) cytotoxic effects in Vero cells.
  • Figs. 9A and B Representative ileal loops from a mouse fed untransformed potato tissues and a plant-CTB immunized mouse are shown in Figs. 9A and B, respectively.
  • V volume of fluid accumulated in ileal loops from immunized and non-immunized animals was measured and expressed as the ratio of volume (V) to loop length (L) [V/L ( ⁇ L/cm)] (Fig. 9C).
  • V volume of fluid accumulated in ileal loops from immunized and non-immunized animals was measured and expressed as the ratio of volume (V) to loop length (L) [V/L ( ⁇ L/cm)] (Fig. 9C).
  • V volume of fluid accumulated in ileal loops from immunized and non-immunized animals was measured and expressed as the ratio of volume (V) to loop length (L) [V/L ( ⁇ L/cm)] (Fig. 9C).
  • mice immunized with 30 ⁇ g of bacterial CTB 42% protection for mice immunized with 1 g of transformed potato tissues
  • 62% protection for mice immunized with 3 g of transgenic potato tissues 64% protection for mice immunized with 3 g of transgenic potato tissues.
  • Ileal loops injected with saline did not accumulate significant amounts of fluid (1.5-3.5 ⁇ L/cm), suggesting that mechanical disturbances during loop ligation do not cause an inflammatory response.
  • Plant-synthesized CTB was shown to specifically bind to G M1 -ganglioside, but not to mono- or di-saccharides such as galactose and sucrose.
  • a G M1 -ELISA was performed to determine if CT neutralization in Vero cell cultures and mouse ileal loops was due to anti-CTB antibody-mediated prevention of CT binding to G M1 -ganglioside.
  • Incubation of CT with pooled antiserum from mice fed 1 g of transgenic potato tissues resulted in 36% less RLU signal in comparison with the same amount of CT incubated with pooled antiserum derived from mice fed 1 g of untransformed potato tissues (Fig. 10, GM1+).
  • the 2 l-amino acid leader peptide of the CTB protein is thought to function as a leader peptide in eukaryotic cells for translocation of nascent CTB polypeptides into the lumen of the plant ER (van Heijne, G., 1985).
  • the ER retention signal (SEKDEL) fused to the C-terminus of the plant CTB may sequester the protein within the plant ER and may increase stability of the protein (Haq, T., et al, 1995; Wandelt, C, et al 1992).
  • the plant ER may function similarly to the periplasmic space of gram-negative bacteria in providing an intracellular environment in which CTB monomers accumulate and assemble into pentamers. It was previously demonstrated that the disassembled plant-synthesized CTB monomer was approximately 3 kDa larger than the bacterial CTB monomer (15 kDa vs. 12 kDa), which may be due to the presence of the hexapeptide ER retention signal and (or) the 2 l-amino acid leader peptide on the CTB molecule. Many infectious agents which invade through mucosal surfaces can be effectively controlled by parenteral immunization, however, oral immunization is more effective for certain enteric diseases, resulting from V. cholerae and salmonellae infections.
  • GM1+ is unlikely to result from competition between mouse anti-CTB antibody and primary rabbit anti-CTB antibody for binding sites on the CT molecule, as there was no sigmficant difference in RLU signal levels when CT was mixed with either immune or nonimmune serum prior to addition to microtiter plate wells not coated with G M1 -ganglioside (Fig. 10, GM1-).
  • immunological protection against CT may be due predominantly to anti-CTB antibody- mediated specific prevention of CT binding to cellular G M ⁇ -ganglioside.
  • the molecular mechanism responsible for inhibition of CT binding to G M1 -ganglioside may rely on conformational changes induced in the B subunit of cholera holotoxin by CTB-specific antibodies.
  • mice fed 1 g of potato tissue exhibited detectably lower systemic and mucosal antibody titers, Vero cell neutralization titers and intestinal protection from cholera toxin in comparison with mice gavaged with 30 ⁇ g of bacterial CTB.
  • plant-delivered CTB is less effective in immune stimulation than the same amount of bacterial CTB, which could be due to differences in antigen delivery i.e., gavage versus chewing and prolonged digestion of plant tissues consumed intermittently over a several hour period.
  • plant tissues may contain factors which interfere with antigen presentation to gut-associated lymphoid tissue (GALT) (Haq, T., et al, 1995).
  • the sodium bicarbonate buffer used for bacterial CTB gavage to neutralize stomach acid might have contributed to reduction in CTB pentamer disassembly, or possibly the presence of trace amounts of holotoxin in commercial CTB preparations may have enhanced immune response.
  • An additional explanation for the differences in immunogenicity in mice, without apparent differences in antigenicity in vitro between bacterial CTB and plant CTB, may be the differences in amino acid sequences between the two CTB forms, especially presence of the hexapeptide sequestration sequence at the C-terminus and possible retention of the 2 l-amino acid leader peptide at the N-terminus of the plant CTB molecule.
  • mice fed 3 g of potato exhibited a higher toxin neutralization titer than mice fed 1 g of potato (Fig. 8B and Table 1).
  • bacterial CTB was expected to provide the highest levels of intestinal protection.
  • mice fed 3 g of potato showed a slightly higher level of intestinal protection than mice given bacterial CTB (Figs.
  • CTB is not a strong adjuvant for co-administered antigens, due to its ability to bind to G M1 -ganglioside on the surface of mammalian intestinal epithelial cells especially M cells of the GALT, it can function as an effective carrier for induction of an increased mucosal immune response to polypeptides to which CTB is chemically or genetically conjugated (Svennerholm, L., 1986; Czerkinsky, C, et al, 1989; Dertzbaugh and Elson, 1993; Holmgren, J., et al, 1994).
  • CTB pentameric CTB in food plants is not necessarily confined to development of an anti-diarrheal vaccine against cholera enterotoxin; CTB may be even more useful for providing safe and cost-effective mucosal immunization against other enteric pathogens which can be effectively controlled by recombinant subunit vaccines.
  • Increased immunogen concentration at the mucosal lymphoid tissues may reduce the requirement for high levels of antigen biosynthesis in food plants.
  • CTB has recently been shown to function as a carrier molecule for conjugated peptides for induction of immunological tolerance (Bergerot, I., et al, 1997; Sun, J., et al, 1996; Sun, J., et al, 1994).
  • food plant CTB-based oral vaccines may open the way for a novel food plant-based therapeutic approach for prevention of autoimmune diseases such as type I diabetes (Bergerot, I., et al, 1997) and encephalomyelitis (Sun, J., et al, 1996).
  • Potato is an excellent species for experimental study in the newly emerging field of edible plant-based oral vaccines, because potato tissues are relatively easily transformed and regenerated into plants.
  • Tubers are relatively rich in proteins (over 4% soluble protein), and experimental animals (i.e., mice) readily consume raw tubers. Humans, however, favor cooked potatoes which may result in extensive destruction of vaccine antigens especially heat- labile proteins such as LTB and CTB.
  • a novel result of these animal immunization experiments is the indication that antibody titers can be boosted by oral administration of additional vaccine food plant tissues when the protective titer declines.
  • food plants grown in tropical and semi- tropical regions of the world can provide a continuous source of oral vaccine for the inevitable booster dose.
  • Application of the CTB pentamer as an effective carrier for conjugated peptides in food plants will move us closer to achievement of a low-cost, convenient, effective, and safe strategy for prevention of infectious enteric diseases in animals and in man, especially in regions of the economically emerging world where conventional vaccines are unaffordable as well as unavailable.
  • the present invention has been described with reference to particular examples for purposes of clarity and understanding.

