Expression of Eimeria sequences in plants and plant-produced vaccines therefor.

Abstract

Vaccines and methods are provided that express Eimeria polypeptides, which when administered to an animal result in a protective response against Eimeria. The vaccine provides expression of Eimeria vaccine protein 3-1e, Gam82, and / or EF-1a polypeptides in a plant or plant part linked to a promoter that directs expression preferentially to the embryonic tissue of the plant or plant part. Further embodiments provide that the polypeptides may be targeted to the apoplast / cell wall or endoplasmic reticulum. Increased expression levels are obtained in the plant or plant part. The plant or plant material in one embodiment may be administered orally.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority under 35 U.S.C. § 119 to U.S. Provisional Patent Application No. 63 / 266,112, filed December 29, 2021, which is incorporated herein by reference in its entirety, including but not limited to the specification, claims, and abstract, and any figures, tables, appendices, or drawings thereof.

[0002] STATEMENT REGARDING FEDERALLY FUNDED RESEARCH This invention was made with Government support under SBIR Grant #2019-33610-29782, Production of a Candidate Vaccine for Avian Coccidiosis in maize, awarded by the USDA-National Institute of Food and Agriculture. The Government has certain rights in the invention.

[0003] Reference to Electronically Submitted Sequence Listing This application contains a Sequence Listing that has been submitted electronically in XML format, which is incorporated herein by reference in its entirety. Said XML copy, created on December 13, 2022, is named "P13608US01_SequenceListing.xml" and is 18,077 bytes in size. [Background technology]

[0004] background Coccidiosis is an infectious disease of poultry, pigs and cattle caused by Apicomplexan parasitic protozoa and represents a problem worldwide. In particular, the health and welfare of poultry is threatened by several pathogens and parasitic protozoa of the genus Eimeria. Infections with Eimeria, which invade and replicate within intestinal epithelial cells, can impair chicken welfare and necessitate expensive treatments. The costs of these treatments, combined with the losses caused by infection, are estimated to cost the poultry industry worldwide more than $2 billion annually.1 Eimeria infection has also been demonstrated to exacerbate the outcome of exposure to other pathogens, such as Clostridium perfringens, which may concomitantly cause necrotizing enterocolitis. 2 .

[0005] Seven species of Eimeria are recognized to infect chickens, which differ in fecundity, virulence, and location of replication in the intestine. 3 In addition, there are three circulating strains that cause the majority of infections: Eimeria acervulina, Eimeria tenella, and Eimeria maxima. Immunization of chickens with live Eimeria oocysts has been effective and has been the basis of live oral coccidiosis vaccines for almost 70 years, but to be fully protected, chickens must be vaccinated with oocysts from each Eimeria species. 4 It is necessary for a live vaccine to contain controlled doses of oocysts for all pathogenic species of Eimeria, and in some cases multiple Eimeria maxima strains make vaccine production logistically cumbersome, as all vaccine strains must be grown separately in chickens under strict specific pathogen-free conditions. Another important consideration is the need for fecal-oral recycling of the vaccine parasite to generate a sufficient level of protective immunity to protect chickens against pathogenic challenge with most Eimeria species. 5 Therefore, a recombinant vaccine that is protective against multiple Eimeria species is highly desirable and should contain multiple antigens derived from different parasite species and life cycle stages.

[0006] Control of Eimeria in commercial chicken production currently relies on a combination of anticoccidiopathies (chemicals and antibiotics) and vaccination using preparations of live virulent or attenuated Eimeria species. Mass administration of anticoccidiopathies has been used as a control method for many years, but drug resistance is now widespread, no new chemical control measures have been developed, and there is growing public / legal pressure to reduce antibiotic use in livestock. Furthermore, existing vaccines are expensive and can cause harmful stress to animals. Addressing this need, plant-produced vaccines can provide large quantities of vaccine protein while minimizing production costs and equipment use. For example, corn-produced vaccines are cost-effective, heat stable, and suitable for direct feeding to a variety of animals.

[0007] Therefore, we sought to produce, optimize and characterize corn lines expressing high levels of 3-1e, EF-1alpha and Gam82 to induce immune responses against one or more of Eimeria acervulina, Eimeria tenella and Eimeria maxima. A secondary objective was to demonstrate that bioencapsulated Eimeria antigens in corn kernels provide an effective vaccine when delivered orally. The commercial goal of this invention is a vaccine that provides greater than 85% protection against challenge with Eimeria species in chickens. Successful completion of this work will lead to the future development of a low-cost, heat-stable, effective oral vaccine for avian coccidiosis that should reduce reliance on antibiotics and the economic losses associated with the disease. Summary of the Invention [Means for solving the problem]

[0008] A brief summary Eimeria vaccines produced from plants are provided. Vaccines and methods for generating a protective response against Eimeria in animals are provided. A protective response is observed when the plant or plant product is orally administered to an animal. An embodiment provides for orally administering a plant or plant product comprising at least one Eimeria vaccine protein expressed in the seeds of said plant at a level of at least 10 mg / kg. The Eimeria vaccine protein comprises 3-1e, Gam82, and / or EF-1a. An embodiment provides a promoter that directs expression preferentially to embryonic tissues of the plant. Further embodiments provide for targeting expression of the Eimeria vaccine protein to the apoplast or endoplasmic reticulum. Further embodiments provide constructs and plasmids for its high level expression in plants. [Brief description of the drawings]

