MXPA02003789A - Plant virus particles with exogenous internal eitopes. - Google Patents

Abstract

The present invention relates to the expression of peptides on viral particles, and more particularly to the expression of peptides on the interior of the viral capsid. Methods are described for modifying viruses so that exogenous epitopes are expressed on the interior of the viral capsid. Viruses that can be modified include (plus;) stranded RNA viruses, especially plant (plus;) stranded RNA viruses such as the cowpea mosaic virus. Internal expression is especially useful for the expression of hydrophobic epitopes. The modified viral particles also find use as vaccines and as such are capable of eliciting an immune response.

Description

VIRAL PARTICLES WITH EXOGENOUS INTERNAL EPITHOPES FIELD OF THE INVENTION The present invention relates to the expression of peptides on viral particles, and more particularly, to the expression of peptides in the internal part of the viral capsid.

BACKGROUND OF THE INVENTION Vaccines are one of the greatest achievements of biomedical science and public health. At the beginning of the 20th century, infectious diseases were widely prevalent in the United States.

America and demanded a huge tribute on the population. For example, in 1900, 21,064 cases of smallpox were reported, and 894 patients died.

In 1920, 469,924 cases of measles were reported, and 7575 patients died; 147,991 cases of diphtheria were reported, and 13,170 patients died. In 1922, 107,473 cases of whooping cough were reported, and died 5099 patients. These diseases have been largely eliminated in the United States of America. Despite this success, more than 5 million infants die worldwide each year because of diseases that could have been prevented by existing vaccines. However, many of the vaccines that currently exist must be refrigerated, which makes their distribution in developing countries difficult. In addition, there are no or no vaccines available for diseases that are associated with significant averages of morbidity or mortality. For example, more than 250 million people are chronically infected with the hepatitis B virus; malaria causes 1 -2 million deaths per year; diarrheal diseases (for example, the infections caused by the rotavirus, Schigella sp., Vibrio cholera, and the toxin produced by E. coli) kill an estimated 4-5 million people annually. The Center for Disease Control has identified several factors to achieve the full potential of vaccines (www.cdc.gov/epo/mmwr/preview/mmwrhtml/ 00056803.htm). These suggestions include the search for new approaches to the delivery and administration of vaccines. A new approach is the development of vaccines that stimulate the two types of immune responses: humoral responses mediated by B cells and cellular responses mediated by helper T cells. However, attempts to create vaccines that stimulate both humoral and cellular immune responses, or that preferably induce a cellular immune response, have encountered difficulties. For example, synthetic peptide vaccines and recombinant protein vaccines are often poorly immunogenic and tend to induce humoral responses and not to induce cellular immune responses. DNA vaccines can induce both humoral and cellular immune responses. However, the question remains as to what the consequences of long-term antigen expression will be. In accordance with the above, what is needed in the * - "• iipmg-j • - 3 - In particular, the administration mechanism should be useful to induce both cellular and humoral immune responses.

BRIEF DESCRIPTION OF THE INVENTION The present invention relates to the expression of the peptides on the viral particles, and more particularly, to the expression of the peptides on the internal part or the viral capsid. In some embodiments, the present invention provides a compound comprising a chimeric viral particle having a capsid, in Wherein the capsid has an inner side and an outer side, the capsid comprising at least one exogenous peptide on the inner side of the capsid. In some preferred embodiments, the viral particle can be assembled into a host cell or tissue. In some embodiments, the viral particle is in the form of an icosahedron. In some modalities Preferred, the viral particle is a comovirus. In some embodiments that are particularly preferred, the viral particle is the cowpea mosaic virus. In some embodiments, the exogenous peptide is inserted into a layer protein of the viral particle. In some preferred embodiments, the exogenous peptide has from 5 to 20 amino acids. In others In 20 preferred embodiments, the exogenous peptide is inserted at a point of 5 to 20 amino acids from the N terminus of a layer protein, so that assembly of the viral particle is not disabled in a host cell. In some embodiments that are particularly preferred, the exogenous peptide is inserted into the VP-S of the cowpea mosaic virus, 25 between a tyrosine residue in the 1 1 position, and a tyrosine residue TFjí duplicated at position 12. In other modalities that are particularly preferred, the exogenous peptide is inserted into the VP-S of cowpea mosaic virus, between a dipeptide comprising a valine residue at position 10, and a residue of tyrosine at the 1 1 position and a duplicated dipeptide comprising a valine residue at position 12, and a tyrosine residue at position 13. In still other preferred embodiments, the exogenous peptide is inserted into the virus VP-S of cowpea mosaic, between a valine residue at position 10, and a duplicate valine residue at position 1 1. In additional embodiments, the viral particle does not contain nucleic acid. In other embodiments, the exogenous peptide encodes an epitope that can recognize the animal's immune system. In some preferred embodiments, the exogenous epitope is a cytotoxic T lymphocyte epitope. In the particularly preferred embodiments, the exogenous peptide contains a cytotoxic T lymphocyte epitope with flanking amino acids that are derived from a source that occurs naturally from the epitope. In some embodiments, the exogenous peptide is an auxiliary cell epitope T. In some preferred embodiments, the exogenous peptide contains an auxiliary cell epitope T with flanking amino acid sequences that are derived from a source that occurs naturally from the epitope. In still other embodiments, the exogenous peptide is a B-cell epitope. In additional embodiments, the exogenous peptide contains an auxiliary cell epitope T with flanking amino acid sequences that are derived from a source that occurs naturally from the epitope. .

In some embodiments, the chimeric viral particle contains a second exogenous peptide that is expressed on the outer surface of the viral capsid. In preferred embodiments, the second exogenous peptide is expressed on the outer surface of the viral capsid, wherein the peptide is inserted into the βC'-βC "cycle of the VP-S of the cowpea mosaic virus, In other preferred embodiments, the second exogenous peptide is expressed on the outer surface of the viral capsid, wherein the peptide is inserted into the βB-βC cycle of the VP-S of the cowpea mosaic virus, In still other preferred embodiments, the second exogenous peptide is expressed on the outer surface of the viral capsid, wherein the peptide is inserted into the ßE-aB cycle of the VP-L of the cowpea mosaic virus In other embodiments, the present invention provides a vaccine composition characterized in that it has an effective of a viral particle comprising a capsid having an inner side and an outer side, the capsid comprising at least one exogenous peptide, wherein the exogenous peptide is on the inter In still other embodiments, the present invention provides a formulation comprising as an active ingredient, a viral particle comprising a capsid having an inner side and an outer side, and the capsid comprising at least one exogenous peptide, wherein the exogenous peptide is on the inner side of the capsid and an adjuvant. In some embodiments, the present invention provides a compound comprising a viral layer protein, wherein the viral layer protein includes an exogenous peptide, the layer protein. atheist tIlM.

V / iilral is configured to be assembled within a viral capsid having an inner side and an outer side, wherein the exogenous peptide is expressed on the inner side of the viral capsid. In additional modalities, the present invention provides a process for preparing a viral particle comprising the provision of a host cell and a nucleic acid encoding a viral particle, the viral particle comprising: i) a viral layer protein, the viral layer protein that it comprises an internal side and an external side, and ii) an exogenous peptide, where the exogenous peptide is inserted on the inner side of the viral layer protein; transfect the host cell with the nucleic acid, so that the viral particles are produced. In still other embodiments, the present invention provides a method for inducing an immune response in an animal that requires this treatment, which method comprises administering to an animal a viral particle comprising a capsid having an inner side and an outer side, the capsid comprising at least one exogenous peptide, wherein the exogenous peptide is on the inner side of the capsid. In still further embodiments, the present invention provides a product that can be obtained by the process comprising the provision of a host cell and the nucleic acid encoding a viral particle, the viral particle comprising: i) a viral layer protein, the viral layer protein comprising an inner side and an outer side, and ii) an exogenous peptide, wherein the peptide exogenous is inserted on the inner side of the viral layer protein; transfect the host cell with the nucleic acid, so that the viral particles are produced. In some embodiments, the present invention provides a commercial package comprising a viral particle comprising a capsid having an inner side and an outer side, the capsid comprising at least one exogenous peptide, wherein the exogenous peptide is on the inner side of the capsid as an active ingredient, together with the instructions for its use. In other embodiments, the present compound comprises a chimeric virus particle that expresses an internal epitope, as described herein in any of the examples. In some embodiments, the present invention provides a vector comprising the nucleic acid encoding a viral particle, the viral particle comprising: i) a viral layer protein comprising an inner side and an outer side, and ii) an exogenous peptide , wherein the exogenous peptide is inserted on the inner side of the viral layer protein. The present invention is not limited to any particular type of vector. In fact, a variety of vectors are contemplated, including, but not limited to, RNA vectors (e.g., the nucleic acid encoding a strand RNA (+)) or DNA vectors (e.g. , the plasmid DNA encoding a strand RNA virus (+)).

Likewise, the present invention is not limited to any particular viral particle. In fact, a variety of viral particles are contemplated, including, but not limited to, viral particles in the form of ^^ ^^ -y ... m ~ & *, *** g¡H ^^^^^^^ rods and viral particles in the form of icosahedron. The present invention is not limited to any particular plant (+) chain virus. In fact, a variety of plant (+) chain RNA viruses find use in the present invention. In some preferred embodiments, the plant (+) chain RNA virus is a comovirus. In the modalities that are particularly preferred, the plant (+) chain RNA virus is a cowpea mosaic virus. In some embodiments, the coat protein is derived from a strand RNA (+) virus. The present invention is not limited to the layer proteins from any particular (+) chain RNA virus. In fact, coat proteins are contemplated from a variety of strand RNA viruses (+). In some preferred embodiments, the coat protein is from a plant (+) chain RNA virus. The present invention is not limited to the insertion of the exogenous peptide in any particular location. In fact, insertion into a variety of locations is contemplated. In some embodiments, the viral layer protein has an N terminus, and the exogenous peptide is inserted at a position of from 5 to 20 amino acids from the N terminus, so that the assembly of that viral layer protein is not impossible. In some preferred embodiments, the viral layer protein is the VP-S of the cowpea mosaic virus and the exogenous peptide is inserted between a thirosin residue at the 1 1 position of the VP-S and a duplicate tyrosine residue that is designs in position 12 of the VP-S. In other preferred embodiments, the viral layer protein is the VP-S of the cowpea mosaic virus and the exogenous peptide is inserted between a dipeptide which it comprises a valine residue at position 10, and a tyrosine residue at position 1 1 of the VP-S, and a duplicated dipeptide comprising a valine residue that is designed at position 12 and a tyrosine residue that is designs at position 13 of the VP-S. The present invention is not limited to exogenous peptides of any particular type. In fact, a variety of exogenous peptides can be expressed in the vectors of the present invention. In some embodiments, the exogenous peptide is hydrophobic. In other modalities, the exogenous peptide is a cytotoxic T lymphocyte epitope. In additional embodiments, the exogenous peptide is an auxiliary T cell epitope. In still other embodiments, the exogenous peptide is a B cell epitope. The present invention is not limited to vectors encoding only a single exogenous peptide. In fact, the present invention contemplates that more than one exogenous peptide can be expressed from the vectors. In some embodiments, the viral layer protein further comprises a second exogenous peptide. In preferred embodiments, the second exogenous peptide is inserted on the outer side of the viral layer protein. In some embodiments that are particularly preferred, the viral layer protein is VP-S having a βC'-βC "cycle and the second exogenous peptide is inserted into the βC'-βC" cycle. In other preferred embodiments, the viral layer protein is VP-L having a ßE-aB cycle and the second exogenous peptide is inserted into this ßE-aB cycle. The vectors of the present invention also include other components, such as regulatory elements. In some embodiments, the nucleic acid further encodes a promoter that operably links to the nucleic acid encoding a viral particle. The present invention is not limited to any particular promoter. In fact, a variety of promoters are contemplated, including, but not limited to, tissue-specific plant promoters and constitutive plant promoters. In other embodiments, the present invention provides methods comprising providing: i) the vector described above and ii) host cells; and b) transfecting the host cells with the vector, to produce transfected host cells under conditions such that the transfected host cells express the viral particle. The present invention is not limited to the transfection of any particular host cell. In fact, transfection of a variety of host cells, including, but not limited to, host cells that are selected from the group consisting of cells in plant, plant tissue culture cells, plant protoplasts, is contemplated. and the cells in the plant tissue. In still other embodiments, the present invention includes host cells that are produced by these methods. In some embodiments, the present invention provides methods comprising: providing a plant transfected with the vector described above and culturing the plant under conditions such that the viral particle is produced. In some preferred embodiments, the methods further comprise the step of purifying the viral particles from the plant.

In further embodiments, the present invention provides compositions comprising a nucleic acid encoding a viral layer protein, comprising an exogenous peptide, the viral layer protein is configured to assemble within a viral capsid having an internal side and an outer side, wherein the exogenous peptide is expressed on the inner side of the viral capsid. The present invention is not limited to any particular type of nucleic acid. In fact, a variety of nucleic acids are contemplated, including, but not limited to, RNA (e.g., nucleic acid encoding a strand RNA (+)) or DNA (e.g., plasmid DNA which encodes a chain RNA virus (+)). Likewise, the present invention is not limited to any particular viral particle. In fact, a variety of viral particles are contemplated, including, but not limited to, viral particles in the form of sticks and viral particles in the form of an icosahedron. The present invention is not limited to any particular plant (+) chain virus. In fact, a variety of chain (+) RNA viruses of plants find use in the present invention. In some preferred embodiments, the plant (+) chain RNA virus is a comovirus. In the embodiments that are particularly preferred, the plant (+) chain RNA virus is a cowpea mosaic virus. In some embodiments, the viral layer protein is derived from a strand RNA virus (+). The present invention is not limited to layer proteins from any particular (+) chain RNA virus. In fact, coat proteins starting from of a variety of chain RNA viruses (+). In some preferred embodiments, the coat protein is from a plant (+) chain RNA virus. The present invention is not limited to the insertion of the exogenous peptide in any particular place. In fact, insertion into a variety of locations is contemplated. In some embodiments, the viral layer protein has an N terminus, and the exogenous peptide is inserted at a position of from 5 to 20 amino acids from the N terminus, so that the assembly of that viral layer protein is not impossible. In some preferred embodiments, the viral layer protein is the VP-S of the cowpea mosaic virus and the exogenous peptide is inserted between a tyrosine residue at the 1 1 position of the VP-S and a duplicate tyrosine residue that is designs in position 12 of the VP-S. In other preferred modalities, the viral layer protein is the VP-S of the cowpea mosaic virus and the exogenous peptide is inserted between a dipeptide comprising a valine residue at position 10, and a tyrosine residue at the 1 1 position of the VP -S, and a duplicated dipeptide comprising a valine residue that is designed at position 12 and a tyrosine residue that is designed at position 13 of the VP-S. The present invention is not limited to exogenous peptides of any particular type. In fact, a variety of exogenous peptides can be expressed in the vectors of the present invention. In some embodiments, the exogenous peptide is hydrophobic. In other embodiments, the exogenous peptide is an auxiliary T cell epitope. In still other embodiments, the exogenous peptide is a B-cell epitope. The present invention is not limited to nucleic acids which they encode only a single exogenous peptide. In fact, the present invention contemplates that more than one exogenous peptide may be expressed by the nucleic acids. In some embodiments, the viral layer protein further comprises a second exogenous peptide. In preferred embodiments, the second exogenous peptide is inserted on the outer side of the viral layer protein. In some embodiments that are particularly preferred, the viral layer protein is VP-S having a βC'-βC "cycle and the second exogenous peptide is inserted into the βC'-βC" cycle. In other preferred embodiments, the viral layer protein is VP-L having a ßE-aB cycle and the second exogenous peptide is inserted into this ßE-aB cycle. The nucleic acids of the present invention also include other components, such as regulatory elements. In some embodiments, the nucleic acid further encodes a promoter that is operably linked to the nucleic acid encoding a viral particle. The present invention is not limited to any particular promoter. In fact, a variety of promoters are contemplated, including, but not limited to, tissue-specific plant promoters and constitutive plant promoters. In still further embodiments, the present invention provides viral particles comprising a capsid having an inner side and an outer side, the capsid comprising at least one exogenous peptide, wherein the exogenous peptide is on the inner side of the capsid. The present invention is not limited to any particular viral particle. Actually, as described above, the present invention encompasses a wide variety of viral particles. In some embodiments, the present invention provides a plant that expresses the viral particles. In further embodiments, the present invention provides viral particles of fruit, leaves, tubers, stems or purified, which are isolated from the plant. In other embodiments, the present invention provides methods for inducing an immune response comprising providing i) viral particles (which are described above) comprising a plurality of layer proteins having an inner side and an outer side, the layer proteins that comprise an exogenous peptide, wherein the exogenous peptide is on the inner side of the layer proteins; and ii) a subject; and b) exposing the subject to the viral particle under conditions such that the subject develops an immune response to the exogenous peptide. In some preferred embodiments, the viral particles are provided from a plant source. In still further embodiments, the present invention provides vectors comprising the nucleic acid encoding a viral layer protein sequence having inserted therein, a sequence of exogenous peptides, the viral layer protein comprising a second site mutation, so that the viral layer protein can be assembled within a viral capsid. The present invention is not limited to any second mutation of a particular site. In fact, a variety of second site mutations are contemplated, including, but not limited to, second site mutations in both the VP-S and the VP-L of the cowpea mosaic virus. In the modalities that are preferred i. A ^ fie ^ - .. »...» m. t- * > In a particular way, the second site mutation is selected from the group consisting of F91 S in VP-S, F180L in VP-S, M177V in VP -S, I 124V in VP-S, R2102K in VP-L, I2045 in VP-L, M177T in VP-S, A2092T in VP-L, G80D in VP-S. In some embodiments, the present invention provides methods for inducing the second site mutations in a viral layer protein comprising providing i) a vector comprising the nucleic acid encoding a viral particle, the viral particle comprising a viral layer protein , the viral layer protein comprising an inner side and an outer side, and ii) a foreign peptide, wherein the foreign peptide is inserted on the inner side of the viral layer protein and ii) a first host plant; infect the first host plant with the vector, so that the viral particle is expressed; monitor the first host plant until late lesions appear on the leaves that were directly infected; isolate the viral particles from the late lesions, to provide isolated viral particles; and inoculate a second host plant to obtain a secondary infection. In some modalities, the methods also include the step of monitoring the second host plant by the appearance of systemic symptoms. In some preferred embodiments, the systemic symptoms appear in a time frame that can be compared with that of the infected plant with the viral control particles. In other embodiments, the methods further comprise the steps of Method of Claim 84, further comprising the steps of isolating the viral particles from the systemic lesions, the viral particles comprising a genome; and sequencing the genome of the viral particles. In some embodiments, the present invention provides the viral particles that the method produces. In some embodiments, the present invention provides a vaccine composition comprising a viral particle comprising a capsid having an inner side and an outer side, the capsid comprising at least one exogenous peptide, wherein the exogenous peptide is on the side internal capsid. In additional embodiments, the present invention provides methods for inducing a humoral immune response, which comprises administering to an animal the vaccine composition. In still other embodiments, the present invention provides methods for improving an immune response to a B cell epitope comprising providing i) a modified viral particle comprising a B cell epitope and an internal CTL epitope in an immunogenic complex; and ii) an animal; and administering the modified viral particle to the animal. The present invention is not limited to a particular CTL epitope. In fact, a variety of CTL epitopes are contemplated including, but not limited to, a mimotope of peptide 2F10 of class "a" determinant of the surface antigen of hepatitis B virus. The present invention also provides methods for improving an immune response to a B cell epitope comprising providing i) a modified viral particle comprising a B cell epitope and an internal CTL epitope in an antigenic complex; and an animal; and b) administering the modified viral particle to the animal. The present invention is not limited to a particular CTL epitope. In fact, a variety of CTL epitopes are contemplated including, but not limited to, a mimotope of peptide 2F10 of class "a" determinant of the surface antigen of hepatitis B virus. In some embodiments, the present invention provides methods for improving an immune response to a B cell epitope comprising providing i) a modified viral particle comprising a B cell epitope and an auxiliary T cell epitope in an immunogenic complex; and ii) an animal; and administering the modified viral particle to the animal. The present invention is not limited to a particular T cell epitope. In fact, a variety of T-cell epitopes including, but not limited to, the epitope of the universal T-helper of tetanus toxoid are contemplated. In further embodiments, the present invention provides methods for improving an immune response to a B cell epitope comprising providing i) a modified viral particle comprising a B cell epitope and an auxiliary T cell epitope in an antigenic complex; and i) an animal; and administering the modified viral particle to the animal. The present invention is not limited to any particular T cell epitope. In fact, a variety of T-cell epitopes including, but not limited to, the epitope of the universal T-helper of tetanus toxoid are contemplated. - l í BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows the sequence of the N terminus of the VP-S protein of the CPMV and illustrates where the foreign peptides can be inserted. Figure 2 shows the results of a CTL assay for mice that were vaccinated with the chimeric virus particles, according to one embodiment of the invention (see Example 6); the release of chromium from the target cells that were loaded with the target peptide derived from the LCMV or the unloaded cells are measured using a BetaMax workstation. Figure 3 shows a restriction map of the vector pCP26 that is used in most of the genetic constructions that are described.

