The present invention relates to vaccines that are made in transgenic soybeans for use in humans, animals of agricultural importance, pets, and wildlife. These vaccines are used as vaccines against viral, bacterial, fungal, parasitic or prion related diseases, cancer antigens, toxins, and autologous or self proteins. The transgenic soybeans of the instant invention also can be used for inducing tolerance to allergens or tolerance to autoimmune antigens, wherein an individual shows hypersensitivity to said allergen or has developed autoimmunity to autologous or self proteins, respectively. The invention also relates to prophylatically treating individuals and/or populations prior to showing hypersensitivity to allergens. Other aspects of the invention include using the transgenic soybeans as an oral contraceptive, and the expression of protein adjuvants in transgenic soybeans.

Description of the Invention

Recombinant techniques using expression cassettes have allowed the incorporation of exogenous genes (as well as endogenous genes) into other organisms. Expression cassettes generally contain a number of regulatory elements. The construction of the expression cassette of the instant invention contains one or more of the following regulatory elements: 1) a promoter to initiate transcription, 2) an enhancer region to enhance transcription and/or translation, dependent upon the promoter used, 3) sequences to target transcription, translation, and/or protein accumulation to specific locations within the plant or cell, 4) a coding region, which determines the specific protein or proteins that will be expressed in transgenic plants, and 5) a polyadenylation recognition site to determine transcription termination and for mRNA stability. In addition, 6) a selectable marker cassette can be included for selection during transformation and in subsequent generations.

The promoter of the instant invention directs transcription and/or directs protein accumulation in the seed component of the plant. However, it should be understood that protein accumulation can be directed to other parts of the soybean. Seed-specific promoters are available and are known to those of skill in the art. In an exemplary embodiment, beta conglycinin is used as a promoter, which is sometimes referred to as the 7S promoter. The 7S promoter has been used to drive expression of synthetic fanC, synthetic SEB, and synthetic bee venom PLA2. Although the 7S promoter was used in the embodiments of the instant invention, it should be understood that other promoters can be used with the ideal promoter being one which results in the highest accumulation of transgenic protein in the desired region of the soybean (for example in the seed) as determined by assays such as Western analysis, ELISA, or direct plant part (e.g., seed) composition assays. Another embodiment contemplates the used of constitutive promoters, such as 35S. Any of a plurality of constitutive promoters can be used depending on where one wants to drive the accumulation of protein. For example, 35 S drives the accumulation of transgenic protein in soybean seeds and leaves.

An example of a subunit antigen expressed in seed driven by the 35S promoter is synthetic fanC. Other antigens that can be driven by the 35S promoter include wild-type and synthetic LT-A and Lt-B, wild-type and synthetic SEB, and wild-type and synthetic PLA2. A plurality of other subunit antigen species genes that cause known diseases can be incorporated into exogenous systems (such as higher plant systems like soybeans and the like) and driven by the 35S promoter or any of a plurality of promoters. Examples of the diseases (and their associated proteins) include hepatitis A (capsid protein and a non-structural protein), AIDS (gp 120 & gp41 Gag & RT), SARS (Spike glycoprotein nucleocapsid), Genital Herpes (gB and gD scaffolding proteins), Smallpox (membrane protein and core proteins), Encephalitis (nsProtein 1-4 spike proteins) and the like.

It is also contemplated that any of a plurality of enhancers can be used in the present invention. An exemplary embodiment of an enhancer is the tobacco etch virus leader sequence (TEV), which enhances translation. Any of a plurality of enhancers can be used for a plurality of constructs, such as the constructs for synthetic fanC, synthetic LT-A and Lt-B, synthetic SEB, and synthetic PLA2.

The present invention also contemplates the use of targeting sequences. Targeting sequences are used to direct MRNA and/or protein to various cellular locations. The reason for doing this would be to increase/optimize protein accumulation and or to optimize immunogenicity of a particular antigen or allergen in a particular region. It is possible that a protein targeted to one compartment may accumulate to higher levels than the same protein targeted to another location within the cell. It is also possible that proteins targeted to different locations may have different immunogenicities, possibly due to post translational modifications such as phosphorylation, glycosylation, etc. Targeting sequences may be located 5', 3' and/or 5' and 3' to the gene of interest (GOI). Examples of how different areas can be targeted using different targeting sequences are shown in Table 1 (the synFanC gene is discussed in more detail below (see Original Patent)).

An example of a sequence targeted to the endoplasmic reticulum would contain the alpha subunit of soybean vegetative storage protein located 5' to the coding region, along with an in-frame 3' retention signal such as one coding for the amino acids KDEL, (SEQ ID NO. 54), HDEL (SEQ ID NO. 55), or RDEL (SEQ ID NO. 56). An example of a sequence used to target a protein to the chloroplast would be the small subunit of the protein Rubisco derived from Pisum sativum (pea), fused in frame and at the amino terminus of the gene of interest. Thus, the instant invention contemplates the use of any of a plurality of targeting sequences based upon where the GOI is to be accumulated.

The coding region is the nucleotide sequence that when expressed, generates a desired protein. Essentially any soybean gene segment(s) designed to encode a single protein, or part of a protein, multiple proteins, multiple parts of a single protein, or multiple parts of multiple proteins optimized for expression in soybean are contemplated and within the scope of the invention. Modifications from the native gene are also contemplated and are within the scope of the present invention. Examples of modifications or segment would include the following: Alteration of the codon usage to employ preferred codon usage in Glycine max (soybean) Alteration of the GC content of the segment to represent similarity to GC content of soybean coding genes Removal of any AT stretches longer than 5 nucleotides to reduce the potential for cryptic polyadenylation and MRNA instability Removal of any MRNA processing signals, including splice site recognition and AT content of introns that may destabilize mRNAs Removal of secondary structures, hairpin loops, etc. that may destabilize mRNA Addition of sequences to direct mRNA or subsequent pre-proteins to desired locations within cells or plant structures (such as seeds, chloroplasts, mitochondria, cytosol, endoplasmic reticulum, etc.).

In an embodiment of the present invention, a synthetic sequence is optimized for expression in soybean and subcloned in the context of other regulatory elements to allow expression and accumulation in soybean. Examples of segments that have been constructed with these criteria for transformation and expression in soybean include synthetic fanC, synthetic SEB (Staph enterotoxin B), and synthetic bee venom phospholipase A2 (PLA2). It is also contemplated and within the scope of the present invention to have multiple protein segments. Although any of a plurality of multiple protein segments are contemplated, an exemplary embodiment of multiple protein segments expressed simultaneously in soybean include synthetic E. coli heat labile toxin subunit A and subunit B (LT-A and LT-B). Another example of multiple segments from multiple proteins may include immunogenic domains from several of the known allergens in bee venom.

An embodiment of the present invention may also include terminators and/or polyadenylation sequences. These elements provide stability. Exemplary embodiments that can be used in the system of the instant invention include the CaMV 35S polyadenylation sequence, and the Nopaline Synthase polyadenylation sequence. Other terminators and/or polyadenylation sequences are known and are considered to be within the scope of the present invention.

Multiple expression cassettes can be introduced into soybean. However, when one limits the number of redundant sequences, expression tends to proceed more efficiently. Multiple expression cassettes may include for example, the use of different promoters, enhancers, targeting signals, and terminator/polyadenylation elements. Moreover, any antigen or toleragen cassette can be stacked with other cassettes to employ beneficial effects to the seeds, such as increased linolenic acid, herbicide tolerance, pesticide tolerance, drought resistance, stress resistance, etc.

The expression of subunit vaccines in soybeans has significant advantages over previously reported strategies. Due to the relatively high protein content of soybeans when compared to other transgenic plants that have been used for the development of edible vaccines (e.g. potatoes and tomatoes), the soybean system permits the highest expression of immunogen per total weight of plant material. Furthermore, the ease of efficiently converting soybeans into formulations which can be consumed is a significant advantage in developing oral immunization strategies. Accordingly, the use of transgenic soybeans as one of the methods of choice for expression of plant-derived vaccines should be apparent to those of skill in the art when read in light of the instant disclosure.

Mice were allowed to eat whole soybeans coupled with an oral adjuvant. The mice were exposed several times, and serum antibody titers were determined about one month later. Mice fed whole soybeans expressing FanC developed high antibody titers when compared to controls. The results of this study provide compelling evidence to support the practicality of expressing edible vaccines in soybeans.

Thus, any of the plurality of above described regulatory elements can be incorporated or omitted from the expression cassettes. Specific examples of how the expression cassettes can be manipulated to generate transgenic organisms to develop oral vaccines are given below. However, first a general overview of the invention is given, then several examples of an overall strategy (using specific examples) are given that describes how one might develop a strategy for oral vaccines.

Subunit Vaccines Containing a Single Protein

Essentially any protein which is contained within a single open reading frame can be expressed in soybeans for use as a vaccine once a synthetic, plant-compatible gene is made and transformation is performed. An example showing E. coli FanC for use as a vaccine in livestock is described below. Another example is SEB.

It is contemplated and within the scope of the present invention that modifications of the original protein sequence may be made. These modifications include:

a) amino acid changes at specific residues in the original protein sequence which might change the stability of the protein, change the immunogenicity of the protein, change the solubility of the protein, change the glycosylation of the protein, or change the phosphorylation of the protein.

b) conserved amino acid changes at specific residues in the original protein sequence to construct similar, but distinct proteins (e.g. a change from glutamic acid to aspartic acid).

c) non-conserved amino acid changes at specific residues in the original protein sequence to construct similar, but distinct proteins (e.g. a change from glutamic acid to glutamine).

d) addition of amino acids at the N-terminus or C-terminus to target proteins to specific cellular locations (for example, chloroplast, endoplasmic reticulum, seed, mitochondria, etc.).

Subunit Vaccines Containing Multiple Proteins

Essentially any protein which is contained within two or more open reading frames can be expressed in soybeans for use as a vaccine once synthetic, plant-compatible genes are made and transformation is performed. However, there are some additional considerations, including the use of the same or different regulatory elements for each of the proteins to be expressed.

