Title:  Oral immunization with transgenic plants

United States Patent:  6,194,560

Assignee:  Texas A & M University System (College Station, TX)

Filed:  November 12, 1998

The oral vaccines and oral vaccine adjuvants of the present invention are produced in transgenic plants and then administered through the consumption of the transgenic plant. DNA sequences both natural and synthetic encoding for the expression of immunogenic agents which are capable of causing an immune response in animals when fed in edible plants, plant tissues, or derived plant materials are constructed and plants transformed for stable or transient expression in plant cells. The present invention provides the first known functional method for immunizing animals via transgenic plants, where the plants express bacterial antigens that act as both immunogens and adjuvants when the transgenic plant material expressing the antigens is fed to animals.

SUMMARY OF THE INVENTION

In order to provide a clear and consistent understanding of the present invention, the following list of terms and their definitions are provided.

An animal is any vertibrate or invertibrate, including, but not limited to humans, birds and fish.

An antigen is a macromolecule that is capable of stimulating the production of antibodies upon introduction into a mammal or other animal including humans. As used in this application, antigen means an antigen per se, an antigenic determinant or the antigen, or a fusion protein containing the antigen or antigenic determinant sometimes referred to a native epitopes.

An antigenic determinant is a small chemical complex that determines the specificity of an antigen-antibody reaction. Colonization and/or virulence antigens of a pathogen contain one or more antigenic determinants.

An amino acid domain is an amino acid sequence within a protein that can be associated with a particular function or sequence homology.

A colonization or virulence antigen is an antigen on the surface of a pathogenic microorganism that is associated with the ability of the microorganism to colonize or invade its host. Discussion and claims may refer to colonization or virulence antigens or antigenic determinants thereof. A pathogen may contain antigens of either colonization or virulence or both and one or more DNA sequences for each or both may be transferred to a vector and used to transform a plant such that it expresses the antigen or antigens.

An immunogenic agent is any antigen that is capable of causing an immune response in animals such as upon oral ingestion of plants carrying vectors that express the antigen.

A chimeric sequence or gene is a DNA sequence containing at least two heterologous parts, i.e., parts derived from, or having substantial sequence homology to pre-existing DNA sequences which are not associated in their pre-existing states. The pre-existing DNA sequences may be of natural or synthetic origin.

A coding DNA sequence is a DNA sequence form which the information for making a peptide moleucle, mRNA or tRNA are transcribed. A DNA sequence may be a gene, combination of genes, or a gene fragment.

A foreign DNA is a DNA that is exogenous to or not naturally found in the microorganisms or plants to be transformed. Such foreign DNA includes viral, prokaryotic, and eukaryotic DNA, and may be naturally occurring, chemically synthesized, cDNA, mutated, or any combination of such DNAs. The foreign DNA of this invention is derived from or has substantial sequence homology to DNA of pathogenic microorganisms and viruses, or is a synthetic gene which encodes a protein which is of similar amino acid sequence to prokaryotic genes.

A fusion protein is a protein containing at least two different amino acid sequences linked in a polypeptide where the sequences were not natively expressed as a single protein. Fusion proteins are frequently the result of genetic engineering whereby DNA sequences from different genes are joined together to encode a single protein composed of amino acid sequences from the originally separate genes.

A gene is a discrete chromosomal region that codes for a discrete cellular product.

An LT-B containing protein is any protein that is substantially homologous in amino acid sequent to, or substantially functionally similar to bacterially derived LT-B protein or CT-B protein. LT-B containing proteins include, but are not limited to proteins with at least one domain with at least about 90% amino acid sequence homology with bacterially derived LT-B or CT-B proteins, proteins that are substantially functionally similar to bacterially derived LT-B or CT-B proteins in binding to GM I gangliosides, and protein fusions in which domains that are substantially similar to bacterially derived LT-B or CT-B are fused to other amino acid sequences either at the N or C terminus of the other amino acid sequences, or fused within the other amino acid sequences.

An LT-A containing protein is any protein that is substantially homologous in amino acid sequence to, or substantially functionally similar to the bacterially derived LT-A protein. LT-A containing proteins include, but are not limited to proteins with at least one domain with at least about 90% amino acid sequence homology with bacterially derived LT-A, and proteins that are substantially functionally similar to bacterially derived LT-A.

A microorganism is a member of one of the following classes: bacteria, fungi, protozoa, or viruses.

A plant tissue is any tissue of a plant in its native state or in culture. This term includes, without limitation, whole plants, plant cells, plant organs, plant seeds, protoplasts, callus, cell cultures, and any group of plant cells organized into structural and/or functional units. The use of this term in conjunction with, or in the absence of, any specific type to plant tissue as listed above or otherwise embraced by this definition is not intended to be exclusive of any other type of plant tissue. Plants suitable for transformation according to the processes of this invention included, without limitation, monocots such as corn, wheat, barley, sorghum, rye, rice, banana, and plantains, and dicots such as potato, tomato, alfalfa, soybean, beans in general , canola, apple, pears, fruits in general, and other vegetables.

