WO1996040229A1

WO1996040229A1 - Vaccines synthesized by transgenic plants

Info

Publication number: WO1996040229A1
Application number: PCT/US1996/009558
Authority: WO
Inventors: Hilary Koprowski; Douglas Craig Hooper; John Hammond; Peter Bruce Mcgarvey; Frank H. Michaels
Original assignee: Thomas Jefferson University; UNITED STATES OF AMERICA, represented by its SECRETARY OF AGRICULTURE
Priority date: 1995-06-07
Filing date: 1996-06-06
Publication date: 1996-12-19

Abstract

A transgenic process for inducing protective immunity in which the gene for an immunogenic protein is transferred to a plant such that the resulting transgenic plant will express the gene in a form capable of inducing a protective immune response in an animal. Also the use of a transgenic plant is used to confer immunologic systemic tolerance in an animal.

Description

VACCINESSYNTHESIZEDBYTRANSGENICPLANTS

Field of the Invention

The invention relates to vaccines synthesized in transgenic plants.

Background

Human and animal diseases are most effectively treated by prevention.

Although sanitary practices such as clean water and effective sewage isolation contribute to general health, the development of safe, efficacious, and long-lasting vaccines is perhaps the single most effective approach available for the prevention of disease.

Conventional vaccine technology, however, has serious limitations for many applications. Because of the advanced technology required for vaccine production, only technologically advanced societies have ready access to the medicines. Further, administration of conventionally produced vaccines requires refrigeration to preserve the potency of the product, skilled para-medical professionals for administration, and repeat visits for sequential booster inoculations. Oral vaccines in particular require repeated administration in order to maintain immunity.

An effective plant-based system would have several advantages over traditional vaccine production systems including: 1) a significant reduction in expense of production of vaccines,

2) the ability to vaccinate recipients through prolonged exposure to the vaccines, a technology not currently practical, 3) the ability to produce vaccine crops locally, obviating the need for high technology production centers and the associated medical infrastructure required for administration,

4) a further advantage is that transgenic plants do not have the added risk of pathogens associated with animal cells used for vaccine production. Many viral vaccines are produced in cells of human or simian origin, and the risk of concomitant culturing of unrecognized pathogens which may be included in the vaccine preparation is a constant threat.

5) Possible increase in effectiveness because oral administration of a vaccine more closely mimics the natural entry route of certain pathogens and first contact with the host's immune system. Thus oral vaccines may prove more effective than injected vaccines at preventing an initial infection through the mucosal surfaces of the body.

C. J. Arntzen et al., PCT application, PCT/US94/02332, reported the transformation of tomatoes with the gene for hepatitis B surface antigen (HBsAg) such that the tomatoes synthesized HBsAg precipitable with anti-HBsAg antibodies.

The transformation of tobacco plants with the gene for HBsAg, such that the plants synthesized HBsAg precipitable with monoclonal anti-HBsAg was demonstrated by H.S. Mann et al. (PNAS 89, pp 11745-11749, 1992; see also R.E. Dittman,

"Eradication, Disease, Trends in Vaccine Development Priorities," Summer 1993, pp40-42).

C. J. Arntzen et al., in Vaccines 94, pp 339-344 (1994) implied that they would eventually publish results describing the oral immunogenicity of receptor- binding E. coli heat-labile enterotoxin accumulated in transgenic plants. Similarly, the intent of another group to test alfalfa, genetically engineered to produce cholera toxin, as a vaccine in mice was described by R. Taylor (The J. of NIH research, 5, pp 49-53, 1993).

Usha et al. in Virology 197, pp 366-374 (1993) showed that cowpea mosaic virus (CPMV) could be engineered to contain an epitope of the viral coat protein of foot-and-mouth disease virus (FMDV). Plants infected with CPMV produced protein that reacted with FMDV-specific antiserum. Similar experiments were reported by G. P. Lomonossoff et al. (PCT/GB92/00589). An intent to test the vaccination capability of such CPMV/FMDV virus particles was implied in a publication "A plant-based system for the production of animal vaccines" (1992) of Agricultural Genetics Company Limited, as was an intent to test in mice CPMV particles expressing a Human Immunodeficiency Virus (HIV) epitope.

R. Curtiss et al. (PCT/US89/03799) transformed E. coli to synthesize surface protein antigen A (SpaA protein), heat-killed and lyophilized the E. coli, and fed the lyophilized bacteria to mice, and then observed elevated levels of anti-SpaA slgA. In none of the above-mentioned references was the administration of a transgenic plant vaccine demonstrated to induce protection against a pathogen.

We disclose a method of vaccine production and delivery based on the expression of antigenic proteins in transgenic plants. In a model system we have developed, the plants express a primary antigenic protein (rabies glycoprotein, Rgp) from rabies virus.

Brief Summary of the Invention

The present invention, in one general aspect, is a transgenic process for inducing protective immunity: A gene for an immunogenic protein that is part of a pathogen is transferred to a plant, thereby creating a transgenic plant that synthesizes the protein, and the plant or subportion thereof is administered to a mammal, bird, or fish, so as to induce protective immunity against the pathogen. In a related aspect of the invention, the transgenic plant is used to confer systemic tolerance in relation to an antigen that triggers a pathological response in a mammal. The induction of systemic tolerance is useful in the treatment of diseases such as multiple sclerosis.

Brief description of the Drawings

Fig. 1. (Composed of Figs. IA, IB, IC, and ID). Schematic drawings of Agrobacterium vectors referred to in the Examples.