As discussed above in the section pertaining to subunit vaccines containing a single protein, modifications of the original protein sequence are considered to be within the scope of the invention and include:

a) amino acid changes at specific residues in the original protein sequence which might change the stability of the protein, change the immunogenicity of the protein, change the solubility of the protein, change the glycosylation of the protein, or change the phosphorylation of the protein.

b) conserved amino acid changes at specific residues in the original protein sequence to construct similar, but distinct proteins (e.g. a change from glutamic acid to aspartic acid).

c) non-conserved amino acid changes at specific residues in the original protein sequence to construct similar, but distinct proteins (e.g. a change from glutamic acid to glutamine).

d) addition of amino acids at the N-terminus or C-terminus to target proteins to specific cellular locations (for example, chloroplast, endoplasmic reticulum, seed, mitochondria, etc.).

Subunit Toleragens (i.e. Allergens or Autoimmune Antigens) Containing a Single Protein

Essentially any protein which is contained within a single open reading frame can be expressed in soybeans for use as a toleragen once a synthetic, plant-compatible gene is made and transformation is performed.

An example of one of these toleragens, bee venom phospholipase A2 for use in humans is given in detail below. Another example would be grass pollen.

As discussed above in the section pertaining to subunit vaccines containing a single protein and multiple proteins, modifications of the original protein sequence are considered to be within the scope of the invention and include:

a) amino acid changes at specific residues in the original protein sequence which might change the stability of the protein, change the immunogenicity of the protein, change the solubility of the protein, change the glycosylation of the protein, or change the phosphorylation of the protein.

b) conserved amino acid changes at specific residues in the original protein sequence to construct similar, but distinct proteins (e.g. a change from glutamic acid to aspartic acid).

c) non-conserved amino acid changes at specific residues in the original protein sequence to construct similar, but distinct proteins (e.g. a change from glutamic acid to glutamine).

d) addition of amino acids at the N-terminus or C-terminus to target proteins to specific cellular locations (for example, chloroplast, endoplasmic reticulum, seed, mitochondria, etc.).

Subunit Toleragens (i.e. Allergens or Autoimmune Antigens Containing Multiple Proteins)

Essentially any protein which is contained within two or more open reading frames can be expressed in soybeans for use as a toleragen once synthetic, plant-compatible genes are made and transformation is performed. However, there are additional considerations, including the use of the same or different regulatory elements for each of the proteins to be expressed.

As discussed above in the section pertaining to subunit vaccines containing a single protein subunit toleragens, modifications of the original protein sequence are considered to be within the scope of the invention and include:

a) amino acid changes at specific residues in the original protein sequence which might change the stability of the protein, change the immunogenicity of the protein, change the solubility of the protein, change the glycosylation of the protein, or change the phosphorylation of the protein.

b) conserved amino acid changes at specific residues in the original protein sequence to construct similar, but distinct proteins (e.g. a change from glutamic acid to aspartic acid).

c) non-conserved amino acid changes at specific residues in the original protein sequence to construct similar, but distinct proteins (e.g. a change from glutamic acid to glutamine).

Subunit Vaccines or Toleragens Containing a Peptide Segment, or Concatemers of Homologous Peptide Segments, or Concatemers of Heterologous Peptide Segments Representing B Lymphocyte Epitopes

The subunit vaccines of the present invention includes concatemers and peptide segments that have been incorporated into transformed soybeans. Concatemers may be a) contiguous or b) separated by a finite number of irrelevant amino acids (e.g., a single glycine residue or a sequence of 6 glycine residues), c) separated by amino acids representing known proteolytic cleavage sites (e.g. trypsin, chymotrypsin, serine protease, caspase, etc.), or d) separated by cysteine residues to allow disulfide bond formation. Generally, these concatemers and peptide segments will present an epitope that can be recognized by B lymphocytes (and thus, serve as a vaccine).

Subunit Vaccines or Toleragens Containing a Peptide Segment, or Concatemers of Homologous Peptide Segments, or Concatemers of Heterologous Peptide Segments Representing CD4+ T Helper Lymphocyte Epitopes

The subunit vaccines of the present invention includes concatemers and peptide segments that have been incorporated into transformed soybeans. Concatemers may be a) contiguous or b) separated by a finite number of irrelevant amino acids (e.g., a single glycine residue or a sequence of 6 glycine residues), c) separated by amino acids representing known proteolytic cleavage sites (e.g. trypsin, chymotrypsin, serine protease, caspase, etc.), or d) separated by cysteine residues to allow disulfide bond formation. Generally, these concatemers and peptide segments will present an epitope that can be recognized by CD4+ T lymphocytes (and thus, serve as a vaccine).

Subunit Vaccines or Toleragens Containing a Peptide Segment, or Concatemers of Homologous Peptide Segments, or Concatemers of Heterologous Peptide Segments Representing CD8+ T Lymphocyte Epitopes

The subunit vaccines of the present invention includes concatemers and peptide segments of homologous or heterologous that have been incorporated into transformed soybeans. Concatemers may be a) contiguous or b) separated by a finite number of irrelevant amino acids (e.g., a single glycine residue or a sequence of 6 glycine residues), c) separated by amino acids representing known proteolytic cleavage sites (e.g. trypsin, chymotrypsin, serine protease, caspase, etc.), or d) separated by cysteine residues to allow disulfide bond formation. Generally, these concatemers and peptide segments will present an epitope that can be recognized by CD8+ T lymphocytes (and thus, serve as a vaccine).

Subunit Vaccines or Toleragens Containing a Peptide Segment, or Concatemers of Homologous Peptide Segments, or Concatemers of Heterologous Peptide Segments Representing Gamma-delta T Cell Epitopes

The subunit vaccines of the present invention includes concatemers and peptide segments of homologous or heterologous that have been incorporated into transformed soybeans. Concatemers may be a) contiguous or b) separated by a finite number of irrelevant amino acids (e.g., a single glycine residue or a sequence of 6 glycine residues), c) separated by amino acids representing known proteolytic cleavage sites (e.g. trypsin, chymotrypsin, serine protease, caspase, etc.), or d) separated by cysteine residues to allow disulfide bond formation. Generally, these concatemers and peptide segments will present an epitope that can be recognized by gamma-delta T cells (and thus, serve as a vaccine).

Subunit Vaccines or Toleragens Containing a Peptide Segment, or Concatemers of Homologous Peptide Segments, or Concatemers of Heterologous Peptide Segments Representing Pattern Recognition Receptor Epitopes

The subunit vaccines of the present invention includes concatemers and peptide segments of homologous or heterologous that have been incorporated into transformed soybeans. Concatemers may be a) contiguous or b) separated by a finite number of irrelevant amino acids (e.g., a single glycine residue or a sequence of 6 glycine residues), c) separated by amino acids representing known proteolytic cleavage sites (e.g. trypsin, chymotrypsin, serine protease, caspase, etc.), or d) separated by cysteine residues to allow disulfide bond formation. Generally, these concatemers and peptide segments will present an epitope that can be recognized by pattern recognition receptors (and thus, serve as a vaccine).

Subunit Adjuvants Containing a Single Protein

The present invention also relates to subunit adjuvants containing a single protein. The single protein adjuvant serves the function of increasing the efficacy of the immune response to the vaccine. Essentially, any protein which is contained within a single open reading frame can be expressed in soybeans for use as an adjuvant once a synthetic, plant-compatible gene is made and transformation is performed.

These single protein subunit adjuvants are expressed in soybeans and can either be co-expressed with the protein of the vaccine, or alternatively, the adjuvant can be expressed in separate soybeans and administered to individuals separately from the soybeans that serve as the edible vaccine. Modifications of these single protein subunit adjuvants are contemplated and are within the scope of the present invention. These modifications include:

a) amino acid changes at specific residues in the original protein sequence which might change the stability of the protein, change the immunogenicity of the protein, change the solubility of the protein, change the glycosylation of the protein, or change the phosphorylation of the protein.

b) conserved amino acid changes at specific residues in the original protein sequence to construct similar, but distinct proteins (e.g. a change from glutamic acid to aspartic acid).

c) non-conserved amino acid changes at specific residues in the original protein sequence to construct similar, but distinct proteins (e.g. a change from glutamic acid to glutamine).

d) addition of amino acids at the N-terminus or C-terminus to target proteins to specific cellular locations (for example, chloroplast, endoplasmic reticulum, seed, mitochondria, etc.).

Subunit Adjuvants Containing Multiple Proteins

In addition to the single protein subunit vaccines disclosed above, the present invention also encompasses subunit vaccines that contain multiple proteins. Almost any protein which is contained within two or more open reading frames can be expressed in soybeans for use as an adjuvant once synthetic, plant-compatible genes are made and transformation is performed. However, there are additional considerations, including, the use of the same or different regulatory elements for each of the proteins to be expressed that should be considered.

In an embodiment discussed in more detail below, an example of a subunit adjuvant that contains multiple proteins is the E. coli heat labile toxin for use as an adjuvant in humans or livestock.

It should be apparent to those of ordinary skill in the art that the subunit vaccines that contain multiple proteins can encompass modifications of the original protein sequence. These modifications include:

a) amino acid changes at specific residues in the original protein sequence which might change the stability of the protein, change the immunogenicity of the protein, change the solubility of the protein, change the glycosylation of the protein, or change the phosphorylation of the protein.

b) conserved amino acid changes at specific residues in the original protein sequence to construct similar, but distinct proteins (e.g. a change from glutamic acid to aspartic acid).

c) non-conserved amino acid changes at specific residues in the original protein sequence to construct similar, but distinct proteins (e.g. a change from glutamic acid to glutamine).

d) targeting sequences to direct one or both proteins to various cellular locations.

Subunit Vaccines or Toleragens Containing a Peptide Segment, or Concatemers of Homologous Peptide Segments, or Concatemers of Heterologous Peptide Segments Co-Expressed with Proteins or Peptides which Target Binding to Receptors on Epithelial Cells to Facilitate Deliver

Essentially any vaccine or toleragen construct can be co-expressed as a fusion protein with proteins or peptides which target binding to epithelial cells in the gastrointestinal tract. For example, the plant lectin protein, Ulex europaeus agglutinin I can be coupled to vaccines or toleragens to facilitate uptake by specialized epithelial cells called "M" cells (Vaccine, 2005 23:3836-42 incorporated by reference in its entirety). Thus, the present invention encompasses any of the vaccines or toleragen constructs described generically or specifically herein wherein the vaccine or toleragen is co-expressed as a fusion protein with proteins or peptides, which target binding to epithelial cells.

