Compositions and methods for in planta production of a porcine circovirus vaccine

Abstract

Compositions and methods for producing porcine circovirus (PCV) vaccines in plants are provided. Compositions comprising PCV antigens, particularly PCV2 antigens, are provided. Compositions include isolated and recombinant polypeptide sequences expressing at least one PCV2 antigen, recombinant and synthetic nucleic acid molecules encoding at least one PCV2 antigen, constructs or expression cassettes comprising at least one nucleotide sequence encoding a PCV2 antigen, and plants, plant cells, plant parts, and seed transformed with the nucleic acid molecules or constructs comprising the same. Animal feed made from the transformed plants, plant parts, and seed are also encompassed.

Description

[0001] COMPOSITIONS AND METHODS FOR IN PLANTA PRODUCTION OF A PORCINE CIRCOVIRUS VACCINE

[0002] CROSS REFERENCE TO RELATED APPLICATIONS

[0003] This application claims priority to U.S. Provisional Application No. 63 / 587,570, filed October 3, 2023, which is herein incorporated by reference in its entirety.

[0004] REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

[0005] The instant application contains a Sequence Listing which has been submitted electronically in XML format and is herein incorporated by reference in its entirety. Said XML copy, created on October 3, 2024, is named “M117885_1050WO_seq_listing.xml” and is 29.9 kilobytes in size.

[0006] FIELD OF THE INVENTION

[0007] The invention is drawn to the production of porcine circovirus (PCV) vaccines in plants.

[0008] BACKGROUND OF THE INVENTION

[0009] Circoviruses (CV) include some of the smallest viruses known. The circoviruses were named after their circularly arranged single-stranded DNA genome with a gene encoding a conserved replicase protein on the sense strand. Circoviruses are widely distributed in mammals, fish, avian species and even insects. Four different CVs have been identified in pigs and named with consecutive numbers based on the order of their discovery: Porcine circovirus 1 (PCV1), Porcine circovirus 2 (PCV2), Porcine circovirus 3 (PCV3) and most recently Porcine circovirus 4 (PCV4). PC Vs are ubiquitous in global pig populations and uninfected herds are rarely found.

[0010] Since the discovery of porcine circovirus type 2 (PCV2) in 1998, this small, circular, nonenveloped DNA virus is recognized as one of the most important pathogens of the pig population worldwide. PCV2 is associated with post-weaning multi-systemic wasting syndrome (PMWS). PMWS is characterized by poor weight gain, wasting and general symptoms such as dyspnea, pallor, diarrhea, and icterus.

[0011] Current vaccines are based on PCV2a or its capsid protein and administered using traditional injection methods. As PCV rapidly mutates there is a continuing need to develop vaccines easy to administer and capable of protecting pigs against disease associated with PCV.

[0012] SUMMARY OF THE INVENTION Compositions and methods for the production of porcine circovirus (PCV) vaccines in plants are provided. Compositions comprising PCV antigens, particularly PCV2 antigens, are provided. Compositions include isolated and recombinant polypeptide sequences comprising at least one PCV2 antigen, recombinant and synthetic nucleic acid molecules encoding at least one PCV2 antigen, constructs or expression cassettes comprising at least one nucleotide sequence encoding a PCV2 antigen, and plants, plant cells, plant parts, and seed transformed with the nucleic acid molecules or constructs comprising the same. Animal feed made from the transformed plants, plant parts, and seed are also encompassed.

[0013] The expressed PCV polypeptide(s) may be targeted to organelles, vacuoles, embryo, or compartmental spaces in a plant, seed, or plant part. Further, once the plant, seed, or plant part is harvested and ready for preparation as animal feed, the PCV2 polypeptides or antigens are available to provoke an immune response in an animal eating the animal feed.

[0014] Transformed plants, plant cells, and seeds, as well as methods for making such plants, plant cells, and seeds are additionally provided. Such transformed plants, plant cells, and seeds are transformed with a nucleic acid molecule encoding PCV polypeptides and may be operably linked to a targeting peptide. In this manner, the polypeptide of interest is directed to the compartments, ER, or vacuoles of the plant cells. Animal feed comprising the transformed plants, plant parts, and / or seed are also encompassed.

[0015] Embodiments of the invention include:

[0016] 1. A method for eliciting an immune response in an animal, said method comprising orally administering to said animal a composition comprising a plant or plant product, wherein said plant or plant product comprises at least one PCV2 antigen.

[0017] 2. The method of embodiment 1, wherein said administering is by oral administration.

[0018] 3. The method of any one of embodiments 1 or 2, wherein said animal is a pig.

[0019] 4. The method of any one of embodiments 1-3, wherein said PCV2 antigen comprises a capsid protein.

[0020] 5. The method of any one of embodiments 1-4, wherein said composition comprises two PCV2 antigens.

[0021] 6. The method of any one of embodiments 1-5, wherein said PCV2 antigen is PCV2a, PCV2d, or PCV2a and PCV2d.

[0022] 7. The method of any one of embodiments 1-6, wherein said composition comprises seed or seed embryos. The method of any one of embodiments 1-7, wherein said plant or plant product has been transformed with at least one expression cassette comprising a nucleotide sequence encoding said PCV2 antigen. The method of embodiment 8, wherein said plant or plant product comprises said expression cassette stably integrated into the plant or plant product. The method of embodiment 8 or 9, wherein said nucleotide sequence encoding a PCV2 antigen has been optimized for expression in said plant. The method of any one of embodiments 8-10, wherein said nucleotide sequence encoding a PCV2 antigen is operably linked to a promoter that is active in a plant cell. The method of embodiment 11, wherein said promoter is a seed-preferred promoter. The method of embodiment 12, wherein said seed-preferred promoter is selected from the group consisting of Ciml (cytokinin-induced message), maize 15 kDa zein, maize 19 kDa zein, 22 kDa zein, 27 kDa zein, gamma-zein, waxy, shrunken 1, shrunken 2, Globulin 1, endl, end2, milps (myo-inositol- 1 -phosphate synthase, Gamma-zein, Globulin (Glb-1), bean P-phaseolin, napin, P-conglycinin, soybean lectin, and cruciferin. The method of embodiment 11, wherein said promoter is an embryo-preferred promoter. The method of embodiment 14, wherein said embryo-preferred promoter is selected from the group consisting of a globulin 1 promoter, and a pr25 promoter. The method of any one of embodiments 8-15, wherein said nucleotide sequence encoding a PCV2 antigen is operably linked to a nucleotide sequence which encodes a peptide that targets the PCV2 antigen to a subcellular compartment. The method of embodiment 16, wherein said subcellular compartment is an apoplast or endoplasmic reticulum. The method of embodiment 17, wherein said peptide that targets the PCV2 antigen to the endoplasmic reticulum is Lys-Asp-Glu-Leu (KDEL) that is operably linked to the C- terminus of the PCV2 antigen. The method of any one of embodiments 16-18, wherein said PCV2 antigen is operably linked at its N-terminus to the barley alpha amylase signal sequence (BAASS). The method of any one of embodiments 8-19, wherein said PCV2 antigen is PCV2a, PCV2d, or PCV2a and PCV2d. The method of embodiment 20, wherein said PCV2a antigen comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 3. 22. The method of embodiment 20 or 21, wherein said nucleotide sequence encoding the PCV2a antigen is a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 1 or 2.

[0023] 23. The method of any one of embodiments 20-22, wherein said PCV2d antigen comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 6.

[0024] 24. The method of any one of embodiments 20-23, wherein said nucleotide sequence encoding the PCV2d antigen comprises a nucleotide sequence having at least 90% sequence identity to the nucleotide sequence set forth in SEQ ID NO: 4 or 5.

[0025] 25. The method of any one of embodiments 1-24, wherein said plant is maize.

[0026] 26. The method of any one of embodiments 1-25, wherein said method comprises at least two administrations.

[0027] 27. The method of any one of embodiments 1-26, wherein said composition comprising a plant or plant product comprises animal feed comprising said plant or plant product.

[0028] 28. A construct comprising at least one expression cassette comprising a nucleotide sequence encoding a PCV2 antigen.

[0029] 29. The construct of embodiment 28, wherein said nucleotide sequence encoding a PCV2 antigen has been optimized for expression in a plant.

[0030] 30. The construct of embodiment 29, wherein said plant is maize.

[0031] 31. The construct of any one of embodiments 28-30, wherein said nucleotide sequence encoding a PCV2 antigen is operably linked to a promoter active in a plant cell.

[0032] 32. The construct of embodiment 31, wherein said promoter is a seed-preferred promoter.

[0033] 33. The construct of embodiment 32, wherein said seed-preferred promoter is selected from the group consisting of Ciml (cytokinin-induced message), maize 15 kDa zein, maize 19 kDa zein, 22 kDa zein, 27 kDa zein, gamma-zein, waxy, shrunken 1, shrunken 2, Globulin 1, endl, end2, milps (myo-inositol- 1 -phosphate synthase, Gamma-zein, Globulin (Glb-1), bean P-phaseolin, napin, P-conglycinin, soybean lectin, and cruciferin.

[0034] 34. The construct of embodiment 31, wherein said promoter is an embryo-preferred promoter.

[0035] 35. The construct of embodiment 34, wherein said embryo-preferred promoter is a globulin 1 promoter or a pr25 promoter.

[0036] 36. The construct of any one of embodiments 28-35, wherein said nucleotide sequence encoding a PCV2 antigen is operably linked to a nucleotide sequence which encodes a peptide that targets the PCV2 antigen to a subcellular compartment. 37. The construct of embodiment 36, wherein said subcellular compartment is an apoplast or endoplasmic reticulum.

[0037] 38. The construct of any one of embodiments 28-37, wherein said PCV2 antigen comprises a capsid protein.

[0038] 39. The construct of any one of embodiments 28-38, wherein said construct comprises at least two nucleotide sequences encoding a PCV2 antigen.

[0039] 40. The construct of any one of embodiments 28-39, wherein said PCV2 antigen is PCV2a, PCV2d, or PCV2a and PCV2d.

[0040] 41. The construct of embodiment 40, wherein said PCV2a antigen comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 3.

[0041] 42. The construct of embodiments 40 or 41, wherein said nucleotide sequence encoding the PCV2a antigen is a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 1 or 2.

[0042] 43. The construct of any one of embodiments 40-42, wherein said PCV2d antigen comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 6.

[0043] 44. The construct of any one of embodiments 40-43, wherein said nucleotide sequence encoding the PCV2d antigen comprises a nucleotide sequence having at least 90% sequence identity to the nucleotide sequence set forth in SEQ ID NO.4 or 5.

[0044] 45. The construct of any one of embodiments 28-44, wherein said construct comprises two expression cassettes, wherein a first expression cassette comprises operably linked nucleotide sequences comprising: a) a first seed preferred promoter; b) a first nucleotide sequence encoding a BAASS peptide; c) a nucleotide sequence encoding a PCV2a antigen; and d) a first terminator sequence; and wherein a second expression cassette comprises operably linked nucleotide sequences comprising: a) a second seed preferred promoter; b) a second nucleotide sequence encoding a BAASS peptide; c) a nucleotide sequence encoding a PCV2d antigen; and d) a second terminator sequence. 46. The construct of embodiment 45, wherein said nucleotide sequence encoding said PCV2a antigen comprises the sequence set forth in SEQ ID NO: 1 or 2.

[0045] 47. The construct of embodiment 45 or 46, wherein said nucleotide sequence encoding said PCVd antigen comprises the sequence set forth in SEQ ID NO: 4 or 5.