Fig. 2. (Composed of Figs. 2A and 2B). Schematic drawings of recombinant constructs referred to in the Examples. Fig. 3. (Composed of Figs. 3 A and 3B). Two recombinant plasmids referred to in the Examples.

Detailed Description

GLOSSARY AND DISCUSSION OF TERMS USED

A "plant" for purposes of this patent application include liverworts (Hepaticae), mosses (Musci), psilopsids (Psilopsida), club mosses (Lycopsida), horsetails (Sphenopsida), ferns and seed plants, and certain fungi specified below. Ferns and seed plants together make up the Pteropsida. Seed plants include gymnosperms (Gymnospermae) and angiosperms (Angiospermae). The great majority of plants used for food are angiosperms. For purposes of this application, the following fungi are considered plants: Basidiomycetes, which include mushrooms. The following are not considered plants for purposes of this application: algae, bacteria, blue-green algae, single-celled eukaryotes, and the following fungi: Phycomycetes, Ascomycetes (yeasts), and Deuteromycetes.

The term "plant tissue" includes any tissue of a plant. Included are whole

plants, any part of plants, plant cells, plants seeds, and plant protoplasts.

A "bird" is a warm-blooded vertebrate of the class Aves.

A "fish" is a cold-blooded aquatic vertebrate, having gills and fins.

A "chimeric protein" is created when two genes that normally code for two separate proteins recombine, either naturally or as the result of human intervention, to make a protein that is a combination of all or part of each of those two proteins.

A "protein of a pathogen" is a protein that is coded for by the genetic material of that pathogen.

A "naturally occurring plant protein" is one that is normally found in a plant, at least one stage in its life cycle, in its natural habitat. "Transforming a plant cell" means adding one or more genes that it does not naturally have.

"Protective immunity" is the ability of an animal, such as a mammal, bird, or fish, to resist, as the result of its exposure to the antigen of a pathogen, disease and/or death that otherwise follows contact with the pathogen. Protective immunity is achieved by one or more of the following mechanisms: mucosal, humoral, or cellular immumty. Mucosal immunity is primarily the result of secretory IgA (sIGA) antibodies on mucosal surfaces of the respiratory, gastrointestinal, and genitourinary tracts. The sIGA antibodies are generated after a series of events mediated by antigen-processing cells, B and T lymphocytes, that result in sIGA production by B lymphocytes on mucosa-lined tissues of the body. Mucosal immunity can be stimulated by an oral vaccine.

"Humoral immunity" is the result of IgG antibodies and IgM antibodies in serum. "Cellular immunity" can be achieved through cytotoxic T lymphocytes or through delayed-type hypersensitivity that involves macrophages and T lymphocytes, as well as other mechanisms involving T cells without a requirement for antibodies. A "derivative cell" derived from a transgenic plant cell is one created as a result of the transgenic plant cell undergoing cell division or a series of cell divisions such that one or more copies of the foreign gene that transformed a plant cell to make a transgenic cell is in the derivative cell.

ASPECTS OF THE INVENTION

In a general aspect, the invention is a transgenic process of inducing protective immunity against a pathogen in an animal (especially a mammal, bird, or fish) the process comprising the steps of:

(1) transforming a plant cell with a gene coding for a protein of a pathogen, thereby creating a transgenic cell;

(2) cultivating the transgenic cell, or a derivative cell derived from said transgenic cell, under conditions where said transgenic cell or derivative cell makes the protein; and (3) administering the protein or an antigenic portion thereof to an animal so as to induce protective immunity response in the animal against the pathogen; wherein, in steps (1) and (2), the transgenic cell or derivative cell may be part of a plant or plant tissue or may be free of other plant cells; and wherein, in step (3), the protein or antigenic portion thereof may be part of the transgenic or derivative cell, part of an extract of such a transgenic or derivative cell or (as a result of protein purification) free of other material normally present in said transgenic or derivative cell.

The protein purification steps would be ones commonly used for the fractionation of tomatoes into its protein components and the separation of individual proteins from other components of the transgenic or derivative cell. Such steps include protection of the native conformation of the transgenic protein by steps such as flash freezing the transgenic protein containing plant material with dry ice or liquid nitrogen. Subsequent steps would include mechanical homogenization of the frozen tissue and solubilization in a cold aqueous solvent containing a non-ionic detergent and compounds which inhibit proteolytic degradation of the proteins. Particulate material is removed by sedimentation, centrifugation and filtration. The solubilized transgenic protein is concentrated by precipitation with ethanol or another appropriate organic solvent, and further purified by either preparative high performance liquid chromatography or immunoaffinity chromatography. The route of administration in step (3) can be parenterally or nonparenterally. If administered parenterally, the protein to be administered to the mammal will preferably be substantially pure of other material found in the plant cells that produced it . An extract would be created by a process comprising mechanical or chemical disruption of a cell. In some cases, additional protein purification steps would be used to make the extract.

In one important embodiment of the invention, in step (3) the protein is part of the transgenic or derivative cell, which are part of a plant or plant product and is fed to the mammal (i.e. oral route of administration). In such a case, it is highly preferable that the plant be edible is raw; i.e., has not been cooked (heated above the temperatures associated with growth, storage, and transport). In the examples described below, the plant (tomato) is not cooked. Animals typically may eat the plant, pieces of the plant, a puree from the plant, or plant juice. It is frequently preferred that in the transgenic production process, step (2) takes place in an edible plant or part of an edible plant.