Subunit Vaccines or Toleragens Containing a Peptide Segment, or Concatemers of Homologous Peptide Segments, or Concatemers of Heterologous Peptide Segments Co-expressed with Proteins which Function as Adjuvants or Co-stimulatory Molecules

The present invention also encompasses concatemers and/or peptide segments that are co-expressed with proteins that function as adjuvants or co-stimulatory molecules. Essentially any vaccine or toleragen can be co-expressed as a fusion protein with proteins or peptides which can function as adjuvants. For example, the adjuvant, E. coli heat labile toxin, can be co-expressed with any vaccine or toleragen. For example, the co-stimulatory molecule CD40 can be co-expressed with any vaccine or toleragen. As another example, the cytokine, IL-2, can be co-expressed with any vaccine or toleragen.

The present invention also encompasses mutants of the concatemers and/or peptide segments that are co-expressed with proteins that function as adjuvants or co-stimulatory molecules. The mutants include:

a) amino acid changes at specific residues in the original protein sequence which might change the stability of the protein, change the solubility of the protein, change the glycosylation of the protein, or change the phosphorylation of the protein.

b) conserved amino acid changes at specific residues in the original protein sequence to construct similar, but distinct proteins (e.g. a change from glutamic acid to aspartic acid).

c) non-conserved amino acid changes at specific residues in the original protein sequence to construct similar, but distinct proteins (e.g. a change from glutamic acid to glutamine).

Soybean Formulations for Soybean Based Vaccines and Adjuvants in Humans and Animals

After transformation, selection and growth of the transgenic soybeans containing any of the above discussed embodiments, one can collect the soybeans and process the soybeans for consumption. The soy formulations can be prepared in the following forms:

Soy Protein Powder Formulations:

1) In the soy protein powder formulation, the formulation involves a process wherein one grinds soybeans, extracts lipids, oils, and carbohydrates using known extractants leaving the soy protein containing the particular antigen or adjuvant of interest (for extracting lipids, oils, and carbohydrates from a solution, see, for example, http://www.fao.org/docrep/t0532e/t0532e04.htm visited on Oct. 12, 2005). Then one dries the soy protein powder using any of a known drying techniques (e.g., rotary evaporation or lyophillization). The optimization of the process for this formulation into soy protein powder of each antigen or adjuvant for human or animal consumption is within the purview of those of skill in the art and should take into account the following factors: a) the stability of antigen or adjuvant b) the suitability for consumption of soy protein powder by humans or animals c) the geographic location for vaccine administration (e.g., third world versus developed countries) which might affect the availability of electricity, storage considerations (e.g., refrigeration), or technical knowledge of the individuals expected to utilize the formulation. Soy Milk Formulations:

1) In the soy milk formulation, this process involves grinding soybeans into a powder, solubilizing soy beans in an aqueous suspension containing the particular antigen or adjuvant, and treating the suspension in a manner (e.g. heating) which makes it compatible for human and/or animal consumption. Optionally, the suspension is pressed through a filter and the soy milk is collected in a vessel, which may or may not contain adjuvant. The optimization process of the generating soy milk is within the purview of one of skill in the art. The person of skill in the art in the process for formulation into soy milk of each antigen or adjuvant for human or animal consumption should take into account: a) the stability of antigen or adjuvant b) the suitability for consumption of soy protein powder by humans or animals c) the geographic location for vaccine administration (e.g. third world versus developed countries) which might affect availability of electricity, storage considerations, or technical knowledge of the individuals expected to utilize the formulation. Dehydrated Soybeans

1) In the dehydrated soybean formulation, the soybeans follow known dehydration procedures such as rotary evaporating the soy milk or lyophillization procedures, which are well within the purview of those of skill in the art. All of these processes must be performed under GFP conditions if human use is intended.

Human, Veterinary, Agricultural, and Wildlife Applications of Soybean Based Vaccines and Adjuvants

As was discussed briefly above, the soy formulations of the instant invention can be used in a plurality of animals including but not limited to humans and other primates, to veterinary uses such as uses in dogs, cats, horses, and/or birds, in agricultural animals including but not limited to cattle, pigs, sheep, horses (donkeys, mules), goats, chickens, ducks, fish (catfish, trout), and for wildlife including but not limited to deer, elk, moose, waterfowl (ducks, geese), birds (blackbirds), and/or fish.

The soybean formulations that are used for the above enumerated animals can be in the form of Protein Subunit vaccines, which serve a plurality of functions including but not limited to viral vaccines, bacterial vaccines, fungal vaccines, parasite vaccines, prion vaccines, microbial Toxin vaccines, zoonotic disease vaccines to treat wildlife (i.e., rabies vaccine for raccoons, foxes, skunks), anti-vector vaccines to target mosquitoes, ticks, fleas, which spread disease, plant toxin vaccines (i.e., Ricin), cancer vaccines, and/or mutant self protein vaccines (heterologous chromosomal expression).

The soybean formulations that are used for the above enumerated animals can also be in the form of Protein Subunit toleragens (desensitization antigens and autoimmune antigens), which can be used to treat individuals against a number of allergens, including but not limited to plant allergens (e.g., pollen), invertebrate allergens (e.g., dust mite allergens), microbial allergens (e.g., mold spore allergens), or for Fc receptor for IgE molecules on mast cells and basophils.

The soybean formulations that are used for the above enumerated animals can also be in the form of protein subunit self-antigens including but not limited to thyroid antigens targeted to combat hyperthyroidism, agonist sites of growth hormone receptors to treat dwarf/stunted growth syndrome or for example, to make farm animals grow bigger, for antagonist sites of "addiction" receptors, for IgE molecule as an immunogen to prevent all allergies, for prostate antigens to combat prostatitis, and or for any organ that a surgeon routinely takes out due to inflammation or dysfunction.

The soybean formulations that are used for the above enumerated animals can also be in the form of protein Subunit adjuvants, co-stimulatory molecules, and/or cytokines.

The soybean formulations of the instant invention have uses as vaccines for third world countries that would not be pursued for commercialization by companies due to the limited potential for profits.

The soybean formulations that are used for the above enumerated animals can also be in the form of oral contraceptive vaccines including but not limited to vaccines which target self proteins necessary for completion of the reproductive cycle. (e.g., LHRH, FSH, sperm antigens and oocyte zona pellucida antigens). The formulation can be used to sterilize farm animals by vaccinating against self proteins (e.g., testicular antigens, ovarian antigens). Moreover, the soybean formulations of the present invention can be used for food supplements, in addition to the desired vaccine effect.

Military Applications for Soybean Based Vaccines and Adjuvants

The soybean formulations that are used in the above enumerated animals can also be used for vaccines which can be stockpiled for long periods of time (i.e., in soybeans) for a wide variety of vaccines against known agents of bio-terrorism (e.g., for smallpox, anthrax, etc.). This would limit threat and possible use by terrorists if the terrorists knew of the existence of such vaccine stockpiles.

The soybean formulations of the instant invention have a plurality of advantages that are discussed throughout this disclosure (for example cost). One other advantage is the fact that the storage of soybean seeds containing a desired antigen or toleragen can be done for prolonged periods of time (and eliminate the needs for refrigeration or any cold chain needed during its manufacture). Soybeans can also be grown in regulated greenhouses (versus outdoors), which will increase the number of generations that can be grown within the year, and also contain the GMOs (genetically modified organisms).

In one example, the strategy stimulates protective memory T helper lymphocyte and memory B lymphocyte activity, coupled with an alternative strategy to stimulate protective memory T helper lymphocyte and memory Cytotoxic T lymphocyte activity.

Having described the uses of the instant invention in general, the following passages look at specific embodiments that flush out these general uses. In a first embodiment, a strategy for development of two different oral vaccines against Hepatitis A virus is given:

Development of a Vaccine to Stimulate a Memory T Helper Lymphocyte Response, and Mucosal IPA and Systemic IgG Antibodies (and Memory B Lymphocytes) Against the Surface Antigens of Hepatitis A.

A subunit immunogen is encoded by a synthetic gene optimized for expression in soybean expressing the complete coding region for Hepatitis A structural (capsid) protein, which is given below (see Original Patent) in the amino acid one letter code.

The gene encoding the above Hepatitis A structural (capsid) protein is synthesized using a nucleotide synthesizer. The inventors note that a plurality of possible nucleotide sequence will give the above amino acid sequence. Thus, it is contemplated and within the scope of the present invention to include any of these nucleotide sequences as the sequence that is exogenously expressed in soybean plants. Likewise, it is contemplated and within the scope of the instant invention to include any site directed mutant of the above sequence as an exogenous gene that is incorporated into soybeans. It is noted that conservative amino acid substitutions are preferred for mutants, with one amino acid substitution being preferred, or one deletion or one addition being the preferred mutants. However, it is contemplated that multiple site directed substitutions can be employed, with conservative amino acid substitutions for all of these multiple amino acids being a preferred embodiment. Preferably, any of these mutants should have 90% or more of the above sequence conserved, more preferably 95% or more of the above sequence conserved, even more preferably 98% or more of the above sequence conserved, and most preferably only one amino acid changed.

After the gene is synthesized, it is incorporated into an expression vector (generally cloned into a binary vector which is transferred into Agrobacterium). Soybean plants are transformed with the expression vector and selected transformants expanded.

The transformed soybeans expressing the Hepatitis A structural (capsid) polyprotein are processed to soy protein powder or soymilk for consumption. Although the vaccine is described with reference to humans, it should be understood by those of skill in the art that these vaccines are also readily available to be used by veterinarians for the treatment of animals. Moreover, putative uses for the transgenic soybean products of the instant will be discussed in some detail below.