DESCRIPTION OF THE INVENTION The present invention relates to the expression of peptides on the viral particles, and more particularly, to the expression of the peptides in the internal part of the viral capsid. The use of chimeric viral particles (CVP's) as vaccines represents an attractive strategy to improve the storage and delivery of vaccines. In the Patents of the United States of North America Nos. 5,874,087 and 5,958,422 (each of which is incorporated herein by reference), the use of CVPs is described. These patents describe the development of the vectors encoding the modified Cowpea Mosaic Virus (CPMV) genomes. containing the inserts of the peptides in the coat protein. The vectors are useful for the production of viral particles in which the peptide is presented on the surface of the viral particle. Following the definitions, the systems for expression of viral particles, the inserts of the peptides and the sites for the insertion, and the uses for the modified viral particles are described.

DEFINITIONS To facilitate understanding of the invention, a number of terms are defined below. The term "viral particle" as used herein, refers to the capsid of a complete or partially assembled virus. A viral particle may or may not contain nucleic acid. The term "viral capsid" as used herein, refers to a protein layer surrounding the viral nucleic acid in a wild-type virus. Viral capsids have internal surfaces and external surfaces. The internal surface of a viral capsid is the surface that is normally exposed to the viral nucleic acid. The external surface of a viral capsid is the surface that is generally exposed to the environment. The term "viral layer protein" as used herein, refers to a protein that interacts with other proteins to assemble and form part of the viral capsid. Examples of viral layer proteins include, but are not limited to, the VP-S and VP-L layer proteins of the cowpea mosaic virus. The inner side of a layer protein viral, is the portion of the layer protein that is exposed on the inner surface of the viral capsid. The outer side of a viral layer protein is the portion of the layer protein that is exposed on the outer surface of the viral capsid. The term "(+) chain RNA virus" as used herein, refers to a virus having an RNA genome, wherein the isolated RNA is directly infectious when introduced to an appropriate host. It is known that chain (+) RNA viruses infect a variety of animal, fungal, and plant hosts. Examples of (+) chain RNA viruses include, but are not limited to, the Picornoviruses (eg, poliovirus), and viruses from the following families: Caulimoviridae, Bromoviridae, Comoviridae, Geminiviridae, Reoviridae, Partitiviridae, Sequiviridae, Tobamovirus, and Tombusviridae. The term "symptoms", when used with reference to plant virus infection, refers to the appearance of indicators of viral infection in the plant. These indicate both local injuries, as systemic symptoms. Local lesions, such as necrotic lesions, chlorotic lesions, and ring spots, occur at or near the site of infection. Systemic symptoms appear throughout the plant and include mosaic patterns, mottled patterns, dwarfism, yellowing and necrosis. The term "systemic infection" as used herein, refers to viral infections that extend from the site of the initial infection. In plants, systemic infection occurs when viruses move from infected cells within the vasculature (eg, phloem) of the plant. In many viruses, this movement is mediated by a movement protein that modifies the plasma desaturate. The term "icosahedron", when used with reference to the viral capsid or viral particle, refers to a capsid exhibiting symmetry in general icosahedron: rotating symmetry in 5 parts through each of the 12 vertices, rotating symmetry in 3 parts around an axis through the center of each of the 20 triangular faces, and rotating symmetry in 3 parts around an axis through the center of each of the thirty edges. The icosahedrons are made up of 60 identical construction units (which may comprise more than one sub-unit) or multiple units of 60 identical construction units. The contacts of the interunit are not exactly identical throughout the capsid; however, the entire union of the interunit includes the same general type of contact, so that the links of the interunit can be described as almost equivalent. It is contemplated that some viruses in icosahedron, especially large icosahedron viruses (eg, adenoviruses), may be deviated from the structural and geometric criteria observed by smaller icosahedron viruses. Examples of viruses in icosahedron include, but are not limited to, polioviruses, adenoviruses, and viruses from the following families: Caulimoviridae, Bromoviridae, Comoviridae, Geminiviridae, Reoviridae, Partitiviridae, Sequiviridae, and Tombusviridae. The term "epitope" as used herein, refers to an antigenic determinant, which is in any region of a macromolecule with the ability or potential to produce, and combined with, the specific antibody (that is, it can bind to an immunoglobulin or specific T cell receptor). The term "hydrophobic", when used with reference to a peptide or epitope, refers to a peptide or epitope having approximately 20 percent or more of hydrophobic amino acid residues (eg, alanine, valine, leucine, isoleucine, proline, phenylalanine, tryptophan, and methionine). The term "cytotoxic T lymphocyte epitope" as used herein, refers to an epitope that can be recognized by a cytotoxic T lymphocyte. The term "helper T cell epitope" as used herein, refers to an epitope that can be recognized by an auxiliary T cell. The term "B cell epitope" as used herein, refers to an epitope that can be recognized by a B cell. The term "immune response" as used herein, refers to the reaction of an animal , mediated by the immune system, to an antigen or immunogen and can be characterized by the production of antibodies and / or the stimulation of cell-mediated or immune tolerance. The term "antigenic complex" as used herein, refers to the complex that contains at least one epitope that can be combined with an immunoglobulin or cell surface receptor. ? ti¡á? í? áJi »itÉt * ?. t, ¡. .-. 1JAla ^ ... L t a.AM. ^ a.1- ^. - - * »..........

The term "immune complex" as used herein, refers to a peptide that is sufficiently structurally similar to an epitope, to induce an immune reaction against that epitope, even though the two sequences do not share any homology or similarity at the level of the amino acids (in the case of a peptide mimotope), or, in the case where the mimotope represents a structural configuration that adopts a non-protein molecule, such as a carbohydrate, the mimotope can react with the immune molecules that they are directed against the non-protein epitope. As used herein, the term "immunoglobulin" refers to the secreted product of the plasma cell (e.g., the cell B activated), comprising two heavy chain polypeptides that are complexed with two light chain polypeptides, which together make a binding site for the proteins. As used herein, the term "group I proteins of major histocompatibility Class I MHC" refers to the proteins encoded by the genes of the major histocompatibility group and which are involved in the effective presentation of the antigens on the CD8 + T lymphocytes. As used herein, the term "MHC class II major histocompatibility proteins" refers to the proteins encoded by the genes of the major histocompatibility group and which are involved in the effective presentation of the antigens on the CD4 + T lymphocytes. The term "plant" as used herein, refers to a plurality of plant cells, which differ greatly in a structure that is present at any stage of the development of a plant. These structures include, but are not limited to, a fruit, shoot, stem, leaf, flower petal, and so on. The term "plant tissue" includes the differentiated and undifferentiated tissues of plants including, but not limited to, roots, shoots, leaves, pollen, seeds, tumor tissue and different types of cells in culture (e.g. unique, protoplasts, embryos, callus, etc.). The plant tissue may be in plant, in organic culture, tissue culture, or cell culture. The term "protoplast" as used herein, refers to the isolated plant cells in which the cell walls have been removed. In general, protoplasts are produced in accordance with conventional methods (See, for example, U.S. Patent Nos. 4,743,548; 4,677,066; 5, 149.645 and 5, 508, 184; all of which are incorporated herein by reference). The plant tissue may be dispersed in an appropriate medium having an appropriate osmotic potential (eg, 3 to 8 weight percent of a sugar polyol) and one or more polysaccharide hydrolases (eg, pectinase, cellulase, etc.) , and the degradation of the cell wall allowed it to proceed for a sufficient time, to provide the protoplasts. After filtration, protoplasts can be isolated by centrifugation and then resuspended for subsequent treatment or use. The regeneration of the protoplasts that are maintained in culture for whole plants is carried out by methods that are known in the art (See, for example, Evans et al., Handbook of Plant Cell Culture, 1: 124-176, MacMillan Publishing Co, New York

[1983], Binding, Plant Protoplasts, p.21-37, CRC Press, Boca Raton

[1985] and Potrykus and Shillito, Methods in Enzymology, volume 1 18, Plant Molecular Biology, A. and H. Weissbach eds., Academic Press, Orlando

[1986] The term "gene" as used herein, refers to a DNA sequence comprising the control and coding sequences necessary for the production of a protein polypeptide or precursor. The polypeptide can be encoded by a full-length coding sequence or by any portion of the coding sequence, provided that the activity of the desired protein is retained. "Nucleoside", as used herein, refers to a compue This consists of a purine base [guanine (G) or adenine (A)] or pyrimidine [thymine (T), uridine (U), or cytidine (C)] that is covalently linked to a pentose, while "nucleotide" refers to a phosphorylated nucleotide in one of its pentose hydroxyl groups. A "nucleic acid" as used herein, is a nucleotide sequence that is covalently linked at the 3 'position of the pentose of a nucleotide that is attached by a group of the phosphodiester to the 5' position of the pentose. of the following, and in which the nucleotide residues (bases) are linked in the specific sequence; that is, a linear order of nucleotides. A "polynucleotide", as used herein, is a nucleic acid that contains a sequence that is greater than about 100 nucleotides in length. An oligonucleotide, as used herein, is a short polynucleotide or a portion of a polynucleotide. An oligonucleotide typically contains a sequence of about two to about two bases. The word "oligo" is sometimes used instead of the word "oligonucleotide". The nucleic acid molecules are said to have a "5 'terminus (5' end) and a" 3 'terminus "(3' end), because nucleophic acid phosphodiester bonds occur at 5 'carbon and carbon 3 'of the pentose ring of the substitute mononucleotides. The end of a nucleic acid in which the new bond would be with the 5 'carbon, is its 5' terminal nucleotide. The end of a nucleic acid in which the new bond would be with the 3 'carbon, is 3' terminal nucleotide. A terminal nucleotide, as used herein, is the nucleotide in the extreme position of the 3'- or 5'- terminus. The DNA molecules are said to have "5 'ends" and "3' ends", because the mononucleotides are reacted to make oligonucleotides in such a way that the 5 'phosphate of a pentose ring of the mononucleotide is bound to the 3 'oxygen from its neighbor in one direction, by means of a phosphodiester bond. Therefore, one end of an oligonucleotide is referred to as the "5 'end" if its 5' phosphate is not bound to the 3 'oxygen of a pentose ring of the mononucleotide, and as the "3' end" if its 3 'oxygen is not linked to a 5' phosphate of a pentose ring of the subsequent mononucleotide. As used herein, it can also be said that a . .,.,.,.,.,.,. nucleic acid sequence, even if they are internal to a longer oligonucleotide or polynucleotide, has 5 'and 3' ends. In a DNA molecule whether linear or circular, discrete elements are referred to as being "upstream", or 5 'of the "downstream" or 3' elements. This terminology reflects the fact that transcription proceeds in a 5 'to 3' fashion along the DNA strand. Typically, the promoter and enhancer elements that direct the transcription of a linked gene are generally located 5 'or upstream of the coding region. However, the enhancer elements can exert their effect even when they are located 3 'of the promoter element and the coding region. The termination of the transcription and the polyadenylation signals are located 3 'or downstream of the coding region. The term "wild type", when made in relation to a gene, refers to a gene that has the characteristics of a gene that is isolated from a naturally occurring source. The term "wild type", when made in relation to a product of the gene, refers to a product of the gene that has the characteristic of a product of the gene that is isolated from a source that occurs naturally. A wild-type gene is one that is observed more frequently in a population and is therefore arbitrarily designated as the "normal" or "wild type" form of the gene. In contrast, the term "modified" or "mutant" when made with reference to a gene or a product of the gene refers, respectively, to a gene or gene product that displays modifications in sequence and / or functional properties (ie, , ! . ??? * 'k *, altered characteristics) when compared to the gene or product of the wild-type gene. It is noted that mutants that occur naturally can be isolated; these are identified by the fact that they have altered characteristics when compared to the gene or product of the wild-type gene. As used herein, the term "over expression" refers to the production of a gene product in transgenic organisms, which exceeds production levels in normal or untransformed organisms. As used herein, the term "cosuppression" refers to the expression of a foreign gene that has substantial homology to an endogenous gene that results in the suppression of both the foreign and the endogenous gene expression. As used herein, the term "altered levels" refers to the production of gene product (s) in transgenic organisms, in amounts or proportions that differ from those of normal or untransformed organisms. The term "recombinant", when made in relation to a DNA molecule, refers to a DNA molecule that is composed of DNA segments joined together by molecular biological techniques. The term "recombinant", when made in relation to a protein or polypeptide, refers to a protein molecule that is expressed using a recombinant DNA molecule. The term "nucleotide sequence of interest" refers to any nucleotide sequence, the manipulation of which could be deemed desirable for any reason (eg, confer improved qualities), by one skilled in the art. These nucleotide sequences include, but are not limited to, the coding sequences of structural genes (eg, reporter genes, selection marker genes, oncogenes, drug resistant genes, growth factors, etc.), and non-regulatory sequences. coding, which do not encode an mRNA or protein product (e.g., promoter sequence, polyadenylation sequence, terminator sequence, enhancer sequence, etc.). As used herein, the term "coding region", when used in relation to the structural gene, refers to the nucleotide sequences encoding the amino acids found in the nascent polypeptide, as a result of the translation of a mRNA molecule. Typically, the coding region is fixed on the 5 'side by the nucleotide "ATG", which encodes the initiating methionine and on the 3' side by a stop codon (eg, TAA, TAG, TGA). In some cases, it is also known that the coding region is initiated by a "TTG" triple nucleotide. As used herein, the terms "complementary" or "complementarity", when used in relation to polynucleotides, refer to polynucleotides that are related by the rules of base pairings. For example, the sequence 5'-AGT-3 'is complementary to the sequence 5'-ACT-3'. The complementarity can be "partial", in which only some bases of the nucleic acids are matched, in accordance with the rules of pareo of bases. Or, there may be "complete" or "total" complementarity between the nucleic acids.