[0046] 48. The construct of any one of embodiments 45-47, wherein said first expression cassette comprises the sequence set forth in SEQ ID NO: 7.

[0047] 49. The construct of any one of embodiments 45-48, wherein said second expression cassette comprises the sequence set forth in SEQ ID NO: 12.

[0048] 50. The construct of any one of embodiments 45-49, wherein each cassette further comprises a nucleotide sequence encoding a polypeptide which targets the antigen to the endoplasmic reticulum operably linked to the nucleotide sequence encoding the PCV2 antigens.

[0049] 51. The construct of embodiment 50, wherein said polypeptide which targets the antigen to the endoplasmic reticulum is KDEL operably linked to the C-terminus of the PCV2 antigen.

[0050] 52. The construct of embodiment 51, wherein said PCV2 antigen is operably linked at its N- terminus to the barley alpha amylase signal sequence (BAASS).

[0051] 53. The construct of embodiment 52, wherein said first expression cassette comprises the sequence set forth in SEQ ID NO: 9 or 10.

[0052] 54. The construct of embodiment 52 or 53, wherein said second expression cassette comprises the sequence set forth in SEQ ID NO: 14 or 15.

[0053] 55. A plant cell comprising any one of the constructs of embodiments 28-54.

[0054] 56. The plant cell of embodiment 55, wherein said construct is stably integrated into the genome of the cell.

[0055] 57. A plant cell comprising at least one nucleic acid molecule comprising a nucleotide sequence encoding a PCV2 antigen.

[0056] 58. The plant cell of embodiment 57, wherein said nucleotide sequence encoding a PCV2 antigen is stably incorporated into the genome of the cell.

[0057] 59. The plant cell of embodiment 57 or 58, wherein said nucleotide sequence encoding a PCV2 antigen has been optimized for expression in said plant cell.

[0058] 60. The plant cell of any one of embodiments 57-59, wherein said nucleotide sequence encoding a PCV2 antigen is operably linked to a nucleotide sequence which encodes a peptide that targets the PCV2 antigen to a subcellular compartment.

[0059] 61. The plant cell of embodiment 60, wherein said subcellular compartment is an apoplast or endoplasmic reticulum. 62. The plant cell of embodiment 61, wherein said peptide that targets the PCV2 antigen to the endoplasmic reticulum is Lys-Asp-Glu-Leu (KDEL) that is operably linked to the C- terminus of the PCV2 antigen.

[0060] 63. The plant cell of any one of embodiments 60-62, wherein said PCV2 antigen is operably linked at its N-terminus to the barley alpha amylase signal sequence (BAASS).

[0061] 64. The plant cell of any one of embodiments 57-63, wherein said PCV2 antigen comprises a capsid protein.

[0062] 65. The plant cell of any one of embodiments 57-64, wherein said plant cell comprises at least two nucleotide sequences encoding a PCV2 antigen.

[0063] 66. The plant cell of any one of embodiments 57-65, wherein said PCV2 antigen is PCV2a, PCV2d, or PCV2a and PCV2d.

[0064] 67. The plant cell of embodiment 66, wherein said PCV2a antigen comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 3.

[0065] 68. The plant cell of embodiments 66 or 67, wherein said nucleotide sequence encoding the PCV2a antigen is a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 1 or 2.

[0066] 69. The plant cell of any one of embodiments 66-68, wherein said PCV2d antigen comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 6.

[0067] 70. The plant cell of any one of embodiments 66-69, wherein said nucleotide sequence encoding the PCV2d antigen comprises a nucleotide sequence having at least 90% sequence identity to the nucleotide sequence set forth in SEQ ID NO.4 or 5.

[0068] 71. The plant cell of any one of embodiments 55-70, wherein said plant cell is a maize cell.

[0069] 72. A plant comprising the plant cell of any one of embodiments 55-71.

[0070] 73. Seed of the plant of embodiment 72.

[0071] 74. Animal feed that comprises the plant of embodiment 72.

[0072] 75. Animal feed that comprises the seed of embodiment 73.

[0073] BRIEF DESCRIPTION OF THE FIGURES

[0074] Figure 1 provides a depiction of each of the 9321, 9322, and 9323 constructs used to express PCV2a and PCV2d in maize.

[0075] DETAILED DESCRIPTION OF THE INVENTION Overview

[0076] The present invention provides compositions and methods for expressing at least one PCV2 antigen in plant cells, plants, plant parts, and plant seeds (collectively plant product). An antigen is a substance (generally, a foreign substance) capable of eliciting an immune response (humoral and / or cell-mediated) when administered to a subject. The presently disclosed methods and compositions utilize monovalent vaccines (only a single antigen type) or bivalent vaccines using antigens (e.g., the capsid protein sequence) from two different PCV2 genotypes. In some embodiments, the capsid proteins or antigens are PCV2a and PCV2d antigens. The expressed PCV antigens (e.g., capsid proteins) may be directed to plant compartmental or subcellular spaces. Compositions of the invention include the transformed plants, plant parts, plant seeds expressing the PCV antigen or polypeptide, animal feed made from the transformed plants, seeds, or plant material, as well as expression cassettes for expressing the PCV nucleotide sequences.

[0077] The PCV2 nucleotide sequences may be operably linked to promoters and targeting sequences to express the polypeptide in tissues of interest and direct storage of the polypeptide into cellular compartments such as vacuoles, organelles, embryo, endoplasmic reticulum, apoplast, and the like. Additionally, the methods encompass co-immunization against other pathogens such as Mycoplasma hypopneumonia, Swine erysipelas, Colibacillosis, Mycoplasmal pneumonia, Atrophic rhinitis, (Bordetella), Pleuropneumonia, Actinobacillus pleuropneumoniae. Porcine proliferative enteritis, Glasser’s disease, Haemophilus parasuis. Salmonellosis, Streptococcus suis. Porcine parvovirus, Pseudorabies, other Porcine circoviruses, Porcine respiratory and reproductive syndrome virus, Porcine epidemic diarrhea, Influenza, Rotavirus, and the like. When the vaccines of the invention are orally administered to the animal, a protective response is observed. The vaccines of the invention may elicit a humoral and mucosal response in the animal and show efficacy in reducing clinical signs associated with diseases caused by PCV2, even if another genotype may be present. Further, it is expected that PCV2a vaccines will provide cross-protection against the most widespread genotypes (PCV2a, PCV2b, and PCV2d).

[0078] Porcine Circovirus

[0079] Circoviruses are widely distributed in mammals, fish, avian species and even insects. In pigs, four different CVs have been identified and named with consecutive numbers based on the order of their discovery: Porcine circovirus 1 (PCV1), Porcine circovirus 2 (PCV2), Porcine circovirus 3 (PCV3) and most recently Porcine circovirus 4 (PCV4). PCVs are ubiquitous in global pig populations and uninfected herds are rarely found. While it is generally accepted that PCV1 is non-pathogenic, PCV2 is considered an important, economically challenging pathogen on a global scale.

[0080] Porcine circovirus type 2 (PCV2) is considered one of the most important pathogens of the pig population worldwide. Porcine circovirus is a non-enveloped, icosahedral virus of the Circoviridae family, with a small, circular, single-stranded DNA genome. Two major open reading frames ORF1 and ORF2, encode a replication associated protein and a capsid protein, respectively. A capsid protein is found on the shell of a virus, enclosing the genetic material, and often forms oligomeric structural subunits to create the viral shell or coat. Four types of PCV have been identified: Type 1, Type 2, Type 3, and Type 4. In general, PCV1 is considered relatively benign, while PCV2 is responsible for substantial losses and is also known as post-weaning multisystemic wasting syndrome (PMWS).

[0081] Asymptomatic infection appears to be common and symptomatic disease may be dependent on the presence of additional stress factors. In addition, animal studies may require co-challenge to see an effect. PCV3 was identified in 2016 and its epidemiology is less clear and challenge models are less well developed. PCV4 is more recently characterized and not yet well understood.

[0082] Despite the description of several different genotypes (PCV2a to PCV2i), there appears to be one single PCV2 serotype so far. Although there is substantial evidence for cross-protection between genotypes of PCV2, this may not extend to cross-protection between PCV2 and PCV3.

[0083] While the methods of the invention encompass the production of a single PCV2 antigen such as PCV2a, PCV2b, or PCV2d in a plant, the invention also encompasses the production of two or more antigens, such as, but not limited to PCV2a and PCV2b, PCV2a and PCV2d, and the like.

[0084] "Codon optimization" may be used to optimize sequences for increased expression in a particular plant of interest. Codon optimization may be used to modify a nucleic acid sequence for enhanced expression in the cells of the plant of interest, e.g., maize, soybean, and the like, by replacing at least one, more than one, or a significant number, of codons of the native sequence with codons that are more frequently or most frequently used in the genes of that plant. See, for example, Campbell and Gowri (1990) Plant Physiol. 92: 1 -11. Various plants exhibit a particular bias for certain codons of a particular amino acid. For example, although nucleic acid sequences of the embodiments may be expressed in both monocotyledonous and dicotyledonous plant species, sequences can be modified to account for the specific codon preferences and GC content preferences of monocotyledons or dicotyledons as these preferences have been shown to differ (Murray et al. (1989) Nucleic Acids Res. 17:477-498). Thus, the maize-preferred codon for a particular amino acid may be derived from known gene sequences from maize. Maize codon usage for 28 genes from maize plants is listed in Table 4 of Murray et al., supra. Methods are available in the art for synthesizing plant-preferred genes. See, for example, U.S. Patent Nos. 5,380,831, and 5,436,391, and Murray et al. (1989) Nucleic Acids Res. 17:477-498. All of these references are herein incorporated by reference.

[0085] As discussed, the polynucleotides, polypeptides, or variants thereof may be utilized in the practice of the invention. By "variants" substantially similar sequences are intended. That is, the nucleotide sequence encoding the PCV2 polypeptide may be modified as long as the encoded product is capable of inducing the generation of a protective response. For nucleotide sequences of the nucleic acid molecules of the invention, conservative variants include those sequences that, because of the degeneracy of the genetic code, encode the amino acid sequence of one of the PCV2 polypeptides of the invention. Variant nucleotide sequences also include synthetically derived nucleotide sequences, such as those generated, for example, by using site-directed mutagenesis but which still encode a PCV2 polypeptide of the invention. Generally, variants of a particular nucleotide sequence of the invention will have at least about 75%, 80%, 85%, preferably at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to that particular nucleotide sequence as determined by sequence alignment programs described elsewhere herein using default parameters.

[0086] By "variant protein or polypeptide" a protein derived from the native protein by deletion (so-called truncation) or addition of one or more amino acids to the N-terminal and / or C-terminal end of the native protein; deletion or addition of one or more amino acids at one or more sites in the native protein; or substitution of one or more amino acids at one or more sites in the native protein is intended. Variant proteins encompassed by the present invention are biologically active, that is, they continue to possess the desired biological activity (inducing the generation of a protective response to PCV). Biologically active variants of a PCV polypeptide of the invention will have at least about 75%, 80%, 85%, preferably at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the amino acid sequence for the PCV polypeptide.

[0087] Methods for the determination of sequence identity between sequences are known in the art. See, for example, W02012 / 030759, herein incorporated by reference. Computer implementations of these mathematical algorithms can be utilized for comparison of sequences to determine sequence identity. Such implementations include, but are not limited to: CLUSTAL in the PC / Gene program (available from Intelligenetics, Mountain View, California); the ALIGN program (Version 2.0) and GAP, BESTFIT, BLAST, FASTA, and TFASTAin the Wisconsin Genetics Software Package, Version 8 (available from Genetics Computer Group (GCG), 575 Science Drive, Madison, Wisconsin, USA). Alignments using these programs can be performed using the default parameters. The CLUSTAL program is well described by Higgins et al. (1988) Gene 73:237-244 (1988). The ALIGN program is based on the algorithm of Myers and Miller (1988) CABIOS 4: 11-17. A PAM 120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used with the ALIGN program when comparing amino acid sequences.