The soy formulation is combined with an adjuvant (for example, mutant, E. coli heat labile toxin, LT, which is described later) and used for oral vaccine against Hepatitis A. This vaccine targets the development of memory T helper cells and memory B lymphocytes at mucosal and systemic sites. However, it is noted that although an adjuvant greatly enhances the effect that an antigen (for example, the Hepatitis A structural (capsid) protein) has at generating an immune response, it is contemplated and therefore, within the scope of the invention that an adjuvant not be used (although incorporating an adjuvant is the preferred embodiment).

The advantages of employing the above vaccine system over those that are known in the art include that the present system:

1) Does not require needles, therefore no danger of needle-associated transmission of diseases.

2) Induces an IgA response. No current vaccine induces an IgA response. Since Hepatitis A enters via a fecal-oral route, this IgA response in the gut could prevent virus from entering the circulatory system. Present vaccines only produce IgG in blood, and therefore rely on viral neutralization after the virus has already entered.

3) Should be useful for children under the age of 2. Current vaccines are not licensed for children under 2 years of age. Because of the well known safety of soy products, the safety of an oral vaccine could extend the age range eligible for vaccination.

4) Is inexpensive. Current vaccines are too expensive to prevent most of the 1.5 million cases of hepatitis A in the world today.

Development of a Vaccine to Stimulate T Helper and Cytotoxic T Lymphocyte Responses Against Internal Antigens of Hepatitis A.

In another embodiment, the subunit immunogen is encoded by a synthetic gene optimized for expression in soybean expressing the complete coding region for Hepatitis A non-structural protein -- see Original Patent.

As described above for the Hepatitis A structural (capsid) protein, the gene encoding the above Hepatitis A non-structural protein is synthesized using a nucleotide synthesizer. The inventors note that a plurality of possible nucleotide sequence will give the above amino acid sequence. Thus, it is contemplated and within the scope of the present invention to include any of these nucleotide sequences as the sequence that is exogenously expressed in soybean plants. Likewise, it is contemplated and within the scope of the instant invention to include any site directed mutant of the above sequence as an exogenous gene that is incorporated into soybeans. It is noted that conservative amino acid substitutions are preferred for mutants, with one amino acid substitution being preferred, or one deletion or one addition being the preferred mutants. However, it is contemplated that multiple site directed substitutions can be employed, with conservative amino acid substitutions for all of these multiple amino acids being a preferred embodiment. Preferably, any of these mutants should have 90% or more of the above sequence conserved, more preferably 95% or more of the above sequence conserved, even more preferably 98% or more of the above sequence conserved, and most preferably only one amino acid changed.

After the gene is synthesized, it is incorporated into an expression vector (generally a binary vector which is transferred into Agrobacterium). Soybean plants are transformed with the expression vector and selected transformants expanded.

The Transformed soybeans expressing the structural polyprotein are processed to soy protein powder or soymilk for consumption.

As described above, the soy formulation is combined with an adjuvant (e.g. mutant LT) and used for oral vaccine against Hepatitis A. This vaccine targets the development of memory T helper cells and memory Cytotoxic T lymphocytes at mucosal and systemic sites. As with the Hepatitis A structural (capsid) protein, this methodology has advantages, some of which include:

1) Needles are not required. Accordingly, there is no danger of needle-associated transmission of diseases.

2) No current vaccine has been shown to induce a cytotoxic T lymphocyte response against Hepatitis A. Since Hepatitis A enters via a fecal-oral route, and then infects intestinal epithelial cells, the most efficient method for killing these virally infected cells is through the induction of memory cytotoxic T lymphocytes. Present vaccines only produce IgG in blood, and therefore rely on viral neutralization after virus has already entered.

3) Once a hepatocyte is infected, the infected liver cell must be killed by the immune response so that it does not become a viral factory, producing Hepatitis A to infect other hepatocytes. The induction of a cytotoxic T lymphocyte memory response would allow for such cellular clearance of virally infected cells. Present vaccines only produce IgG in blood, and therefore rely on viral neutralization after virus has already entered.

4) Current vaccines are not licensed for children under 2 years of age. Accordingly, the present invention is advantageous in that the safety of an oral vaccine extends the age range eligible for vaccination.

5) The vaccine is inexpensive. Current vaccines are too expensive to prevent most of the 1.5 million cases in the world today.

The nucleotide sequences for Hepatitis A proteins can be found in Rizzetto, M., Purcell, R. H., Gerin, J. L. and Verme, G. (Eds.); Viral Hepatitis And Liver Disease: 313-316; Edizioni Minerva Medica, Torino (1997), which is herein incorporated in its entirety by reference.

Thus, the above proposed methods are a novel common strategy for vaccine development against a variety of microbes. Mucosal and systemic antibody (memory B cell) responses are targeted to the outer proteins (e.g. capsid proteins) of a microbe following expression of these antigens in soybeans. Antibodies bind to the surface of the microbe and prevent binding to cells or target the microbe for destruction by the immune response. It is contemplated and therefore within the scope of the invention that a concomitant (or separate) immunization strategy uses internal proteins (e.g. nonstructural proteins) of a microbe to target development of a memory cytotoxic T lymphocyte response. In this manner, infected cells present these epitopes to Cytotoxic T lymphocytes, which are then targeted for lysis.

Although the above general method is described for hepatitis A proteins and their associated proteins, it should be understood that the above described method is a general method that can apply to a plurality of other viral diseases and their associated proteins. Other viral diseases and their potential targets for memory T helper cell, memory B lymphocyte, and memory Cytotoxic T lymphocyte responses are given in the below Table 2 -- see Original Patent.. Moreover, the availability of the sequences for these proteins and/or nucleotide sequences are given below the table -- see Original Patent.

One HIV strain protein and/or DNA sequence(s) is described in Fang et al., Recombination following Superinfection by HIV-1, AIDS, 18 (2), 153-159 (2004), which is herein incorporated in its entirety by reference.

The SARS Coronavirus protein and/or DNA sequence(s) is described in He et al., Analysis of multimerization of the SARS coronavirus nucleocapsid protein, Biochem. Biophys. Res. Commun. 316 (2), 476-483 (2004), which is herein incorporated in its entirety by reference.

The Herpes simplex 2 protein and/or DNA sequence(s) is described in McGeoch et al., DNA sequence and genetic content of the HindIII 1 region in the short unique component of the herpes simplex virus type 2 genome: identification of the gene encoding glycoprotein G, and evolutionary comparisons, J. Gen. Virol. 68 (PT 1), 19-38 (1987), which is herein incorporated in its entirety by reference.

The smallpox sequence is not published due to accessibility to terrorists. However, the sequences can be readily obtained by those who need them for legitimate research purposes.

The West Equine Encephalitis protein and/or DNA sequence(s) is described by Netolitzky et al., which involved a direct submission on 08 Dec. 1999 to the Medical Countermeasures Section, Defence Research Establishment Suffield, P.O. Box 4000, Stn Main, Medicine Hat, Alberta T1A 8K6, Canada. All of the above references are incorporated in their entirety by reference.

Other possible subunit vaccines include polio and human ETEC toxins.

Thus, with the above description, it should be apparent that the above described protocol for generating subunit vaccines using higher plants, and in particular, soybeans is a generic method that can be employed on any of a plurality of immunogens associated with viral diseases.

Likewise, similar methodology can be applied on bacterial disease related proteins. One example of an overall strategy to develop oral vaccines that stimulate protective memory T helper lymphocyte and memory B lymphocyte activity against mutant bacterial toxins is given below.

Those of skill in the art will recognize that genes encoding mutant toxins are synthesized to encode proteins which do not have toxicity but still retain their ability to stimulate a protective response against the native toxin. Typically, at least two separate point mutations are made in the mutant toxin. A particular example is given using Staphylococcus Enterotoxin B (SEB) as a prototype, but it should be understood by those of skill in the art that the following protocol is a generic method that can be employed for a plurality of bacterial diseases proteins.

Strategy for Development of an Oral Vaccine Against Mutant Staphylococcus Enterotoxin B (SEB)

The following describes a general protocol for the development of a vaccine to stimulate a memory T helper lymphocyte response, and mucosal IgA and systemic IgG antibodies (and memory B lymphocytes) against mutant Staphylococcus Enterotoxin B (SEB). A more detailed protocol occurs further below. This general procedure is presented to show that the method of incorporating the exogenous gene and expressing bacterial related immunogens is a generic procedure that can be adapted to use any of a plurality of these bacterial related immunogens.

In an exemplary embodiment, a version of mutant SEB is constructed by a gene synthesizer and incorporated into a higher plant (for example, into soybeans). The soybean plants are transformed and selected transformants expanded. The transformed soybeans expressing this mutant SEB is processed to soy protein powder or soymilk for consumption. The soy formulations are combined with an adjuvant (e.g. mutant LT) and used for oral vaccine against mutant SEB. Although the preferred embodiment uses an adjuvant, it should be understood by those of skill in the art that the present invention encompasses embodiments wherein no adjuvant is used.

Vaccine Targeting the Development of Memory T Helper Cells and Memory B Lymphocytes at Mucosal and Systemic Sites

The vaccine, as described above, targets the development of memory T helper cells and memory B lymphocytes at mucosal and systemic sites. This provides several advantages over the vaccines that are currently in use. These advantages include:

1) There is no need for the use of needles, and therefore there is no danger of needle-associated transmission of diseases.

2) There is no current vaccine for SEB. Thus, the transgenic soybeans of the instant invention provide a vaccine for SEB that does not exist.

It was noted above that the above process is a generic process that can accommodate a plurality of bacterial related disease. Additional examples of vaccines against mutant bacterial toxins are included in table 3 below -- see Original Patent.

Development of a Vaccine to Stimulate a Memory T Helper Lymphocyte Response, and Mucosal IgA and Systemic IgG Antibodies (and Memory B Lymphocytes) Against the Surface Antigens of Enteropathogenic E. coli. FanC

In an exemplary embodiment, a version of a subunit immunogen is encoded by a synthetic gene optimized for expression in soybean gene expressing the complete coding region for FanC. Soybean plants are transformed and the selected transformants expanded. The transformed soybeans expressing this surface antigen are processed to soy protein powder or soymilk for consumption. The soy formulation is combined with an adjuvant (e.g., mutant E. coli heat labile toxin, LT) and used as an oral vaccine against E. coli infection. This vaccine targets the development of memory T helper cells and memory B lymphocytes at mucosal and systemic sites.