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Abstract

Selon cette invention, un gène codant la protéine de la sous unité de la toxine B du choléra (CTB) est introduit dans un vecteur d'expression végétal. Dans un mode préféré de réalisation, le gène CTB est fusionné à un signal de retenue (SEKDEL) de l'ergastoplasme jouxtant le promoteur de la mannopine synthase P2, et possède un gène marqueur luciférase bactérien (lux F) lié à un promoteur P1. Des explants de feuilles de pommes de terre transformés par Agrobacterium tumefaciens portant ce vecteur et les plants résistant à la kanamycine sont régénérés. Le CTB végétal est indiscernable du CTB bactérien sur le plan antigénique, et les molécules CTB oligomériques (Mr ∩ 50 kDa) sont les espèces moléculaires dominantes isolées à partir des tissus et des tubercules transgéniques de la feuille de pomme de terre. La quantité de CTB maximale détectée dans des tissus transgéniques, induits par auxine, des feuilles et des tubercules de pommes de terre représente environ 0,3 % de la protéine végétale soluble totale. Les anticorps sériques et intestinaux spécifiques au CTB sont induits chez la souris immunisée par voie orale. Le titre d'anticorps dans les muqueuses diminue progressivement après la dernière immunisation mais se reconstitue avec un stimulant oral à base de pomme de terre transgénique. L'expression du CTB oligomérique avec des propriétés biochimiques et immunologiques identiques à celles du CTB endogène chez les plantes comestibles permet d'établir des vaccins oraux peu onéreux à base de plantes, protégeant contre le choléra et d'autres agents pathogènes sévissant dans les régions endémiques à travers le monde.
PCT/US1998/021237 1997-10-07 1998-10-07 Expression de la sous-unite de la toxine b du cholera chez les plantes transgeniques et son efficacite dans les vaccins oraux Ceased WO1999018225A1 (fr)