Animals vary as regards which food is edible. Plants of greatest interest include potatoes, tomatoes, peas, beans, alfalfa, citrus fruits (e.g., oranges, lemons, grapefruit), grapes, carrots, strawberries, blueberries and other berries, bananas, rice, wheat, corn ,barley, oats, rye, dates, cabbage, Brassel sprouts, cauliflower, turnips, cucurbits, papaya, guava, apples, cherries, apricots, and pears.

In another important embodiment of the invention, the protein of interest is extracted in purified form from the plant and administered as a substantially pure protein (possibly with an adjuvant or other compounds needed to facilitate or improve vaccine administration.)

As in the case of the rabies virus G protein (Rgp protein), the protein of the pathogen may be one that is a glycoprotein in the pathogen. In this case, the plant is expected to add a sugar moiety to the protein, but probably a moiety substantially different from those added during post-translational processing in mammalian cells.

In a preferred aspect of the transgenic production process, step (1) is achieved by means of a DNA plasmid that comprises the gene coding for the protein and a promoter that is functional in the plant cell.

A particularly preferred process is one in which step (1) is mediated by Agrobacterium tumefaciens (A. tumefaciens) .

In a preferred embodiment, the transformation of the plant cell in step (1) results in the gene coding for the protein of the pathogen being under the control of a promoter such that the promoter activates the gene only under conditions of fruit ripening. (Ripening is defined as the final phase in the development of the fruit organ, associated with a sharp change in metabolism. Specific changes associated with ripening include increased respiration, autocatalytic ethylene production, conversion of starch to sugars, and increased activity of cell wall degrading enzymes.

Ethylene plays an important part in regulating the ripening of climacteric fruits such as tomato, and ethylene biosynthesis increases rapidly at the onset of tomato fruit ripening (Deikman and Fischer, EMBO J. 7:3315-3320, 1988).) Examples of such a promoter are the E8 promoter in tomatoes, which is ethylene-responsive in an organ-specific fashion. E8 mRNA is abundant in ripe tomato fruit, but is not detected in leaf, root or stem organs; at the onset of fruit ripening, E8 mRNA concentration and the level of ethylene increase concurrently. Exposure of unripe tomato fruit to ethylene results in rapid accumulation of E8 mRNA (Lincoln and Fischer, Mol. Gen.

Genet. 212:71-75 (1988); Deikman and Fischer, EMBO J. 7:3315-3320 (1988)).

When such a promoter is used, the unripe fruit can be stored in a more biologically and mechanically stable form than ripe fruit. Expression of the gene or genes can then be induced at will. Also the ability to focus transgene expression to a specific part of the plant aids control of the environmental and commercial distribution of the plant product. Ethylene-responsive promoters such as the E8 promoter are particularly suitable.

In another general aspect, the invention is a transgenic process for producing an immunogen, the process comprising the steps of:

(2) cultivating the transgenic cell, or a derivative cell derived from said transgenic cell, under conditions where said cell or derivative cell makes the protein, such that the protein is capable of inducing protective immunity against the pathogen in an animal (especially a mammal, bird, or fish).

In step (1) of the above noted processes, the plant cell can be in a plant, a piece of a plant, a plant seed, in tissue culture. If in tissue culture, the plant cell can be either attached or unattached to other plant cells.

Pathogens of interest

A pathogen is any organism such as a virus, bacterium, fungus, and parasites or a protein which is capable of self-replication such as a prion and capable of inducing disease in an animal.

Pathogens against which transgenic plant vaccines are effective are those including but not limited to bacteria of the genera streptococci and staphylococci, as well as the mycoplasma, rickettsia and spirochetes. The following viral groups of the parvoviridae, papovaviridae, adenoviridae, herpesviridae, poxviridae, iridoviridae, picornaviridae, caliciviridae, togaviridae, caliciviridae, flaviviridae, coronaviridae, ortho- and paramyxoviridae, rhabdoviridae, bunyaviridae, reoviridae, birnaviridae, and the retroviruses all contain representative members with application for transgenic plant vaccines.

The primary result of protective immunity is the destruction of the pathogen or inhibition of its ability to replicate itself.

Pathogens against which transgenic plant vaccines are expected to be particularly useful are rabies, respiratory syncytial virus, cholera, typhoid fever, herpes simplex types I and II, tuberculosis, pathogenic pneumococci, human immunodeficiency virus-1 (HIV-1) and human immunodeficiency virus-2 (HIV-2). The spectrum of pathogens includes those of veterinary significance and includes parvoviridae, papovaviridae, adenoviridae, herpesviridae, poxviridae, iridoviridae, picornaviridae, caliciviridae, togaviridae, caliciviridae, flaviviridae, coronaviridae, ortho- and paramyxoviridae, rhabdoviridae, bunyaviridae, reoviridae, birnaviridae, and the retroviruses. Both gram negative and gram positive bacteria and spirochetes are also expected to be pathogens which may be clinically affected by the transgenic plant vaccines.

Methods for transferring genes to plants Genes may be transferred by any of a variety of means, which means include but are not limited to:

1) Agrobacterium plasmid-mediated gene transfer, for example as described herein. This method has been applied to many species including but not limited to potatoes (F. T. Truve et al., Bio /Technology U, 1048 (1993); tobacco (W. R. Streber et al., Bio/Technology 7, 811 (1989); grapes (M. G. Mullins et al., Bio/Technology

8, 1041 (1990); plums (S. Mante et al., Bio/Technology 9, 853 (1991); melon (J. Z.