The degree of complementarity between the nucleic acid strands has significant effects on the efficiency and resistance of the hybridization between the nucleic acid strands. This is of particular importance in the amplification reactions, as well as the detection methods that depend on the binding between the nucleic acids. A "complement" of a nucleic acid sequence, as used herein, refers to a nucleotide sequence whose nucleic acids show a complete complementarity to the nucleic acids of the nucleic acid sequence. The term "homology", when used in relation to nucleic acids, refers to a degree of complementarity. There may be partial homology or complete homology (that is, identity). "Sequence identity" refers to a measure of correspondence between two or more nucleic acids or proteins, and is given as a percentage with reference to the total comparison length. The identity calculation takes into account those nucleotide or amino acid residues that are identical and in the same relative positions in their respective longer sequences. Identity calculations can be performed using algorithms contained within computer programs, such as "GAP" (Genetics Computer Group, Madison, Wis.) And "ALIGN" (DNAStar, Madison, Wis.). A partially complementary sequence is one that at least partially inhibits (or competes with) a completely complementary sequence from hybridizing to a target nucleic acid and is referenced therein using the functional term i. *. A * mHtaM [t¡ || i na [., ji »» a. , ^ _ ... fc ... i *. ? t? V? tíff ** m * «? jBtíJÍ. "substantially homologous". The inhibition of hybridization of the sequence completely complementary to the target sequence can be examined using a hybridization assay (Southern or Northern blot, solution hybridization and the like), under conditions of low stringency. A substantially homologous sequence or probe will compete for, and inhibit the binding (i.e., hybridization) of a sequence that is completely homologous to a target, under conditions of low stringency. This does not mean that the conditions of low stringency are such as to allow non-specific fixation; low stringency conditions require that the binding of two sequences to one another be a specific (ie, selective) interaction. The absence of non-specific binding can be proven by the use of a second objective, which lacks even a partial degree of complementarity (eg, less than about 30 percent identity); in the absence of non-specific binding, the probe will not hybridize with the second non-complementary target. When used with reference to a double-stranded nucleic acid sequence, such as a cDNA or genomic clone, the term "substantially homologous" refers to any probe that can hybridize to either or both of the nucleic acid sequence chains double chain, under conditions of low stringency as described below. The conditions of low stringency, when the hybridization of the nucleic acid is used with reference, comprise equivalent conditions to the fixation or hybridization at 42 ° C in a solution consisting of 5X SSPE (43.8 g / l NaCl, 6.9 g / l NaH2PO4 » H2O and 1.85 g / l EDTA, pH adjusted to 7.4 with NaOH), 0.1 percent SDS, 5X Denhardt's reagent [50X Denhardt's contained per 500 ml: 5 g Ficoll (Type 400, Pharmacia), 5 g BSA (Fraction V; Sigma)] and 100 μg / ml denatured salmon sperm DNA, followed by washing in a solution comprising 5X SSPE, 0.1 percent SDS at 42 ° C when using a probe of approximately 500 nucleotides in length. High stringency conditions, when nucleic acid hybridization is used with reference, comprise conditions equivalent to binding or hybridization at 42 ° C in a solution consisting of 5X SSPE (43.8 g / l NaCl, 6.9 g / l NaH2PO4 «H2O and 1.85 g / l EDTA, pH adjusted to 7.4 with NaOH), 0.5% SDS, 5X Denhardt reagent and 100 μg / ml denatured salmon sperm DNA, followed by washing in a solution comprising 0.1X SSPE, 1.0 percent SDS at 42 ° C when using a probe approximately 500 nucleotides in length. When used with reference to nucleic acid hybridization, the art knows well that numerous equivalent conditions can be employed to understand stringency conditions either low or high; factors such as length and nature (DNA, RNA, base composition) of the probe and the nature of the target (DNA, RNA, base composition, present in the solution or immobilized, etc.) and the concentration of the salts and other components (for example, the presence or absence of formamide, sulfate jlh¿ ntAA ..? atoá .. m * JümAie- * • ^^^ "- • ^ k. ^ ,, ^, ^,. ^. ^ .. ^. ^^ of dextran, polyethylene glycol), and the hybridization solution can vary to generate stringency hybridization conditions either low or high, different from, but equivalent to, the conditions that are listed above.Stringency, when used with reference to nucleic acid hybridization, typically occurs in a range of from about Tm-5 ° C (5 ° C below the Tm of the probe) to about 20 ° C to 25 ° C below Tm As will be understood by those skilled in the art, it can be used stringent hybridization to identify or detect identical polynucleotide sequences or to identify or detect similar or related polynucleotide sequences Under "stringent conditions", a nucleic acid sequence of interest will hybridize with its exact complement and sequences that are related in a manner It is said that e polypeptide molecules have an "amino terminus" (N-terminus) and a "carboxy-terminus" (C-terminus), because the peptide bonds occur between the amino group of the base structure of a first amino acid residue and the group carboxyl of base structure of a second amino acid residue. Typically, the term of a polypeptide in which the novel linkage would be to the carboxy terminus of the growing polypeptide chain, and the polypeptide sequences, are written from left to right starting with the amino terminus. As used herein, the terms "exogenous peptide" or "foreign peptide" refer to a peptide that is not in its natural environment (that is, it has been altered by the hand of man). For example, a id. . «Bfa ^» ^ .-. . . . J? .ai? Fc ** - ^ ijL * á ^. * ^ 4i? * Ti * jhá js & ála ??? exogenous peptide gene includes a peptide that has been inserted into another polypeptide or that has been added or fused with a polypeptide. As used herein with reference to an amino acid sequence or a protein, the term "portion" (as in "a portion of an amino acid sequence"), refers to the fragments of that protein. The fragments can range in size from four amino acid residues to the complete amino acid sequence, minus one amino acid. As used herein, the term "fusion protein" refers to a chimeric protein that contains the protein of interest (e.g., the viral layer protein), bound to an exogenous protein fragment (e.g., an epitope). hydrophobic). The term "isolated" when used in relation to a nucleic acid, as in "an isolated nucleic acid sequence"), refers to a nucleic acid sequence that is identified and separated from at least one contaminating nucleic acid with which it is ordinarily associated in its natural source. The isolated nucleic acid is the nucleic acid present in a form or environment that is different from that in which it is found in nature. In contrast, non-isolated nucleic acids are nucleic acids such as DNA and RNA that are in the state in which they exist in nature. For example, a given DNA sequence (e.g., a gene) is located on the chromosome of the host cell in proximity to neighboring genes; RNA sequences, such as the specific mRNA sequence that encodes a specific protein, are found in the cell as a mix with many other mRNAs that encode a multitude of proteins.

However, an isolated nucleic acid sequence comprising SEQ ID NO: X includes, by way of example, those nucleic acid sequences in cells that ordinarily contain SEQ ID NO: X, wherein the nucleic acid sequence is in a chromosomal or extrachromosomal location different from that of natural cells, or otherwise flanked by a nucleic acid sequence different from that found in nature. The isolated nucleic acid sequence can be present in the form of a single chain or double chain. When you are going to use an isolated nucleic acid sequence to express a protein, the nucleic acid sequence will contain at least one portion of the sense or coding strand (that is, the nucleic acid sequence can be a single strand). Alternatively, it could contain the chains both sense and anti-sense (that is, the nucleic acid sequence could be double-stranded). As used herein, the term "purified" refers to molecules or additions of molecules (eg, viral particles), either nucleic or amino acid sequences, which are removed from their natural environment, isolate or separate . An "isolated nucleic acid sequence" is therefore a purified nucleic acid sequence. "Substantially purified" molecules are at least 60 percent free, preferably at least 75 percent free, and most preferably at least 90 percent free of other components with which they naturally associate.

As used herein, the terms "vector" and "carrier" are used interchangeably with reference to nucleic acid molecules that transfer the DNA segment (s) from one cell to another.

Vectors may include plasmids, bacteriophages, viruses, cosmids, and the like. The term "expression vector" or "expression cartridge", as used herein, refers to a recombinant DNA molecule that contains a desired coding sequence and the appropriate nucleic acid sequences necessary for the expression of the sequence of coding that is operably linked in a particular host organism. The nucleic acid sequences necessary for expression in prokaryotes, usually include a promoter, an operator (optional), and a ribosome binding site, often together with the other sequences. It is known that eukaryotic cells use promoters, enhancers, and termination and polyadenylation signals. The terms "target vector" or "target construct" refer to oligonucleotide sequences comprising a gene of interest flanked on each side by a recognition sequence, which is capable of homologous recombination of the DNA sequence that it is located between the flanking recognition sequences. The terms "in operable combination", "in operable order" and "operably linked", as used herein, refer to the linking of nucleic acid sequences in such a way as to produce a nucleic acid molecule which can direct the transcription of a given gene and / or the synthesis of a desired protein molecule. The term also refers to the linkage of amino acid sequences such that a functional protein is produced. The transcriptional control signals in eukaryotes comprise the elements "promoter" and "enhancer". Promoters and enhancers consist of short configurations of DNA sequences that interact specifically with cellular proteins in transcription (Maniatis et al., Science, 236: 1237, 1987). The promoter and enhancer elements have been isolated from a variety of eukaryotic sources including the genes of yeast, insects, mammals and plant cells. Promoter and enhancer elements from viruses have also been isolated and analogous control elements, such as promoters, are also found in prokaryotes. The selection of a particular promoter and enhancer depends on the type of cell that is used to express the protein of interest. Some eukaryotic promoters and enhancers have a broad host range, while others are functional in a limited subset of cell types (for review, see Voss et al., Trends Biochem. Sci., 11: 287, 1986; and Maniatis et al. collaborators, supra, 1987). The terms "promoter element", "promoter", or "promoter sequence" as used herein, refer to a sequence of DNA that is located in the 5 'end protein coding region (that is, precedes) a DNA polymer. The location of most , .. ^ k. • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • in nature, they precede the region that was transcribed. The promoter works like a switch, which activates the expression of a gene. If the gene is activated, it is said to be transcribed or involved in transcription. Transcription includes the synthesis of mRNA from the gene. The promoter, therefore, serves as a transcriptional regulatory element and also provides a site for the initiation of gene transcription within the mRNA. The promoters can be tissue-specific or cell-specific. The term "tissue-specific" as applied to a promoter, refers to a promoter that can direct the selective expression of a nucleotide sequence of interest, to a specific type of tissue (e.g., seeds) in the relative absence of expression of the same nucleotide sequence of interest, in a different type of tissue (e.g., leaves). The tissue specificity of a promoter can be evaluated by, for example, operably linking a reporter gene to the promoter sequence, to generate a reporter construct, which introduces reporter construction within the genome of a plant so that reporter construction is integrated into each tissue of the resulting transgenic plant, and which detects the expression of the reporter gene (for example, which detects the mRNA, protein, or activity of a protein that encoded the reporter gene) in different tissues of the transgenic plant . The detection of a higher level of expression of the reporter gene in one or more tissues in relation to the level of expression of the reporter gene in other tissues, shows that the promoter is specific for tissues in which higher levels are detected expression. The term "cell type specific" as applied to a promoter, refers to a promoter that can direct the selective expression of a nucleotide sequence of interest in a specific type of cell, in the relative absence of expression thereof. nucleotide sequence of interest, in a different type of cell, within the same tissue. The term "cell-type specific" when applied to a promoter also means a promoter that can promote the selective expression of a nucleotide sequence of interest in a region within a single tissue. The specificity of the cell type of a promoter can be evaluated, using methods well known in the art, for example, immunohistochemical staining. Briefly, sections of tissue are soaked in paraffin, and the paraffin sections are reacted with a primary antibody, which is specific for the polypeptide product that was encoded by the nucleotide sequence of interest, whose expression is controlled by the promoter. A labeled secondary antibody (eg, peroxidase conjugate), which is specific for the primary antibody, is allowed to attach to the excised tissue and specific binding is detected (eg, with avidin / biotin) by microscopy. The promoters can be constitutive or regulable. The term "constitutive" when made with reference to a promoter, means that the promoter can direct the transcription of an operably linked nucleic acid sequence, in the absence of a stimulus (e.g., heat shock, chemicals). , light, etc.). Typically, constitutive promoters can direct the expression of B. A &* T? «F-t a i * t * M, A. j ^» jj. * Jiiuito¡ttá ».a¿ita» ..JAi.il > i a transgene in substantially any cell and any tissue. Exemplary constitutive plant promoters include, but are not limited to, the Cauliflower Mosaic Virus SD promoters (CaMV SD, see for example, U.S. Patent No. 5,352,605, which is incorporated herein by reference). reference), mannopine synthase, octopine synthase (oes), superpromotor (see, for example, WO 95/14098), and ub3 (see, for example, Garbarino and Belknap, Plant Mol. Biol. 24: 1 19-127

[1994]). These promoters have been used successfully to direct the expression of heterologous nucleic acid sequences in transformed plant tissue. In contrast, an "adjustable" promoter is one that can direct a level of transcription of a nucleic acid sequence that is operably linked in the presence of a stimulus (e.g., heat shock, chemicals, light, etc.). ), which is different from the level of transcription of the nucleic acid sequence that is operably linked, in the absence of the stimulus. As used herein, the term "regulatory element" refers to a genetic element that controls some aspect of the expression of the nucleic acid sequence (s). For example, a promoter is a regulatory element that facilitates the initiation of transcription of a coding region that is operably linked. Other regulatory elements are splicing signals, polyadenylation signals, termination signals, and so on. The enhancer and / or promoter can be "endogenous" or "exogenous" or "heterologous". An "endogenous" enhancer or promoter is one that is binds naturally with a given gene in the genome. An "exogenous" or "heterologous" enhancer or promoter is one that is placed in juxtaposition with a gene through genetic manipulation (ie, molecular biological techniques), so that transcription of the gene is directed by the enhancer or linked promoter. . For example, an endogenous promoter in operable combination with a first gene can be isolated, removed, and placed in operable combination with a second gene, thereby making it a "heterologous promoter" in operable combination with the second gene. A variety of these combinations are contemplated (for example, the first and second genes can be of the same species, or of different species). The presence of "splice signals" on an expression vector frequently results in higher levels of expression of the recombinant transcript in eukaryotic host cells. The splicing signals mediate the removal of introns from the primary RNA transcript and consist of a splice donor and receptor site (Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press, New York).

[1989] pages 16.7-16.8). A commonly used splice donor and receptor site is the splice junction from the SV40 16S RNA. The efficient expression of the recombinant DNA sequences in eukaryotic cells requires the expression of the signals that direct the efficient termination and polyadenylation of the resulting transcript. The transcription termination signals are ... ¿* f ^ > J, ^, i < M ^, a > jA ^, tji MfcB ^ ..m¿ ^. they are generally downstream of the polyadenylation signal and are a few hundred nucleotides in length. The term "poly (A) site" or "poly (A) sequence" as used herein, denotes a DNA sequence that directs both transcription and polyadenylation of the incipient RNA transcript. Efficient polyadenylation of the recombinant transcript is desirable, since transcripts lacking a poly (A) tail are unstable and degrade rapidly. The poly (A) signal that is used in an expression vector can be "heterologous" or "endogenous". An endogenous poly (A) signal is one that is naturally found at the 3 'end of the coding region of a given gene in the genome. A heterologous poly (A) signal is one that has been isolated from one gene and placed 3 'to another gene. A heterologous poly (A) signal that is commonly used is the poly (A) signal of SV40. The SV40 poly (A) signal is contained in a Bam / Bcl / 237 bp restriction fragment and directs both termination and polyadenylation (Sambrook, supra, 16.6-16.7). The terms "infect" and "infection" with a bacterium refer to the co-incubation of an objective biological sample (e.g., cell, tissue, etc.) with the bacterium, under conditions such that the nucleic acid sequences contained therein. of the bacteria, are introduced into one or more cells of the target biological sample. The terms "bombard", "bombardment", and "biolistic bombardment" refer to the process to accelerate the particles towards an objective biological sample (for example, cell, tissue, etc.), to effect a wound of the cellular membrane of a cell in the sample biological objective and / or the entry of the particles into the objective biological sample. Methods for biolistic bombardment are known in the art (e.g., U.S. Patent No. 5,584,807, the content of which is incorporated herein by reference), and are commercially available (e.g., microprojectile-driven accelerator by helium gas (PDS-1000 / He, BioRad)). The term "microherir" when made with reference to plant tissue, refers to the introduction of microscopic wounds in that tissue. The microheride can be achieved by, for example, the bombardment of particles as described herein, or by means of scoring the tissue. The term "transfection" as used herein, is required upon the introduction of foreign DNA into eukaryotic cells. Transfection can be achieved by means of a variety of elements known in the art, including coprecipitation of DNA with calcium phosphate, DEAE-dextran-mediated transfection, polybrene-mediated transfection, electroporation, microinjection, liposome fusion, lipofection, protoplast fusion, retroviral infection, and biolistics. The term "transgenic", when used with reference to a cell, refers to a cell that contains a transgene, or whose genome has been altered by the introduction of a transgene. The term "transgenic", when used with reference to a tissue or a plant, refers to a tissue or plant, respectively, that comprises one or more cells that contain a transgene, or whose genome has been altered by introducing a transgene. Tranegenic cells, tissues and plants can be produced by different methods including the introduction of a "transgene" comprising nucleic acid (usually DNA) within a target cell or the integration of the transgene within a chromosome of a target cell by mediated human intervention, such as by the methods described herein. The term "foreign gene" refers to any nucleic acid (for example, the gene sequence) that is introduced into the genome of a cell by means of experimental manipulations and can include sequences of genes found in that cell, provided that the gene that is introduced contains some modification (for example, a point mutation, the presence of a selectable marker gene, etc.) in relation to the gene that occurs naturally. The term "transformation" as used herein, refers to the introduction of a transgene within a cell. The transformation of a cell can be stable or transient. The term "transient transformation" or "transiently transformed", refers to the introduction of one or more transgenes within a cell, in the absence of integration of the transgene within the genome of the host cell. Transient transformation can be detected by, for example, the enzyme linked immunosorbent assay (ELISA), which detects the presence of a polypeptide encoded by one or more of the transgenes. Alternatively, the transient transformation can be detected by detecting the activity of the protein (e.g., β-glucuronidase) encoded by the transgene. The term "transient transformant" refers to a cell that has transiently incorporated one or more transgenes. In contrast, the term "stable transformation" or "stably transformed" refers to the introduction and integration of one or more transgenes within the genome of a cell. Stable transformation of a cell can be detected by hybridizing Southern blot of the genomic DNA of the cell, with nucleic acid sequences that can bind to one or more of the transgenes. Alternatively, stable transformation of a cell can also be detected by the polymerase chain reaction of the genomic DNA of the cell, to amplify the transgene sequences. The term "stable transformant" refers to a cell that has stably integrated one or more transgenes into genomic DNA. In this manner, a stable transformant is distinguished from a transient transformant because, while the genomic DNA from the stable transformant contains one or more transgenes, the genomic DNA of the transient transformant does not contain a transgene.