[0088] Expression Cassettes

[0089] Polynucleotides encoding the pesticidal proteins provided herein can be provided in expression cassettes for expression in a plant of interest. The cassette will include 5' and 3' regulatory sequences operably linked to a polynucleotide encoding a PCV2 polypeptide that allows for expression of the antigen. The cassette may additionally contain at least one additional gene or genetic element or antigen to be cotransformed into the organism. Where additional genes or elements are included, the components may be operably linked. The term "operably linked" as used herein refers to a functional linkage between a genetic element and the polynucleotide expressing the polypeptide of interest.

[0090] Alternatively, the additional gene(s) or element(s) can be provided on multiple expression cassettes. The expression cassettes of the invention are provided with a plurality of restriction sites and / or recombination sites for insertion of the polynucleotides to be under the transcriptional regulation of the regulatory regions. The expression cassette may additionally contain a selectable marker gene, targeting or regulatory sequences, stabilizing or leader sequences, introns etc.

[0091] The expression cassette will include in the 5'-3' direction of transcription, a transcriptional and translational initiation region (i.e., a promoter), a PCV2 polynucleotide of the invention, and a transcriptional and translational termination region (i.e., termination region) functional in a plant. The expression cassette may include at least a second genetic element comprising in the 5'-3 ' direction of transcription a promoter, another PCV2 polynucleotide of the invention, and a termination region functional in a plant.

[0092] The regulatory regions (i.e., promoters, transcriptional regulatory regions, targeting peptides, and translational termination regions) may be endogenous or heterologous to the plant cell or to each other. Further, the nucleic acid molecule(s) encoding a PCV antigen can be heterologous to the cell (e.g., plant cell) in which it has been introduced. As used herein, "heterologous" in reference to a sequence is a sequence that originates from a foreign species, or, if from the same species, is substantially modified from its native form in composition and / or genomic locus by deliberate human intervention. As used herein, a chimeric gene comprises a coding sequence operably linked to a transcription initiation region that is heterologous to the coding sequence.

[0093] Convenient termination regions are available from the Ti-plasmid of A. lumefaciens. such as the octopine synthase and nopaline synthase termination regions. See also Guerineau et al. (1991) Mol. Gen. Genet. 262: 141-144; Proudfoot (1991) Cell 64:671-674; Sanfacon et al. (1991) Genes Dev. 5: 141-149; Mogen etal. (1990) Plant Cell 2: 1261-1272; Munroe etal. (1990) Gene 91 : 151- 158; Ballas et al. (1989) Nucleic Acids Res . 17:7891-7903; and Joshi et al. (1987) Nucleic Acids Res. 15:9627-9639. Another terminator includes the pin / / terminator from the protease inhibitor II gene from potato (An et al. (1989) Plant Cell 1, 115-122. See also, Guerineau et al. (1991)Afo / . Gen. Genet. 262: 141-144; Proudfoot (1991) Cell 64:671-674; Sanfacon et al. (1991) Genes Dev . 5: 141-149; Mogen et al. (1990) Plant Cell 2 :1261-1212; Munroe et al. (1990) Gene 91 : 151-158; Ballas et al. (1989) Nucleic Acids Res . 17:7891-7903; and Joshi et al. (1987) Nucleic Acid Res. 15:9627-9639.

[0094] Additional regulatory signals may be used and include, but are not limited to, transcriptional initiation start sites, operators, activators, enhancers, other regulatory elements, ribosomal binding sites, an initiation codon, termination signals, and the like. See, for example, U.S. Patent Nos. 5,039,523 and 4,853,331; EPO 0480762A2; Sambrook et al. (1992) Molecular Cloning: A Laboratory Manual, ed. Maniatis et al. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.) (hereinafter "Sambrook 11"); Davis et al, eds. (1980) Advanced Bacterial Genetics (Cold Spring Harbor Laboratory Press), Cold Spring Harbor, N. Y, and the references cited therein.

[0095] The expression cassettes may additionally contain 5' leader sequences. Such leader sequences can act to enhance translation. Translation leaders are known in the art and include: picornavirus leaders, for example, EMCV leader (Encephalomyocarditis 5' noncoding region) (Elroy-Stein et al. (1989) roc. Natl. Acad. Sci. USA 86:6126-6130); potyvirus leaders, for example, TEV leader (Tobacco Etch Virus) (Gallie et al. (1995) Gene 165(2): 233-238), MDMV leader (Maize Dwarf Mosaic Virus), human immunoglobulin heavy-chain binding protein (BiP) (Macejak et al. (1991 ) Nature 353: 90-94); untranslated leader from the coat protein mRNA of alfalfa mosaic virus (AMV RNA 4) (Jobling et al. (1987) Nature 325: 622-625); tobacco mosaic virus leader (TMV) (Gallie et al. (1989) in Molecular Biology of RNA, ed. Cech (Liss, New York), pp. 237-256); and maize chlorotic mottle virus leader (MCMV) (Lommel et al. (1991 ) Virology 81 : 382-385). See also, Della-Cioppa et al. (1987) Plant Physiol. 84: 965-968.

[0096] Targeting or signal sequences may also be included in the expression cassette and operably linked to the PCV2 polynucleotide. Signal sequences are known in the art. See, for example Becker et al. (1992) Plant Mol. Biol. 20:49; Knox et al. (1987) Plant Mol. Biol. 9:3-17; Lerner et al. (1989) Plant Physiol. 91 : 124-129; Fontes et al. (1991) / awt Ce / Z 3:483-496; Matsuoka et al. (199V) Proc. Natl. Acad. Sci. USA 88:834; Gould et al. (1989) J. Cell. Biol. 108: 1657; Creissen et al. (1991) Plant J. 2: 129; Kalderon et al. (1984) “A short amino acid sequence able to specify nuclear location,” Cell 39:499-509; Steifel et al. (1990) “Expression of a maize cell wall hydroxyproline- rich glycoprotein gene in early leaf and root vascular differentiation,” Plant Cell 2:785-793. When targeting the protein to the cell wall, use of a signal sequence is necessary. One example is the barley alpha-amylase signal sequence, such as that disclosed as SEQ ID NO: 17. Rogers, J.C. (1985) “Two barley alpha-amylase gene families are regulated differently in aleurone cells” J. Biol. Chem. 260: 3731-3738.

[0097] In those instances where it is desirable to have the expressed polypeptide directed to a particular organelle, particularly the plastid, amyloplast, or to the endoplasmic reticulum, or secreted at the cell's surface or extracellularly to the apoplast, the expression cassette can further comprise a coding sequence for a transit peptide. Such transit peptides are well known in the art and include, but are not limited to, the transit peptide for the acyl carrier protein, the small subunit of RUBISCO, plant EPSP synthase, Zea mays Brittle- 1 chloroplast transit peptide (Nelson et al. (1998) Plant Physiol 117(4): 1235-1252; Sullivan et al. Plant Cell 3(12): 1337 -48; Sullivan et al. (1995) Planta 196(3):477-84; Sullivan et al. (1992) J. Biol. Chem. 267(26): 18999-9004) and the like.

[0098] One skilled in the art will readily appreciate the many options available in expressing a product to a particular organelle or plant subcellular compartment. Use of transit peptides is well known (e.g., see U.S. Patents Nos. 5,717,084; 5,728,925). A protein may be targeted to the endoplasmic reticulum of the plant cell. This may be accomplished by use of a localization sequence, such as KDEL. This sequence (Lys-Asp-Glu-Leu) contains the binding site for a receptor in the endoplasmic reticulum. See, Munro et al. (1987) “A C-terminal signal prevents secretion of luminal ER proteins.” Cell. 48:899-907. There are a wide variety of endoplasmic reticulum retention signal sequences available to one skilled in the art and the KDEL sequence is one example. Another example is HDEL (His-Asp-Glu-Leu). See, for example, Kumar et al. which discuses methods of producing a variety of endoplasmic reticulum proteins. Kumar et al. (2017) “prediction of endoplasmic reticulum resident proteins using fragmented amino acid composition and support vector machine” Peer J. doi: 10.7717 / peerj.3561.

[0099] Signal peptides may be used to transport a protein to the plant vacuole. See, for example, W02012 / 030759, herein incorporated by reference. Several vacuolar sorting signals / vacuolar sorting determinants have been described in plants and they can be divided into three major groups: ctVSS [C-terminal VSS (vacuolar sorting signals)], ssVSS (sequence-specific VSS) and physical structure VSS. The sequence specific sorting signals (ssVSD) require a conserved sequence of Asn- Pro-Ile-Arg (NPIR) or similar, which do not tolerate significant changes. Usually, this type of signal is located at the N-terminal of the protein directed to the lytic vacuole. The second main group comprises the signals present at the C-terminal region of the protein (ctVSD) and normally designates proteins destined to the protein storage vacuole. These signals do not have any homologous sequence or size set, having in common that they are rich in hydrophobic amino acids and need to be exposed at the C-terminus to operate. Finally, the third group corresponds to signals dependent on the tertiary (physical) structure of proteins (psVSD) and is most common in storage proteins. These sorting signals are present in the protein sequence, and may correspond to more than one polypeptide that function as whole when the protein acquires the native conformation. See, Neuhaus J.-M., Rogers J. (1998) Sorting of proteins to vacuoles in plant cells. Plant Mol. Biol. 38: 127-144; Zouhar J., Rojo E. (2009) Plant vacuoles: Where did they come from and where are they heading? Curr. Opin. Plant Biol. 12:677-684; and, Jolliffe N.A., Craddock C.P., Frigerio L. (2005) Pathways for protein transport to seed storage vacuoles. Biochem. Soc. Trans. 33: 1016- 1018.

[0100] As noted, signal sequences to target to the plant vacuole are well known. For example, Raikhel (U.S. Patent No. 5,360,726) shows a vacuole signal sequence as does Warren et al. (U.S. Patent No. 5,889,174). Vacuolar targeting signals may be present either at the amino-terminal portion, (Holwerda et al. (1992) The Plant Cell, 4:307-318, Nakamura el al. (1993) Plant Physiol., 101 : 1-5), carboxy -terminal portion, or in the internal sequence of the targeted protein. (Tague et al. (1992) The Plant Cell, 4:307-318, Saalbach et al. (1991) The Plant Cell, 3:695-708). Additionally, amino-terminal sequences in conjunction with carboxy-terminal sequences are responsible for vacuolar targeting of gene products (Shinshi et al. (1990) Plant Molec. Biol. 14:357-368).