Dong et al., BioTechnology 9, 859 (1991); lettuce (L. Penarrubia, Bio/Technology

10, 561); tomatoes (L. Penarrubia, Bio/Technology 10, 561); and sugarbeets (K.

D'Halluin, Bio/Technology 10, 309 (1992). 2) Microparticle bombardment, for example as described for wheat by V. Vasil et al. , Bio/Technology 9, 743 (1991) and generally in Bio/Technology 10, 286 (1992).

3) Electroporation, for example as described for lettuce by M. C. Chupeau et al., Bio/Technology 7, 503 (1989).

4) Liposome fusion with protoplasts (A. Deshayes et all., EMBO J. 4, p2731- 2737(1985)).

5) Polyethylene glycol-mediated transformation (I. Potrykus et al., Mol. Gen Genetics, 197, 183-188).

6) Microinjection (R. Griesbach, Biotechnology 3, p348-350; C.K. Shewmaker Mol. Gen. Genetics, 202 p. 179-185 (1986)). 7) Viruses; e.g. tobacco mosaic virus (N. Takematsu et al., EMBO J. 6 p 307-

311) and geminivirus (A. Ward et al., EMBO J. 7 1583-1587 (1988)).

It may be desirable to target protein expression to different plant tissues (i.e. fruit and leaves) and to different intracellular locations (i.e. chloroplast, vacuole, plasma membrane) in order to enhance protection of the transgenic proteins from degradation in the gastrointestinal tract and also to present the antigen to the gut as part of larger structure for uptake by the M cells of the Peyers patch.

It may be desirable to combine presentation of the primary antigens with a biologically active molecule that will stimulate and or enhance an immune response and serve as an adjuvant. Since transgenic plants can be crossed genetically, plants expressing an adjuvant protein and a primary antigen can be produced separately and combined to deliver antigen and adjuvant in one plant.

Examples

Media and solutions used in Examples

Media For Tomato Transformation CM (Callus Media): 1 x MS salts; m-inositol at 100 mg/L ; 2,4-D (2,4- dichlorophenoxyacetic acid) at 0.2 mg/L; thiamin at 1.5 mg/L; kinetin at 0.2 mg/L; KH₂PO₄ at 200 mg/L; sucrose at 30 g/L; Phytagel (Sigma) at 2.5 g/L; pH 5.8.

MSO: 1 x MS Salts; 1 x Nitsch & Nitsch vitamin solution; sucrose at 30 g/L; pH 5.8 (for solid add Phytagel at 2.5 g/L)

SMK (Shoot Media + Kanamycin): 1 x MS salts; sucrose at 30 g/L; 1 x Nitsch & Nitsch vitamin solution; Zeatin at 2.0 mg/L; GA₃ at 0.2 mg/L; Phytagel (Sigma) at 2.5 g/L; Kanamycin 100 mg/L (for Kanamycin resistant vectors only); Antibiotics (carbenicillin and cefotaxime at 500 mg/L or 100 mg/L); pH 5.8.

PRK (Prerooting Media + Kanamycin): 0.5 x MS salts; sucrose at 15 g/L; 1 x Nitsch & Nitsch vitamin solution (J.P. Nitsch and C. Nitsch, Science 163 p. 85 (1969))

Zeatin 2.0 mg/L;

GA₃ (Gibberellic acid) at 0.2 mg/L; Phytagel (Sigma) at 2.5 g/L; Kanamycin at 100 mg/L (for Kanamycin resistant vectors only);

Antibiotics (carbenicillin and cefotaxime at 100 mg/L); pH 5.8.

Root Media 1: 0.5 x MS salts; sucrose at 5 g/L; 1 x Nitsch & Nitsch vitamin solution; IBA (indole-3-butyric acid) at 1 mg/L; Phytagel (Sigma) at 3.0 g/L; Antibiotics (carbenicillin and cefotaxime at 50 mg/L); pH 5.8.

Root Media 2: 0.5 x MS salts; sucrose at 5 g/L; 1 x Nitsch & Nitsch vitamin solution; Phytagel (Sigma) at 3.0 g/L; Kanamycin at 25 mg/L; Antibiotics (carbenicillin and cefotaxime at 50 mg/L); pH 5.8.

In preparing media, add salts, sucrose, Phytagel, and water, then adjust pH and autoclave. After cooling, add vitamins, hormones, and antibiotics. "pH 11.5 buffer" for immunoaffinity column elution is 50 mM triethanolamine,

0.15 M NaCl, 1 % Triton X-100, pH about 11.3.

"Complete Freunds adjuvant" is a well known mixture of light weight mineral oil and heat killed Mycobacte um tuberculosis.

ELISA wells (Immunolon 4 plates from Dynatech) are coated with a 10 microgram/ml solution of UV-inactivated rabies virus or purified rabies glycoprotein in phosphate buffered saline.

"Normal saline" is physiological saline: 0.9% sodium chloride in water.

Media for BHK cells is Dulbeccos modified Eagles Minimal Essential Medium containing 10% heat-inactivated fetal calf serum, supplemented with L-glutamine and antibiotics.

EXAMPLE 1 Construction of Vectors and Method of Plant Transformation

Construction of Agrobacterium Cloning Vectors.