A. Viral Particle Expression Systems To produce virus particles modified in accordance with this invention, the viral nucleic acid is modified by introducing a nucleotide sequence encoding the foreign peptide (eg, an animal virus antigen). ) in the part of the viral genome that codes for a portion of the layer protein exposed to the rf r - - **? - «•« * - internal part of the viral capsid, infecting the host cells or organisms with the modified viral nucleic acid, and harvesting the assembled particles of the modified virus. This procedure is best performed by direct manipulation of the virus DNA in the case of DNA viruses or by manipulation of a cDNA corresponding to the RNA of an RNA virus. In accordance with the foregoing, in some embodiments, the present invention provides the vectors that encode a viral particle that has been modified, by way of expressing an exogenous or foreign peptide on the inner surface of the viral capsid. In the particularly preferred embodiments, the sequence of nucleic acids encoding a viral layer protein is modified by inserting a sequence encoding an exogenous peptide, so that when the viral layer protein is assembled within a capsid, the exogenous peptide is presented on the inner surface of the capsid. In the additional embodiments, the sequence encoding the exogenous peptide is inserted into a portion of a viral layer protein, so that the assembly of the viral layer protein within a capsid is not substantially disrupted or disrupted. A large variety of viral particles find use in the present invention. It is contemplated that both DNA and RNA viruses are suitable for modification, by the methods described herein. In the modalities that are particularly preferred, the modified viral particle is a plant virus. In further preferred embodiments, plant viruses are preferably viruses in icosahedron. To date, all plant viruses with symmetry in icosahedron for which crystal structures have been elucidated, they are characterized by the presence of a canonic eight-chain barrel conformation. Therefore, it is likely that this is a configuration common to all plant icosahedron viruses. In this way, the plant virus in the preferred icosahedron can be selected from the following virus families: Caulimoviridae, Bromoviridae, Comoviridae, Ge iniviridae, Reoviridae, Partitiviridae, Sequiviridaß, and Tombusviridae; and the following genera of viruses: Luteovirus, Marafiviris, Sobemovirus, Tymovirus, Enamovirus, and lldeavirus. In the modalities that are particularly preferred, the modified viral particle belongs to the family Comoviridae. The comoviruses are a group of at least fourteen plant viruses, which predominantly infect legumes. Their genomes consist of two molecules of positive RNA of a single chain of different sizes, which are encapsulated separately in isometric particles of approximately 28 nm in diameter. The two types of nucleoprotein particles are called intermediate (M) and lower (B) components, as a consequence of their behavior in the cesium chloride density gradients, the particles of RNAs inside the particles being known as RNA M and B, respectively. The two types of particles have an identical protein composition, consisting of 60 copies of a large-layer protein (VP37; VP-L) and a small one (VP23; VP-S). In addition to the nucleoprotein particles, the preparations of comovirus contain a variable amount of empty capsids (only protein), which are known as components higher (T). In the case of the type member of the group of the comovirus, the cowpea mosaic virus (CPMV), it is known that both RNA M and B are polyadenylated and have a small protein (VPg) that binds covalently with its term 5 '. The more limited studies on other viruses, suggest that these characteristics are shared by ARNs of all the members of the group. The two RNAs from the CPMV have been sequenced and shown to consist of 3481 (M) and 5889 (B) nucleotides, which exclude poly (A) tails (van Wezenbeek et al., EMBO J. 2: 941 -46

[1983]; Lomonossoff and Shanks, EMBO J. 2: 2253-2258

[1983]).

The two RNAs contain a single, long open reading frame, the expression of the viral gene products that occur through the synthesis and the subsequent dissociation of large precursor polypeptides.

Although both RNAs are required for infection of whole plants, the larger B RNA is capable of independent replication in the protoplasts, although no virus particle is produced in this case (Goldbach et al., Nature 286: 297- 300

[1980]). This observation, which is coupled with previous genetic studies, established that the layer proteins are encoded by M RNA. An electron density map of 3.5 angstroms CPMV, shows that there is a clear relationship between CPMV and plant viruses T = 3 such as tombusvirus, in particular the tomato dwarf tomato plant (TBSV) and the so-called virus, in particular southern bean mosaic (SBMV). The capsids of these latter viruses are composed of 180 subunits of identical layer proteins, each consisting of t ^ l || É g £ ^^ of a single barrel domain ß. These can occupy three different positions, A, B, and C, within the virions. It was shown that the two layer proteins of the CPMV consisted of three different β-barrel domains, two of them being derived from VP37 and one from VP23. In this way, in common with the T = 3 viruses, each particle of the CPMV is made with 180 barrel structures ß. The single domain from VP23 occupies a position analogous to that of the A-type subunits of the TBSV and the SBMV, while the N- and C- terminal domains of the VP37 occupy the positions of the type C and B units, respectively. X-ray diffraction analysis of CPMV crystals and another member of the bean pod variegated virus (BPMV) group show that the 3-D structures of BPMV and CPMV are very similar and are typical of the comovirus group in general. In the structures of the CPMV and the BPMV, each barrel ß consists mainly of 8 antiparallel ß sheet chains that are connected by cycles of variable length. The sheets ß are called sheets B, C, D, E, F, G, H and I, and reference is made to the cycles of connection such as the cycles ßB-ßC, ßD-ßE, ßF-ßG, and ßH- ßl. The comoviruses are also structurally related to the animal picornaviruses. The picornavirus capsids consist of 60 copies of each of the three different layer proteins VP1, VP2, and VP3, each consisting of a unique β-barrel domain. As in the case of the comoviruses, these layer proteins are released by the dissociation of a precursor polyprotein and are synthesized in the order VP2-VP3-VP1. The comparison of the three-dimensional structure of the CPMV ***** "'•'" »MtttMfcifa-Í '** fc i¿M &JW * ¿IU? * With that of the picornaviruses, has shown that the N- and C- terminal domains of the VP37, are equivalent to VP2 and VP3 respectively, and that VP23 is equivalent to VP1. The equivalence between the structural position and the order of the gene suggests that the VP37 corresponds to a non-dissociated form of the two picornavirus capsid proteins, VP2 and VP3. One of the main differences between the comovirus and the picornavirus is that the protein subunits of the comovirus lack the large insertions between the chains of the β-barrels that are found in the picornaviruses, although the fundamental architecture of the particles is very similar. The four cycles (ßB-ßC, ßD-ßE, ßF-ßG, and ßH-ßl) between the ß sheets, are not critical to maintain the structural integrity of the virions, but, according to this invention, are used as expression sites of foreign peptide sequences, such as antigenic sites from animal viruses. An advantage of Comoviridae is that the capsid contains 60 copies of each of the two constituent layer proteins, allowing for the same to occur 60-180 copies of a peptide per virion, where the individual viral layer protein domains they have been manipulated so as to express the inserted peptides. Within the Comoviridae family, cowpea mosaic virus and bean pod variegated virus are preferred. CPMV is a bipartite RNA virus and with the aim of manipulating the genome of any RNA virus to express the foreign peptides, it is desirable to use the clones of the RNA cDNA. From In accordance with the foregoing, in some embodiments, the present invention provides the cDNA vectors that encode a vira particle. modified to express an exogenous peptide in the inner part of the viral capsid. Clones of the full-length cDNA of the two CPMV RNA molecules are available, which can be manipulated to insert the oligonucleotide sequences encoding an exogenous peptide. In some modalities that are particularly preferred, the vector is CP26. In other embodiments, the vector contains RNA B or M RNA or a variant or homologue of RNA B or M RNA. In some embodiments, the variant or homologue can hybridize to an upper or lower strand of B RNA or M RNA, under conditions of low to high stringency. In the embodiments that are particularly preferred, the variant or homolog contains a sequence encoding an exogenous peptide. In some embodiments, cDNA is used to generate in vitro transcripts that are infectious when inoculated into plants.

In accordance with the above, in some embodiments, the present invention provides RNA vectors that encode a viral particle that is modified to express an exogenous peptide in the internal part of the viral capsid. However, the transmission of the transcripts is significantly less than that of the natural virion RNAs, probably as a result of the presence of non-viral residues at the end of the transcripts. Difficulties may also be caused by the exposure of the transcripts to degenerative agents during inoculation. For this reason, the transcripts are usually stabilized by J?. ^^ at, Í £ -.fa fc »» a, ltt »3.«? ... , ". ¿,. .JI? ¿A.? ^ - ^ ** '-. - * ^ - measured to cover their ends 5 '. In still other preferred embodiments, the viral particles that were modified also include an exogenous peptide that occurs on the outer surface of the viral capsid. Methods for presenting the exogenous peptides on the outside of the viral capsid are provided in U.S. Patent Nos. 5,874,087 and 5,958,422, each of which is incorporated herein by reference. In additional embodiments, the cDNA is used to directly inoculate the plants. In these embodiments, the sequences encoding the modified viral particle are operably linked to a promoter that is expressed in the plant tissue. Promoters that find use in the present invention include, but are not limited to, the Cauliflower Mosaic Virus SD promoters (CaMV SD, see for example, U.S. Patent No. 5,352,605, which is incorporated herein by reference). present as reference), mannopine synthase, octopine synthase (oes), superpromotor (see, for example, WO 95/14098), and ubi3 (see, for example, Garbarino and Belknap, Plant Mol. Biol. 24: 1 19-127

[1994]). This technique overcomes some of the problems encountered with the use of transcripts that are generated in vitro and is applicable to all plant RNA viruses. In the case of a DNA virus, the DNA itself is introduced inside the plant. In this way, the foreign peptide is expressed Initially as part of the capsid protein and produced by the same as part of the complete virus particle. The peptide can be produced in this way as a conjugated molecule that is proposes for use as such. Alternatively, the genetic modification of the virus can be designed with the aim of allowing the release of the desired peptide, by applying the appropriate agents, which will effect dissociation from the virus particle. With the aim of producing modified viruses on a commercial scale, it is not necessary to prepare ineffective inocula (transcribed from DNA or RNA) for each batch of viral production. In some embodiments an initial inoculum can be used to infect plants and the resulting modified virus can be passed into the plants, to produce complete virus or viral RNA as inoculum for subsequent batches. In some embodiments, the viral capsid does not contain nucleic acid. The methods are known in the art for the selective enrichment and purification of "empty" virions (See, for example, van Kammen and Jaeger, Cowpea Mosaic Virus, In: CMI / AABN Description of Plant Viruses 197, Commonwealth Agricultural Bureaux [ 1978], and WO 98/56933, the description of which is incorporated herein by reference).

B. Expression of Exogenous Peptides in the Inner Part of the Viral Capsid One of the limitations of the viral expression technologies described above is the fact that the positively charged peptides or the hydrophobic peptides that are inserted in one of the superficial cycles of the layer proteins, eliminate viral contagion, due to a folding of disturbed protein, aggregation of - ~ * * • * &* "** l ttí &M * ¿¿¿¿** ¿- * - * particles, or a disturbed viral transport.The non-viability of these particles is a great impediment to the expression of some epitopes The epitopes that are positively charged can be compensated for by the expression of some additional amino acids (eg, plMM8, plMM9, Bendalm-nane et al., J. Mol. Biol 290 (1): 9 -20

[1999]) The expression of hydrophobic residues on the surface, however, has been very difficult so far, the use of alternative insertion sites on the surface of the virus does not solve the problem. epitopes in the C-terminus of VP-S, which is on the surface as well, generally gives similar characteristics.This limitation of technology makes it difficult to express most T-cell epitopes. Insertion sites: 1. Insertion Sites In general, exogenous RNA or DNA can be inserted into the genome of plant viruses in a variety of configurations. For example, it can be inserted as an addition of! existing nucleic acid or as a substitution for part of the existing sequence, the selection being determined mostly by the structure of the capsid protein and the simplicity with which additions or replacements can be made, without interference with the capacity of the modified virus in a genetic way to assemble in plants. The determination of the allowable and most appropriate size of addition or elimination for the purposes of this invention can be achieved in each particular case by the experiment in light of the present description. It seems that the use of the inserts of addition offers more flexibility than the inserts of 7 replacements in some cases. The present invention demonstrates that the insertion of epitopes into the viral layer proteins so that they are expressed on the inner surface or side of a viral capsid. In general, any portion of a coat protein turns! that is exposed on the internal surface of an assembled viral capsid, is a candidate site for the insertion of the epitope. In some embodiments of the present invention, these sites are selected by analyzing high resolution structures (eg, crystal structure analysis) of the viral capsids. In additional embodiments of the present invention, the viral layer protein at the identified site is modified by inserting an epitope. Vectors (e.g., cDNA or RNA vectors) that encode the modified virus are then used to infect an appropriate host (e.g., protoplasts, plant tissue, or whole plants). If the infection occurs (for example, as it was tested by the appearance of local lesions in a plant), then the site is useful for the expression of an epitope. In some cases, the serial selection of infectious viruses identifies mutations that lead to a greater contagion, including viral particles capable of systemic infection (discussed later in more detail). The present invention is exemplified by the insertion of a foreign peptide into the VP-S of the CPMV. In some modalities, the insertion site is in the N-term of the VP-S. In modalities 'afeafa ***'!. * - *. »» _. ^^ J, JftijM.t..a¿? faa A1..i ¡».

In addition, the foreign peptide is inserted at a point between amino acids 5 and 20 of the N-terminus, preferably between amino acids 7 and 15 of the N-terminus, and more preferably between amino acids 9 and 12 of the N-terminus. In preferred embodiments , the insertion of the foreign peptide does not disturb the function (for example, the assembly) of the virus in vivo. The term N is, in accordance with the high resolution structure, inside the virion, rather than on the external surface.

The present invention is not limited to a particular mechanism of action. In fact, an understanding of the mechanism of action for practicing the invention is not necessary. However, as a strategy for the expression of the epitopes in the N-terminus of the VP-S of the CPMV, it seems desirable to insert the epitope between a duplication of Y1 1 or V10Y1 1. The term N plays a role in the viral life cycle, because it is recognized by the viral protease during polyprotein processing. The exact size of the recognition site is not known, but in analogy with the recognition site between the VP37 and the MP (movement protein), is probably 10-1 1 amino acids (Verver et al., Virology 242: 22-27

[1998]). The first 10 amino acids of the VP-S project from the β-barrel of the VP-S and do not interact with any other residue. Y1 1 marks the boundary between the N-terminus of the VP-S and the rest of the small-layer protein and is in a hydrophobic package that is formed by Q73, R 165 and H71. the insertion in Y1 1 apparently does not interrupt the process of the polyprotein. Other factors also make this a desirable site. The distribution of RNA within the CPMV is unknown. However, in the i * f * ~ - ^ * -, -i ^ iJ ?? ] t ^ μ + .tl *. • - ^ * ^ '- ^^ - ^. ^ - ^: ^ jgj ^ jj M ^ ^ ¡¡¡j ^ intermediate component of the bean pod variegated asvirus, some RNA was observed in the crystal structure. Interactions of the main RNA protein take place at the N-terminus of VP37. This could very well be the case for the CPMV. The photographs Crío E.M. of the CPMV show that there is probably a small empty space in the five-part symmetry axes, just below the protein shell. In addition, the N-terminus of the VP-S contains two negatively charged residues, which makes it unlikely that there is an interaction with the sugar-phosphate base structure of the RNA as well. Thus, insertion into the N-terminus of the VP-S is not likely to interfere with RNA interactions and there is a space to accommodate the foreign peptide. Additionally, the N-term of the VP-S is probably well-structured, and the term five of the symmetry that is related to the VP23 molecules in the virion forms a ring (Lin et al., Journal of Virology 74 ( 1): 493-504

[1991]). B factors indicate that amino acids 1-9 are flexible. Inserts in this region will most likely disturb the ring, but will probably not affect the folding of the barrel. It may be relevant that the pepscan experiments indicate that the N-terminus of the VP-S in the CMPV and a number of other plant viruses in the icosahedron represent one of the strongest B-cell epitopes. This is consistent with the notion that this domain (normally buried within the capsid) is exposed temporarily through the dynamic behavior (ie, "breathing") of the virion that was assembled, such as the CVPs. In accordance with the above, in some modalities, the ^ t,. ^,. * .. ^ .... ^ (í.í M ^ lf] i | í i. * «M¡ ~ J ^ b. * a» a ^ Jk? iá > SújLJu £ The present invention provides a modified CPMV, having a foreign peptide inserted into Y1 1. In still other embodiments, the foreign peptide inserted between a duplication of Y1 1 of VP-S In other embodiments, the present invention provides a Modified CPMV having a foreign peptide inserted between a VPS V10Y1 1 duplication The present invention is not limited to any particular mechanism of action In fact, an understanding of the mechanism of action for practicing the invention is not necessary. , it is contemplated that flanking a foreign peptide with a duplication of Y1 1 or V10Y1 1, retains the hydrophobic context of Y1 1. The duplication of Y1 1 represents a designed modification of the viral vector that has been made to facilitate the accommodation of foreign peptides in of the CPMV capsid., other changes to the amino acid sequence of CPMV have proven difficult to design, since many de novo observed mutations that occur in particles that display peptides in the external aspect of the particle have been limited to changes within the same foreign peptide. Many epitopes were inserted successfully in the Y1 1 position, giving reason to a very good infection in the cowpea plants. Many constructs had a duplication of V10Y1 1, which in two cases (pNLAL7 / pMAL8 and pMV14 / pMV15) were found to be useful to avoid mutations in the epitope, or to make the construct infectious. In one construction (pTr4), only V10 doubled (because the epitope starts and ends with Val), and in this case the construction was also infectious. These results make it advisable to double at least V10, and if V10Y1 1 is possible in new constructions. 2. Peptides The foreign peptides that can be incorporated into the plant viruses according to this invention, can be of highly different types and are subject only to the limitation of the nature and size of the foreign peptide, and the site in which it is detected. This one, or in the virus particle, does not interfere with the ability of the modified virus to assemble when grown in vitro or in vivo. In a broad concept, modified viruses can be formed from any biologically useful peptides (usually polypeptides), whose function requires a particular conformation for their activity. This can be achieved by associating the peptide with a larger molecule (for example, to improve its stability), or the mode of presentation in a particular biological system. Examples of those peptides are peptide hormones; enzymes; growth factors; antigens of protozoal, viral, bacterial, or fungal origin; antibodies that include antiidiotypic antibodies; immunoregulators and cytokines (e.g., interferons and interleukins); receivers; adhesins; and parts of precursors of any of the above types of peptides. The peptide preferably contains more than 5 amino acids. The present invention allows the expression of a wide variety of foreign peptides on the inner surface of viral capsids. In some embodiments, the peptide is 5-20 amino acids, preferably 7-15 amino acids, and most preferably 8-12 amino acids. amino acids. However, it is contemplated that the limit on the foreign size is limited only by the ability of the chimeric virus to accommodate a foreign peptide and still be able to assemble within an infectious virus in plant. In the preferred embodiments, the foreign peptide has immunological properties. In accordance with the foregoing, in some embodiments, the foreign peptide is an antigen or 11 immunogen. In the following Table 1 the examples of the epitopes that have been inserted successfully are provided. In some embodiments, the epitope is a B cell epitope. In other embodiments, the epitope is a T cell epitope. In some preferred embodiments, the foreign peptide is a cytotoxic T lymphocyte epitope, which is reactive towards cytotoxic T lymphocytes. . In general, the T cell epitopes are hydrophobic. In some embodiments, the epitopes have a pl of more than 7.0 (eg, pH BV16, pLCMV2, PVSVI) and are hydrophobic, or contain long hydrophobic tracts (eg, pLCMV2 and pHBV16). Some of these epitopes have been previously inserted in the ßBßC cycle of VP-S, giving good symptoms (epitope short HBV), moderate (epitope LCMV), or none (peptide 2F10). In some preferred embodiments, the peptide corresponds to SEQ ID NOs: 4-17. In other embodiments, the peptide is encoded by a nucleic acid sequence corresponding to SEQ I D NOs: 18-31.