A plant transformation vector is a plasmid or viral vector that is capable of transforming plant tissue such that the plant tissue contains and expresses DNA not pre-existing in the plant tissue.

A food stuff or edible plant material is any plant material that can be directly ingested by animals or humans as a nutritional source or dietary complement.

A pre-existing DNA sequence is a DNA sequence that exits prior to its use, in toto or in part, in a product of method according to this invention. While such pre-existence typically reflects a natural origin, pre-existing sequences may be of synthetic or other origin.

An immune response involves the production of antibodies, which are proteins called immunoglobulins. The antibodies circulate in the bloodstream and permeate the other body fluids, where they bind specifically to the type of foreign antigen that induced them. Binding by antibody inactivates viruses and bacterial toxins (such as tetanus or botulinum toxin) frequently by blocking their ability to bind to receptors on target cells. Antibody binding also marks invading microorganisms for destruction, either by making it easier for a phagocytic cell to ingest them or by activating a system of blood proteins, collectively called complement, that kills the invaders. Cell-mediated immune responses, the second class of immune responses, involve the production of specialized cells that react with foreign antigens on the surface of other host cells. The reacting cell can kill a virus-infected host cell that has viral proteins on its surface, thereby eliminating the infected cell before the virus has replicated. In other cases the reacting cell secretes chemical signals that activate macrophages to destroy invading microorganisms.

A secretory immune response (SIR) is a specific type of immune response. It involves the formation and production of secretory IgA antibodies in secretions that bathe the mucosal surfaces of human and other animals and in secretions form secretory glands. An agent which causes the formation and production of such antibodies is considered to stimulate secretory immunity or to elicit a SIR. Secretory immunity is also sometimes referred to as mucosal immunity.

A substantial sequence homology is a functional and/or structural equivalence between sequences of nucleotides or amino acids. Functional and/or structural differences between sequences having substantial sequence homology is frequently de minimus

A transgenic plant is a plant that contains and expresses DNA that was not pre-existing in the plant prior to the introduction of the DNA into the plant.

Transgenic plant material is any plant matter, including, but not limited to cells, protoplasts, tissues, leaves, stems, fruit and tubers both natural and processed, containing and expressing DNA that was not pre-existing in the plant prior to the introduction of the DNA into the plant. Further, plant material includes processed derivatives thereof including, but not limited to food products, food stuffs, food supplements, extracts, concentrates, pills, lozengens, chewable compositions, powders, formulas, suryps, candies, wafers, capsules and tablets.

An edible plant material includes a plant or any material obtained from a plant which is suitable for ingestion by mammal or other animals including humans. This term is intended to include raw plant material that may be fed directly to animals or any processed plant material that is fed to animals, including humans. Materials obtained from a plant are intended to include any component of a plant which is eventually ingested by a human or other animal.

An endoplasmic reticulum (ER) retention sequence is any DNA sequence that codes for an amino acid sequence known to result in the retention of a given protein at or associates with the endoplasmic reticulum such as DNA sequences coding for the amino acids KDEL, HDEL, SEKDEL, and SEHDEL.

An ER signal sequence is any DNA sequence that codes for an amino acid sequence known to result in the recognition of a given protein by the signal recognition particle on the endoplasmic reticulum resulting in the localization of the protein with the ER. Examples of signal sequences which direct newly synthesized proteins to the endoplasmic reticulum in plant cells include barley lectin (Dombrowski, J E, Schroeder M R, Bednarek S Y, Raikhel N V, 1993, Plant Cell 5:587-596), barley aleurain (Holwerda B C, Padgett H S, Rogers J C, 1992 Plant Cell 4:307-318), sweet potato sporamin (Matsuoka K, Nakamura K, 1991, Proc. Natl. Acad. Sci. USA 88:834-838), patatin (Sonnewald U, Braur M, von Schaewen A, Stitt M, Willmitzer L, 1991, Plant J. 1:95-106), soybean vegetative storage proteins (Mason H S, Guerrero, F, Boyer J, Mullet J, 1988, Plant Mol. Biol. 11:845-856), and .beta.-fructosidase (Faye L, Chrispeels M J, 1989, Plant Physiol. 89:845-851).

A LT holotixin is a protein complex produced by enterotoxic E. coli or by transgenic organisms containing the gene for LT-A and LT-B, such as the enterotoxin itself or proteins that contain replacement codons to facilitate plant transcription through the replacement of bacterial preferred amino acid codons for plant preferred amino acid codons.

A CT holotoxin is a protein complex produced by V. cholerae or by transgenic organisms containing the gene for CT-A and CT-B, such as the cholera toxin itself or proteins that contain replacement codons to facilitate plant transcription through the replacement of bacterial A and T rich preferred amino acid codons for plant preferred amino acid codons.