A binary Agrobacterium Ti vector was modified for use as a transformation vector and is shown schematically in Fig. 1. The vector contains the 35S promoter from Cauliflower Mosaic Virus (CaMV) which is highly expressed in most plant tissues; a multiple cloning site; a transcription termination site; and the neomycin phosphotransferase gene (NPTII) which confers kanamycin resistance when expressed in plants. The vector also contains the 3-glucuronidase (GUS) gene under control of the 35S promoter and is almost identical to the vector pBI121 (R. A. Jefferson et al., EMBO J., 6, 3901 (1987) except that the 35S-GUS cassette is in the reverse orientation in relation to pB121. The RG-2 vector was modified as follows: The GUS gene was excised by digestion with Smal and Sstl, treated with T4 DNA polymerase to remove overhanging ends and religated to form the vector RG-G (see Fig. 1). The E8 promoter is only active in ripening tomato fruit (J. Deikman and R.L.

Fischer, EMBO J., 7 p 3315-3320 (1988)). The plasmids pE8mutRN2.0(+) and pE8mutRN2.0(-), both shown schematically in Fig. 3, containing 2 kb of the E8 promoter cloned between the EcoRI and BamHI site of PUC118 were obtained from Dr. Robert Fischer of the University of California, Berkeley, and are described in Fig. 3. The (+) construct has an Ncol site (absent in the (-) construct) which contains an AUG translation initiation codon used to initiate translation from this construct and its derivatives. The (-) construct uses an AUG in the cloned insert. The two E8 promoter constructs were engineered into Ti- vectors as follows. The plasmids pE8mutRN2.0( +) and pE8mutRN2.0( -) were cut with EcoRI and Pstl and the E8 promoter fragment ligated into the plasmid Bluescript II SK(+) (commercially available from Stratagene, La Jolla, California) cut with the same enzymes to form pE8(+)blue and pE8(-)blue. The E8 promoter was then removed from pE8(+)blue and pE8(-)blue by digestion with Hindlll and BamHI and put into RG-G in place of the 35S promoter to form RGE8(+) and RGE8(-) (see RGE8(+/-), figure 1). In addition, the E8 promoter was removed from pE8(+)blue and pE8(-)blue by digestion with Hindlll and Sstl and put into RG-2 in place of the 35S promoter and GUS to form RGE8(+)B and RGE8(-)B (see RGE8(+/-)B, figure 1).

Cloning of Rabies Glvcoprotein into Ti-vectors.

A cDNA of the rabies glycoprotein (Rgp) from the ERA strain of rabies virus was obtained in the plasmid pTG155 (M. P. Kieny et al., Nature 312, p 163-166 (1989)). The entire DNA sequence coding for Rgp was removed by digestion with Bglll, inserted into the compatible BamHI sites of pGA643, RG-G, RGE8(+) and RGE8(-) to make the plasmids pGARgp+, RGRgp+, RGE8(+)Rgp+, and RGE8(-)Rgp+ respectively (see figure 2). The orientation of the inserts was confirmed by restriction mapping.

The recombinant Ti vectors RGRgp + , RGE8( + )Rgp + , and RGE8( - )Rgp + , were all transformed into the Agrobacterium tumefaciens strain LBA4404 by direct transformation following treatment with calcium chloride and the addition of 1 microgram of engineered plasmid as referenced in G. An et al., Plant Molecular Biology Manual, S. B. Gelvin and R. Schilperoort eds., Kluwer Academic, Doerdrecht Netherlands, (1988). Incorporation of the plasmids was confirmed by restriction enzyme digests of plasmid DNA purified from transformed Agrobacterium cultures.

Plant Transformation.

Tomato tissue (Lycopersicon esculentum Mill var. UC82b) was transformed using Agrobacterium-mediated plant transformation by a modification of the methods of Fillatelli (J.J. Fillatelli et al., Biotechnology, 6, p726-730 (1987)) and McCormick (S. McCormick et al., Plant Tissue Culture Manual, B6 ppl-9, Kluwer Academic Publishers, Netherlands (1991) outlined as follows:

1) Seeds were sterilized with bleach and germinated 7-10 days on MSO media plus agar (Phytagel).

2) Cotyledons were cut at the tip and stem in MSO media and placed on CM media previously covered with sterile filter paper, sealed with time tape , and stored on a lab bench under fluorescent lights 24-48 hours.

3) Recombinant Agrobacterium were inoculated into 5 -10 ml cultures with selective antibiotic (Kanamycin for RG constructs, tetracyclin for PGA constructs) and grown 24-48 hours at 28° C with shaking. 4) The bacterial cultures were gently pelleted, resuspended in an equal volume of LB Medium (pancreatic digested casein at 10 g/L, autolysed yeast extract at 5g/L, and sodium chloride at 10 g/L) which had been pH-adjusted to 4.5, and to which had been added 2 microliters per/ml of a 0.1 M solution of acetosyringone in dimethyl sulfoxide (DMSO) and the culture was returned to the shaker for 5 hours.

5) The cotyledon tissue from the CM plates was immersed in a 1:20 dilution of the induced bacteria in MSO media for 15-30 minutes.

6) The tissue was removed from the bacterial suspension, gently blotted dry with sterile filter paper, and returned to the CM plates for 24-48 hours. 7) The tissue was transferred from CM plates to SMK plates to induce shoots.