[0009] [Figure 1] FIG. 1 is a graph showing a schematic of Eimeria antigen expression constructs. Four plasmid constructs were designed to variably express Eimeria antigens 3-1e, Gam82 and / or EF-1 polypeptides (collectively referred to as "Eimeria vaccine proteins") in maize. The four constructs include construct EIA (expressing 3-1e and Gam82), construct EIB (expressing EF-1a), construct EID (expressing 3-1e and EF-1a), and construct EIE (expressing Gam82). EIA and EIB target the apoplast / cell wall, while EID and EIE target the endoplasmic reticulum. A cell / organelle targeting sequence (BAASS:cell wall / apoplast) was included in all constructs. A C-terminal sequence (KDEL:ER) was included in the codon-optimized EID and EIE constructs. An embryo-preferred promoter (Pr44: seed embryo) was used to drive high gene transcription and subsequent high-yield protein production in all constructs. To increase mRNA stability, the untranslated 3' region of potato protease inhibitor II (pinII) was also added to all constructs as a transcription termination element.

[0010] [Diagram 2] Figure 2 is a Western blot of seed grain expression of 3-1e in constructs EIA and EID. Seeds were harvested and analyzed by Western blot to detect 3-1e. Each lane represents seeds from one plant from an independent insertion event in either EIA or EID. Lane 1 is a 3-1e standard showing a specific band at the expected MW of approximately 27 kD. Notably, the band migrating larger than expected in lane 1 is due to 3-1e aggregation and / or post-translational modifications. Lane 2 is a standard ladder. Lane 3 (HiII) is a non-transformed negative control. Lanes 4-7 represent the EIA construct, with lanes 4, 5, and 7 showing a specific band at the expected MW of approximately 27 kD. Lanes 8-10 represent the EID construct, with lanes 8 and 10 showing a specific band at the expected MW of approximately 27 kD.

[0011] [Diagram 3] Figure 3 is a Western blot of seed grain expression of EF-1a in constructs EIB and EID. Seeds were harvested and analyzed by Western blot to detect EF-1a. Each lane represents seeds from one plant from an independent insertion event in either EIB or EID. Lane 1 is EF-1a standard and lane 2 is standard ladder. Lane 3 (HiII) is a non-transformed negative control. Lanes 4-7 represent EIB constructs and lanes 8-10 represent EID constructs. Lanes 4-10 show a specific band at the expected MW of approximately 51 kD. The band may appear to migrate larger due to post-translational modifications.

[0012] [Figure 4]Figure 4 is a Western blot of seed grain expression of Gam82 in constructs EIA and EIE. Seeds were harvested and analyzed by Western blot to detect Gam82. Each lane represents seeds from one plant from an independent insertion event in either EIA or EIE. Lanes 4-7 represent the EIA construct and lanes 8-10 represent the EIE construct. Lane 1 is a Gam82 standard showing a specific band at the expected MW of approximately 67 kD (see arrow). Lane 2 is a standard ladder. Lane 3 (HiII) is a non-transformed negative control. Lanes 4-10 show a specific band at the expected MW of approximately 67 kD. The band may appear to migrate larger due to post-translational modifications.

[0013] [Diagram 5] FIG. 5 is the plasmid map of EIA pr44-BAASS:3-1e pr44-BAASS:Gam82.

[0014] [Figure 6] FIG. 6 is a plasmid map of EIB pr44-BAASS:EF-1a.

[0015] [Figure 7] FIG. 7 is a plasmid map of EID pr44-BAASS:3-1e KDEL pr44-BAASS:EF-1a KDEL.

[0016] [Figure 8] FIG. 8 is a plasmid map of EIE pr44-BAASS:Gam82 KDEL.