[0101] A number of promoters may be used in the practice of the invention, including promoters that are active in a plant cell. The promoters can be selected based on the desired outcome. The nucleic acids can be combined with constitutive, tissue-preferred, or other promoters for expression in the plant. Of interest are tissue specific promoters. Tissue specific promoters which may be used in the expression cassette include seed-preferred promoters, embryo-preferred promoters, and the like. "Seed-preferred" promoters include "seed-specific" promoters (those promoters active during seed development such as promoters of seed storage proteins). See Thompson et al. (1989) BioEssays 10: 108. Seed-preferred promoters include, but are not limited to, Ciml (cytokinin- induced message); cZ19B 1 (maize 19 kDa zein); milps (myo-inositol- 1 -phosphate synthase) (see WO 00 / 11177 and U.S. Patent No. 6,225,529). Another seed preferred promoter includes the phaseolin promoter (Bustos et al. (1989) The Plant Cell 1, 839-853). Gamma-zein is an endospermspecific promoter. Globulin-1 (Glb-1) is a representative embryo- specific promoter. For dicots, seed-specific promoters include, but are not limited to, bean P-phaseolin, napin, P-conglycinin, soybean lectin, cruciferin, and the like. For monocots, seed-specific promoters include, but are not limited to, maize 15 kDa zein, 19kDa zein, 22 kDa zein, 27 kDa zein, gamma-zein, waxy, shrunken 1, shrunken 2, an Ltpl (See, for example, US Patent No. 7,550,579), an Ltp2 (Opsahl-Sorteberg, H- G. et al., (2004) Gene 341 :49-58 and US Patent 5,525,716), and promoters from oleosin genes. See also WO 00 / 12733, where seed-preferred promoters from endl and end2 genes are disclosed.

[0102] Seed-preferred promoters also include those promoters that direct gene expression predominantly to specific tissues within the seed such as, for example, the endosperm-preferred promoter of y-zein, the cryptic promoter from tobacco (Fobert et al. (1994) “T-DNA tagging of a seed coat-specific cryptic promoter in tobacco” Plant J. 4: 567-577), the P-gene promoter from corn (Chopra et al. (1996) “Alleles of the maize P gene with distinct tissue specificities encode Myb-homologous proteins with C-terminal replacements” Plant Cell 7: 1149-1158, Erratum in Plant Cell (1997) 1 : 109), the globulin-1 promoter from com (Belanger and Kriz (1991) “Molecular basis for Allelic Polymorphism of the maize Globulin-1 gene” Genetics 129: 863-972 and GenBank accession No. L22344), promoters that direct expression to the seed coat or hull of com kernels, for example the pericarp-specific glutamine synthetase promoter (Muhitch et al., (2002) “Isolation of a Promoter Sequence From the Glutamine Synthetase Gene Capable of Conferring Tissue-Specific Gene Expression in Transgenic Maize” Plant Science 163:865-872 and GenBank accession number AF359511) and to the embryo (germ) such as that disclosed at US Patent No. 7,169,967. When referring to a seed or an embryo preferred promoter is meant that it expresses an operably linked sequence to a higher degree in seed or embryo tissue that in other plant tissue. It may express during seed or embryo development, along with expression at other stages, may express strongly during seed or embryo development and to a much lesser degree at other times.

[0103] The globulin-1 promoter has been characterized and previously used in transgenic maize seeds. Storage promoters besides globulin-1 that are also active in cereal seed tissues may be used. The principal tissue types in maize seeds are the embryo, the endosperm including a surrounding aleurone cell layer and the maternally derived pericarp. Of these, the endosperm and to a lesser extent the embryo, comprise most of the volume of the seed. Thus, endosperm and embryo promoters are particularly important for modifying seed characteristics and content. The relatively high soluble protein content of the embryo makes it a particularly good target for foreign protein expression. The proximal 1.1 kb of the maize 27 kD g-zein promoter and the proximal 1.4 kb of the maize globulin-1 promoter (GenBank accession L22344) are prominent examples of seed preferred promoters that have been used to express transgenes in endosperm and embryo tissues, respectively, of monocots. Cereal seeds consist principally of two tissue types, the embryonic plant or embryo and the surrounding endosperm. The endosperm is comprised almost entirely of nutritional reserves, primarily of complex carbohydrate and aqueous insoluble protein, but the embryo also contains considerable stores, mainly of oils and aqueous soluble proteins. Globulin-1 is one of the most abundant proteins in maize embryo tissue. It is largely limited to this tissue and becomes particularly concentrated in the scutellum late in embryo development. Given the high concentration of this protein observed in embryo tissues the maize, the globulin-1 promoter was previously identified as being a good candidate to direct high levels of transgene expression in the embryo. An approximately 1.4 kb extent of this promoter has been cloned (Genbank accession L22344) and used to drive high levels of transgene expression specifically in maize seed. Longer versions up to 3 kb of the globulin-1 promoter may be used. A maize globulin-1 pr25 promoter set forth as SEQ ID NO: 19 can be used to express the PCV antigen.

[0104] For embryo specific promoters, see for example Streatfield et al. (2010) Identification of maize embryo-preferred promoters suitable for high-level heterologous protein production, GM Crops, 1 :3, 162-172, DOI: 10.4161 / gmcr. l.3.12816. Other seed preferred promoters include those disclosed in US Patent No. 6,528,704; 6,403,862; 8,049,000; 9,227,794; 9,383,543; 9,703,754; 9,267,147; W02000 / 011177; and W02000 / 040710. Embryo specific or preferred promoters also include the promoters from the embryo-specific genes Zm.13387, Zm.66589, Zm.85502, Zm.3896, and Zm.2941 (See, Liu et al. (2014) Plant Biotech J 12(9): 1286-1296, herein incorporated by reference.)

[0105] Suitable constitutive promoters for use in a plant host cell include, for example, the core promoter of the Rsyn7 promoter and other constitutive promoters disclosed in WO 99 / 43838 and U.S. Patent No. 6,072,050; the core CaMV 35S promoter (Odell et al. (1985) Nature 313: 810- 812); rice actin (McElroy et al. (1990) Plant Cell 2: 163-171 ); ubiquitin (Christensen et al. (1989) Plant Mol. Biol. 12: 619-632 and Christensen et al. (1992) Plant Mol. Biol. 18: 675-689); pEMU (Last et al. (1991 ) Theor. Appl. Genet. 81 : 581 -588); MAS (Velten et al. (1984) PMBO J. 32123- 2730); ALS promoter (U.S. Patent No. 5,659,026), and the like. Other constitutive promoters include, for example, those discussed in U.S. Patent Nos. 5,608,149; 5,608,144; 5,604,121; 5,569,597; 5,466,785; 5,399,680; 5,268,463; 5,608,142; and 6,177,611.

[0106] Any inducible promoter may be used. See, Ward et al. (1993) Plant Mol. Biol. 22: 361-366. Exemplary inducible promoters include ecdysone receptor promoters, U.S. Patent No. 6,504,082; promoters from the ACE1 system which responds to copper (Mett et al. (1993) PNAS 90: 4567- 4571); In2-1 and In2-2 genes from maize which respond to benzenesulfonamide herbicide safeners (US Patent No. 5,364,780; Hershey etal., (1991) Mol. Gen. Genetics 227: 229-237 and Gatz et al., (1994) Afo / . Gen. Genetics 243: 32-38) Tet repressor from TnlO (Gatz et al., (1991) Mol. Gen. Genet. 227: 229-237; or from a steroid hormone gene, the transcriptional activity of which is induced by a glucocorticosteroid hormone. Schena et al. (1991) roc. Natl. Acad. Sci. USA 88: 10421; the maize GST promoter, which is activated by hydrophobic electrophilic compounds that are used as pre-emergent herbicides; and the tobacco PR-la promoter, which is activated by salicylic acid. Other chemical-regulated promoters of interest include steroid-responsive promoters (see, for example, the glucocorticoid-inducible promoter in Schena et al. (1991) Proc. Natl. Acad. Sci. USA 88: 10421-10425 and McNellis etal. (1998) Plant J. 14(2):247-257) and tetracyclineinducible and tetracycline-repressible promoters (see, for example, Gatz et al. (1991) Mol. Gen. Genet. 227:229-237, and U.S. Patent Nos. 5,814,618 and 5,789,156).

[0107] The termination region can be native with the promoter nucleotide sequence can be native with the DNA sequence of interest or can be derived from another source. Convenient termination regions are available from the Ti-plasmid of A, tumefaciens, such as the octopine synthase (MacDonald et al. (1991) Nuc. Acids Res. 19(20)5575-5581) and nopaline synthase termination regions (Depicker et al. (1982) Mol. andAppl. Genet. 1 :561-573 and Shaw et al. (1984) Nucleic Acids Research 12(20):7831-7846. Examples of various other terminators include the pin II terminator from the protease inhibitor II gene from potato (An et al. (1989) Plant Cell 1 : 115-122. See also, Guerineau et al. (1991) Mol. Gen. Genet. 262: 141-144; Proudfoot (1991) Cell 64:671- 674; Sanfacon et al. (1991) Genes Dev. 5: 141-149; Mogen et al. (1990) Plant Cell 2:1261-1272; Munroe et al. (1990) Gene 91 : 151-158; Ballas et al. (1989) Nucleic Acids Res. 17:7891-7903; and Joshi et al. (1987) Nucleic Acids Res. 15:9627-9639.

[0108] Many variations on the promoters, selectable markers, signal sequences, leader sequences, termination sequences, introns, enhancers, and other components of the vector are available to one skilled in the art.

[0109] In preparing the expression cassette, the various DNA fragments may be manipulated, so as to provide for the DNA sequences in the proper orientation and, as appropriate, in the proper reading frame. Toward this end, adapters or linkers may be employed to join the DNA fragments or other manipulations may be involved to provide for convenient restriction sites, removal of superfluous DNA, removal of restriction sites, or the like. For this purpose, in vitro mutagenesis, primer repair, restriction, annealing, resubstitutions, e.g., transitions and transversions, may be involved.

[0110] Transformed Plants and Animal Feed

[0111] Compositions of the invention include transformed organisms, such as a plant, including whole plants, as well as plant organs (e.g., leaves, stems, roots, etc.), seeds, plant cells, propagules, embryos and progeny of the same. Plant cells can be differentiated or undifferentiated (e.g., callus, suspension culture cells, protoplasts, leaf cells, root cells, phloem cells, pollen). "Transgenic plants" or "transformed plants" or "stably transformed" plants or cells or tissues refers to plants that have incorporated or integrated a polynucleotide encoding at least one PCV2 polypeptide of the invention. It is recognized that other exogenous or endogenous nucleic acid sequences or DNA fragments may also be incorporated into the plant cell.

[0112] Methods for plant transformation are known in the art. See, for example, US2018 / 0066277; US2020 / 0399324; US2022 / 0282273; WO2014 / 018853; WO2015 / 023846; US2021 / 0108223; W02020 / 056070; and CA3093297. All of which are herein incorporated in their entirety. Agrobacterium-and biolistic-mediated transformation remain the two predominantly employed approaches. However, transformation may be performed by infection, transfection, microinjection, electroporation, microprojection, biolistics or particle bombardment, electroporation, silica / carbon fibers, ultrasound mediated, PEG mediated, calcium phosphate co-precipitation, polycation DMSO technique, DEAE dextran procedure, Agro and viral mediated (Caulimoriviruses, Geminiviruses, RNA plant viruses), liposome mediated and the like.

[0113] Transformation protocols as well as protocols for introducing polypeptides or polynucleotide sequences into plants may vary depending on the type of plant or plant cell, i.e., monocot or dicot, targeted for transformation. Methods for transformation are known in the art and include those set forth in US Patent Nos: 8,575,425; 7,692,068; 8,802,934; 7,541,517; each of which is herein incorporated by reference. See, also, Rakoczy-Trojanowska, M. (2002) Cell Mol Biol Lett. 7:849- 858; Jones et al. (2005) Plant Methods 1 :5; Rivera etal. (2012) Physics of Life Reviews 9:308- 345; Bartlett et al. (2008) Plant Methods 4: 1-12; Bates, G.W. (1999) Methods in Molecular Biology 111 :359-366; Binns and Thomashow Annual Reviews in Microbiology 42:575- 606; Christou, P. (1992) The Plant Journal 2:275-281; Christou, P. (1995) Euphytica 85: 13-27; Tzfira c / al. (2004) TRENDS in Genetics 20:375-383; Yao et al. (2006) Journal of Experimental Botany 57:3737-3746; Zupan and Zambryski (1995) Plant Physiology 107: 1041-1047; Jones et al. (2005) Plant Methods 1 :5.