* ¡T¡ai ^? * - r *? .... aM ^ »* ^ ..., -., I. ^.? ^ T. "Jfc ^ afi¿ ^, ^ B &., T ..) ^, .. < RaMi fcttt * .. Hl «t, .MftOh? ti¡ÍÉÍ-« '^ - ^ ».«? áa¡Í¡ «M ^ fa1fe1if? iail lfc The use of the insertion site in the N-term of VP-S had clear advantages over the insertion sites that were used before, in the sense that it is now possible to express hydrophobic or basic epitopes.

This opens up a whole new range of possibilities for the expression of foreign epitopes in the CPMV. Since the new insertion site is inside the virus particle, a strong antibody response to the inserted epitope is not likely. However, the hidden N term of many plant viruses turns out to be among the most immunogenic parts of the virus. The most probable explanation for this phenomenon is the dynamic behavior (respiration) of the particles. For this reason, the response of B cells to peptide 2F10 in BBV16 was tested. The absence of any of the a-2F10 antibodies clearly indicates that the epitope in this construct is hidden. 3. Mutations in Second Site Surprisingly, it has been found that chimeric virus particles that contain a foreign peptide in the N-terminus of VP-S (i.e., an internally deployable particle) are particularly ..t .- *, *** *** .-: ^ aa &a ^^ = ^ ii ^ a ^^ ft ^ t aa ^ M ^ iafeMi tiifíilhi ^ ^ ^^ = s ^^^ aat prone to selection for mutations that affect the sequence of in-layer coat proteins at sites other than the insertion itself. These de novo mutations are characterized by the following characteristics: the new mutations occur spontaneously and are selected in the host plant; and a very large proportion of the mutations occur at distant positions and close to the insertion of the foreign peptide. Interestingly, many of the mutations effect changes in the amino acid sequences in the layer proteins located in domains that are believed to be involved in the protein-protein interaction between capsid subunits. By infecting a host plant with a novel chimeric virus particle construct containing an internalized epitope, it is possible to select and isolate novel virus particles in which amino acids different from those present in the insert are altered. Furthermore, it is clear that de novo mutations at a given position in a given coat protein can arise independently of time, and bring back mutations induced and selected separately at precisely the same position in proteins comprising the capsid. In addition, the fact that a given amino acid residue can be altered to different amino acids (ie, non-conservative changes), suggests that it is more important to remove from the structure the restriction exerted by the original amino acid, than to establish a particular amino acid substitution. . In addition, amino acid substitutions are naturally limited to those that are the result of mutations of (a single) point in non-wobbling positions in the codons. Accordingly, a method is provided for identifying positions in the CPMV capsid, permissive for the selection of compensatory mutations ("hot spots"). The method is independent of the expressed internalized peptide and its precise point of insertion within that capsid. Still more outstandingly, mutations of second site, third site and others can be selected in CPMV layer proteins, by infecting a host plant with a chimera that internally expresses a foreign peptide. Where these third-site and other mutations occur, the infectivity and productivity of the novel chimeric virus particle is improved over that of its counterpart with only a de novo mutation (second-site only). Furthermore, if the genome of the altered virus is used to present a different peptide, both the infectivity and the productivity of that virus particle is greater than those seen with the wild-type virus vector expressing the same peptide. In this way, the invention provides a means to generate improved viral vectors, capable of higher productivity in the plant than wild type consanguineous CPMV vectors. In accordance with the above, in some embodiments, the present invention also provides vectors containing second and third site mutations, which improve the infectivity of viral particles containing epitope insertions, and methods for selecting those mutations. In some modalities, the mutations are in VP-S, whereas in other modalities, the mutations are in the VP-L. In the following Table 2, seven mutations of the second site and two mutations of the third site are described. In accordance with the foregoing, the present invention also provides a vector library that contains a variety of mutated viral vectors that can be selected for expression of the epitope. In Example 12 a detailed protocol for the selection and identification of these mutations is provided.

The present invention is not limited to a particular mechanism of action. In fact, an understanding of the andfaj mechanism of action for using the present invention. However, most mutated amino acids are probably involved in protein-protein intermolecular interactions. Phe91 is at the interface of two neighboring VP23 molecules. The F91 S mutation will definitely weaken this interaction. Phe180 is at the interface of a molecule VP23 and VP37, and interacts with Phe2045 and Ala2047. The F180L mutation inhibits these interactions. Met177 has a hydrophobic interaction with the Arg97 side chain of VP37 domain C. Both the M 177V and the M177T mutation make this interaction impossible. Arg 2102 is probably close in space to the N terminus of a neighboring VP37. There is an intramolecular interaction with E2121, which is impossible when the R2102K mutation occurs. Some of the mutated residues, however, are hidden in one of the layer proteins, and the structural consequences are less likely to be predicted. One possible explanation for at least some of the mutations is that there is a need for reduced particle stability to ensure efficient virus depletion. Since the N-VP-S terms form a ring, the inserted epitopes can interact with each other, and stabilize the virion particle. It seems that some epitopes have a much stronger tendency to do this than others. Therefore, there is a clear application for second site mutations. In some embodiments of the present invention, the mutations can be used in constructs that by themselves are not infectious (eg, pSEN2, see Table 2 above). It is contemplated that this will increase the range of possibilities for the use of the new site of insertion. In accordance with the above, a useful approach is the production of a bank of vectors with all the mutations of second and third sites that have been observed, in which the new epitopes can be cloned. In some modalities, the selection for the most viable vector can take place in the plant. 4. Expression in Combination with External Epitopes The present invention also encompasses viral particles and vectors that encode viral particles that express epitopes both inside and outside of the viral capsid. These particles are called chimeric amphysipply virus particles (ADCVPs). The present invention is not limited to any particular mechanism of action. In fact, an understanding of the mechanism to practice the invention is not necessary. However, it is contemplated that the coexpression of the T cell and B cell epitopes in the viral particle may lead to an improved immune response to the B cell epitope. In accordance with the foregoing, in some embodiments, the present invention comprises a viral particle (or vector encoding a viral particle) that expresses an epitope of B cells outside the viral capsid, and a T cell epitope within the viral capsid. In some preferred embodiments, the viral particle is CMPV and the T-cell epitope is inserted into the N-terminus of VP-S. In still other embodiments, the T-cell epitope is inserted via a duplication of Y1 1 or V10Y1 1, and the B-cell epitope is inserted either into the βBβC cycle, the βββCC cycle, or the carboxyl terminus. of VP-S or the ßEßA cycle of VP-L Examples of ADVCPs are given in Example 7.

C. Uses of Modified Viral Particles The vectors and viral particles of the present invention have many uses. In some embodiments, the viral particles are used to induce a response either immunogenic or antigenic in an animal. Therefore, viral particles are useful in the protection of animals, including humans, against diseases caused by pathogens. In other modalities, the particles find use as vaccines against cancer. In additional embodiments, the viral particles are used to make a vaccine composition. In preferred embodiments, the vaccine composition comprises a modified viral particle and an adjuvant (e.g., QS-21). The vaccine is then administered to the animal as is known in the art.

EXAMPLES The following examples serve to illustrate certain preferred embodiments and aspects of the present invention, and will not be construed as limiting the scope thereof. In the description that follows, the following abbreviations apply: M (molar); mM (millimolar); μM (micromolar); nM (nanomolar); mol (moles); mmol (millimoles); μmol (micromoles); nmol (nanomoles); gm (grams); mg (milligrams); μm (micrograms); pg (picograms); L (liters); ml (milliliters); μl (microliters); cm (centimeters); mm (millimeters); μm (micrometers); nm (nanometers); ° C (degrees Celsius); CDNA (copy or complementary DNA); DNA (acid deoxyribonucleic); SsDNA (single-stranded DNA); DsDNA (double-stranded DNA), dNTP (deoxyribonucleotide triphosphate); CPMV (Cowpea Mosaic Virus); ADVCP (chimeric amphysipply virus particle); CTL (cytotoxic T lymphocyte); CVP (Chimeric Virus particle); ELISA (enzyme-linked immunosorbent assay); MP (Movement Protein); VLP (Virus-like Particle); VP-L (large virus protein [37 kDa layer protein]); VP-S (small virus protein [23 kDa layer protein]). The vector that was used in all the experiments described below is pCP26. This is derived from pCP7 described in (Dalsgaard et al., Nat. Biotech 15, 248-252

[1997]) and consists of: the commercially available vector Bluescript p BS 1 1 BS SK + (Stratagene, CA) having been cloned in a cartridge comprising the plant promoter of the cauliflower mosaic virus (CAMV 35S) bound to a cDNA molecule corresponding to the RNA2 of the infectious cowpea mosaic virus, in which the nucleotides encoding the proteins have been deleted. 24 N-terminal amino acids, and a mutation of nt3295 (T- »A), which introduces a restriction endonuclease site Psfl, has been designed. In the sequence of the base structure of the vector, the following point mutations have been designed to create new restriction endonuclease sites: Eco47lll (T- »C in nt960) and Sa / I, (T-> C in nt1005) . The polylinker sequence between Sa / I and Kpn \ has been deleted. Since the majority of the polylinker has been removed in this construct, there is a unique EcoO 1091 restriction site, near the Nhe site, which is normally used for inserts in the βBßC cycle. of VP-S. This is shown in Figure 1, which indicates the cloning strategy for the insertion of epitopes by means of a duplication of TyrH of VP-S or CPMV. The two restriction sites are used to insert oligonucleotides encoding different epitopes. Unless stated otherwise, the inoculation of host plants with cDNA encoding chimeric virus particles, described in the following examples, was performed essentially as outlined in Daisgaard et al., Supra, and Dessens and Lomonossoff, J. Gen. Virol. 74, 889-892

[1993]). To achieve the simultaneous expression of N-terminal combinations plus surface epitopes in CPMV VP-S, two plasmids are digested, each encoding an epitope at a different insertion site, with Nhel and FíamHI, resulting in 1.3 kb fragments. and 5 kb. The smallest fragment contains the sequence encoding the N-terminus of VP-S, and the largest fragment contains sequences encoding the ßBßC cycle, the ßB'ßC "cycle and the C-terminus of VP-S. Epitopes are made by purifying the fragments containing the inserted epitopes, and linking them together using standard molecular biology techniques (Sambrook, J., Fritsch, EF and Maniatis, T. Molecular Cloning-A laboratory manual, 2nd edition, Cold Spring Harbor Laboratory Press

[1989]).

Example 1 This example demonstrates the expression of a peptide within a viral capsid. In particular, the peptides derived from a mimotope of the class "a" determinant of the hepatitis B surface antigen are inserted into the VP-S of the CPMV. It is reported that a sequence derived from the anti-idiotypic monoclonal antibody against the hepatitis B surface antigen (H BsAg), AVYYCTRGYHGSSLY (SEQ ID NO: 33), and a highly hydrophobic octometer, derived from this same sequence (GYHGSSLY; SEQ ID NO: 34) are effective to mount a cognate of immune response with that generated by the determinant "a" of the surface antigen of H BV itself (See, for example, U.S. Patent Nos. 5,531, 990; 5,668,253; 5,744,135; and 5,856,087, each of which is incorporated herein by reference). The same peptide generates a T helper response specific to HBV. Therefore, the peptide, designated 2F10, and its derivatives are mimotopes of the "a" determinant. The pentadecamer and the octomer derived from 2F10 can be expressed on the outer surface of the CPMV particles. The octomer is expressed in the ßBßC cycle of the VP-S, and in the ßEßCA cycle of VP-L (Brennan et al., Microbiology 145: 21 1 -220

[1991]) generating chimeric particles designated respectively, H BV7 and HBV14. Both CVPs are capable of mounting an infection in cowpea plants after inoculation. The pentadecamer can be expressed differently in the βBßC of the VP-S, to generate a construct known as H BV2; in the C term of VP-S to produce H BV8; and in the ßEaB cycle of VP-L (HBV3). For these three constructions the symptoms are restricted to the inoculated leaves, even after the passage of the purified virus to other plants. In other words, there is no in-plant selection for viruses J é .. * Í ?? »*» J '> ••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••• • N of the VP-S The octamer (HBV 15) and the pentadecamer (H BV 16) are inserted in half of Tyr1 1 and Ser 12. This position is chosen because the term N is involved in the binding of a viral polyprotein protease. which is believed to require at least the ten amino acids with N-terminal. Therefore, a desired consequence of inserting a foreign peptide within the N-terminus of VP-S, is to avoid ablation of the protease binding, in order to perform its function Since the native N-terminal domain includes a tyrosine residue in the 1 1 position, it seems important to maintain the presence of this residue in a contiguous amino acid motif, since the peptides themselves each end with a tyrosine residue , it is not necessary duplication of Tyr1 1 for the purpose of expressing them internally in this case (see also Example 2 below). In all plants inoculated with cDNA, H BV 15 produces symptoms in the host plant indistinguishable from those produced in a wild-type CPMV infection. The particles purified from 62 grams of sheet, after the standard procedure for the purification of CPMV, Daisgaard et al., Supra, yielded 64.5 mg. HBV 16 produces symptoms in 3 out of 5 plants inoculated with cDNA. However, in plants that were subsequently inoculated with purified viruses from the initial infection cycle, symptoms similar to a wild type infection are observed. He Purified virus yield of these plants are 13 mg of 23 grams (0.57 milligrams / gram of plant material) after a standard procedure for the purification of CPMV (as mentioned above). Both viruses are analyzed in a 15 percent denaturing polyacrylamide gel. The small-layer protein shows no dissociation of these internal peptides as can be seen sometimes for the peptides deployed in the surface cycles of the CPMV.

Example 2 This Example demonstrates the expression and internal deployment of a hydrophobic peptide corresponding to a CTL epitope (MAL 7) derived from the circumsporozoite protein of Plasmodium berghei. P. berghei is a causal agent of unicellular protozoan malaria in man. A peptide corresponding to an epitope of the circumsporozoite protein with the amino acid sequence SYIPSAEKI, can be expressed in the ßBßC cycle of the VP-S (giving rise to a chimeric virus particle designated Mal 4), and in two different positions in the C term of VP-S (to generate, respectively, Mal 5 and Mal 6). The same peptide can be expressed at the N-terminus of VP-S between "a duplication of a tyrosine residue at the 1 1 position on the protein (Tyr1 1; cf. Example 1) .This construct is designated MAL 7. Initially after of the inoculation of cDNA encoding the modified MAL 7 CPMV on the leaves of the host cowpea plants, the construction does not generate systemic symptoms, however, after 21 days, very clear local lesions are visible on the primary leaves of two of five plants, indicating the establishment of an infection mediated by the inoculated CVP. The virus purified from these local lesions is transferred directly to two groups, each of three young cowpea plants. The local lesions became visible within the next 5 days, and the subsequent systemic infection follows within a week. This highly improved viability in all likelihood indicates the selection of a virus from within the population that carries a de novo mutation in this genome. RNA from the purified virus 8 days after inoculation in the second two groups of cowpea plants is subjected to RT-PCR and sequencing of the resulting cDNA is performed. The analysis reveals a point mutation, which is observed to have arisen independently in both sets of plants, and which is verified in each case by sequencing in the chains as much as less than that produced by the cDNA in the RT-PCR reaction. However, this inversion is not present in 100% of the isolated viruses, since in this position a mixture of two nucleotides is observed in the chromatographs of the automated sequencer of each isolate. However, notably, in each case an adenosine is changed to a guanosine in the nucleotide sequence, causing a mutation of Glu to Gly in the peptide, at the amino acid level. When the upper leaves of the plants are analyzed 3 weeks after inoculation, a mixture of viral genomes is still observed, but the mutated genome is present in numbers apparently greater than the original sequence, as judged by the chromatograph examination. This indicates that the mutated VP-L has an advantage competitive on the non-mutated chimeric particle in its ability to mount and progress a systemic spread of the virus inside its host. Due to the mutated sequence of the peptide, this construct can not be used for an immunological analysis of the epitope of interest. However, it exemplifies two elements of the internalization of the peptide system. Firstly, the usefulness of a duplication of the tyrosine residue in the native sequence of the CPMV genome between which the insertions of the foreign peptides are demonstrated. In some cases this is required to maintain the putative binding site for the viral polyprotein protease. Secondly, the ability to select in vivo altered chimeric virus particles whose characteristics produce them ready for infection and systemic dissemination inside a cowpea plant is demonstrated. In this particular case, the alteration in the recombinant viral genome directly affects the inserted foreign peptide itself. Mutations that affect the peptides deployed externally in the CPMV can be seen in cases where in-plant selection pressure arises because the peptide has charge characteristics that unbalance the pl (see Definitions) of the global capsid protein cortex . The result described here is not anticipated because the fundamental reason behind the internalization of the peptides is that the surface charge inherent in the peptide in question should not represent a restriction once the peptide is deployed inside the virion. The following example identifies a completely unanticipated consequence of the internalization of the peptides into chimeric CPMV particles.