Similar to the oral vaccine against mutant SEB, the transgenic soybean containing FanC has similar advantages, such as:

1) There is no need for needles and therefore, there is no danger of needle-associated transmission of diseases.

2) There are no current vaccines that induce an IgA response against FanC. Thus, this is the first vaccine against FanC.

3) The current vaccines are more expensive and therefore are not used for agriculture purposes as often as they could be.

The above protocol describes the protocol for how an oral vaccine against mutant SEB is prepared. Similarly, a general protocol is described below that shows the development of a vaccine to stimulate memory T helper lymphocyte responses against internal antigens of Mycobacterium tuberculosis.

Some bacteria are not extracellular pathogens like E. coli, but are intracellular pathogens that can live inside cells (i.e. macrophages). The causative agent for tuberculosis is such a bacterium which can live and hide inside macrophages while the disease develops. An effective immune response against such an intracellular bacterium induces T helper lymphocytes to activate the macrophage response. Therefore, this embodiment of the invention discloses vaccines that combat intracellular bacteria wherein the target is a helper T lymphocyte response.

As described above the requisite gene sequences are synthesized by a gene synthesizer (which is described in more detail below) and incorporated through a vector into the desired higher plant (e.g., soybean). As an example, a subunit immunogen is encoded by a synthetic gene optimized for expression in soybean that expresses Antigen 85 complex (Ag85 A-C) internal antigens expressed by mycobacterium tuberculosis (XX):

The DNA and/or protein sequences for Ag85-A can be found in Cole et al., Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence, Nature 393 (6685), 537-544 (1998), which is herein incorporated in its entirety by reference. The DNA and/or protein sequences for Ag85-B can be found in Matsuo et al., Cloning and expression of the Mycobacterium bovis BCG gene for extracellular alpha antigen, J. Bacteriol. 170 (9), 3847-3854 (1988), which is herein incorporated in its entirety by reference. The DNA and/or protein sequences for Ag85-C can be found in Content et al., The genes coding for the antigen 85 complexes of Mycobacterium tuberculosis and Mycobacterium bovis BCG are members of a gene family: cloning, sequence determination, and genomic organization of the gene coding for antigen 85-C of M. tuberculosis, Infect. Immun. 59 (9), 3205-3212 (1991), which is herein incorporated in its entirety by reference.

After the gene has been synthesized and incorporated into the correct vector, the soybean plants are transformed with the vector and the selected transformants are expanded. The transformed soybeans expressing the Ag85 complex is processed to soy protein powder or soymilk for consumption. The soy formulation is combined with an adjuvant (e.g. mutant LT) and used for oral vaccine against Mycobacterium tuberculosis. As above, this embodiment can be used with or without an adjuvant. However, the presence of the adjuvant is preferred. This vaccine targets the development of memory T helper cells at mucosal and systemic sites.

The vaccine as described has a plurality of advantage over currently available vaccines.: These include:

1) There is no need for needles, and therefore there is no danger of needle-associated transmission of diseases.

2) No current vaccine has been shown to induce an effective helper T lymphocyte response. BCG (live attenuated Mycobacterium bovis BCG) represents the only vaccine currently available against tuberculosis. It is the most widely administered of all vaccines in the WHO Expanded Programme for Immunization, but has been estimated to prevent only 5% of all potentially vaccine preventable deaths due to TB. It has been shown to protect against disseminated and meningeal TB in young children, and to provide some protection against leprosy, but its efficacy in preventing adult pulmonary TB, which carries the major burden of morbidity and mortality from this disease, has varied dramatically in carefully conducted studies throughout the world--from 77% in the UK to 0% in Chingleput, India. As a result of this variability in efficacy, the impact of BCG on the global TB epidemic has been negligible. Moreover, the use of BCG vaccine is not recommended for use in the US and some northern European countries because of its low efficacy and its interference with skin test screening.

Thus, it should be apparent to those of skill in the art that this general procedure to generate oral vaccines can be used on any of a plurality of bacterial related diseases, including bacterial related diseases that are not enumerated here as long as there is a target antigen that can be used. Similar to bacterial related disease, the instant invention also encompasses the development of vaccines against tumor antigens.

Development of Vaccines to Stimulate a Memory T Helper Lymphocyte Responses, Memory B Lymphocyte Responses, and/or Memory Cytotoxic T Lymphocyte Responses Against Tumor Antigens

The same general procedure as used for the above disclosed viral and bacterial related diseases can be used for tumor antigens. Generally, this procedure involves synthesizing the gene encoded the desired tumor antigen(s), incorporating the gene into an appropriate vector and transforming the higher plant (preferably soybean) with the vector containing the gene of interest. Mutants that have undergone site directed mutagenesis are considered to be within the scope of the present invention. Moreover, mutants that have a plurality of conservative amino acid substitutions are considered within the scope of the present invention. Preferably, these mutants should have 90% or more homology with the wild type, more preferably 95% or more of the above sequence conserved, even more preferably 98% or more of the above sequence conserved, and most preferably only one amino acid changed. The below table 4 (see Original Patent) enumerates several antigens that are known to be cancer antigens, in what type of cancer they are found, and where the DNA and/or protein sequences for these cancers can be found.

It should be apparent to those of skill in the art that the above procedure is a generic procedure for generating vaccines that can be used on any of a plurality of tumor antigens, including those tumor antigens that are known that are not enumerated here (as well as those that are not yet known).

Expression of Allergens or Autoimmune Antigens in Soybeans for use in the Induction of Mucosal or Systemic Tolerance:

The above describes using transgenic plants (in particular, soybeans) in the formation of vaccines from antigens that are related to viral related diseases, bacterial related diseases and tumor antigens. Transgenic higher plants such as soybeans containing a desired exogenous gene can also be used to induce tolerance in individuals prior to showing hypersensitive sensitivity to allergens. Prior to this invention, this prophylactic approach had not been used for the widespread prevention or treatment of allergic reactions and autoimmune diseases. Presently, immunotherapy may be used once an individual has demonstrated a significant hypersensitivity against a particular allergen or autoimmune disease. However, it has seemed impractical to suggest that one might be able to induce tolerance toward specific allergens and autoimmune antigens in individuals even before they demonstrate hypersensitivity or autoimmune disease. Thus, in another embodiment of the present invention, the widespread induction of tolerance as a viable method for preventing and/or treating the development of potentially life threatening allergic reactions or autoimmune diseases is presented. This embodiment encompasses giving soy protein powder or soy milk formulations derived from transgenic soybeans expressing allergens or autoimmune antigens to individuals prior to showing a hypersensitive reaction.

As an exemplary embodiment, venom phospholipase A2 is illustrated briefly here and discussed in more detail later. Venom phospholipase A2 is one of the antigens present in stings from honey bees. The general procedure is comparable to the procedure outlined above for generating vaccines. The gene of interest (in this instance, venom phospholipase A2) is synthesized on a gene synthesizer and incorporated into an expression vector. Soybeans are transformed with the expression vector, which also contains a selection marker on the plasmid backbone. The soybeans are selected using the selection marker and grown. The soybeans can be processed into soy powder and/or soy milk for consumption. Tolerance is induced by giving small quantities a number of times to the individual. The procedure is a general procedure that can be performed on any of a plurality of allergens and or antigens that may be related to autoimmune diseases. Tables 5 and 6 (see Original Patent) list a series of the allergens and autoimmune antigens, respectively that can be performed using the above enumerated general procedure. Tables 5 and 6 (see Original Patent) also contain information as to where the DNA and/or protein sequences can be found for each of the respective allergens and autoimmune antigens.

Thus, it should be apparent to those of ordinary skill in the art that any of the above nucleotide sequences of the allergens and/or autoimmune antigens can be incorporated into soybeans, which expresses the correlated protein.

In an embodiment of the invention, minor variants to the protein sequence of allergens and/or autoimmune antigens can be made. A single mutation can be made or alternatively several amino acids can be substituted. Generally, conserved amino substitutions are preferred. Moreover, the mutants should have preferably 90% or more of the wild-type sequence of the above sequence conserved, even more preferably 98% or more of the wild-type sequence conserved, and most preferably only one amino acid changed from the wild-type.

Development of Vaccines to Stimulate a Memory T Helper Lymphocyte, Memory B Lymphocyte, and/or Memory Cytotoxic T Lymphocyte Mucosal and Systemic Responses Against Diseases of Animals of Agricultural Importance

The same general procedure as disclosed above for preparing vaccines to viral and bacterial related diseases, tumor antigen(s) and autoimmune related diseases can also be used for developing vaccines that stimulate responses against diseases in animals of agricultural importance. The process as disclosed above should be followed. The process involves incorporating the gene of interest (after synthesis) into an appropriate vector and transforming the higher plant (preferably soybean) with the vector containing the gene of interest. The transformed soybeans are selected, grown and then collected. The soybean in a purified or unpurified form can be fed to animals in a single dose or in multiple doses to stimulate the desired response.

A series of antigens are known that are important in these animal related diseases and are thus the target of the instant invention. One of skill in the art should note that mutants that have undergone site directed mutagenesis are considered to be within the scope of the present invention. Moreover, mutants that have a plurality of conservative amino acid substitutions are considered within the scope of the present invention. Preferably, these mutants should have 90% or more homology with the wild type, more preferably 95% or more of the above sequence conserved, even more preferably 98% or more of the above sequence conserved, and most preferably only one amino acid changed. Table 7 (see Original Patent) enumerates several antigens and the microbes that contain the antigen, and where the DNA and/or protein sequences for these antigens can be found.