[0114] Transformation may result in stable or transient incorporation of the nucleic acid into the cell. "Stable transformation" is intended to mean that the nucleic acid molecule introduced into a host cell integrates into the genome of the host cell and is capable of being inherited by the progeny thereof. "Transient transformation" is intended to mean that a polynucleotide is introduced into the host cell and does not integrate into the genome of the host cell.

[0115] The cells that have been transformed may be grown into plants in accordance with conventional ways. See, for example, McCormick et al. (1986) Plant Cell Reports 5:81- 84. These plants may then be grown, and either pollinated with the same transformed strain or different strains, and the resulting hybrid having constitutive expression of the desired phenotypic characteristic identified. Two or more generations may be grown to ensure that expression of the desired phenotypic characteristic is stably maintained and inherited and then seeds harvested to ensure expression of the desired phenotypic characteristic has been achieved. In this manner, the present invention provides transformed seed (also referred to as "transgenic seed") having a nucleic acid molecule of the invention, for example, an expression cassette of the invention, stably incorporated into their genome.

[0116] In specific embodiments, the sequences provide herein can be targeted to specific sites within the genome of the plant cell. Such methods include, but are not limited to, meganucleases designed against the plant genomic sequence of interest (D'Halluin et al. 2013 Plant Biotechnol J); CRISPR-Cas9, TALENs, and other technologies for precise editing of genomes (Feng et al. (2013) Cell Research 23: 1229-1232, Podevin et al. (2013) Trends Biotechnology, online publication, Wei et al. (2013) J Gen Genomics, Zhang et al (2013) WO 2013 / 026740); Cre-lox site-specific recombination (Dale et al. 77:649-659; Lyznik et al. (2007) Transgenic Plant J 1 : 1-

[0117] 9; FLP-FRT recombination (Li et al. (2009) Plant Physiol 151 : 1087-1095); Bxbl-mediated integration (Yau et al. (2011) Plant J 701 : 147-166); zinc-finger mediated integration (Wright et al. (2005) Plant J 44:693-705),' Cai et al. (2009) Plant Mol Biol 69:699-709); and homologous recombination (Lieberman-Lazarovich and Levy (2011) Methods Mol Biol 701 : 51-65); Puchta (2002) Plant Mol Biol 4 : 173-182).

[0118] While any plant can be transformed, (monocots and dicots), and used according to the methods of the invention, of special interest are feed grains, including corn, sorghum, barley, and oats as well as legumes, including soybean, beans, peas, alfalfa, peanuts, and the like.

[0119] Once transformed, plants can be selected, propagated, crossed, and used to obtain a plurality of plants that contain the PCV2 polypeptide(s) or polynucleotide(s) of the invention. The plant, plant part, or plant seed expressing the recombinant PCV2 polypeptide(s) can be used or processed for animal feed. Accordingly, the invention encompasses the animal feed with the recombinant polypeptide(s) of the invention. Such animal feed includes but is not limited to feedstuffs, including corn meal soybean meal (SBM), palm kernel meal, copra meal, and sesame meal.

[0120] In another aspect, determining whether the molecules provided protection against PCV includes determining the presence or absence of challenge disease in a test animal by electron microscopy or antibody or assays such as the fluorescent focusing neutralizing (FFN) test or Western blot assay may be used. PCR methods may be used to determine if the protective molecule is present. Northern blotting can detect the presence of diagnostic sequences. In another aspect, an ELISA or similar assay, such as a hemagglutinin inhibition assay are the types of many varied assays that can determine if the protective molecule is effective. In general, antigens solubilized in a buffer are coated on a plastic surface. When serum is added, antibodies can attach to the antigen on the solid phase. The presence or absence of these antibodies can be demonstrated when conjugated to an enzyme. Adding the appropriate substrate will detect the amount of bound conjugate which can be quantified. A common ELISA assay is one which uses biotinylated anti- (protein) polyclonal antibodies and an alkaline phosphatase conjugate. For example, an ELISA used for quantitative determination of protein levels can be an antibody sandwich assay, which utilizes polyclonal rabbit antibodies obtained commercially. The antibody is conjugated to alkaline phosphatases for detection. In another example, an ELISA assay to detect trypsin or trypsinogen uses biotinylated anti-trypsin or anti-trypsinogen polyclonal antibodies and a streptavidin-alkaline phosphatase conjugate. Many such methods are available to one skilled in the art to ascertain if the molecule provides protection and provides protection at the levels administered to the animal.

[0121] The nucleic acid molecule, polypeptide, or fragment thereof, plant or plant part, or animal feed comprising the same, when administered to the animal (e.g., orally), produces a protective response to PCV. A protective response is elicited in the animal. The animal may or may not produce antibodies in response, but the animal will have decreased morbidity or mortality resulting from administration of the vaccine, and as described further herein. The terms "protecting", "protection", "protective immunity" or "protective immune response," as used herein, are intended to mean that the host animal mounts an active immune response (humoral and / or cell-mediated) to the vaccine or polypeptides of the present invention, such that upon exposure to disease challenge, the animal is able to combat the infection. Thus, a protective immune response will decrease the incidence of morbidity and mortality among host animals. The animal will be protected from subsequent exposure to the disease-causing agent. In an embodiment, the animal may be protected by treating the animal which has already been exposed to the disease-causing agent by administration of the vaccine or polypeptide after such exposure. In such an instance there is also shown to be a lessening of morbidity and mortality. Those skilled in the art will understand that in a commercial animal setting, the production of a protective immune response may be assessed by evaluating the effects of vaccination on the herd as a whole, e.g., there may still be morbidity and mortality in a minority of vaccinated animals. Furthermore, protection also includes a lessening in severity of any gross or histopathological changes and / or of symptoms of the disease, as compared to those changes or symptoms typically caused by the isolate in similar animals which are unprotected (i.e., relative to an appropriate control). Thus, a protective immune response will decrease the symptoms of the disease compared to a control pig.

[0122] The following examples are offered by way of illustration and not by way of limitation. EXAMPLES

[0123] Construct Preparation

[0124] Three constructs for expression of the capsid protein from PCV2a and PCV2d in maize were prepared as described in the list below. The sequence of the PCV2a capsid protein was a consensus of multiple GenBank entries, while the sequence of the PDV2d capsid protein was from GenBank NC24897. The sequence was optimized using the algorithm developed by either GenScript or ThermoFisher Scientific for maize codon usage. Previously characterized signal sequences for subcellular targeting to the cell wall / apoplast or endoplasmic reticulum (ER) were added. An additional amino acid (valine) was added to the predicted N-terminus of the protein to increase blend stability once signal sequence is cleaved. The nucleotide sequence of the coding region was outsourced for commercial gene synthesis by GenScript or ThermoFisher Scientific. In all constructs the capsid protein coding region was synthesized for transfer into the maize transformation vector pSBll using an Ncol site overlapping the initiating ATG and a PacI restriction site in the terminator region to add the coding region downstream of the promoter. All constructs incorporated the capsid coding region under control of a promoter derived from the maize globulin-1 gene, pr25, which targets expression to the embryo. Each transcription unit also incorporated the terminator from potato proteinase inhibitor II. Subcellular localization and terminator sequences were as previously described (C. A. Hayden et al., Production of highly concentrated, heat-stable hepatitis B surface antigen in maize. Plant Biotechnology Journal 10, 979-984 (2012)). To produce constructs with two plant transcription units with both PCVa and PCVd capsid proteins, standard restriction digests and ligation with partially overlapping Asci and Mlul restriction sites were used. All constructs also include an expression cassette with a maize codon usage optimized phosphinothricin-acetyl- transf erase gene under control of a cauliflower mosaic virus 35S promoter for herbicide selection using bialaphos. Figure 1 provides a depiction of each of the 9321, 9322, and 9323 constructs.

[0125] Plant transformation

[0126] Maize transformation is carried out as previously described with modifications (Y. Ishida et al., High efficiency transformation of maize (Zea mays L.) mediated by Agrobacterium tumefaciens. Nat Biotechnol 14, 745-750 (1996)). In brief, the constructs are transferred into the LBA4404-pSB- JT Agrobacterium strain containing the vector pSBl by a triparental mating procedure (T. Komari, Y. Hiei, Y. Saito, N. Murai, T. Kumashiro, Vectors carrying two separate T-DNAs for cotransformation of higher plants mediated by Agrobacterium tumefaciens and segregation of transformants free from selection markers. Plant Journal 10, 165-174 (1996)). The cointegrate DNA is then electroporated into Agrobacterium tumefaciens strain EHA101 (E. E. Hood, G. L. Helmer, R. T. Fraley, M. D. Chilton, The hypervirulence of Agrobacterium tumefaciens A281 is encoded in a region of pTiBo542 outside of T-DNA. J Bacteriol 168, 1291-1301 (1986)). Hill maize embryos 1.5 to 3 mm in length are mixed with A. tumefaciens EHA101 with the appropriate vector for transformation (Y. Ishida et cd.. High efficiency transformation of maize (Zea mays L.) mediated by Agrobacterium tumefaciens. Nat Biotechnol 14, 745-750 (1996)). Plants from events selected on bialaphos are grown to maturity in the greenhouse and pollinated with Hill to produce T1 generation seed.

[0127] Many modifications and other embodiments of the inventions set forth herein will come to mind to one skilled in the art to which these inventions pertain having the benefit of the teachings presented in the foregoing descriptions and the associated drawings. Therefore, it is to be understood that the inventions are not to be limited to the specific embodiments disclosed and that modifications and other embodiments are intended to be included within the scope of the appended claims.

[0128] All publications and patent applications mentioned in the specification are indicative of the level of those skilled in the art to which this invention pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

[0129] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be obvious that certain changes and modifications may be practiced within the scope of the appended claims.