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WO2001052886A1 (fr) * 2000-01-21 2001-07-26 Alfred Hospital Strategie de vaccination « prime-boost » (primo-immunisation-rappel)
WO2001072959A3 (fr) * 2000-03-01 2002-05-10 Univ Auburn Proteines pharmaceutiques, agents therapeutiques humains, albumine serique humaine, insuline, et toxique b de cholera natif soumis a des plastes transgeniques
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WO2005096806A1 (fr) * 2004-04-09 2005-10-20 Toudai Tlo, Ltd. Plant de riz comportant un gène de vaccin transféré dans celui-ci
US7041296B1 (en) * 1999-11-12 2006-05-09 The United States Of America As Represented By The Department Of Health And Human Services Methods of treating inflammatory bowel disease using cholera toxin B subunit
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US7741536B2 (en) 1997-08-07 2010-06-22 University Of Central Florida Research Foundation, Inc. Expression of human serum albumin in plastids
CN102580118A (zh) * 2012-02-24 2012-07-18 重庆大学 志贺样毒素Stx1B口服疫苗的制备方法及其产品
US9080180B2 (en) 2007-07-03 2015-07-14 Idemitsu Kosan Co., Ltd. Transgenic plants expressing STX2EB protein for use as a pig edema disease vaccine
US9657302B2 (en) 1998-05-15 2017-05-23 The Trustees Of The University Of Pennsylvania Expression of human interferon in transgenic chloroplasts
US10689633B2 (en) 2008-02-29 2020-06-23 The Trustees Of The University Of Pennsylvania Expression of β-mannanase in chloroplasts and its utilization in lignocellulosic woody biomass hydrolysis

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Cited By (16)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7741536B2 (en) 1997-08-07 2010-06-22 University Of Central Florida Research Foundation, Inc. Expression of human serum albumin in plastids
US9657302B2 (en) 1998-05-15 2017-05-23 The Trustees Of The University Of Pennsylvania Expression of human interferon in transgenic chloroplasts
US7041296B1 (en) * 1999-11-12 2006-05-09 The United States Of America As Represented By The Department Of Health And Human Services Methods of treating inflammatory bowel disease using cholera toxin B subunit
WO2001052886A1 (fr) * 2000-01-21 2001-07-26 Alfred Hospital Strategie de vaccination « prime-boost » (primo-immunisation-rappel)
WO2001072959A3 (fr) * 2000-03-01 2002-05-10 Univ Auburn Proteines pharmaceutiques, agents therapeutiques humains, albumine serique humaine, insuline, et toxique b de cholera natif soumis a des plastes transgeniques
EP1522585A1 (fr) * 2003-10-09 2005-04-13 Plant Research International B.V. Molécules porteuses chimères pour la production de vaccins absorbés au niveau des muqueuses
WO2005033317A1 (fr) * 2003-10-09 2005-04-14 Plant Research International B.V. Molecules porteuses chimeres utilisees dans la production de vaccins des muqueuses
WO2005096806A1 (fr) * 2004-04-09 2005-10-20 Toudai Tlo, Ltd. Plant de riz comportant un gène de vaccin transféré dans celui-ci
JPWO2005096806A1 (ja) * 2004-04-09 2008-02-21 独立行政法人農業生物資源研究所 ワクチン遺伝子導入イネ
JP4769977B2 (ja) * 2004-04-09 2011-09-07 独立行政法人農業生物資源研究所 ワクチン遺伝子導入イネ
EP1685848A1 (fr) * 2005-01-26 2006-08-02 Plant Research International B.V. Vaccins oraux pour poissons
US9080180B2 (en) 2007-07-03 2015-07-14 Idemitsu Kosan Co., Ltd. Transgenic plants expressing STX2EB protein for use as a pig edema disease vaccine
EP2169054B1 (fr) * 2007-07-03 2015-12-23 Idemitsu Kosan Co., Ltd. Vaccin pour maladie de l' edème porcine
US10689633B2 (en) 2008-02-29 2020-06-23 The Trustees Of The University Of Pennsylvania Expression of β-mannanase in chloroplasts and its utilization in lignocellulosic woody biomass hydrolysis
CN102580118A (zh) * 2012-02-24 2012-07-18 重庆大学 志贺样毒素Stx1B口服疫苗的制备方法及其产品
CN102580118B (zh) * 2012-02-24 2014-03-12 重庆大学 志贺样毒素Stx1B口服疫苗的制备方法及其产品

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