Thus, using the above described process of gene synthesis, exogenous gene incorporation into soybeans, selecting transformed soybeans, growing and collecting the soybeans, one can treat BVDV. BVDV has a high prevalence in the cattle population. Currently, it is mandatory that BVDV vaccination be done to decrease potential losses due to BVDV infection. The use of killed or modified-live vaccines is the method currently used and can provide protection by decreasing the consequences of acute infections. However, it is questionable whether killed or modified-live vaccines provide complete fetal protection from the development of in utero fetal infections. Moreover, the vaccine of the present invention will put less stress on cattle because the cattle can simply eat the soybeans rather than having to receive vaccinations.

Likewise, the above described methodology for BVDV can be used for Equine herpesvirus. An inactivated carbomer-adjuvated EHV-1/EHV-4 vaccine (DUVAXYN.RTM.) is a currently used vaccine. This vaccine has had its efficacy tested for reducing abortions caused by EHV-1 in a small group of mares. Pregnant mares were vaccinated 3 times during pregnancy and control mares were left unvaccinated. At 4 weeks after the third vaccination, the mares were exposed to a strain of EHV-1 that is known to cause abortions in pregnant mares. All of the control mares aborted between days 15 and 16 post infection. Of the vaccinated mares, 20% aborted and 80% maintained their pregnancies and delivered healthy foals. Thus, it was concluded that an inactivated, adjuvant vaccine based on EHV-1 and EHV-4 antigens was safe for pregnant mares and significantly reduces the incidence of EHV-1 induced abortions. The vaccine of the present invention will put less stress on cattle because the cattle can simply eat the soybeans rather than having to receive vaccinations.

Similarly, the above described methodology can be used for Pasteurella. The Pasteurella bacteria, even though they all are derived from the same species, are not all the same. It is know that there are as many as 15 different strains of Pasteurella haemolytica. Thus, a vaccine made with one strain does not always protect against other strains. For these reasons, the popularity of Pasteurella vaccines has decreased in recent years. However, farmers in a particular region may find a Pasteurella vaccine that works for the strain that is in their particular area and use it. The vaccines are currently manufactured with killed bacteria. At least two live avirulent REPLICATING Pasteurella vaccines are on the market. However, objections to their use generally include: 1) high cost; 2) adverse reactions which include injection site pain, swelling or abscesses, and muscle stiffness; 3) the administration of antibiotics or sulfa drugs at the same time as vaccination is thought to kill the live vaccine organisms and cause vaccination failure; and 4) in one case, the vaccine was recommended only for use in calves over three months of age. Since many shipped cattle are routinely administered antibacterial drugs on arrival, the use of these particular vaccines in recently shipped cattle has been questioned. Thus, it should be apparent to those of ordinary skill in the art that the instant invention using transgenic soybeans that have expressed the Pasteurella antigen has many advantages over the vaccines in current use. The cost is less, the cattle do not suffer from adverse reactions from injections (because the vaccine is eaten), and a dosage can be prepared that is very high (still inexpensively) so that even if cattle are treated with antibiotics or sulfa drugs, the vaccine may still be effective. Moreover, cattle younger than 3 months can likely be treated.

Similar to the above-enumerated cattle diseases, Mycoplasma, which affects chickens can also be treated. Mycoplasma infections are intracellular infections and, therefore one must target cell mediated immunity. Mycoplasma infection is a major cause of production loss in chickens all aroLuLd the world. The currently available vaccines partially protect breeder chickens from clinical disease, but do not eliminate the pathogen. The extensive use of antibiotics has lead to severe resistance problems and utmost care must be given to select the right antibiotic to ensure a successful treatment.

Development of Oral Contraceptive Vaccines for Animals of Agricultural Importance, Pets, and for Wildlife

The above enumerated methods for generating vaccines can also be used in the development of oral contraceptives for animals of agricultural importance, pets and for wildlife. One instance where this might apply is in a given deer population wherein the deer population in a given location is too high. By feeding deer transgenic soybeans that have an antigen that functions as an oral contraceptive, the deer population can be reduced preventing a plurality of problems (such as loss of crops due to mastication by large deer populations, large deer die-offs in the winter, etc.). In this embodiment of the invention, a subunit immunogen will be encoded by a synthetic gene optimized for expression in soybean expressing the complete coding region for proteins expressed on sperm or eggs that are necessary for gametogenesis or fertilization.

The general method involves transforming the Soybean plants, selecting the transformed soybeans, and then expanding the selected transformants. The selected transformants are grown and collected. The Transformed soybeans expressing this protein are processed so they are used as seeds or processed to soy powder, or alternatively, they are left unprocessed. The processed or unprocessed transgenic soybeans can then be fed to animals and used as an oral contraceptive. Alternatively, the soy formulation can be combined with adjuvant seeds or soy powder (e.g. mutant E. coli heat labile toxin, LT) and used with seeds or powder expressing antigen. The vaccine targets the development of memory T helper cells and memory B lymphocytes at systemic sites.

The normal, cellular prion protein (PrP.sup.C) is encoded by the PRNP gene and is found within cells in the central nervous system. However, a variant form of the prion protein referred to as abnormal, scrapie prion (PrP.sup.Sc) can transmit prion-mediated diseases when consumed.

Bovine Spongiform Encephalopathy and Chronic Wasting disease are prion diseases of cattle and free range ruminants (e.g., elk and deer), respectively. It is thought that these diseases are transmitted by eating feed containing prion proteins which can cause disease. Since humans consume these animals, there have been some reports of spread of prion-related diseases following consumption of contaminated meat. The neurological disease which humans get following consumption of contaminated meat is called Variant Creutzfeldt-Jakob Disease. It is thought that approximately 155 humans in the United Kingdom have presented with this disease.

Some investigators have used parenteral immunizations of mice with prion proteins to limit development of prion-like disease in rodent models. However, it is not clear how practical such immunizations would be for wildlife (e.g., elk and deer) since delivery to such animals would be a significant problem. Furthermore, since only a small percentage of cattle (>1%) contract prion related diseases, the cost-effectiveness of such widespread vaccinations would be questioned. An advantage of a soybean-based vaccine against abnormal prion proteins is the ability to deliver vaccine to wildlife or agricultural animals in a cost-effective manner.

Having described the general process and a background of prion based diseases, the following table 8 (see Original Patent) discloses the antigens and a prion protein that can be expressed by the above method as well as where the gene sequences can be found.

Although the above antigens in Table 8 are given for a particular species (for example, pig Gonadotrophin releasing hormone), it should be understood by those of ordinary skill in the art that other species also possess the same antigens (for example deer Gonadotrophin releasing hormone). Many of the gene sequences of these antigens are known and are thereby considered to be part of the present invention.

Prophylactic Therapy Using Phospholipase A2

As was mentioned above, general prophylactic therapy is within the scope of the instant invention. As a specific example of prophylactic therapy, the prevention of development of hypersensitivity to the bee venom allergen, phospholipase A2 (Api ml) in neonatal and adolescent mice is disclosed. Generally, to accomplish prophylactic therapy, an allergen can be expressed in soy protein and in soy milk which can be given orally to weanling mice to induce systemic tolerance. Specifically, one can express the bee venom allergen (phospholipase A2) in transgenic soybeans. Soy protein and soy milk preparations from these transgenic soybeans can then be given orally to weanling mice prior to sensitization. The ability to induce allergic reactions in these tolerized animals can then be assessed using the methodology described above for the mice that ingest the fanC transgenic soybeans.

Insertion of Phospholipase A2 into Soybeans

Hypersensitivity to hymenoptera (e.g. bee, wasp, hornet) venom in the human population has been reported to occur in approximately 1% to 5% of the population, with about 1 death per million people attributed to this allergic reaction. Interestingly, studies indicate that as many as 25% to 30% of the population are sensitized to hymenoptera venoms as indicated by the presence of detectable IgE antibodies, though most of these individuals are not classified as being hypersensitive. The risk of bee venom allergy increases with the degree of exposure, so that beekeepers are at a high risk for such hypersensitivity.

In an embodiment of the instant invention, a focus on hypersensitivity to honeybee (Apis mellifera) venom can be studied, as this is the most common insect sting which results in allergic reactions. Honeybee venom is a complex mixture of proteins (Hoffman, D. R. 1996. Hymenoptera venom proteins. Adv Exp Med Biol 391:169, which is herein incorporated by reference in its entirety), however several of these proteins as major allergens can be identified. These include phospholipase A2 (Api ml), hyaluronidase (Api m2), acid phosphatase (Api m3), and melittin (Api m4), as well as other recently identified allergens (Winningham, K. M., C. D. Fitch, M. Schmidt, and D. R. Hoffman. 2004. J Allergy Clin Immunol 114:928; Tavares, B., F. Rodrigues, C. Pereira, G. Loureiro, and C. Chieira. 2005. Allerg Immunol (Paris) 37:171, both of which are incorporated by reference in their entirety). In an exemplary embodiment of the present invention, one of the major allergens in honeybee venom, phospholipase A2, can be studied. In addition to this being a major allergen, a significant amount of information about this protein and gene sequence is known. Such information includes its nucleotide sequence (Muller, U. R. 2002. Recombinant Hymenoptera venom allergens. Allergy 57:570. Moreira, L. A., J. Ito, A. Ghosh, M. Devenport, H. Zieler, E. G. Abraham, A. Crisanti, T. Nolan, F. Catteruccia, and M. Jacobs-Lorena. 2002. Bee venom phospholipase inhibits malaria parasite development in transgenic mosquitoes. J Biol Chem 277:40839. both of which are incorporated by reference in their entirety), and an enzymatically inactive mutant (H34Q) which still retains its ability to stimulate a hypersensitivity response in allergic patients (Wymann, D., C. A. Akdis, T. Blesken, M. Akdis, R. Crameri, and K. Blaser. 1998. Enzymatic activity of soluble phospholipase A2 does not affect the specific IgE, IgG4 and cytokine responses in bee sting allergy. Clin Exp Allergy 28:839, which is incorporated in its entirety by reference). In addition, a murine model for hypersensitivity to bee venom phospholipase A2 has been previously used to question the efficacy of various types of immunotherapy (Akdis et al. 1996. Epitope-specific T cell tolerance to phospholipase A2 in bee venom immunotherapy and recovery by IL-2 and IL-15 in vitro. J Clin Invest 98:1676; Astori, et al. 2000. Inducing tolerance by intranasal administration of long peptides in naive and primed CBA/J mice. J Immunol 165:3497. von Gamier, et al. 2000. Allergen-derived long peptide immunotherapy down-regulates specific IgE response and protects from anaphylaxis. Eur J Immunol 30:1638, all of which are incorporated by reference in their entireties). In the present invention, we can express the enzymatically inactive mutant of honeybee venom phospholipase A2 (H34Q) (Wymann, et al. 1998. Enzymatic activity of soluble phospholipase A2 does not affect the specific IgE, IgG4 and cytokine responses in bee sting allergy. Clin Exp Allergy 28:839, which is herein incorporated by reference in its entirety) for use as an allergen in a mouse model of hypersensitivity that has been previously described (Akdis et al. 1996. Astori, et al. 2000. von Gamier, et al. 2000).