The plates were incubated under a combination of white fluorescent and pink GrowLux fluorescent lights (150-200 lux) at 26° C with a 16 hr light and 8 hr dark photoperiod. The tissue was maintained on SMK plates until shoots form in 2 to 10 weeks. The tissue was transferred to fresh media once a week for two weeks and then once every two weeks. The concentration of carbenicillin and cefotaxime was 500ug/ml each the first week and reduced to lOOug/ml in following weeks if bacterial growth is not a problem.

8) After shoots formed, the shoot and some callus at its base were transferred to PRK plates and development allowed to continue for 1-2 weeks or until they touched the top of the petri plates, whichever occurred first.

9) The shoots were cut with a sharp scalpel at a 45° angle and placed in Root Media 1. Roots developed in 2 to 10 weeks.

10) After the roots developed die tissue was transferred to Root Media 2 and maintained until the plant grew to fill container (2-4 inches). 11) The plant was transferred to wet potting soil and covered with plastic bag under lights for one week. The plant was kept moist but not saturated. After one week the bag was removed without the plant wilting.

Expression of Recombinant Constructs in Transgenic Plants.

Primary transgenic plants regenerated from tissue culture on selective media were screened for the presence of the recombinant genes by PCR amplification of total nucleic acid extracts, using specific primers. (P. McGarvey et al., Biotechniques H pp 428-432.) The primers used to detect the presence of the recombinant constructs in the plant tissues were

5'-CGTTCACATGAGGATGACACC specific for rabies glycoprotein, 5'-CACTGACGTAAGGGATGACG selected to hybridize with 35S CAMV promoter, and 5'-CACCAACGCTGATCAATTCC which was selected to anneal with the E8 promoter. Plants were checked for expression of RNA by Northern blots and protein by Western blots. The location of the recombinant proteins was determined by electron microscopy using immuno-gold labeling.

We have produced transgenic tomato plants containing genes for the rabies glycoprotein (Rgp) under the control of both constitutive and fruit-specific promoters. The plants transformed with the gene for rabies G protein demonstrated expression of that gene as RNA in Northern blots and protein in Western blots of immunoprecipitated protein. Immuno-gold labeling of electron microscope sections from the leaf tissue shows that the Rgp is localized in the cell wall area and golgi bodies of transgenic but not unengineered tomato plants. EXAMPLE 2

Immunization and testing protocol for recombinant rabies glycoprotein isolated from transgenic tomatoes

Approximately 100 micrograms of formalin inactivated whole rabies virus of the CVS fixed strain was emulsified in 1 ml total volume in complete Treunds adjuvant and injected into two para-scapular sites in a rabbit. The animal received a booster injection of another 100 micrograms of fixed virus 3 weeks later, and bi- weekly collections of 50 ml of whole blood commenced 2 weeks later. Serum was collected from the blood collections and stored at -20 degrees F. until used. The serum was enriched for the immunoglobulin fraction by ammonium sulfate precipitation: one volume of whole rabbit serum was mixed with 0.4 volumes of a saturated solution of ammonium sulfate. The precipitated immunoglobulin was dialyzed against PBS to remove the ammonium sulfate, and bound to activated cyanogen bromide sepharose 4B beads. The unbound immunoglobulins were removed by extensive washing with PBS, and a column prepared using the immunoglobulin bearing beads. The tomato or tomato leaf extract previously prepared was applied to the column, and recycled for immunoaffinity trapping of the transgenic protein. The column was then washed with a solution containing 0.1 M tris-HCl, 0.15 M NaCl, and 1 % triton X-100, and adjusted to a pH of 7.6 to remove tomato constituents not specifically recognized by the rabbit antisera. (See Current Protocols in Molecular Biology, F. M. Ausubel et al., eds., John Wiley & Sons, New York (1989)). The proteins specifically bound to the immunoaffinity column are eluted by the application of a pH 11.5 buffer, and the eluate collected.

That the transgenic tomato fruit and somatic tissue were producing protein translated from the mRNA derived from the transgene was demonstrated by electrophoresing the immunoaffinity concentrated extract, followed by blotting to a nitrocellulose membrane. Authentic rabies glycoprotein and an extract from tomatoes that had not been genetically engineered was also electrophoresed and blotted on the same gels. The membrane was then exposed to a diluted monoclonal antibody specific for rabies glycoprotein and die immunoreaction product visualized witii a peroxidase- conjugated anti-mouse antibody. More specifically, ten grams of fresh leaf or 1.0 g of freeze dried ripe fruit pericarp were frozen with liquid nitrogen, ground with mortar and pestle, and homogenized with a Brinkman Polytron in ice cold TSA buffer (TSA= 0.05 M Tris:HCl pH 8.0, 0.15 M NaCl, 0.025% NaN₃) containing 0.5% polyvinylpolypyrrolidone-40, 1.0 ug/ml aprotinin, and, 1.0 mM phenylmethylsulfonylfluoride (PMSF). Leaf tissue was homogenized in 4 ml/gram and freeze dried fruit in 15 ml/gram. One volume of cold TSA containing 2% Triton X-100 was added and mixed on ice for 1 hr. The mixture was filtered through a layer of Miracloth and centrifuged at 4,000 x g for 10 min. at 4°C. 0.1 volumes of a 10% sodium deoxycholic acid solution was added to the supernatant, mixed on ice for 10 min. , and centrifuged at 100,000 x g for 1 hr in a Beckman SW28 rotor. The protein concentration of the supernatant was determined using a Bio-rad protein determination kit (approximately 5 mg/ml in leaf and 2.5 mg/ml in fruit) and die extract stored at -80°C. For immunoprecipitation 40 ml of extract was first precleared with 30 ul of normal rabbit sera and 300 ul of a 10% solution of fixed S. aureus cells washed with TSA as described (Molecular Cloning. (1989), Sambrook, J., Fritsch. E.F., and