[0130] SEQUENCES

[0131] SEQ ID NO: 1 PCV2a capsid nucleotide sequence (GenScript)

[0132] GTCACCTACCCGCGCAGGCGCTACCGGAGGAGGAGGCACAGGCCAAGGTCGCACCTC GGCCAGATCCTGCGGAGGAGGCCGTGGCTGGTGCACCCAAGGCACAGGTACCGCTGG CGCAGGAAGAACGGCATCTTCAACACCAGGCTGTCCCGCACCTTCGGCTACACGGTGA AGGCTACCACGGTCAGGACCCCCAGCTGGGCGGTGGACATGATGCGCTTCAACATCGA CGACTTCGTGCCGCCGGGCGGCGGCACCAACAAGATCTCCATCCCCTTCGAGTACTAC AGGATCCGCAAGGTGAAGGTCGAGTTCTGGCCGTGCTCCCCCATCACGCAGGGCGACA GGGGCGTGGGCAGCACCGCCGTCATCCTCGACGACAACTTCGTGCCCAAGGTCCCGGC CCAGACCTACGACCCCTACGTGAACTACTCCAGCAGGCACACGATCCCGCAGCCCTTC TCCTACCACAGCCGCTACTTCACCCCGAAGCCCGTCCTCGACAGCACGATCGACTACTT CCAGCCCAACAACAAGCGCAACCAGCTCTGGCTGAGGATCCAGACGAGCAGGAACGT GGACCACGTGGGCCTGGGCACGGCGTTCGAGAACAGCATCCACGACCAGGACTACAA

[0133] CATCAGGGTGACCATGTATGTGCAGTTCCGCGAGTTCAACCTCAAGGACCCGCCCCTG

[0134] AAGCCG

[0135] SEQ ID NO: 2 PCV2a capsid nucleotide sequence (ThermoFisher)

[0136] GTTACTTACCCGAGGCGCAGATATAGGCGCAGAAGGCATAGGCCAAGATCCCACCTGG

[0137] GCCAGATTCTGAGGCGCAGGCCTTGGCTTGTGCATCCGAGACATAGATACAGGTGGCG

[0138] CCGCAAGAACGGCATCTTCAACACCAGGCTGAGCAGGACCTTCGGCTACACCGTGAAG

[0139] GCTACCACTGTGAGGACCCCAAGCTGGGCCGTTGACATGATGAGGTTCAACATCGACG

[0140] ACTTCGTGCCGCCAGGCGGTGGCACCAACAAGATCTCTATCCCGTTCGAGTACTACAG

[0141] GATCCGCAAGGTGAAGGTCGAGTTCTGGCCGTGCAGCCCGATCACTCAGGGCGATAGA

[0142] GGCGTTGGCTCTACCGCTGTGATCCTGGACGACAACTTCGTCCCAAAGGTGCCGGCGC

[0143] AGACCTACGATCCGTACGTGAACTACAGCAGCAGGCACACTATCCCGCAGCCGTTCTC

[0144] TTACCACAGCCGCTACTTCACCCCGAAGCCAGTGCTGGACAGCACCATCGACTACTTC

[0145] CAGCCGAACAACAAGAGGAACCAGCTGTGGCTGAGGATCCAGACCTCTAGGAACGTG

[0146] GACCATGTTGGCCTGGGCACCGCTTTCGAGAACAGCATCCACGACCAGGACTACAACA

[0147] TCAGGGTGACCATGTACGTGCAGTTCCGCGAGTTCAACCTGAAGGACCCGCCGCTGAA

[0148] GCCA

[0149] SEQ ID NO: 3 PCV2a capsid protein

[0150] VTYPRRRYRRRRHRPRSHLGQILRRRPWLVHPRHRYRWRRKNGIFNTRLSRTFGYTVKAT

[0151] TVRTPSWAVDMMRFNIDDFVPPGGGTNKISIPFEYYRIRKVKVEFWPCSPITQGDRGVGST

[0152] AVILDDNFVPKVPAQTYDPYVNYSSRHTIPQPFSYHSRYFTPKPVLDSTIDYFQPNNKRNQL

[0153] WLRIQTSRNVDHVGLGTAFENSIHDQDYNIRVTMYVQFREFNLKDPPLKP

[0154] SEQ ID NO: 4 PCV2d capsid nucleotide sequence (GenScript)

[0155] GTCACCTACCCGCGCAGGCGCTTCAGGAGGAGGAGGCACAGGCCAAGGTCGCACCTG

[0156] GGCCAGATCCTCCGGAGGAGGCCGTGGCTGGTGCACCCAAGGCACAGGTACCGCTGG

[0157] CGCAGGAAGAACGGCATCTTCAACACCAGGCTCTCCCGCACCATCGGCTACACGGTGA

[0158] AGAAGACCACGGTCAGGACGCCGAGCTGGAACGTGGACATGATGCGCTTCAACATCA

[0159] ACGACTTCCTCCCGCCGGGCGGCGGCTCCAACCCGCTCACCGTCCCCTTCGAGTACTAC

[0160] AGGATCCGCAAGGTGAAGGTCGAGTTCTGGCCGTGCTCCCCCATCACGCAGGGCGACA

[0161] GGGGCGTGGGCAGCACCGCCGTCATCCTGGACGACAACTTCGTGACCAAGGCCAACGC

[0162] GCTCACGTACGACCCCTACGTGAACTACTCCAGCAGGCACACCATCACGCAGCCCTTC TCCTACCACAGCCGCTACTTCACGCCCAAGCCGGTGCTGGACGGCACCATCGACTACT

[0163] TCCAGCCGAACAACAAGAGGAACCAGCTCTGGCTCAGGCTCCAGACGACCGGCAACG

[0164] TGGACCACGTGGGCCTCGGCACCGCGTTCGAGAACAGCATCTACGACCAGGACTACAA

[0165] CATCAGGATCACGATGTATGTGCAGTTCCGCGAGTTCAACCTGAAGGACCCGCCCCTC AACCCCAAG

[0166] SEQ ID NO : 5 PCV2d capsid nucleotide sequence (ThermoFisher)

[0167] GTTACTTACCCGAGGCGCAGATTTCGCAGGCGTAGACATAGGCCAAGAAGCCACCTGG

[0168] GCCAGATCCTGAGGCGCAGGCCTTGGCTTGTTCACCCAAGGCATAGATACAGGTGGCG

[0169] CCGCAAGAACGGCATCTTCAACACCAGGCTGAGCAGGACCATCGGCTACACCGTGAA

[0170] GAAAACCACCGTGAGGACCCCGAGCTGGAACGTGGACATGATGAGGTTCAATATCAA

[0171] CGACTTCCTGCCGCCAGGCGGCGGTTCTAACCCACTGACTGTTCCGTTCGAGTACTACA

[0172] GGATCCGCAAGGTGAAGGTCGAGTTCTGGCCGTGCAGCCCGATCACTCAGGGCGATAG

[0173] AGGCGTTGGCTCTACCGCTGTGATCCTGGACGACAACTTCGTGACCAAGGCCAACGCG

[0174] CTGACCTACGATCCGTACGTGAACTACAGCAGCAGGCACACCATCACGCAGCCGTTCT

[0175] CTTACCACAGCCGCTACTTCACCCCGAAGCCAGTGCTGGATGGCACCATCGACTACTTC

[0176] CAGCCGAACAACAAGAGGAACCAGCTGTGGCTGAGGCTGCAGACCACCGGCAACGTT

[0177] GACCATGTTGGCCTGGGCACCGCTTTCGAGAACAGCATCTACGACCAGGACTACAACA

[0178] TCAGGATCACCATGTACGTGCAGTTCCGCGAGTTCAACCTGAAGGACCCGCCGCTGAA CCCGAAG

[0179] SEQ ID NO: 6 PCV2d capsid protein

[0180] VTYPRRRFRRRRHRPRSHLGQILRRRPWLVHPRHRYRWRRKNGIFNTRLSRTIGYTVKKTT

[0181] VRTPSWNVDMMRFNINDFLPPGGGSNPLTVPFEYYRIRKVKVEFWPCSPITQGDRGVGSTA

[0182] VILDDNFVTKANALTYDPYVNYSSRHTITQPFSYHSRYFTPKPVLDGTIDYFQPNNKRNQL

[0183] WLRLQTTGNVDHVGLGTAFENSIYDQDYNIRITMYVQFREFNLKDPPLNPK

[0184] SEQ ID NO: 7 PCV2a BAAS S: Cap nucleotide sequence (GenScript)

[0185] ATGGCCAACAAGCACCTCTCCCTGAGCCTCTTCCTGGTGCTGCTCGGCCTCTCGGCCAG

[0186] CCTGGCCTCCGGCGTCACCTACCCGCGCAGGCGCTACCGGAGGAGGAGGCACAGGCC

[0187] AAGGTCGCACCTCGGCCAGATCCTGCGGAGGAGGCCGTGGCTGGTGCACCCAAGGCA

[0188] CAGGTACCGCTGGCGCAGGAAGAACGGCATCTTCAACACCAGGCTGTCCCGCACCTTC

[0189] GGCTACACGGTGAAGGCTACCACGGTCAGGACCCCCAGCTGGGCGGTGGACATGATG

[0190] CGCTTCAACATCGACGACTTCGTGCCGCCGGGCGGCGGCACCAACAAGATCTCCATCC CCTTCGAGTACTACAGGATCCGCAAGGTGAAGGTCGAGTTCTGGCCGTGCTCCCCCAT

[0191] CACGCAGGGCGACAGGGGCGTGGGCAGCACCGCCGTCATCCTCGACGACAACTTCGTG

[0192] CCCAAGGTCCCGGCCCAGACCTACGACCCCTACGTGAACTACTCCAGCAGGCACACGA

[0193] TCCCGCAGCCCTTCTCCTACCACAGCCGCTACTTCACCCCGAAGCCCGTCCTCGACAGC

[0194] ACGATCGACTACTTCCAGCCCAACAACAAGCGCAACCAGCTCTGGCTGAGGATCCAGA

[0195] CGAGCAGGAACGTGGACCACGTGGGCCTGGGCACGGCGTTCGAGAACAGCATCCACG

[0196] ACCAGGACTACAACATCAGGGTGACCATGTATGTGCAGTTCCGCGAGTTCAACCTCAA

[0197] GGACCCGCCCCTGAAGCCG

[0198] SEQ ID NO: 8 PCV2a BAAS S: Cap protein

[0199] MANKHLSLSLFLVLLGLSASLASGVTYPRRRYRRRRHRPRSHLGQILRRRPWLVHPRHRYR

[0200] WRRKNGIFNTRLSRTFGYTVKATTVRTPSWAVDMMRFNIDDFVPPGGGTNKISIPFEYYRIR

[0201] KVKVEFWPCSPITQGDRGVGSTAVILDDNFVPKVPAQTYDPYVNYSSRHTIPQPFSYHSRYF

[0202] TPKPVLDSTIDYFQPNNKRNQLWLRIQTSRNVDHVGLGTAFENSIHDQDYNIRVTMYVQFR

[0203] EFNLKDPPLKP

[0204] SEQ ID NO : 9 PCV2a BAASS:Cap:KDEL nucleotide sequence (GenScript)

[0205] ATGGCCAACAAGCACCTCTCCCTGAGCCTCTTCCTGGTGCTGCTCGGCCTCTCGGCCAG

[0206] CCTGGCCTCCGGCGTCACCTACCCGCGCAGGCGCTACCGGAGGAGGAGGCACAGGCCA

[0207] AGGTCGCACCTCGGCCAGATCCTGCGGAGGAGGCCGTGGCTGGTGCACCCAAGGCACA

[0208] GGTACCGCTGGCGCAGGAAGAACGGCATCTTCAACACCAGGCTGTCCCGCACCTTCGG

[0209] CTACACGGTGAAGGCTACCACGGTCAGGACCCCCAGCTGGGCGGTGGACATGATGCGC

[0210] TTCAACATCGACGACTTCGTGCCGCCGGGCGGCGGCACCAACAAGATCTCCATCCCCTT

[0211] CGAGTACTACAGGATCCGCAAGGTGAAGGTCGAGTTCTGGCCGTGCTCCCCCATCACGC

[0212] AGGGCGACAGGGGCGTGGGCAGCACCGCCGTCATCCTCGACGACAACTTCGTGCCCAA

[0213] GGTCCCGGCCCAGACCTACGACCCCTACGTGAACTACTCCAGCAGGCACACGATCCCG

[0214] CAGCCCTTCTCCTACCACAGCCGCTACTTCACCCCGAAGCCCGTCCTCGACAGCACGAT

[0215] CGACTACTTCCAGCCCAACAACAAGCGCAACCAGCTCTGGCTGAGGATCCAGACGAGC

[0216] AGGAACGTGGACCACGTGGGCCTGGGCACGGCGTTCGAGAACAGCATCCACGACCAG

[0217] GACTACAACATCAGGGTGACCATGTATGTGCAGTTCCGCGAGTTCAACCTCAAGGACCC

[0218] GCCCCTGAAGCCGAAGGACGAGCTG

[0219] SEQ ID NO: 10 PCV2a BAAS S: Cap :KDEL nucleotide sequence (ThermoFisher) ATGGCGAACAAGCACCTGAGCCTGAGCCTCTTCCTGGTGCTGCTTGGCCTGTCTGCCTC