EXAMPLE 3 This example demonstrates the generation in vivo and the subsequent isolation of de novo mutations in the genomes of chimeric viruses that confer a selective advantage over the genome of chimeric wild-type virus cognate in the progression of infection and the systemic spread of CVPs that internally express the MAL peptides 7. The resulting mutant chimera described in Example 2 (above) is, surprisingly, viable with respect to the ability of the recombinant virus to mount and progress a "natural" infection cycle, including systemic spread within the host plant. However, in an attempt to eliminate any structural constraints that, through selection pressure, could result in the mutation reported in previous MAL7, the insertion site is modified. The inspection of the glass structure temperature factor shows that Val10 is in a relatively rigid conformation, while the Asp9 residue is very flexible. This suggests that it should be possible to duplicate Val10Tyr1 1 in order to simultaneously preserve the protease binding site, and to change potentially destabilizing charged residues inside the MAL7 peptide, to a position with more C terminals at the N-terminus of VP-S, with respect to Val 10Tyr1 1 original. The resulting chimera is designated MAL8. The MAL8 construct is used to infect host cowpea plants in a separate study. The profile of the initial infection is as seen for MAL 7 in Example 2. In detail, one plant each five inoculated with cDNA encoding MAL 8 produced a local lesion after 21 days, indicating limited infection without apparent systemic spread of the virus. The virus purified from the local lesion is used to inoculate fresh cowpea plants. Symptoms of systemic dissemination of the chimeric virus particle inside the plant are detectable after 5 days. This is indicative of improved infectivity, most likely the consequence of a mutation in the viral genome. The genomes of viruses isolated from the second round of infection with MAL 8 are produced in cDNA, using RT-PCR and the gene encoding VP-S is sequenced along both chains. A de novo mutation is confirmed at nucleotide position 2931, by altering a thymidine to a cytosine residue, generating by the same an amino acid change from phenylalanine at position 191, to a serine. This non-conservative change occurs at a point in the small-layer protein, VP-S, which is located at the interface between neighboring VP-S proteins in the virion. This mutation is apparently permissive for the viable assembly in vivo and the systemic dissemination of a chimeric CPMV particle, in which a peptide is internally expressed. These data indicate that different permissive de novo mutations can be selected for the in-plant assembly of CVPs, capable of internally expressing a foreign peptide, and capable of mounting the infection from a systemic spread within a cowpea host plant, using CVPs that express the same peptide internally. This further demonstrates that the location of the mutation inside the virus particle, and that it is presumably activated by the structural constraints generated by the inserted peptide, depends to some degree on the precise insertion site that is used, and not merely on the peptide itself. .

Example 4 This Example demonstrates an in vivo procedure for the selection and isolation of de novo mutations in chimeric virus genomes that confer a selective advantage over the genome of cognate wild type chimeric virus, in the progression of infection and systemic spread of CVPs that internally express peptides. In order to regulate the possibility that the phenomenon described in Examples 2 and 3 is restricted to CVPs that internally express malaria peptides, a procedure was followed to select chimeric virus particles that internally expressed peptides where second site mutations occurred. putative Permissive mutations can be selected for viability (meaning assembly within recombinant virions in plant), infectivity, systemic dissemination or improved productivity, as described below. For the purposes of the demonstration of the method, the method of enrichment and selection with reference to a peptide, APGNYPAL, which defines a CTL epitope derived from the nucleoprotein of the Sendai virus is described. Briefly, the cDNA is designed to encode a chimeric virus particle in which the peptide (APGNYPAL, SEQ ID NO: 10) is inserted between tyrosine residues in, respectively, the amino terminal of position 1 1, and in the carboxy terminal. from position 20 to the peptide once inserted in VP-S of the CPMV. Tyrosine at position 20 represents a duplication of tyrosine at the 1 1 position at the native VP-S, and is used to maintain the putative polyprotein protease binding site (as described above). The resulting construction is designated pSEN 1. After the inoculation of the host cowpea plants with SEN 1, the symptoms of viral infection are slow to appear. After 18 days systemic infection is visible in only one out of every five plants inoculated. The symptoms in the other four plants are restricted to local lesions in the inoculated leaves. Virus particles are isolated from the four plants in which the infection is restricted to local lesions on the leaves, with the viruses being isolated from a single lesion. The genomic RNA as it is isolated ex plant is reverse transcribed by RT-PCR. Subsequent sequencing in both strands of the cDNA thus produced indicates that the genomes of the viruses isolated from the four plants contained unchanged sequences. After another 7 days (on day 25 after infection), systemic spread was observed in 3 out of 5 inoculated plants. The virus was purified from the systemically infected leaves and the reverse transcribed RNA. Sequence analysis of the resulting cDNA reveals a de novo mutation in nucleotide 3199, changing a guanosine residue for wild-type thymidine. Consequently, a leucine residue is incorporated at position 180 of the small layer protein (VP-S) instead of phenylalanine. This mutated genomic sequence correlates with the successful infection of cowpea plants by Chimeric CVP that internally expresses a peptide derived from the Sendai virus. This exemplifies the broad utility of the plant-host virus interaction as a dynamic means to enrich and select novel CPMV genomes that encode chimeric virus particles capable of accommodating foreign peptides expressed within the capsid. Taken together with Examples 2 and 3, this example demonstrates that the in vivo selection process can function in, and be applied to, CVPs that internally express many different peptides, and that permissive mutations for better infectivity and systemic dissemination of CVPs can occur in many different sites inside the small layer protein of the CPMV.

Example 5 This example demonstrates the expression internally of a CTL epitope derived from lymphocytic choriomeningitis virus (LCMV). The examples outlined above demonstrate that peptides with physical-chemical properties that are not conducive to the external expression of CVPs can be expressed internally. However, there is likely to be a size restriction in the peptides that can be inserted to be expressed internally in the virion of the CPMV.

Therefore, it is unlikely that foreign peptides subject to internal expression are greater than 20 residues in length (although it is not impossible that empirical experimentation can identify longer peptides that are capable of being displayed internally). A class of peptides that fall within this size range are the »« »AiMj ^. Mtaiaa ^ - * ^ iA * ^ np «« aB »J ^ g ^^» -'- "- ^^ - Jí ^? I 1 called cytotoxic T-cell lymphocyte (CTL) epitopes Importantly, epitopes CTL, in addition to being typically 6-9 residues in length, are independent of conformation.In fact, there is strong evidence that CTL epitopes function as linear epitopes.Therefore, these epitopes must be widely receptive to insertion internally. in CVPs One of these epitopes (RPQASGVYMGNLTAQ; SEQ ID NO: 6) is found in lymphocytic choriomeningitis virus.When deployed externally in CVPs this sequence presents problems due to its high positive charge.Therefore, three amino acids are added extra (glutamic acid, glycine, and alanine) to its N-terminus, in an effort to generate a peptide with a more neutral surface charge when it is expressed externally in a CVP The symptoms produced after infection of a host cowpea plant by This constr Particularly, they are variable and unpredictable, since the chimeric virus produced is intrinsically unstable. In addition, there is severe dissociation of the epitope externally presented, and in mice no specific T cell response could be measured for the chimeric virus particles. In order to confirm that CTL epitopes constitute an important class of foreign peptides capable of expression internally in CVPs, DNA encoding this known CTL epitope of lymphocytic choriomeningitis virus is inserted.

(RPQASGVYMGNLTAQ; SEQ ID NO: 6) within the N term of the VP-S. The same epitope (without the additional amino acids Glu, Gly and Ala) is inserted at the N-terminus of the VP-S, at a position between Tyr1 1 and a duplicate tyrosine residue, immediately downstream of the "-ttF'- - - *" "- foreign peptide in a small-layer protein construct, known as LCMV2 DNA inoculation results in viral symptoms in 3 out of 5 inoculated plants. By sequencing the product cDNA verifies that the sequence of the chimeric construction is not altered, and corresponds to that inoculated inside the plant.The yield of the virus is 25 milligrams of 29 grams of leaves.The analysis by electrophoresis in a gel of 15 percent denaturation polyacrylamide, confirms that there is no dissociation of the LCMV peptide as anticipated for an internalizing peptide.Therefore, it is demonstrated that a representative peptide of a large and immunologically important class of epitopes, the epitopes of cytotoxic T lymphocyte (CTL), can be expressed internally in a chimeric virus particle.On the other hand, the same particular peptide confirms that the The ternalization of positively charged peptides in CVPs represents a technical solution to the unpredictability of the behavior and potential instability of CVPs that internally express peptides with these characteristics. In addition, this demonstrates that a peptide of 15 amino acid residues in length can be successfully accommodated within a CVP, without the requirement of second site mutations.

EXAMPLE 6 This example demonstrates the immunological efficacy of the CTL epitopes expressed internally in the chimeric virus particles. The CVP construction described above, LCMV2, which expresses a CTL epitope of lymphocytic choriomeningitis virus within the particle, is used to immunize mice. On day 0 and day 14, 100 μg was injected subcutaneously, with or without an adjuvant, QS-21. As a control, wild-type CPMV was also inoculated into test animals, with and without QS21. On day 42 spleens were removed, and CTL assays were performed 8 days later (see Current Protocols in Immunology, Volume 1, section 3.1 1.4 and ff). Purified cytotoxic T cells from mice infected with LCMV2 in QS21, promoted the lysis of up to 46 percent of the target cells (Figure 2). No response of CTL to LCMV2 was seen in the absence of adjuvant. In mice inoculated with wild-type CPMV, no specific CTL response was observed, with or without the adjuvant. Apart from the CTL response, a T helper response specific to the very strong epitope was observed in cells purified from the spleens of mice injected with LCMV2. It is clear from the immunological performance of the LCMV2 construct that CVPCs with epitopes deployed inside the particles can induce both a CTL response to a specific peptide and a T helper response specific to the strong peptide. No CTL response was observed, however, in mice immunized with MAL8, VSV1, or SEN 1. This may have to do with the difficulties in processing the epitope in the cells that present the antigen.

Example 7 This example demonstrates the synthesis of virus particles chimeric amphysipple (ADCVPs), which are individual chimeric virus particles that simultaneously express epitopes internally and externally in a single virion.The ability to express peptides within stable CPVs, taken together with the ability (separately) to express a large range of peptides externally in stable CVPs, it gives rise to the possibility that at least two peptides can be present simultaneously in a single particle, one internally, the other externally.In particular, the presence internally of a helper cell epitope T can improve the immune response The epitope concept (GVSTAPDTRPAPGSTA; SEQ ID NO: 35) associated with a variant form of the polymorphic epithelial mucin protein that is predominantly found in the cells of the epitope is used to prove this. solid tumors The immunological profile of this epitope in CVPs is well characterized. The combinations of peptides which are known to be reactive in animal models are inserted into selected sites by molecular genetic manipulation CPMV genome. To date, three external sites are established as viable insertion points in VP-S for the external deployment of the peptides in CVPs: the ßBßC cycle, the ßB'ßC "cycle, and the carboxyl term, while a quarter is represented External insertion site in the CVPs by the ßEaB cycle of the VP-L To test the efficacy of the combination of epitopes internally with epitopes externally in a single CVP, combinations are made as outlined in the following Table 3.

The following constructions induce good symptoms (see above): MUC39, HCG 16, MUC41, MUC42, and MUC47. These results indicate that inserts in the ßBßC cycle or the ßB'ßC "cycle of VP-S can be combined more effectively with small insertions (8 amino acids) in the N-term of VP-S, within the range of sizes of previously shown insertion for this internal CVP site. ^ sa ^ n t ^ fa ^^ ia. However, insertions in the C term of VP-S apparently are not sensitive to the size of the insertions in the N term within the established range of insert sizes.

EXAMPLE 8 This Example demonstrates the immunological efficacy of chimeric amphiskopie virus particles (ADCVPs) to produce specific helper T responses. The stimulatory effect of a T-cell helper epitope on the N-terminus on the immune response to a B-cell epitope inserted elsewhere was investigated, by immunologically comparing a CVP that expresses an helper cell epitope T externally, with two different amphidropion particles expressing the same helper cell epitope T internally, in conjunction with different B cell epitopes. H BV15 is a CVP that expresses an octamer derived from peptide 2F10 at an internal site (see Example 1 above); M UC 39 expresses the same octamer internally together with a peptide derived from human variant mucin associated with solid tumors, MUC1 p (MUC 14) expressed externally in the βBßC cycle; and MUC42 simultaneously expresses the same mimotope of 2F10 internally, and a peptide derived from MUC1 p (MUCL) externally in the C-terminus of VP-S (see Example 7 above). Three groups, each of 5 mice, were immunized with 5 pg of HBV15, MUC39 or MUC42, in the presence of QS-21, on days 0 and 21.

Serum was collected on days 21, 28 and 42, and examined for specific antibodies to both 2F 10 and MUC 1, by ELISA. In The mean titres of antibodies specific for the mucin peptide are summarized in Table 4.

At each time point, mice immunized with CPMV-MUC39 generated higher anti-MUC antibody titers (approximately a higher dilution) than mice immunized with CPMV-MUC14, suggesting that there may be a stimulatory effect of the helper T epitope on the response of anti-MUC B cells. Of the mice immunized with CPMV-MUC42, one in five produced the specific antibody to MUC1 p; whereas when 2F10 was not co-expressed with an helper T cell epitope, no mice produced the specific antibody to MUC1. This reaction was at a modest level on day 28, and the titer declines by day 42. Thus, the presence of 2F10, an helper cell epitope T, can improve the response to a B cell epitope exemplified by the Mud peptide p. When the responses of specific T cells to 2F10 (Table 5) were examined, the spleen cells of the mice Fibrocyanin immunized with CPMV-MUC39 proliferated in response to peptide 2F10. Consistent with the relatively low levels of Mud p peptide-specific antibodies produced by the MUC42 construct (Table 5), there is no apparent stimulation of specific cytotoxic T cell proliferation by the 2F10 peptide in this assay.

These data indicate that the co-stimulation of an immune response with a CTL epitope can be used to activate an improved response to a peptide presented jointly but not related.

Example 9 This Example demonstrates the expression and internalization of a powerful universal helper T epitope, through the selection of additional de novo mutations, at sites other than the inserted peptide. It is known that an epitope of helper T, derived from tetanus toxoid (VDDALI NSTKIYSYFPSV; SEQ I D NO: 15) has a strong immunostimulatory effect in a wide range of organisms, and with a correspondingly large range of haplotypes. In order to further improve the immunogenic properties of CPMV as a system presenting the epitope, tetanus toxoid can be incorporated into the chimeric virus particles. In order to achieve this, the valine in position 10 (VaM O) is replaced with the epitope itself. Since the tetanus toxoid epitope begins and ends with a valine residue, there are by default ten "native" amino acids in the N terminus of the mutated virus, which are likely to be sufficient to maintain the putative polyprotein protease binding site. This resulting construction is designated TT4. After inoculation of the cDNA directly on the cowpea plants, Daisgaard et al., Supra, the TT4 construct shows infection symptoms from day 14 forward, in the form of local lesions on the inoculated leaves. No systemic infection of the host plants took place. Viruses purified from these local lesions are transferred directly onto young cowpea plants in a second-round infection. Local lesions become visible within 5 days of infection, with systemic infection evident within about 7 additional days. This improved viability in all likelihood indicates the selection within the virus population in which a de novo mutation has occurred in the viral genome. Virus RNA purified 10 days after the inoculation of the second group of cowpea plants is subjected to RT-PCR, and sequencing of the resulting cDNA is performed. The analysis reveals many individual point mutations, which are verified by sequencing the cDNA in the opposite strand. Together they are observed iá ..- i..¡ ¿-..... AiAtA - ,. , -! 1,. **, .. »,, .. *, * £. ***. .? -? Jtatt? Í. . .. ^ ^ Jt ^. six de novo mutations in the different clones: -G2388A, which results in an Arg2102Lys mutation in the VP-L protein. (This mutation is observed arising independently in three separate clones); -A3188G, which leads to a Met177Val mutation in the protein VP-S; -A3029G, which leads to a 124Val lle mutation in the VP-S protein; and, -G2388A which results in a lle2045Met mutation in the VP-S protein. In order to test whether these mutations are sufficient to produce the infectious TT4 construct in a primary infection, 3 new constructs were generated, using as the base structure of the vector genomes of novel chimeric virus particles containing respectively, the G2388A mutations (Arg2102Lys in VP-L), LA A3188G (Met177Val in VP-S) and A3029G (lle124Val). Plants inoculated with these new clones showed local symptoms of infection 6 days after inoculation, and systemic symptoms within another 4 days. This is a clear indication that second-site, individual mutations are sufficient to produce the revised infectious TT4 construct. This construction provides a demonstration of many features of the insertion of the epitopes on the inner surface of the CPMV. In the first case, this demonstrates that helper T as well as CTL epitopes can be presented as displayed peptides ^^^ .s..tos ^ < ji ^ s ¿A¡ * a¿ aikH ^, - fc ^^^ ¡t¡?, i t. * internally in the CVPS. In addition, it is clear that Tyr1 1 does not have to be duplicated in order to generate CVPs capable of mounting a viable infection in plants. The presence of 10 naturally occurring amino acids in the N-terminus of the CPMV is sufficient for viability and infectivity. It is also clear that there are many different second site mutations that can greatly improve the viability of a construct with an epitope inserted in the N-terminus of the VP-S of the CPMV, and that these mutations can occur either in the VP-S itself , or in the VP-L (the large layer protein). The fact that particular mutations are found independently in separate clones emphasizes that certain second site mutations are selected more rapidly than others. This represents a means to identify mutational hot spots in the utility viral vector, to create novel chimeric virus particles that clone vehicles capable of generating novel CVPs to display a wide range of foreign peptides.