Maniatis, T. Editors, Cold Spring Harbor Laboratory Press, N.Y.). The precleared extract was immunoprecipitated using 10 ul of rabbit sera specific for rabies virus glycoprotein. The solution plus sera was gently mixed on ice for 12 h and the IgA fraction precipitated with 150 ul of S. aureus cells. The cells were washed once with cold TSA containing 1 % triton X-100, once with TSA, and once witii 10 mM

Tris:HCL (pH 7.5) 1 mM EDTA. The cell pellet was suspended in 30 ul SDS loading buffer, electrophoresed through a 7.5% SDS-PAGE gel, and electroblotted onto a nitrocellulose membrane as described (Molecular Cloning. (1989), Sambrook, J., Fritsch. E.F., and Maniatis, T. Editors, Cold Spring Harbor Laboratory Press, N.Y.). For western blotting the membrane was blocked with 5% non-fat dehydrated

milk in 1 x PBS overnight, incubated for 1 h in the same solution containing a 1:500 dilution of a mouse monoclonal antibody to rabies virus glycoprotein and developed using an anti-mouse Vectastain ABC kit and 3,3 'Diainobenzidine tetrahydrochloride. The results of these experiments showed that a specific immunoreactive product of approximately the same molecular weight was present in the extracts of transgenic tomato fruit and the somatic tissue, and in the lanes which had received authentic rabies glycoprotein. The product was absent in the extracts of tomato fruit and tissue from non-engineered plants. Following the demonstration of immunoreactive protein in the eluate, the positive fractions are pooled, and concentrated by lyophilization.

An aliquot of the concentrated fraction representing approximately 1 microgram of the immunoaffinity purified rabies glycoprotein transgenic fruit extract is emulsified with an equal 50 microliter volume of complete Freunds adjuvant and is administered by intraperitoneal injection to each of several outbred Swiss Webster mice. After 7-14 days, an additional intraperitoneal injection of approximately the same amount as before of the immunoaffinity purified protein is given to each mouse as a booster. Following a 21-day immunization period, each animal is bled for 0.25 ml of blood, and the serum fraction tested for the presence of anti-rabies G protein antibodies (IgG) by ELISA as described in Example 3, and by the ability of serial dilutions to neutralize infectious rabies virus in an in vitro assay. The serum neutralization assay is performed as follows:

Infectious rabies virus is serially diluted starting with a concentration of the pathogen previously determined to have a titer of 100 TCID₅₀ (50% tissue culture infectious doses). Serial 1:3 dilutions are placed in a volume of 0.2 ml in sterile tubes, and a fixed volume of 10 microliters of mouse serum added to each tube. The mixtures are incubated for 1 hour at 37°, and then added to cultures of BHK cells which support rabies virus replication in vitro. After addition of virus/serum mix, the cultures are incubated for 20 hours at 37°C, fixed with acetone, and stained with fluorescein-conjugated rabies virus specific antibodies. The cultures are read with a fluorescent microscope and neutralizing antibody titer is determined by the reduction in the percentage of fluorescent cells and international units of virus neutralizing activity are determined by comparison with the results obtained with Standard Reference Serum.

EXAMPLE 3

Testing the immune status of mice following oral administration of rabies protein transgenic tomatoes.

Experiments are done using groups of 5 Swiss Webster outbred mice. Test groups of mice are administered recombinant or native tomato by gastric tube, and by simply permitting the test animals unrestricted access to the tomato tissue. For those animals receiving tomato by gastric tube, doses will range from 50 microliters to 250 microliters of homogenized tomato fruit per mouse every third day for a total of 5 doses. One set of test mice is used to test live rabies virus protection, and other groups of animals is used to quantify the mucosal and systemic immune response.

Mucosal immunity can be quantified by a number of means including: 1) Direct measurement of specific Ig in intestinal secretions by ELISA; 2) Quantitation of specific Ig producing cells from the Peyer's patches and lamina propria of the gut. Intestinal secretions can be harvested from mice using the technique of Elson, et al. (J. Immunol. Methods, 67, 101-108 1984) or by washing intestine through with a solution containing soybean trypsin inhibitor, EDTA, PEG, Na Azide and buffers. In either case the secretions are clarified by centrifugation and specific antibody content assessed in ELISA using plates coated with inactivated rabies virus (10 ug/ml in PBS) to trap the antibody and peroxidase-conjugated anti-mouse IgA (isotype specific) and tetramethyl-benzidine dihydrochloride as a substrate. The reaction is stopped with H₂SO₄ and titers are recorded as the inverse of the dilution giving half- maximal O.D. at 450 nm. For analysis of cells producing specific antibody, Peyer's patches are excised from intestine (cells may be prepared from lamina propria by enzyme digestion once Peyer's patch have been removed, the rest of the method is the same) and cultured overnight at different numbers in wells with a nitrocellulose bottom (Millipore) which have been precoated with the specific antigen, rabies virus. After 18-24 hours the cells are washed from the plates and spots where specific antibody has been bound are developed using peroxidase-conjugated anti-Ig and diaminobenzidine tetrahydrochloride as a substrate. Each spot is representative of one antigen specific antibody producing B cell and various immunized and non-immune animals can be compared by enumerating the number of specific B cells detected per 10⁶ cultured cells.