[0220] TCTGGCTTCTGGTGTTACTTACCCGAGGCGCAGATATAGGCGCAGAAGGCATAGGCCAA

[0221] GATCCCACCTGGGCCAGATTCTGAGGCGCAGGCCTTGGCTTGTGCATCCGAGACATAGA

[0222] TACAGGTGGCGCCGCAAGAACGGCATCTTCAACACCAGGCTGAGCAGGACCTTCGGCT

[0223] ACACCGTGAAGGCTACCACTGTGAGGACCCCAAGCTGGGCCGTTGACATGATGAGGTT

[0224] CAACATCGACGACTTCGTGCCGCCAGGCGGTGGCACCAACAAGATCTCTATCCCGTTCG

[0225] AGTACTACAGGATCCGCAAGGTGAAGGTCGAGTTCTGGCCGTGCAGCCCGATCACTCA

[0226] GGGCGATAGAGGCGTTGGCTCTACCGCTGTGATCCTGGACGACAACTTCGTCCCAAAGG

[0227] TGCCGGCGCAGACCTACGATCCGTACGTGAACTACAGCAGCAGGCACACTATCCCGCAG

[0228] CCGTTCTCTTACCACAGCCGCTACTTCACCCCGAAGCCAGTGCTGGACAGCACCATCGA

[0229] CTACTTCCAGCCGAACAACAAGAGGAACCAGCTGTGGCTGAGGATCCAGACCTCTAGG

[0230] AACGTGGACCATGTTGGCCTGGGCACCGCTTTCGAGAACAGCATCCACGACCAGGACT

[0231] ACAACATCAGGGTGACCATGTACGTGCAGTTCCGCGAGTTCAACCTGAAGGACCCGCC

[0232] GCTGAAGCCAAAGGACGAGCTG

[0233] SEQ ID NO: 11 PCV2a BAAS S: Cap :KDEL protein

[0234] MANKHLSLSLFLVLLGLSASLASGVTYPRRRYRRRRHRPRSHLGQILRRRPWLVHPRHRYR

[0235] WRRKNGIFNTRLSRTFGYTVKATTVRTPSWAVDMMRFNIDDFVPPGGGTNKISIPFEYYRIR

[0236] KVKVEFWPCSPITQGDRGVGSTAVILDDNFVPKVPAQTYDPYVNYSSRHTIPQPFSYHSRYF

[0237] TPKPVLDSTIDYFQPNNKRNQLWLRIQTSRNVDHVGLGTAFENSIHDQDYNIRVTMYVQFR

[0238] EFNLKDPPLKPKDEL

[0239] SEQ ID NO: 12 PCV2d BAAS S: Cap nucleotide sequence (GenScript)

[0240] ATGGCCAACAAGCACCTCTCCCTGAGCCTCTTCCTGGTGCTGCTCGGCCTGTCGGCCAG

[0241] CCTGGCCTCCGGCGTCACCTACCCGCGCAGGCGCTTCAGGAGGAGGAGGCACAGGCCA

[0242] AGGTCGCACCTGGGCCAGATCCTCCGGAGGAGGCCGTGGCTGGTGCACCCAAGGCACA

[0243] GGTACCGCTGGCGCAGGAAGAACGGCATCTTCAACACCAGGCTCTCCCGCACCATCGG

[0244] CTACACGGTGAAGAAGACCACGGTCAGGACGCCGAGCTGGAACGTGGACATGATGCGC

[0245] TTCAACATCAACGACTTCCTCCCGCCGGGCGGCGGCTCCAACCCGCTCACCGTCCCCTT

[0246] CGAGTACTACAGGATCCGCAAGGTGAAGGTCGAGTTCTGGCCGTGCTCCCCCATCACGC

[0247] AGGGCGACAGGGGCGTGGGCAGCACCGCCGTCATCCTGGACGACAACTTCGTGACCAA

[0248] GGCCAACGCGCTCACGTACGACCCCTACGTGAACTACTCCAGCAGGCACACCATCACG

[0249] CAGCCCTTCTCCTACCACAGCCGCTACTTCACGCCCAAGCCGGTGCTGGACGGCACCAT

[0250] CGACTACTTCCAGCCGAACAACAAGAGGAACCAGCTCTGGCTCAGGCTCCAGACGACC GGCAACGTGGACCACGTGGGCCTCGGCACCGCGTTCGAGAACAGCATCTACGACCAGG

[0251] ACTACAACATCAGGATCACGATGTATGTGCAGTTCCGCGAGTTCAACCTGAAGGACCCG CCCCTCAACCCCAAG

[0252] SEQ ID NO: 13 PCV2d BAAS S: Cap protein

[0253] MANKHLSLSLFLVLLGLSASLASGVTYPRRRFRRRRHRPRSHLGQILRRRPWLVHPRHRYR

[0254] WRRKNGIFNTRLSRTIGYTVKKTTVRTPSWNVDMMRFNINDFLPPGGGSNPLTVPFEYYRI

[0255] RKVKVEFWPCSPITQGDRGVGSTAVILDDNFVTKANALTYDPYVNYSSRHTITQPFSYHSR

[0256] YFTPKPVLDGTIDYFQPNNKRNQLWLRLQTTGNVDHVGLGTAFENSIYDQDYNIRITMYV QFREFNLKDPPLNPK

[0257] SEQ ID NO: 14 PCV2d BAAS S: Cap :KDEL nucleotide sequence (GenScript)

[0258] ATGGCCAACAAGCACCTCTCCCTGAGCCTCTTCCTGGTGCTGCTCGGCCTGTCGGCCAG

[0259] CCTGGCCTCCGGCGTCACCTACCCGCGCAGGCGCTTCAGGAGGAGGAGGCACAGGCCA

[0260] AGGTCGCACCTGGGCCAGATCCTCCGGAGGAGGCCGTGGCTGGTGCACCCAAGGCACA

[0261] GGTACCGCTGGCGCAGGAAGAACGGCATCTTCAACACCAGGCTCTCCCGCACCATCGG

[0262] CTACACGGTGAAGAAGACCACGGTCAGGACGCCGAGCTGGAACGTGGACATGATGCGC

[0263] TTCAACATCAACGACTTCCTCCCGCCGGGCGGCGGCTCCAACCCGCTCACCGTCCCCTT

[0264] CGAGTACTACAGGATCCGCAAGGTGAAGGTCGAGTTCTGGCCGTGCTCCCCCATCACGC

[0265] AGGGCGACAGGGGCGTGGGCAGCACCGCCGTCATCCTGGACGACAACTTCGTGACCAA

[0266] GGCCAACGCGCTCACGTACGACCCCTACGTGAACTACTCCAGCAGGCACACCATCACG

[0267] CAGCCCTTCTCCTACCACAGCCGCTACTTCACGCCCAAGCCGGTGCTGGACGGCACCAT

[0268] CGACTACTTCCAGCCGAACAACAAGAGGAACCAGCTCTGGCTCAGGCTCCAGACGACC

[0269] GGCAACGTGGACCACGTGGGCCTCGGCACCGCGTTCGAGAACAGCATCTACGACCAGG

[0270] ACTACAACATCAGGATCACGATGTATGTGCAGTTCCGCGAGTTCAACCTGAAGGACCCG CCCCTCAACCCCAAGAAGGACGAGCTG

[0271] SEQ ID NO: 15 PCV2d BAAS S: Cap :KDEL nucleotide sequence (ThermoFisher)

[0272] ATGGCGAACAAGCACCTGAGCCTGAGCCTCTTCCTGGTGCTGCTTGGCCTGTCTGCCTC

[0273] TCTGGCTTCTGGTGTTACTTACCCGAGGCGCAGATTTCGCAGGCGTAGACATAGGCCAA

[0274] GAAGCCACCTGGGCCAGATCCTGAGGCGCAGGCCTTGGCTTGTTCACCCAAGGCATAG

[0275] ATACAGGTGGCGCCGCAAGAACGGCATCTTCAACACCAGGCTGAGCAGGACCATCGGC

[0276] TACACCGTGAAGAAAACCACCGTGAGGACCCCGAGCTGGAACGTGGACATGATGAGGT

[0277] TCAATATCAACGACTTCCTGCCGCCAGGCGGCGGTTCTAACCCACTGACTGTTCCGTTC GAGTACTACAGGATCCGCAAGGTGAAGGTCGAGTTCTGGCCGTGCAGCCCGATCACTC

[0278] AGGGCGATAGAGGCGTTGGCTCTACCGCTGTGATCCTGGACGACAACTTCGTGACCAA

[0279] GGCCAACGCGCTGACCTACGATCCGTACGTGAACTACAGCAGCAGGCACACCATCACG

[0280] CAGCCGTTCTCTTACCACAGCCGCTACTTCACCCCGAAGCCAGTGCTGGATGGCACCAT

[0281] CGACTACTTCCAGCCGAACAACAAGAGGAACCAGCTGTGGCTGAGGCTGCAGACCACC

[0282] GGCAACGTTGACCATGTTGGCCTGGGCACCGCTTTCGAGAACAGCATCTACGACCAGG

[0283] ACTACAACATCAGGATCACCATGTACGTGCAGTTCCGCGAGTTCAACCTGAAGGACCCG

[0284] CCGCTGAACCCGAAGAAAGACGAGCTG

[0285] SEQ ID NO: 16 PCV2d BAAS S: Cap :KDEL protein

[0286] MANKHLSLSLFLVLLGLSASLASGVTYPRRRFRRRRHRPRSHLGQILRRRPWLVHPRHRYR

[0287] WRRKNGIFNTRLSRTIGYTVKKTTVRTPSWNVDMMRFNINDFLPPGGGSNPLTVPFEYYRI

[0288] RKVKVEFWPCSPITQGDRGVGSTAVILDDNFVTKANALTYDPYVNYSSRHTITQPFSYHSR

[0289] YFTPKPVLDGTIDYFQPNNKRNQLWLRLQTTGNVDHVGLGTAFENSIYDQDYNIRITMYV QFREFNLKDPPLNPKKDEL

[0290] SEQ ID NO: 17 BAASS

[0291] MANKHLSLSLFLVLLGLSASLASG

[0292] SEQ ID NO: 18 KDEL

[0293] KDEL

[0294] SEQ ID NO: 19 pr25 promoter

[0295] CGGTATGAATTTGGAAACAAATTCAGTACTTTTAAAAAAATTTGTTGTAGGGAGCAAA

[0296] TAATACATAAAATAATTTATGCATTATTTTATTTTTTATTTGTAATAATATGCTTGAAAC

[0297] GATAATTCAGTATGCATGTTGTGCCAGTGTACTACACGGGCGGGGGGAGGGGATTGAG

[0298] TGGGCCAGCGCGGTGCGTAGGGTAGATGGGCTGAAATTGATAACTCAAGTCCGACTAG

[0299] GTTCTCTTTTTATTTCCCTTCCTTTTCTATTTTCCTTTCTTTTAATTTTCATGCTTTCAAAC

[0300] TAAATTCAAATTCGAGTTTTGAATTTCAGCTTCTAAATTGTACACTAAAATTATATGAT

[0301] AAGGTAACCCCTACTATTACTTTTAATTTTTTTATTCTACCCCATATTGTTTACTTAGGG

[0302] GAGAATAATTGACTTAATCACATTCTTCCTAGGTTTCAATTCTCAATCTTTCAAATCCA

[0303] CATTTTTAGATTTCTATTTTGAATTTAAATACCAGTTTGGATTTAGAGTTCAATTTCAAA

[0304] ATACACAACCAAAATACCAGCATGAATGCAAATATATTTTATGTTTATGTATTTACTTT

[0305] TCTTTTATACTTTGCTCAAAATAGTTATTTTCATGTATGAAACTCAATAAGCAAGGAAC TCACGTTATTATATAACCTAATAGGAATAATTTAGGTAACATAATTTATCATCCTCTTG