Example 10 This Example demonstrates the expression and internalization of a powerful universal helper T epitope derived from tetanus toxoid in combination with a B cell epitope inserted in a VP-L cycle in the external CPMV. Since tetanus toxoid is a universal T helper epitope, it is worth investigating the possibility of combining the deployment of this epitope within the CPMV, as described in Example 9, in such a way that it is coexpressed in a * .. ,,,. fe ^ jna * ^,. «. ,, - jifr nr-A ---». ^ .J ^ A., *. ** ^. ^. ^. ^. ^^^. ^^.; single particle with epitopes presented on the external surface of a CVP. To this end, a construct is made in which two peptides derived from Pseudomonas aerugiosa are inserted in tandem in the ßEaB cycle of domain B of the VP-L (peptides 9 and 10 of the outer membrane protein, TDAYNQKLSERRAGADNATAEGRAI N RRVEAE; SEQ ID NO: 36; Brennan et al., Supra), while the tetanus toxoid epitope is inserted into the N-terminus of the VP-S. This construction is designated pPAE14. After inoculation of the cDNA directly on the cowpea plants, the PAE14 construct showed symptoms of infection (local lesions on the inoculated leaves) from day 14 onwards. However, the systemic infection of the host plants did not follow. The virus purified from these local lesions is transferred directly onto young cowpea plants, in order to start a secondary infection. Local lesions became visible within the next 5 days, and subsequent systemic infection followed within the next week. This indicates the selection of a novel virus whose genome has accumulated a de novo mutation. The RNA of the purified viruses was subjected 10 days after the inoculation of the second group of cowpea plants, to RT-PCR and the sequencing of the resulting cDNA was performed. The analysis reveals many mutations of a single point in the population, which were formed by sequencing the cDNA of the opposite strand. The mutations observed in the different clones are: -A3029G, which leads to the mutation Me 124Val in the VP-S (Cf. Example 9 where a different mutation occurs in this same one position); and -T3189C, which results in the Met177Thr mutation. (As above, in Example 9 a different mutation is reported in this same position). This example indicates that it is possible to simultaneously express an epitope within a chimeric plant virus such as CPMV, in combination with an epitope in VP-L, such that it is presented externally. The second site mutations that were observed indicate that similar mutations can be found for different constructs (eg, A3029G in Example 9 and in the present), and that different mutations thus selected in a single amino acid position can occur, leading to a CVP with greatly improved affectivity.

* Example 1 1 This Example demonstrates the expression and internalization of a powerful universal helper T epitope, derived from tetanus toxoid, in combination with a B cell epitope presented at the external CPMV, inserted in a VP-S cycle. Since it is possible to combine the expression of a universal T helper epitope from an internal surface of the CPMV, with an epitope on the external surface of the virus, by using the ßEaB cycle of the B domain of the VP-L (Example 10) ), it is worth trying to combine the expression of the tetanus toxoid epitope with the expression of epitopes in the ßBßC cycle of VP-S. In Example 7 a similar approach with the mimotope is described 2F 10 of hepatitis B virus. Therefore, a construct was made in which the tetanus toxoid epitope is inserted into the N-terminus of VP-S, as described in Example 9, while a peptide of mucin (GVTSAPDTRPAPGSTA; SEQ ID NO: 37) in the ßBßC cycle of VP-S, between Ala22Pro23 essentially as described in Daisgaard et al., supra. This construction is designated pMUC51. After inoculation of the cDNA on the cowpea plants, the MUC51 showed no symptoms of infection, even after 21 days. For this reason, a cDNA construct, identical to pMUC51, was made, except that a second site mutation is encoded as reported in Example 9 (A3188G: Met177Val) in the chimeric virus vector. This novel construction is designated pMUC53. After inoculation of the cDNA on the cowpea plants, the MUC53 construct showed no symptoms of infection until day 14. Systemic infection of the host plants did not occur. Viruses purified from these local lesions are transferred directly onto young cowpea plants to prime a second cycle of infection. The local lesions became visible within the next 5 days, and rapidly followed a subsequent systemic infection within the next 7 days. This probably indicates the selection of another mutation in the genome of the virus. The viruses were purified from the plants 10 days after inoculation, and the genomic RNA was produced in cDNA by RT-PCR. The analysis of the sequence of both complementary chains confirmed the occurrence of additional de novo mutations: . * JfcMt ^ t J ».fJ * - * - ^ a > B_Mltrf > fcAflftjfc > ^^? ^ a ^ -A »'* Z- & í '»- > -G2357A, which leads to Ala492Thr in the VP-L; and -G2898A, which causes GlydOAsp in the VP-S. These experiments show many useful features of epitope internalization. It is clear that it is possible to combine the insertion of epitopes in the ßBßC cycle of the VP-S, with epitopes in the N-terminus of the VP-S, even if the last epitope is 18 amino acid residues in length. Furthermore, these show that in some cases the second site mutations alone are not sufficient to generate an infectious construct, but that third site mutations need to be selected to provide chimeric virus particles capable of mounting an infection. In Example 12 below, a method for selecting those higher order mutations, as well as second site mutations is described.

Example 12 The following Example presents a protocol for the selection of novel chimeric virus particle genomes, capable of the internalization and / or amplexing of the peptides. Consideration of the above examples provides a major reason for the in vivo selection of novel plant virus particles, capable of internally accommodating refractive peptides to internalization or amphipanexpression using wild-type CVPs as vectors. Therefore, the following protocol and variants thereof can be used to select novel CVP vectors. 1 . A sequence encoding a peptide is cloned into i ^ AAit ^ x. , ^ j,. ^ .. ^? ^^^ - ^ .. of an infectious cDNA molecule encoding CPMV-RNA (e.g., pCP7 or pCP26), by ligation of two or more hybridized oligonucleotides or a DNA fragment from a foreign source. Restriction sites that are adjacent to the sequence encoding the N-terminus of VP-S (for example, the unique? / 7β1 and Eco01091 sites) can be used. The exact location in which the peptide is preferably inserted is preferably between VaH O and Tyr1 1, between a duplication of Val10Tyr1 1, or between a duplication of Tyr1 1. It is also possible to use the cDNA clone in which the peptide has already been inserted (for example, βBβC of VP-S), in such a way that many epitopes can be encoded and displayed simultaneously in a particle. 2. In the case of the cowpea mosaic virus, cowpea plants of about 10-14 days of age (or at any other time during the growth of the plant and before the onset of flowering *) are inoculated with the clone as described above. constructed in step 1, in combination with a cDNA clone encoding CPMV-RNA 1. (Any other susceptible host for CPMV can be infected at an appropriate time before the start of flowering *). [* Viruses are capable of mounting a systemic infection in the appropriate host plant until the growth phase has stopped, and in the case of flowering plants, until flowering occurs]. 3. The plants are monitored closely to see the appearance of infection symptoms. For a good replica particle, local lesions on the inoculated leaves can be expected after 4-6 days, although these are not always clear. You can wait for the . ÜHH lf | niMinijjf iji AMi systemic symptoms after 10-14 days after infection. If there are clear signs of systemic infection, the plants are harvested 3-4 weeks after infection, and the virus is purified (step 5). If 14 days or more are taken before the first local lesions appear, and there are no systemic symptoms or there are very few up to 3 weeks after infection, it is likely that spontaneous in-plant mutations have occurred. In this case, the virus needs to be transferred to fresh plants, as in step 4. If there are no detectable symptoms at all, additional mutations may be required (see steps 7 and 8). 4. If the only symptoms are local lesions instead of systemic lesions, which become evident 2 weeks or more after infection (and before the onset of flowering, or the cessation of the growth phase of the plant), these will be cut from the leaves and transferred individually to a test tube. To each test tube is added some water or any pH regulator suitable for the storage of the virus particles, and the leaf fragments are crushed. The resulting suspension is used to inoculate fresh young cowpea plants (or other appropriate host plant), as described in step 2. This will lead to local lesions approximately 5 days after infection, and to systemic symptoms 3-6 days later. The leaves are harvested. The virus can be purified from the leaves, for example, by extraction with chloroform / butanol, followed by PEG precipitation (van Kammen and Jaeger Cowpea mosaic virus, In: CMI / AAB Description of Plant Viruses 197, bÉ ', á ^ -r .állíi.t ?? pr ^ Commonwealth Agricultural Bureaux

[1978]). Samples from each individual plant were purified for sequence analysis (Brennan et al., Supra). 6. Viral particles are used in a standard RT reaction with a primer that is capable of specific hybridization to, and reverse transcription priming of either the VP-S gene or the VP-L gene. The RT product is amplified by PCR, using primers that amplify either the VP-S gene or the VP-L gene, or both. The PCR products are sequenced in such a way that the sequence of the VP-S or the VP-L can be determined in both chains. If there are no mutations in the inserted epitopes, the construct can be used for, among other things, immunological analyzes. 7. If second site mutations are identified, they can be introduced into novel derivatives of the cDNA clones described in step 1, either by site-directed mutagenesis, or by cutting and filling the cDNA fragments from step 6 inside the infectious clone. 8. If in step 4 there are no symptoms at all, a mutation of the second known site identified in a different construct, or any other mutation that can generate an infectious clone, can be introduced into the cDNA clone made in step 1 and modified as in step 6. With the new construction repeat the entire infection procedure, following steps 2-5. If there are good symptoms in step 4, the introduced mutation may be sufficient for infection and replication of CVPs in plant. If there are only local injuries, it is likely that mutations of third place took place. This can be investigated or confirmed by following steps 5 and 6. 9. A bank of virus vectors can be made with different third-site or third-site (or additional site) mutations, within which one can link an epitope, as described in step 1 above. In this case it is important to proceed with the transfer of a local lesion in step 4, to ensure that the systemically infected plants contain a single clone of the virus.

Example 13 This Example demonstrates the application of a de novo second site mutation to improve infectivity and replication of a virus vector expressing a foreign peptide different from the peptide whose internal expression generated the pressure for selection of the mutation in vivo. A peptide derived from the nucleocapsid protein of the Sendai Virus (with the amino acid sequence, HGEFAPGNYPALWYSA; SEQ ID NO: 1 1) is inserted into the N-terminus of the VP-S of the CPMV, at a site between the residues of duplicated tyrosine (that is, the amino terminal Tyr1 1 to the inserted peptide and a second tyrosine residue immediately from the carboxyl terminus to the inserted sequence). This construction is designated pSEN2. After inoculation of the cDNA in the cowpea plants, the SEN2 did not show any symptoms after 21 days. For this reason, a construct was made that was similar to pSEN2, but differed in that the chimeric virus vector contained a second site mutation, Phe91 Ser, selected in a construction that . «.., .... * -« -t ^ HÉf-? titftri -jrttr? i 't * 2j ** ~ ** * i **. * s & i * expressed an epitope of malaria (MAL8, see Example 3). This construction is designated pSEN3. After inoculation of cDNA in cowpea plants, the SEN3 construct showed symptoms of infection in the inoculated leaves from as early as day 6, and the systemic symptoms appeared 4 days later in 4 out of 5 inoculated plants. The plants were harvested 21 days after infection. The virus was purified from the inoculated leaves, and a yield of 50 mg of virus of 47 g of leaves. The RNA of the purified virus was subjected 21 days after the inoculation to RT-PCR, and the sequencing of the resulting cDNA in both chains was performed. The analysis revealed that the sequence did not change from the sequence of the pSEN3 construct that was used to inoculate the plants. This clearly shows that it is possible to apply a mutation of a second particular site, selected in a construct that expresses a peptide internally, to facilitate expression internally of another unrelated peptide inserted in essentially the same position. This indicates that the second site mutations have wide utility in the expression internally of peptides, and in principle in the construction of other amphide-folding particles, according to Examples 7 and 8. Therefore it is possible to follow the means delineated in the Examples 12 and 13 herein, to construct a library of vectors that can be tested to see the expression of peptides inside the virus, without undue experimentation.

Example 14 This Example demonstrates the expression of a T cell epitope within a plant virus particle, to counteract the exposure of that epitope to the elements of the humoral immune response (eg, circulating antibodies) and immunomodulation by route of presentation of immunogen or antigen. The purpose of many peptide-based vaccines is the induction of a cellular rather than humoral (antibody) response to the epitope being presented. In particular, therapeutic intervention in cancer is increasingly being recognized as being the most effective if a cellular immune response can be stimulated. However, since antibodies circulating in the serum can also recognize the epitope of interest presented, there is always the possibility that the immunogen introduced to stimulate a desired immune response may induce a humoral rather than cellular response to the presented peptide. In addition, the accidental presence of circulating antibodies capable of binding the introduced immunogenic or antigenic composition may preclude the appropriate and effective stimulation of a desired immune response by clearing the complex. In fact, this is potentially a problem with any peptide display system in which the peptides are deployed on the outside of a macromolecular carrier such as orifice limpet hemociasin (KLH). On the other hand, the expression of a peptide such as an epitope inside a particle, for example, in a plant chimeric virus, protects that peptide from the binding of antibodies. This not only iMifi-fÉm líií 'rr - íiiinii li ajiíí ?? '.1 iilitil.lilitiirilrii ffilti Mil? F- IJíiTl "prevents an unwanted humoral response to the epitope, but also helps to allow the peptide to survive the clearance and proteolysis until it has been presented to, and processed by the cells of antigen presentation (APCS) of the immune system For these reasons, the internalization of epitopes in CPMV or other plant viruses has applicability for the expression of epitopes that can be cycled with circulating antibodies, and consequently can be used to direct the type of immune response of a humoral response to a cell phone This is also valid for the expression of peptide mimotopes For immunotherapeutic applications with peptides the induction of particularly cytotoxic T cells is required For this to happen, epitopes capable of produce a cytotoxic T cell response, and present through the cells, in such a way that that the epitope of the peptide is processed and present in the MHC 1 1 molecules. The presentation by this route counteracts a direct antibody response and, instead, produces the stimulation of specific cytotoxic T lymphocyte populations. Those lymphocytes subsequently target the cells that deploy the peptide in question directly, leading to cell lysis or to the evacuation of the target cells or antigens after opsonification. It is known that a peptide derived from a protein, mucin, which is found in large quantities on the surface of human breast cancer cells (and others), is capable of inducing CTL responses in mice when coupled to a carrier. The mucin polypeptide, as it is found in cancer cells, differs from the ubiquitous form of the protein found on the surface of many non-cancer cells, in the sense that it modifies post-translationally differently. In attempts to immunize test animals with a vaccine containing mucin peptide, it was found that cross-reactive Mud p antibodies change the immune response produced from a cell to a humoral one. When the same immunogen is used to vaccinate humans, an antibody response follows instead of a cellular response, since the antigenic peptide reacts transversely with the antibodies against the Gal alpha (1, 3) Gal epitope, normally present in the determinants of some blood groupings in humans, but it is not found in mice. A technical solution is to express the same peptide as an insertion at the N-terminus of the VP-S of the CPMV, between a duplication of Tyr1 1. When these particles are used to immunize humans, the antibodies can not be attached to the epitope inserted directly. A cellular response to the epitope preferably occurs through the uptake and presentation of the Mud p peptide in the antigen presenting cells (APCs). The stimulation of specific CTL subpopulations for the Mud p peptide epitope results in the lysis or evacuation of target cells that carry essentially the same epitope on their cell membranes.

Example 15 This Example demonstrates the production of a CPV that íiL-? .you. > dtt * »**» ** «-« - * "« - »• M ^ ^ i. ^, ^^ j ^? - 1 l contains an epitope of CTL of measles virus, a CTL epitope of virus was inserted of measles (LDRLVRLIG; SEQ ID NO: 13), which was positively charged, at the N-terminus of VP-S, between a duplication of Y1 1. This construct is pMV14.The plants inoculated with this construct did not show any symptoms. inserted the same epitope between a duplication of V10Y1 1 (= pMV15), and now symptoms were observed in two out of five inoculated plants.When the purified virus was transferred to young cowpea plants, very good symptoms were observed. of layer protein completely, and they were found to be correct.

Example 16 This Example demonstrates the production of a CPV containing a CTL epitope of vesicular stomatitis virus. A CTL epitope of vesicular stomatitis virus (RGYWQGL; SEQ ID NO: 12) has been successfully expressed in TY particles, and these particles induced very good CTL responses in mice (Layton et al., Immunology 87: 171-178

[1996]) . This same epitope was inserted between duplicate Y1 1 in VP-S of CPMV. This construction is PVSVI. Good symptoms were seen in 4 out of 5 plants inoculated with this construction. The virus gave very good yields (0.79 milligrams / gram of leaves).

Example 17 This Example demonstrates the construction of oligoalanin-containing vectors by flipping a CTL epitope. It is known that for appropriate processing of the CTL epitopes by the antigen presenting cells, the residues flanking the epitope are crucial. Very little is known, however, of which residues are optimal in conjunction with certain epitopes. It has been observed that the insertion of short stretches of alanins at any site of the epitope may be helpful in improving the response to a CTL epitope inserted into a protein carrier (Del Val et al., Cell 66: 1 145-1 153 [1991 ]). A vector with five alanines has been made between a duplication of V10Y1 1, while there is a unique Notl site in this insert (= pP35). The vector itself was infectious for cowpea plants, although the viral symptoms were suppressed with respect to the WT virus. The insertion of epitopes in this oligo-A tract may be useful to study the optimization of the processing of the CTL epitope, in case the epitopes only give weak immune responses. A malaria epitope was inserted into the Notl site to make pMAL1 1. This construction gave good symptoms in the plants. Since no CTL response was observed in the measles virus epitope of pMV15 in mice (see above), an additional construct was made in which this epitope was flanked by many alanins on either side. This construct, pMV16, was not infectious in cowpea plants. In analogy to pSEN3 (see above), a second site mutation of an unrelated construct was applied to pMV16 (M177V, derived from pTT4). This construction, which was called pNW17, did not give any symptoms in the plants. The following useful plant viral vectors are in deposit in the American Type Culture Collection (ATCC), Rockville, Md., USA, under the terms of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Procedure and Patent Regulations under the same: pTB2 (ATCC Number 75280) and pTBU5 (ATCC 75281). The construction details of these plasmids are disclosed in U.S. Patent No. 5,589,367 incorporated herein by reference. All publications and patents mentioned in the above specification are incorporated herein by reference. Different modifications and variations of the described compositions and methods of the invention will be apparent to those skilled in the art., without departing from the scope and spirit of the invention. Although the invention has been described in connection with particular preferred embodiments, it should be understood that the claimed invention should not be unduly limited to those specific embodiments. In fact, it is intended that various modifications of the modes described for carrying out the invention, which are apparent to those skilled in the art and fields related thereto, are within the scope of the following claims.