For the 1%-5% of the population that have a previous medical history of hypersensitivity to hymenoptera stings, desensitization immunotherapy is presently the only allergen-specific treatment option. For the "conventional" desensitization treatment, diluted bee venom (ALK Pharmalgen) is injected subcutaneously in patients beginning with a dose (less than 10 micrograms) that is unlikely to cause much of a systemic effect. Typically, this dose is used weekly for one month, followed by an increase in dose for the next several months until a maintenance dose of 100 micrograms is reached. During the next 3 to 5 years, the maintenance dose is given every 3 to 6 months. Such treatments are performed by medical personnel due to the possibility of side effects. In an effort to limit the time required for desensitization, there are also "rush" and "ultrarush" treatments where increasing doses of bee venom are given in an accelerated fashion. The risk of side effects from such accelerated desensitization therapy is significant enough that such therapy should be performed under close medical supervision (Birnbaum, et al. 2003. Hymenoptera ultra-rush venom immunotherapy (210 min): a safety study and risk factors. Clin Exp Allergy 33:58. Wenzel, et al. 2003. Safety of rush insect venom immunotherapy. The results of a retrospective study in 178 patients. Allergy 58:1176, both of which are incorporated by reference in their entireties.).

Desensitization immunotherapy is an effective treatment to limit hypersensitivity reactions to bee venom in most patients who receive this treatment (Golden, et al. 1996. J Allergy Clin Immunol 97:579. Ross, et al. 2000. Clin Ther 22:351. Valentine, et al. 1990. N Engl J Med 323:1601. Hunt, et al. 1978. N Engl J Med 299:157, all of which are incorporated by reference in their entireties.). Success rates for desensitization therapy have been reported to be 75% to 85% effective for honeybee immunotherapy when a maintenance dose of 100 micrograms is reached in adults (Golden, D. B. 2005. J Allergy Clin Immunol 115:439, which is herein incorporated by reference in its entirety.). An increase to a maintenance dose of 150 to 250 micrograms of bee venom has been reported to improve efficacy for those adults not protected by 100 microgram doses (Rueff, et al. 2001. J Allergy Clin Immunol 108:1027, which is herein incorporated by reference in its entirety.).

There are side effects and risks associated with this therapy despite reports of the relative safety of such immunotherapies (Golden, D. B. 2005. Birnbaum, et al. 2003, Clin Exp Allergy 33:58, which is incorporated by reference in its entirety). Side effects include: patches on the skin, itching, reddening of the skin's surface, swelling at the site of injection, raised patches on the skin at sites systemic to the injection site, inflammation of the mucosal membranes in the nose, mild or moderate difficulty in breathing, and swelling of the eyes, lips, or tongue. In a small percentage of cases, an anaphylactic reaction has been observed following immunotherapy, which included difficulty in breathing, airway obstruction, facial swelling, etc. which requires medical intervention to reverse these symptoms.

The present day immunotherapy for bee venom allergy have limitations. These limitations can be summarized as follows. 1) Time. Often greater than 20 subcutaneous injections with bee venom allergen preparations (ALK, Pharmalgen) are required over a period of 3 to 5 years in order to establish and maintain the desensitized state. This requires a significant commitment by the patient to travel to an appropriate medical facility on numerous occasions over the course of several years to comply with the particular immunotherapy regimen. 2) Cost. The cost of travel to medical clinics or hospitals, the cost of medical personnel to administer the injections, and the costs of bee venom (ALK, Pharmalgen) for multiple treatments over a period of years is a significant financial commitment for immunotherapy patients. 3) Necessity for medical supervision. As noted above (Section B3), these injections must be performed under medical supervision, and if "ultrarush" regimens are used, close medical supervision or hospitalization has been recommended. This requirement places significant limitations on where such treatments can be performed. 4) Side effects. While the side effects associated with any single injection is low, the possibility that an individual patient may have one or more side effects during one of the numerous injections over a 3 to 5 year period increases proportionately. 5) Efficacy. Although desensitization using hymenoptera venom injection is one of the most successful applications for specific immunotherapy that is presently practiced with efficacy levels of 75% to 85% for honeybee venom therapy being reported, there is a percentage of patients wherein this therapy is not effective. An increase in the maintenance dose (150 to 250 micrograms) has been suggested in patients who are not desensitized using standard doses (100 micrograms). Furthermore, efficacy in children is not altogether clear. A recent study (Golden, et al. 2004. Outcomes of allergy to insect stings in children, with and without venom immunotherapy. N Engl J Med 351:668, which is herein incorporated by reference in its entirety) demonstrated that children (age 8+3 years) having a mild to severe hypersensitivity to bee sting early in life do not always outgrow such an allergy, but can have symptoms into adulthood. Furthermore, for those children that received venom immunotherapy, it did reduce the risk of having a systemic response when they were stung by a bee with a mean of 21 years later (+5 years). However this protection into adulthood was not complete. So, immunotherapy in children appears somewhat successful, but is not absolute. 6) Unknown hypersensitivities to bee venom. Only those individuals who have already experienced a hypersensitivity reaction to a honeybee sting are indicated for immunotherapy. Diagnostic tests to identify those individuals who might have an adverse reaction to bee venom are limited by the fact that 25% to 30% of the population shows reactivity in a RAST test. Furthermore, some individuals who have a negative RAST or skin test can still have an allergic reaction to a bee sting (Reisman, R. E. 2001. Insect sting allergy: the dilemma of the negative skin test reactor. J Allergy Clin Immunol 107:781, which is herein incorporated by reference in its entirety). More troubling is the fact that about half of the deaths attributed to fatal sting reactions could not have been prevented since there was no previous indication that these individuals had any hypersensitivity (Barnard, J. H. 1973. Studies of 400 Hymenoptera sting deaths in the United States. J Allergy Clin Immunol 52:259. Hoffman, D. R. 2003. Fatal reactions to hymenoptera stings. Allergy Asthma Proc 24:123, both of which are incorporated by reference in their entireties.), and therefore they would not have been candidates for immunotherapy. 7) Noncompliance of patients for which immunotherapy is indicated. Once a patient presents with a hypersensitivity to bee stings, it is not a certainty that the individual will choose to receive such therapy. The reasons for deciding not to participate in immunotherapy are likely some combination of the problems listed above, including cost, inconvenience, the use of needles, and/or side effects associated with such therapy. In one study (Golden, D. B., A. Kagey-Sobotka, P. S. Norman, R. G. Hamilton, and L. M. Lichtenstein. 2004. Outcomes of allergy to insect stings in children, with and without venom immunotherapy. N Engl J Med 351:668, which is incorporated by reference in its entirety), of 345 children that had a moderate to severe systemic reaction to a bee sting, 99 (or 29%) chose not to undergo immunotherapy even though they were advised to do so.

Thus, despite the successes of venom-based immunotherapies, there remain some significant problems with the practicality and safety of performing these treatments. This fact is underscored by recent investigations which have sought technological advances in the field of immunotherapy in an attempt to overcome some of these limitations (Jilek, et al. 2001. J Immunol 166:3612. Muller, et al. 1998. J Allergy Clin Immunol 101:747. Muller, U. R. 2003. Curr Opin Allergy Clin Immunol 3:299. Alexander, et al. 2002. Curr Drug Targets Inflamm Allergy 1:353, which are incorporated by reference in their entirety). Thus, one embodiment of the present invention is to prophylactically apply oral allergen therapy to prevent the development of hypersensitivity in children who have not yet shown clinical symptoms. This can be a cost-effective, safe, and efficacious treatment option.

There is little doubt that high levels of allergen can be expressed in a stable form in transgenic soybeans for pennies a dose. There have been several excellent review articles documenting the successful expression of proteins in transgenic plants and the above studies with transgenic fanC show this. The ability to express foreign proteins in transgenic plants has been demonstrated, and it is clear that the technology exists to transform plants for human use. In fact, proteins that retain their enzymatic activity or functionality have been expressed in a variety of plants, indicating the potential utility of this technology for a variety of applications. The advantages of transgenic plants for production of proteins of importance to human health have also been discussed (Goldstein, et al. 2004. Qjm 97:705. Peterson, et al. 2004. Trends Biotechnol 22:64. Ma, J. K. 2000. Nat Biotechnol 18:1141. Larrick, et al. 2001. Curr Opin Biotechnol 12:411. Giddings, G. 2001. Curr Opin Biotechnol 12:450, all of which are incorporated by reference in their entireties.). The feasibility of expressing foreign proteins (but not allergens) in transgenic soybeans has also been demonstrated (Zeitlin, et al. 1998. Nat Biotechnol 16:1361. Sojikul, et al. 2003. Proc Natl Acad Sci U S A 100:2209. Smith, et al. 2002. Biotechnol Bioeng 80:812. Hatic, et al. 2001. Anal Biochem 292:171, all of which are incorporated by reference in their entireties) and the disclosure in the present invention of E. coli FanC shows that subunit allergens can be expressed in soybeans. When targeted to the cytoplasm, expression levels of FanC approaches 0.5% of total protein. Therefore, it is postulated that well over 1% of total protein expression of an antigen can be obtained when it is specifically targeted to expression in soybean seeds (Sato, et al. 2004. Crop Sci 44:646, which is herein incorporated by reference in its entirety.).