[0306] ATTTAAAAGAGATATGCCTCCAGAATAAGACACATACTAAAAATAACTCTAATATTGA

[0307] ATAACTAAAGTCGTACAAATCTCTACTATTATTCCTATAAAATAATAAAGAACTAGCT

[0308] ACAACTTCTTTAAGGCATTATTCAGGGTTTACAGCTTGAGAGGCATGAACCCATCCTGT

[0309] ATACTCCTGGACTTGGAAGACAAAATGTCAACCAAAGTGAAAGGTTTTCTTATGGTTG

[0310] CTGCTAAGAGATAGATTGAACACTAGATCTCTCCTAAGACGTCAGGGCATGCGTTTAG

[0311] ACTCCTACACATGCGAAAACTGCATCTTACAGTTGGAAGAAACTATATCTCACCACTTC

[0312] CTGCGGTGTAACTTTGCCCAAAGATGTTGGCTCACTGTTGGAATCACTCCGCCCCGAAC

[0313] TTTGGATCTAACGCTTGCAGTGCTACATATTAGAGCAAGACTAACAATGCCGTGGAGA

[0314] ATGGAAGGTATTATAACCATGTCATGGTGCATATGGAAATGTCGAAATAACTGGATAT

[0315] TCGAAAACATACCGCCAACGGTGGCGGCCTGCAAGGAAATGTTCAAGACTGAAATGA

[0316] ACTACATCTGCTACCAAGTTAAGCTCGAGACAGGAGCTAAAAGTAGAAACTGGATACA

[0317] ACACTTTGTAACATAGTGACACTCCCCTTTTCCTTTCTTTTACCTTAGAACTATACATAC

[0318] AATCCACATTCAATAAAAATTTGTAGGTACGCCATACACACTACCGGAATCCGGCTCT

[0319] TTGCCGAGTGTGAGGCGCTTTGTCGAGTGCTTTTTGTCCAGCACTCGGCAAAAAAGTCT

[0320] TTGCCATGTGCCGCACTCGGCAAAGTCCTGCTCTCGGTAACGACCGCGTTTACCGAGA

[0321] GCAGGACTCTCGACACAGAAATACACTCGACAAAGAAATCTTTGCCGAGAGCCAAAC

[0322] ACTCGGCGAACGGCAGCGCTCGGCAAAGGGTCGTCAGCCGCCGTCTAAAGCTGACGGT

[0323] CGTTATCTTTGTCGAGTGCCCCCTCGTCCGACACTCAGTAGAGCAAGCTTGCCGAGTGC

[0324] CATCCTTGGACACTCGATAAAGTATATTTTATTTTTTTTTATTTTGCCAACCAAACTTTT

[0325] TGTGGTATGTTCCTACACTATGTAGATCTACATGTACCATTTTGGCACAATTACAAAAA

[0326] TGTTTTCTATAACTATTAGATTTAGTTCGTTTATTTGAATTTCTTCGGAAAATTCACATA

[0327] TGAACTGCAAGTCACTCGAAACATGAAAAACCGTGCATGCAAAATAAATGATATGCAT

[0328] GTTATCTAGCACAAGTTACGACCGAATTCAGAAGCAGACCAGAATCTTCAAGCACCAT

[0329] GCTCACTAAACATGACCGTGAACTTGTTATCCAGTTGTTTAAAAATTGTATAAAACACA

[0330] AATAAAGTCAGAAATTAATGAAACTTGTCCACATGTCATGATATCATATATAGAGGTT

[0331] GTGATAAAAATTTGATAATGTTTCGGTAAAGTTGTGACGTACTATGTGTAGAAACCTA

[0332] AGTGACCTACACATAAAATCATAGAGTTTCAATGTAGTTCACTCGACAAAGACTTTGT

[0333] CAAGTGTCCGATAAAAAGTATTCAGCAAAGAAGCCGTTGTCGATTTACTGTTCGTCGA

[0334] GATCTCTTTGCCGAGTGTCACACTAGGCAAAGTCTTTACGGAGTGTTTTTCAGGCTTTG

[0335] ACACTCGGCAAAGCGCTCGATTCCAGTAGTGACAGTAATTTGCATCAAAAATAGCCGA

[0336] GAGATTTAAAATGAGTCAACTAATAGACCAACTAATTATTAGCTATTAGTCGTTAGCTT

[0337] CTTTAATCTAAGCTAAAACCAACTAATAGCTTATTTGTTGAATTACAATTAGCTCAACG

[0338] GAATTCTCTGTTTTTTCTATAAAAAAAAGGGAAACTGCCCCTCATTTACAGCAAACTGT CCGCTGCCTGTCGTCCAGATACAATGAACGTACCTAGTAGGAACTCTTTTACACGCTCG

[0339] GTCGCTCGCCGCGGATCGGAGTCCCAGGAACACGACACCACTGTGGAACACGACAAA

[0340] GTCTGCTCAGAGGCGGCCACACCCTGGCGTGCACCGAGCCGGAGCCCGGATAAGCAC

[0341] GGTAAGGAGAGTACGGCGGGACGTGGCGACCCGTGTGTCTGCTGCCACGCAGCCTTCC TCCACGTAGCCGCGCGGCCGCGCCACGTACCAGGGCCCGGCGCTGGTATAAATGCGCG

[0342] CCACCTCCGCTTTAGTTCTGCATACAGCCAACCCAACACACACCCGAGCATATCACAG TGACAGACACTACAC

[0343] SEQ ID NO: 20 pinll terminator CCTAGACTTGTCCATCTTCTGGATTGGCCAACTTAATTAATGTATGAAATAAAAGGATG

[0344] CACACATAGTGACATGCTAATCACTATAATGTGGGCATCAAAGTTGTGTGTTATGTGTA

[0345] ATTACTAGTTATCTGAATAAAAGAGAAAGAGATCATCCATATTTCTTATCCTAAATGA

[0346] ATGTCACGTGTCTTTATAATTCTTTGATGAACCAGATGCATTTCATTAACCAAATCCAT

[0347] ATACATATAAATATTAATCATATATAATTAATATCAATTGGGTTAGCAAAACAAATCT AGTCTAGGTGTGTTTTGCGAAT

Claims

WHAT IS CLAIMED IS:

1. A method for eliciting an immune response in an animal, said method comprising, orally administering to said animal a composition comprising a plant or plant product, wherein said plant or plant product comprises at least one PCV2 antigen.

2. The method of claim 1, wherein said animal is a pig.

3. The method of claim 1, wherein said antigen comprises a capsid protein.

4. The method of claim 1, wherein said composition comprises two PCV2 antigens.

5. The method of claim 4, wherein said PCV2 antigen is PCV2a and PCV2d.

6. The method of claim 5, wherein said plant or plant product has been transformed with at least one expression cassette comprising a nucleotide sequence encoding a PCV2a and a nucleotide sequence encoding a PCV2d antigen.

7. The method of claim 6, wherein said nucleotide sequence encoding a PCV2 antigen has been optimized for expression in said plant.

8. The method of claim 6, wherein said nucleotide sequence encoding a PCV2 antigen is operably linked to a seed-preferred promoter.

9. The method of claim 6, wherein said nucleotide sequence encoding a PCV2 antigen is operably linked to an embryo-preferred promoter.

10. The method of claim 9, wherein said embryo-preferred promoter is a globulin 1 promoter or a pr25 promoter.

11. The method of claim 6, wherein said nucleotide sequence encoding a PCV2 antigen is operably linked to a nucleotide sequence which encodes a peptide that targets the PCV2 antigen to a subcellular compartment.

12. The method of claim 6, wherein said PCV2a antigen comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 3.

13. The method of claim 6, wherein said PCV2d antigen comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 6.

14. The method of claim 1, wherein said plant is maize.

15. The method of claim 1, wherein said method comprises at least two administrations.

16. A construct comprising at least one expression cassette comprising a nucleotide sequence encoding a PCV2 antigen, wherein said PCV2 antigen is PCV2a or PCV2d.

17. The construct of claim 16, wherein said nucleotide sequence encoding a PCV2 antigen has been optimized for expression in a plant.

18. The construct of claim 16, wherein said plant is maize.

19. The construct of claim 16, wherein said nucleotide sequence encoding a PCV2 antigen is operably linked to a promoter active in a plant cell.

20. The construct of claim 19, wherein said promoter is a seed-preferred promoter or an embryo-preferred promoter.

21. The construct of claim 16, wherein said nucleotide sequence encoding a PCV2 antigen is operably linked to a nucleotide sequence which encodes a peptide that targets the PCV2 antigen to a subcellular compartment.

22. The construct of claim 16, wherein said PCV2a antigen comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 3.

23. The construct of claim 16, wherein said PCV2d antigen comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 6.

24. The construct of claim 16, wherein said construct comprises two expression cassettes,wherein a first expression cassette comprises operably linked nucleotide sequences comprising: a) a first seed preferred promoter; b) a first nucleotide sequence encoding a barley alpha amylase signal sequence (BAASS) peptide; c) a nucleotide sequence encoding a PCV2a antigen; and d) a first terminator sequence; and wherein a second cassette comprises operably linked nucleotide sequences comprising: a) a second seed preferred promoter; b) a second nucleotide sequence encoding a BAASS peptide; c) a nucleotide sequence encoding a PCV2d antigen; and d) a second terminator sequence.

25. The construct of claim 24, wherein said nucleotide sequence encoding said PCV2a antigen comprises the sequence set forth in SEQ ID NO: 1 or 2 and said nucleotide sequence encoding said PCVd antigen comprises the sequence set forth in SEQ ID NO: 4 or 5 and wherein said nucleotide sequences are each operably linked to a nucleotide sequence encoding a KDEL sequence.

26. A plant cell comprising the construct of claim 16.

27. A plant cell comprising at least one nucleic acid molecule comprising a nucleotide sequence encoding a PCV2 antigen.

28. The plant cell of claim 27, wherein said nucleotide sequence encoding a PCV2 antigen has been optimized for expression in said plant cell.

29. The plant cell of claim 27, wherein said nucleotide sequence encoding a PCV2 antigen is operably linked to a nucleotide sequence which encodes a peptide that targets the PCV2 antigen to a subcellular compartment.

30. The plant cell of claim 29, wherein said PCV2 antigen comprises a capside protein.

31. The plant cell of claim 27, wherein said PCV2 antigen is PCV2a, PCV2d, or PCV2a and PCV2d.

32. The plant cell of claim 31, wherein said PCV2a antigen comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 3.

33. The plant cell of claim 31, wherein said PCV2d antigen comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 6.

34. The plant cell of any one of claims 26-33, wherein said plant cell is a maize cell.

35. A plant comprising the plant cell of claim 34.

36. Seed of the plant of claim 35.

37. Animal feed that comprises the plant of claim 35.

38. Animal feed that comprises the seed of claim 36.

Images