T cell proliferation is quantified in response to antigen challenge in vitro. Because of the involvement of T lymphocytes in the immunoglobulin response, and because of the desirability for a competent T cell immune compartment in host protection, it is important to document the presence of specifically reactive T lymphocytes. This is done essentially as described in D. Craig Hooper et al., Proc. Natl. Acad.Sci. (U.S.A.), 91, ppl0908-10912 (1994). Briefly, single cells suspensions are aseptically prepared from spleen and lymph node by teasing the organs through 100 gauge stainless steel mesh. T cell are then isolated by panning on anti-Ig coated plates to remove B cells and adherent macrophages. T cells (250,000) and an equal number of irradiated (1000 rads) spleen cells as antigen presenting cells are cultured in 200 μl volumes in the wells of a microtiter plate. Replicates of 3 to 4 wells are used per test antigen or mitogen. Con A is used as a mitogen to demonstrate cell viability, and as a control for adequate culture conditions; UV-inactivated rabies virus or purified rabies glycoprotein are used for antigen-specific stimulation. Irrelevant antigens are also used as controls. Test wells receive 20 ug of the appropriate test antigen, either rabies G protein or an unrelated antigen. Cultures are incubated and replicate wells pulsed with 1 μC of ³H-thymidine for 4 hours on days 2 to 6. Incorporated label is quantified by scintillography. Statistical significance is determined using Student t calculation.

Resistance to challenge with lethal rabies virus is determined by inoculating groups of 5 mice previously fed transgenic tomato fruit or non-recombinant fruit as described above. Two routes of administration are used, one route for 5 mice fed transgenic tomatoes and for 5 mice fed nonrecombinant fruit, another route for 5 other mice fed transgenic tomatoes and for 5 mice fed nonrecombinant tomatoes. To specifically test the immunity of the immunized animals, each animal is administered a dose of pathogenic rabies virus calculated to equal 10 lethal doses for 50% of test animals contained in 20 microliters of normal saline and instilled into the nasal passage or in a second group of animals, into an intramuscular site. The same dose of challenge virus is used as is used in the nasal route, but suspended in volume of 50 microliters. Control animals are groups of mice which have received a diet of non-transgenic tomato. Following challenge, mice are inspected daily, and the appearance of clinical signs associated with rabies virus infection noted. These signs include general listlessness, piloerection, incontinence, weight loss, paralysis morbidity and death. The health of the test animals is evaluated daily, and the number of moribund or dead animals recorded. The results of the trial are expressed as the percentage of animals surviving at 30 days post-infection.

Claims

WHAT IS CLAIMED IS:

1. A transgenic process of inducing protective immunity against a pathogen in an animal, the process comprising the steps of: (1) transforming a plant cell with a gene coding for a protein of a pathogen, thereby creating a transgenic cell;

(2) cultivating the transgenic cell, or a derivative cell derived from said transgenic cell, under conditions where said transgenic cell or derivative cell makes the protein; and (3) administering the protein or an antigenic portion thereof to an animal so as to induce protective immumty response in the animal against the pathogen; wherein in steps (1) and (2), the transgenic cell or derivative cell may be part of a plant or plant tissue or may be free of other plant cells; and wherein, in step (3), the protein or antigenic portion thereof may be part of the transgenic or derivative cell, part of an extract of such a transgenic or derivative cell or free of other material normally present in said transgenic or derivative cell.

2. A process of Claim 1 wherein the transgenic cell or derivative cell is administered to the mammal while said cell or derivative cell is part of a plant or plant product that is normally eaten by the mammal.

3. A process of Claim 1 wherein in step (2) the transgenic cell or derivative cell takes place in a plant, or part of a plant, edible by the mammal.

4. A process of Claim 1 wherein the protein is extracted in purified form from the plant and administered as a protein substantially free of other compounds of the plant.

5. A process of Claim 1 wherein the pathogen is a rhabdovirus.

6. A process of Claim 1 wherein the protein of the pathogen comprises an antigenic portion of the rabies G protein.

7. A process of Claim 1 wherein step (1) is mediated by Agrobacterium

tumefaciens.

8. A process of Claim 1 wherein the plant cell in step (1) is a tomato plant cell.

9. A process of Claim 1 wherein step (1) is achieved by means of a DNA plasmid that comprises the gene coding for the protein and a promoter that is functional in the plant cell.

10. A process of Claim 9 wherein the promoter is ethylene-responsive.

11. A process of Claim 1 wherein the animal is a mammal, bird, or fish.

12. A process of Claim 11 wherein the animal is a mammal or bird.

13. A process of Claim 12 wherein the animal is a mammal.

14. A process of Claim 3 wherein the animal is a mammal, bird, or fish.

15. A process of Claim 14 wherein the animal is a mammal or bird.

16. A process of Claim 15 wherein the animal is a mammal.

17. A process of Claim 1 wherein prior to step (3) the protein is not part of a cooked plant or cooked plant product.

18. A transgenic process for producing an immunogen, the process comprising the steps of:

(2) cultivating the transgenic cell, or a derivative cell derived from said transgenic cell, under conditions where said cell or derivative cell makes the protein, such that the protein is capable of inducing protective immunity against the pathogen in an animal.

19. A process of Claim 18 wherein the animal is a mammal, bird, or fish.

20. A process of Claim 19 wherein the animal is a mammal or bird.

21. A process of Claim 20 wherein the animal is a mammal.