Presently, soybean yields are .about.40 bushels/acre with a typical price of .about.$6 per bushel. Since soybeans contain .about.38% protein, even with 1% expression of a particular allergen, oral therapy would be pennies a dose. Therefore, the cost of numerous exposures to any particular oral toleragen in a large population of children is not a limitation to using this technology.

As are discussed herein, soy milk formulations for infant and adult consumption are safe to consume and have significant nutritional benefit (Messina, M. J. 1999. Am J Clin Nutr 70:439S. Slavin, J. 1991. J Am Diet Assoc 91:816, both of which are incorporated by reference in their entireties.). In fact, soy milk formulations are so safe that they are routinely fed to infants with little side effects (Motil, K. J. 2000. Curr Opin Pediatr 12:469. Seppo, et al. 2005. Am J Clin Nutr 82:140. Badger, et al. 2002. J Nutr 132:559S, both of which are incorporated by reference in their entireties.). Such safety also seems to apply to transgenic plants in general. In a recent review, it was noted that " . . . more than two trillion transgenic plants have been grown between 1999 and 2000 alone, with no overt documented adverse food reactions being reported, indicating that genetic modification through biotechnology will not impose immediate, significant risks such as food allergen sources beyond that of our daily intake of foods from crop plants." (Helm, R. M. 2003. Ann Allergy Asthma Immunol 90:90, which is incorporated by reference in its entirety.). Such safety further supports the notion that widespread therapy with allergen-containing soy milk formulations would not pose any significant risk to the majority of infants or adolescents, even if those individuals might never develop hypersensitivity to that particular allergen.

Making soybeans into forms that are palatable for human consumption are established and are numerous (Friedman, et al. 2001. J Agric Food Chem 49:1069. Lusas, et al. 1995. J Nutr 125:573S, both of which are incorporated by reference in their entireties). Furthermore, novel processing methods are presently being sought to improve existing technologies (Kitts, et al. 2003. Curr Pharm Des 9:1309, which is incorporated by reference in its entirety.).

Tofu is a well known form of soybeans that is generated by the fermenting soy protein. It is contemplated and within the scope of the invention that the transgenic soy of the instant invention can be processed in such a way. It is believed that the fermenting process may proteolize the proteins, yet nevertheless, these proteins may possess the requisite epitopes necessary to elucidate an immune response. Consequently, the instant invention contemplates that transgenic soybean that has been made into tofu is within the scope of the instant invention.

Without fermentation of soy products, a process can be readily identified that maintains allergen structure, and at the same time forms transgenic soybeans into a consumable product. The above disclosure demonstrates that soybeans expressing the bacterial protein, FanC, can be processed into soy powder and soy milk suitable for consumption and still contain intact FanC capable of stimulating a mucosal response. These studies support the notion that formulating soybeans into soy milk maintains allergen structure for use in the induction of oral tolerance.

Recent reviews (Wu, et al. 2003. Immunol Res 28:265. Mayer, L., et al. 2004. Nat Rev Immunol 4:407, both of which are incorporated by reference in their entireties.) summarize the efficacy of inducing tolerance to a variety of antigens following their oral administration. Animal models of autoimmune disease have shown some of the most promising results, especially when oral antigens are given prior to sensitization of animals to the auto-antigen. In addition, feeding of allogeneic cells or MHC proteins has shown efficacy in animal models of transplant rejection (Stepkowski, et al. 1999. Transplant Proc 31:1557. Zavazava, et al. 2000. J Leukoc Biol 67:793, both of which are incorporated by reference in their entireties.).

There have been a few attempts to use autoimmune antigens expressed in transgenic plants to limit the development of autoimmune disease in mouse models. Of note, others have used transgenic tobacco to express a diabetes associated antigen and prevent diabetes in an animal model following oral administration (Ma, et al. 2004. Proc Natl Acad Sci U S A 101:5680. Ma, et al. 1997. Nat Med 3:793, both of which are incorporated by reference in their entireties.). Other researchers have used transgenic potatoes expressing a cholera toxin B subunit-insulin fusion protein for oral tolerance induction in a mouse model of diabetes (Arakawa, et al. 1998. Nat Biotechnol 16:934, which is incorporated by reference in its entirety.).

There have also been a few attempts to induce oral tolerance to allergens. There has been some success using increasing doses of food allergens given orally to desensitize patients (Patriarca, et al. 2003. Aliment Pharmacol Ther 17:459, which is incorporated by reference in its entirety.). Unfortunately some patients react to even small doses to oral food allergens, making side effects possible in highly reactive patients. Oral tolerance has also been observed when certain pollen extracts are given to mice (Aramaki, et al. 1994. Immunol Lett 40:21. Kim, et al. 2001. Arch Pharm Res 24:557, which are incorporated by reference in their entireties). However, to the inventors knowledge this is the first studies on using transgenic soybeans to express allergens. Furthermore, the inventors believe that this is the first suggestion that one can prophylactically treat children with plant-derived allergens in an attempt to prevent the development of hypersensitivities.

Thus, the present invention shows that the technology is available to express allergens in soybeans. Furthermore, the efficacy of inducing tolerance by oral administration in pre-sensitized animals is quite compelling. To the inventors knowledge, this is the first time that one has shown that it is not necessary to purify allergens from transgenic soybeans. However, the inventors also note that soy milk formulations from such plants should also be able to be used to induce tolerance when given orally to neonatal or adolescent mice. Widespread consumption of soy formulations containing allergens to induce systemic tolerance should thus be a viable therapy for preventing the development of immediate type hypersensitivity reactions.

There are many advantages to using a preventative therapy utilizing allergens expressed in transgenic soybeans.

As noted above, the low costs of expressing allergens in transgenic soybeans for prophylactic therapy makes such treatments feasible for almost anyone. The high protein content of soybeans makes it possible to express high amounts of allergen per soybean, which is a significant advantage over other plants such as tobacco, bananas, potato and tomato previously used to express antigens.

Also noted above, the safety of soybean formulations for humans, like soy milk, is well recognized. Thus, the purification of soy-derived allergens is not a necessity. For example, previous work (Ma, et al. 2004. Proc Natl Acad Sci U S A 101:5680. Ma, et al. 1997. Nat Med 3:793) expressed a diabetes antigen in tobacco to limit autoimmune diabetes in a mouse model. While this was a significant accomplishment, this antigen would have to be purified from such plants for use in humans. Thus, the present invention is advantageous in that the safety of soy formulations permits minimal processing prior to use by humans.

Moreover, due to its safety, the oral delivery of allergens in soy milk formulations does not require medical personnel for such administration. This greatly simplifies treatment and contribute to a low cost. Further, consumption of soy milk formulations would likely be preferable to injections, especially for children. The low cost, safety and ease of administration of soy milk formulations would likely increase compliance with immunotherapeutic regimens that require several years duration to complete. The possibility exists that oral immunotherapy may produce less side effects (Helm, R. M. 2003. Ann Allergy Asthma Immunol 90:90, which is herein incorporated by reference in its entirety.) than those observed with present day injectable immunotherapy. If such a difference exists, it is likely that the route of administration (i.e. systemic versus gastric) would be responsible for the limited side effects.

Soybeans were selected for expression of our oral allergen, bee venom phospholipase A2, for several key reasons. First, soybean has relatively high protein content when compared to other transgenic plants such as tobacco, bananas, potato and tomato previously used to express antigens. The typical composition of a soybean is 38% protein, 30% carbohydrate, 18% oil, and 14% moisture and other components. Therefore, it is possible to express a toleragenic dose of allergen in one or two soybean seeds. Second, procedures for processing soybeans into forms that are palatable for human consumption are established and are numerous:

Production and purification of soybean includes 1) mass production in a factory of soy milk or soy powder containing the appropriate amount of vaccine; 2) individual, disposable vaccine extractors which might be sold over the counter, 3) and other similar systems. A novel processing method is shown in FIG. 17 (see Original Patent), which shows an extract-a-vac. The extract-a-vac employs a concept to extract vaccines from soybeans or from soy powder. The extract-a-vac works as follows.

As pictured, the concept of an extract-a-vac has two essential functions: 1) to produce a single dose of vaccine; and 2) to produce it using a disposable extractor. The extractor can be made so it is reusable. If the extractor is reusable, it should be made of a material that can withstand thorough cleaning (and possibly even being autoclaved). Alternatively, the extract-a-vac can be used for a single vaccine dose followed by disposing of the extract-a-vac. As an extracting solution one would preferably use an aqueous solution that might contain some excipients such as sucralose or some other sugar to add palatability the extraction product. The extract generally should be heated to an appropriate temperature in order to maximize the solubility of the immunogen in the aqueous extraction solvent yet not so high as to cause the degradation of the immunogen (e.g. 75.degree. C., or that temperature empirically determined for each immunogen to be optimal for extraction). Once heated the tab as shown in FIG. 17 should be pulled to mix the extracting solution with soy protein powder containing immunogen and optionally also containing an adjuvant. In a preferred embodiment, the processing of the transgenic soybeans to soy protein powder and quantification of immunogen dose would be done prior to putting the material into each extract-a-vac. After a period of time with gentle shaking to allow solubilization of proteins, the liquid would be pressed through a filter as shown. The cup containing the soymilk formulation could then be consumed by drinking.

The extract-a-vac shows a process that can readily be used that maintains antigenicity, while formulating transgenic soybeans into a consumable product.

Many food products made for human consumption already contain soy protein suggesting that adverse reactions to orally administered soy formulations would be limited. Stated simply, immunogens would not have to be purified from soy preparations because tolerance to soy proteins would be maintained when edible immunogens derived from these plants are used.

The presence of immunogens in seeds of crop plants is advantageous due to antigen stability in seeds and transportability of these crops. Soybean seeds are likely to be stable to antigens.
 

Claim 1 of 16 Claims

1. An immunogen comprising a transgenic soybean and a mutant cholera toxin adjuvant, wherein the transgenic soybean is transformed with an exogenous nucleotide sequence that expresses recombinant SEB (Staphylococcus enterotoxin B) and wherein said immunogen produces a protective immunogenic response upon oral administration to an animal.
 

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