An LT fusion protein is a protein where portions of either the LT-A subunit or the LT-B subunit or both subunits of the LT toxin have been modified to include coding regions for amino acid sequences derived from other antigenic proteins or synthetic proteins or other synthetic molecules designed mimic the immunogenic response of other antigenic proteins or agents. A CT fusion protein is analogously defined except that the fusions occur on either one or both of the CT subunits. Preferred fusion proteins are those fusions to the LT-B or CT-B subunit that do not interfere with the LT-B or CT-B subunit oligomerization into pentamer and/or LT-A or CT-A fusions that preserve that portion of the LT-A or CT-A protein that associates with the pentameric B subunit to form the intact holotoxin in the unmodified form.

The present invention provides an edible vaccine comprising a transgenic plant comprising or expressing at least a DNA sequence encoding an LT-B or CT-B containing protein where the vaccine is designed to elicit immune responses in animals including humans. This invention also provides transgenic plants comprising or expressing at least a DNA sequence encoding an LT-B or a CT-B containing protein which can be used to produce vaccines with enhanced immune responses in animals. The transgenic plants of this invention are especially designed as vaccines or vaccine adjuvants for oral administration and stimulation of immune responses in the GALT of animals including humans.

The present invention also provides a transgenic plant capable of enhancing the immune responses to antigens where the transgenic plant comprises or expresses a DNA sequence encoding an LT-B or a CT-B containing protein. The transgenic plants may also include genetic elements encoding other desired antigens capable of eliciting a secretory immune response in animals. Such other antigens, when presented with transgenic plants containing adjuvantic bacterial toxin antigens can further be enhanced in their immunogenicity via the adjuvant effect of the plant-produced transgenic bacterial toxin antigens.

The present invention also provides for methods for administering the vaccines of this invention, methods for producing the vaccines of the present invention, and methods for preparing the transgenic plants of this invention. This includes the significant and unexpected improvements of causing the production of increased levels of the antigenic protein and by compartmentalization in microsomal vesicles of transgenic plants of an orally active LT-B protein which is highly immunogenic, and by the increased expression of such transgenic bacterial antigens in plants through i) alterations in the plant promoters to increase the level of expression of transgenic proteins in plants, ii) alteration of 3' messages and polyadenylation signals to increase the production of transgenic proteins in plants and iii) production of synthetic genes encoding bacterial antigens where the codon usage has been changed to increase the usage by plants thus increasing the level of transgenic proteins produced in plants. Further, this includes the instant and unexpected discovery that the compartmentalized foreign protein expression in edible transgenic plant tissues allows expression and delivery of additional, desired antigens of value as oral vaccines, since the microsome-encapsulated LT-B serves as an oral adjuvant.

The method for preparing the transgenic plants of this invention includes transforming a target plant with a plant transformation vector containing at least a DNA sequence that codes for a LT-B or CT-B containing protein. The DNA sequence may also contain DNA elements coding for one or more colonization antigens, a virulence antigens, or a antigenic determinant of virulence or colonization antigen of pathogenic microorganisms.

This invention further provides for plant transformation vectors capable of stable incorporation of at least an LT-B or CT-B containing protein gene and promoter regions into target plants. The transforma-tion vectors are prepared by inserting a DNA sequence coding for an LT-B or CT-B containing protein and, optionally, for one or more DNA sequences coding for other antigens where the LT-B or CT-B gene acts as an oral adjuvant and is designed to enhance immune responses to the optionally expressed antigen. The DNA sequences may be natural or synthetic and may comprise entire genes or fragments of genes which code for antigens. Additionally, the DNA sequences may contain plant functional promoters and genetic tags designed to make identification and selection of the transformed plant cells easier, and sequences for selected compartmentalization in desired plant tissues or cellular constituents.

The present invention also provides for transgenic plants usable as oral vaccines or oral vaccine adjuvants wherein the plants comprise or express a DNA sequence encoding an LT-B or CT-B containing protein, sequences encoding proteins containing components of LT-A or CT-A or protein components of LT-A or CT-A, and sequences encoding cellular signal and retention polypeptides or proteins, where the LT-B, CT-B, LT-A, and/or CT-A proteins may include fusions of other antigenic agents. Additionally, the DNA sequence can include encoding elements for the coordinate expression of other non-LT or non-CT antigens. Further, the present invention also provides for the coexpression and/or coprovision of other antigens, such as viral antigens, along with LT-B antigen such that the additional antigens can benefit from the adjuvant effect of the transgenic LT-B. In addition, the present invention provides for the coexpression of both the LT-A and LT-B subunits such that the holotoxin can be assembled in the plant tissue to act as both an immunogen and an adjuvant.

The present invention also provides for a food composition useful for eliciting immune responses comprising a transgenic plant or material derived from the plant. The food composition can include the transgenic plant itself, processed food products of the plant, nutrients, vitamins, coloring, and/or flavoring.

Claim 1 of 1 Claim

We claim:

1. A synthetic E. coli gene which encodes LT-B, wherein said gene comprises the DNA sequence optimized for plant codon usage as presented in Table 3, SEQ ID NO:20.

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