A, á. * A * J *? t? L .. -. ^ - ^ ^ - 10Í LIST OF SEQUENCES < 110 > Hellendoom, Koen < 120 > Viral Particles with Exogenous Internal Epitopes < 130 > DOW-04661 < 150 > GB9924352.9 < 151 > 1999-10-14 < 160 > 37 < 170 > Patentln version 3.0 < 210 > 1 < 211 > 10 < 212 > PRT < 213 > Artificial / Unknown < 220 > < 221 > msc_characteristic < 222 > () •• () < 223 > Synthetic < 400 > 1 Gly Pro Val Cys Ala Glu Ala Ser Asp Val 1 10 < 210 > 2 < 211 > 11 < 212 > PRT < 213 > Artificial / Unknown < 220 > ^^ i ^ ^^ jlH ^ I ^^ JÜ ^^ j ^ j go & iB? B wt gMi? Á-ü < 221 > misc_caracteristic < 222 > () •• () < 223 > Synthetic < 400 > 2 Gly Pro Val Cys Ala Glu Ala Ser Asp Val Tyr 1 5 10 < 210 > 3 < 211 > 9 < 212 > PRT < 213 > Artificial / Unknown < 220 > < 221 > misc_caracteristic < 222 > 0-0 < 223 > Synthetic < 400 > 3 Gly Pro Val Cys Ala Glu Ala Ser Asp 1 5 < 210 > 4 < 211 > 7 < 212 > PRT < 213 > Artificial / Unknown < 220 > < 221 > mise feature T? A? *. i? ^ t ~ M? ^. * ^^^. ^^ J ^^? ^? m ^. ^ *, * ^^, .....? Jk LA, < 222 > () - • () < 223 > Synthetic < 400 > 4 Gly Tyr His Gly Ser Ser Leu 1 5 < 210 > 5 < 211 > 14 < 212 > PRT < 213 > Artificial / Unknown < 220 > < 221 > misc_caracteristic < 222 > () •• () < 223 > Synthetic < 400 > 5 Wing Val Tyr Tyr Cys Thr Arg Gly Tyr His Gly Ser Ser Leu 1 5 10 < 210 > 6 < 211 > 15 < 212 > PRT < 213 > Artificial / Unknown < 220 > < 221 > misc_caracteristic < 222 > () - () lffBtttjA * 'J' * 4'Í- - ^ - KM '- »- *. -.- z-tM? & c? Atd .í. < 223 > Synthetic < 400 > 6 Arg Pro Gln Ala Ser Gly Val Tyr Met Gly Asn Leu Thr Ala Gln 1 5 10 15 < 210 > 7 < 211 > 9 < 212 > PRT < 213 > Artificial / Unknown < 220 > < 221 > misc_característíca < 222 > () •• () < 223 > Synthetic < 400 > 7 Ser Tyr lie Pro Ser Ala Glu Lys lie 1 5 < 210 > 8 < 211 > 9 < 212 > PRT < 213 > Artificial / Unknown < 220 > < 221 > misc_caracteristic < 222 > () - • () < 223 > Synthetic < 400 > 8 Ser Tyr lie Pro Ser Ala Gly Lys lie 10 < 210 > 9 < 211 > 16 < 212 > PRT < 213 > Artificial / Unknown < 220 > < 221 > misc_caracteristic < 222 > 0-0 < 223 > Synthetic < 400 > 9 Wing Wing Wing Being Tyr Me Pro Wing Being Glu Lys lie Wing Wing Ala Ala 1 5 10 15 < 210 > 10 < 211 > 8 < 212 > PRT < 213 > Artificial / Unknown < 220 > < 221 > mise feature ?? A + *?.? JL á. ? H ^ ^ X ~ ^ mM ^. < 222 > 0-0 < 223 > Synthetic < 400 > 10 Wing Pro Gly Asn Tyr Pro Wing Leu 1 5 < 210 > 1 1 < 21 1 > 16 < 212 > PRT < 213 > Artificial / Unknown < 220 > < 221 > misc_caracteristic < 222 > 0-0 < 223 > Synthetic < 400 > eleven His Gly Glu Phe Wing Pro Gly Asn Tyr Pro Wing Leu Trp Ser Tyr Ala 1 5 10 15 < 210 > 12 < 211 > 8 < 212 > PRT < 213 > Artificial / Unknown < 220 > < 221 > misc_caracteristic < 222 > 0-0 < 223 > Synthetic < 400 > 12 Arg Gly Tyr Val Tyr Gln Gly Leu 1 5 < 210 > 13 < 211 > 9 < 212 > PRT < 213 > Artificial / Unknown < 220 > < 221 > misc_caracteristic < 222 > 0-0 < 223 > Synthetic < 400 > 13 Leu Asp Arg Leu Val Arg Leu lie Gly 1 5 < 210 > 14 < 211 > 15 < 212 > PRT < 213 > Artificial / Unknown < 220 > HÉJÜ 'hÉÉilhÉtfl tiltll iVitiitií? Mr? I? If? T? Íitlrii? Hl ?? h? 1? Ln? Flt¡Pii'-f - *' - ^ «- ^ - * - < 221 > misc_caracteristic < 222 > 0-0 < 223 > Synthetic < 400 > 14 Ala Ala Ala Leu Asp Arg Leu Val Arg Leu Me Gly Ala Ala Wing 1 5 10 15 < 210 > 15 < 211 > 18 < 212 > PRT < 213 > Artificial / Unknown < 220 > < 221 > misc_caracteristic < 222 > 0-0 < 223 > Synthetic < 400 > fifteen Val Asp Asp Ala Leu He Asn Ser Thr Lys Me Tyr Ser Tyr Phe Pro 1 5 10 15 Ser Val < 210 > 16 < 211 > 13 < 212 > PRT < 213 > Artificial / Unknown < 220 > < 221 > misc_caracteristic < 222 > 0-0 < 223 > Synthetic < 400 > 16 Met Gln Trp Asn Ser Thr Thr Phe His Gln Thr Leu Gln 1 5 10 < 210 > 17 < 211 > 5 < 212 > PRT < 213 > Artificial / Unknown < 220 > < 221 > mísc_característica < 222 > 0-0 < 223 > Synthetic < 400 > 17 Ala Ala Ala Ala Ala 1 5 < 210 > 18 < 211 > 21 < 212 > DNA ] T ?? ?? ?? lt lt lt lt ---- ---- ---- ---- ---- ---- ---- ---- ---- ---- ---- ---- ---- ---- ---- ---- ---- ---- ---- ---- ---- ---- ---- ---- ---- ---- ---- ---- ---- ---- ---- ---- ---- ----? * * - '** - - ** ^ * ^^ ** ** t < 213 > Artificial / Unknown < 220 > < 221 > misc_característíca < 222 > 0-0 < 223 > Synthetic < 400 > 18 ggttatcatg gttctagttt g 21 < 210 > 19 < 211 > 42 < 212 > DNA < 213 > Artificial / Unknown < 220 > < 221 > misc_caracteristic < 222 > 0-0 < 223 > Synthetic < 400 > 19 gctgtttatt attgtactag aggttatcat ggttctagtt tg 42 < 210 > 20 < 21 1 > 45 < 212 > DNA < 213 > Artificial / Unknown < 220 > < 221 > mise feature 'Í-ÉI:? ~ fffrfr "" - * '"' - '" "» ^'! '^ fcJj' * »t * ^.,. gLi.. &?.? -? <222> 0-0 < 223 > Synthetic < 400 > 20 agacctcaag cttctggtgt ttatatgggt aatttgactg ctcaa 45 < 210 > 21 < 21 1 > 27 < 212 > DNA < 213 > Artificial / Unknown < 220 > < 221 > misc_caracteristic < 222 > 0-0 < 223 > Synthetic < 400 > 21 tcttatattc cttctgctga aaagatt 27 < 210 > 22 < 21 1 > 48 < 212 > DNA < 213 > Artificial / Unknown < 220 > < 221 > misc_caracteristic < 222 > 0-0 < 223 > Synthetic < 400 > 22 gcagcggcct cttatattcc ttctgctgaa aagattgcgg ccgctgct 48 < 210 > 23 < 21 1 > 24 < 212 > DNA < 213 > Artificial / Unknown < 220 > < 221 > misc_caracteristic < 222 > 0-0 < 223 > Synthetic < 400 > 23 gctcctggta attatcctgc tttg 24 < 210 > 24 < 211 > 48 < 212 > DNA < 213 > Artificial / Unknown < 220 > < 221 > misc_caracteristic < 222 > 0-0 < 223 > Synthetic < 400 > 24 catggtgaat ttgctcctgg taattatcct gctttgtggt cttatgct 48 < 210 > 25 < 211 > 23 < 212 > DNA < 213 > Artificial / Unknown < 220 > < 221 > misc_caracteristic < 222 > 0-0 < 223 > Synthetic < 400 > 25 agaggttatg tttatcaagg ttg 23 < 210 > 26 < 211 > 27 < 212 > DNA < 213 > Artificial / Unknown < 220 > < 221 > misc_caracteristic < 222 > 0-0 < 223 > Synthetic < 400 > 26 ttggatagat tggttagatt gattggt 27 < 210 > 27 < 211 > 45 < 212 > DNA < 213 > Artificial / Unknown < 220 > < 221 > misc_caracteristic < 222 > 0-0 < 223 > Synthetic < 400 > 27 gcagcggcct tggatagatt ggttagattg attggggccg ctgct 45 < 210 > 28 < 211 > 54 < 212 > DNA < 213 > Artificial / Unknown < 220 > < 221 > misc_caracteristic < 222 > 0-0 < 223 > Synthetic < 400 > 28 gtggatgatg ctttgattaa ttctactaag atttatagtt attttccttc tgtt 54 < 210 > 29 < 211 > 39 < 212 > DNA < 213 > Artificial / Unknown < 220 > < 221 > misc_caracteristic < 222 > 0-0 w- A "tr ir * < 223 > Synthetic < 400 > 29 atgcaatgga actctactac ttttcatcaa actttgcaa 39 < 210 > 30 < 211 > 1 5 < 212 > DNA < 213 > Artificial / Unknown < 220 > < 221 > misc_caracteristic < 222 > 0-0 < 223 > Synthetic < 400 > 30 gcagcggccg ctgct 1 5 < 210 > 31 < 21 1 > 9 < 212 > PRT < 213 > Artificial / Unknown < 220 > < 221 > misc_caracteristic < 222 > 0-0 < 223 > Synthetic < 400 > 31 Tyr Ser Pro Cys Met He Wing Being Thr < 210 > 32 < 211 > 10 < 212 > PRT < 213 > Artificial / Unknown < 220 > < 221 > misc_caracteristic < 222 > 0-0 < 223 > Synthetic < 400 > 32 Val Tyr Ser Pro Cys Met Me Ala Ser Thr 1 5 10 < 210 > 33 < 211 > 15 < 212 > PRT < 213 > Artificial / Unknown < 220 > < 221 > misc_caracteristic < 222 > 0-0 < 223 > Synthetic < 400 > 33 Wing Val Tyr Tyr Cys Thr Arg Gly Tyr His Gly Ser Ser Leu Tyr 10 15 < 210 > 34 < 21 1 > 8 < 212 > PRT < 213 > Artificial / Unknown < 220 > < 221 > misc_caracteristic < 222 > 0-0 < 223 > Synthetic < 400 > 34 Gly Tyr His Gly Ser Ser Leu Tyr 1 < 210 > 35 < 21 1 > 16 < 212 > PRT < 213 > Artificial / Unknown < 220 > < 221 > misc_caracteristic < 222 > 0-0 < 223 > Synthetic < 400 > 35 Gly Val Ser Thr Wing Pro Asp Thr Arg Pro Wing Pro Gly Ser Thr Ala 1 5 10 15 < 210 > 36 < 21 1 > 32 < 212 > PRT < 213 > Artificial / Unknown < 220 > < 221 > misc_caracteristic < 222 > 0-0 < 223 > Synthetic < 400 > 36 Thr Asp Ala Tyr Asn Gln Lys Leu Ser Glu Arg Arg Ala Gly Wing Asp 1 5 10 15 Asn Wing Thr Wing Glu Gly Arg Wing He Asn Arg Arg Val Glu Glu wing 20 25 30 < 210 > 37 < 21 1 > 16 < 212 > PRT < 213 > Artificial / I did not know < 220 > < 221 > misc_caracteristic < 222 > 0-0 < 223 > Synthetic < 400 > 37 Gly Val Thr Ser Wing Pro Asp Thr Arg Pro Wing Pro Gly Ser Thr Ala 1 5 10 15 11"-rr1'íitrf '" ii'i *' itfc "" * tf "" 'ik' "Ji" "'sfeA

Claims (1)

1. CLAIMS 1. A compound comprising a chimeric viral particle having a capsid, wherein the capsid has an inner side and an outer side, the capsid comprising at least one exogenous peptide on said inner side of the capsid. 2. A chimeric viral particle according to claim 1, which is capable of being assembled in a host cell or tissue. 3. A chimeric virus particle according to claim 1 or 2, wherein the virus is icosahedron. 4. A chimeric virus particle according to any of claims 1 to 3, wherein the virus is a comovirus. 5. A chimeric virus particle according to claim 4, wherein the virus is cowpea mosaic virus. 6. A chimeric virus particle according to any of claims 1 to 5, wherein the exogenous peptide is inserted into a coat protein. 7. A chimeric virus particle according to any of claims 1 to 6, wherein the exogenous peptide has from 5 to 20 amino acids. 8. A chimeric virus particle according to any of claims 1 to 7, wherein the exogenous peptide is inserted at a point of -5 to 20 amino acids from the N-terminus of a layer protein, the viral particle assembly is not impossible in a host cell. 9. A chimeric virus particle according to any of claims 1 to 8, wherein the exogenous peptide is inserted into VP-S of the cowpea mosaic virus, between a tyrosine residue at the 1 1 position and a duplicate tyrosine residue at the 12 position. 10. A chimeric virus particle according to any of claims 1 to 8, wherein the exogenous peptide it is inserted in VP-S of the cowpea mosaic virus, between a dipeptide comprising a valine residue in position 10 and a tyrosine residue in the 1 1 position, and a duplicated dipeptide comprising a residue of valine in the position 12 and a tyrosine residue in the 13 position. 1 1. A chimeric virus particle according to any of claims 1 to 8, wherein the exogenous peptide is inserted into VP-S of the cowpea mosaic virus, between a valine residue at position 10 and a duplicate valine residue at the position 1 1. 12. A chimeric virus particle according to any of claims 1 to 11, wherein the viral particle does not contain nucleic acid. 13. A chimeric virus particle according to any of claims 1 to 12, wherein the exogenous peptide encodes an epitope that can be recognized by an animal immune system. 14. A chimeric virus particle according to any of claims 1 to 13, wherein the exogenous epitope is a cytotoxic T lymphocyte epitope. 15. A chimeric virus particle according to any of claims 1 to 14, wherein the exogenous peptide contains a cytotoxic T lymphocyte epitope with flanking amino acids derived from a naturally occurring source of the epitope. 16. A chimeric viral particle according to any of claims 1 to 12, wherein the exogenous peptide is an helper cell epitope T. 17. A chimeric viral particle according to any of claims 1 to 12 and claim 16, wherein the Exogenous peptide contains a helper T cell epitope, with flanking amino acid sequences derived from a naturally occurring source of the epitope. 18. A chimeric viral particle according to any of claims 1 to 12, wherein the exogenous peptide is a B cell epitope. 19. A chimeric viral particle according to any of claims 1 to 12 and claim 18, wherein the exogenous peptide contains a helper T cell epitope, with flanking amino acid sequences derived from a naturally occurring source of the epitope. 20. A chimeric viral particle according to any of claims 1 to 19, which contains a second exogenous peptide expressed on the outer surface of the viral capsid. 21. A chimeric virus particle according to any of claims 1 to 20, which contains a second exogenous peptide expressed on the outer surface of the viral capsid, wherein said peptide is inserted into the ßC '-ßC "cycle of the virus's VP-S of cowpea mosaic 22. A chimeric viral particle according to any of the Uk. ~, * A? 1 claims 1 to 20, which contains a second exogenous peptide expressed on the outer surface of the viral capsid, wherein said peptide is inserted into the ßB-ßC cycle of VP-S of the cowpea mosaic virus. 23. A chimeric virus particle according to any of claims 1 to 20, which contains a second exogenous peptide expressed on the outer surface of the viral capsid, wherein said peptide is inserted into the ßE-aB cycle of VP-L of the mosaic virus of cowpea. "***" * "falMka - '« * "" * »* ** - ** - **** - *** - RFSUMFN The present invention relates to the expression of peptides in viral particles, and more particularly with the expression of peptides within the viral capsid Methods for modifying the viruses are described, such that the exogenous epitopes are expressed inside the viral capsid.Virus that can be modified includes strand RNA viruses ( +), especially plant (+) chain RNA viruses, such as cowpea mosaic virus Internal expression is especially useful for the expression of hydrophobic epitopes Modified viral particles also find use as vaccines, and as such they are capable of producing an immune response.