[0017] [Figure 9A]Figures 9A-C show the results of a proof-of-concept study for an Eimeria vaccine in broiler chickens. Figure 9A shows the lesion score results for E. acervulina in four treatment groups (24 birds per group). Significant vaccine efficacy was observed in the high dose group. Figure 9B shows the adjusted feed conversion ratios 12 days after challenge (study days 27-33) in four treatment groups (48 birds per group). The high dose vaccine group had significantly better feed conversion than the positive control and low dose vaccine groups. Figure 9C shows the percent reduction in oocyst shedding 6 days after challenge in the four treatment groups. The results show a reduction in shedding for both the high dose vaccine group and the low dose vaccine group. [Figure 9B] Figures 9A-C show the results of a proof-of-concept study for an Eimeria vaccine in broiler chickens. Figure 9A shows the lesion score results for E. acervulina in four treatment groups (24 birds per group). Significant vaccine efficacy was observed in the high dose group. Figure 9B shows the adjusted feed conversion ratios 12 days after challenge (study days 27-33) in four treatment groups (48 birds per group). The high dose vaccine group had significantly better feed conversion than the positive control and low dose vaccine groups. Figure 9C shows the percent reduction in oocyst shedding 6 days after challenge in the four treatment groups. The results show a reduction in shedding for both the high dose vaccine group and the low dose vaccine group. [Figure 9C] Figures 9A-C show the results of a proof-of-concept study for an Eimeria vaccine in broiler chickens. Figure 9A shows the lesion score results for E. acervulina in four treatment groups (24 birds per group). Significant vaccine efficacy was observed in the high dose group. Figure 9B shows the adjusted feed conversion ratios 12 days after challenge (study days 27-33) in four treatment groups (48 birds per group). The high dose vaccine group had significantly better feed conversion than the positive control and low dose vaccine groups. Figure 9C shows the percent reduction in oocyst shedding 6 days after challenge in the four treatment groups. The results show a reduction in shedding for both the high dose vaccine group and the low dose vaccine group. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0018] explanation Eimeria offers several advantages as a model parasitic pathogen for successful vaccine development. We have produced, optimized, and characterized corn lines expressing high levels of Eimeria proteins to advance vaccine development. Eimeria antigens were produced together in the kernels of a single corn line and used to induce immune responses against Eimeria acervulina, Eimeria tenella, and Eimeria maxima in animal subjects. The use of plant-produced proteins and the simultaneous use of multiple Eimeria antigens is predicted to provide greater protective efficacy than previous studies. This simple, cost-effective method of production and administration of Eimeria vaccine proteins represents a major advance in vaccine development in chickens and other animals.

[0019] A preferred embodiment of the invention uses corn kernels as the basis for the production of subunit vaccines based on the Eimeria antigens 3-1e, Gam82 and EF-1a. In an embodiment, the subunit vaccine may be based on immunogens / antigens derived from avian protozoan antigens, including antigens from Eimeria acervulina such as 3-1e (Lillehoj, HS et al., 2005; Ding, X. et al., 2004), antigens from Eimeria maxima such as Gam82 (antigens from Belli, S. et al., 2004), and antigens from Eimeria tenella such as Eimeria tenella elongation factor-1a (EF-1a) (Wu, S. et al., 2004; Pogonka, T. et al., 2003). High expression levels of at least 1 mg / kg whole seed are obtained. One embodiment provides an average range of about 10-600 mg / kg. Further embodiments provide expression at or above or between 11 mg / kg, 12 mg / kg, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40 mg / kg whole seed.

[0020] Oral administration of a plant, plant part, or product produced from a plant part, such as a seed, grain, flour, or other edible composition comprising a plant, plant part, or product produced therefrom, containing an Eimeria protein (e.g., 3-1e, Gam82, and / or EF-1a) can provide protection from challenge to a subject animal. The subject animal may or may not produce detectable antibodies in response, but when the animal is exposed to a disease challenge, morbidity or mortality due to administration of a vaccine is reduced so that the animal can fight off infection. The compositions of the invention can also induce unexpected serum antibody responses as well as mucosal responses. The serum antibody response in one embodiment ranges from more than 2-fold to more than 100-fold over control. In another embodiment, the response can be 5-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 35-fold, 40-fold, 45-fold, 50-fold, 55-fold, 60-fold, 65-fold, 70-fold, 75-fold, 80-fold, 85-fold, 90-fold, 95-fold, or more, or any amount in between, that of unvaccinated control animals.

[0021] As used herein, the terms "animal" or "subject" or "subject animal" are intended to include chickens and other animals, including humans.

[0022] As used herein, the term nucleic acid or polynucleotide refers to deoxyribonucleotides or ribonucleotides and their polymers, either in single-stranded or double-stranded form. The sequences used to make the vaccine may be obtained from any source, such as, for example, from a biological source when isolated from a biological sample, or may refer to sequences that are synthetically produced based on sequences obtained from said sample. As such, these terms include RNA and DNA, which may be genes or parts thereof, cDNA, synthetic polydeoxyribonucleic acid sequences, or the like, and may be single-stranded or double-stranded, as well as DNA / RNA hybrids. Furthermore, these terms are used herein to include naturally occurring nucleic acid molecules that can be isolated from cells, as well as synthetic molecules that can be prepared, for example, by methods of chemical synthesis or by enzymatic methods, such as, for example, polymerase chain reaction (PCR). Unless specifically limited, these terms encompass nucleic acids that contain known analogs of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants (e.g., degenerate codon substitutions) of the nucleic acid sequence, as well as complementary sequences, as explicitly indicated. Specifically, various degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is replaced with a mixed base residue and / or a deoxyinosine residue (Batzer et al., (1991) Nucleic Acid Res. 19:5081; Ohtsuka et al., (1985) J. Biol. Chem. 260:2605-2608; Cassol et al., (1992); Rossolini et al., (1994) Mol. Cell. Probes, 8:91-98). The term nucleic acid is used interchangeably with gene, cDNA, and mRNA encoded by gene.

[0023] Nucleic acid as used herein includes nucleic acid encoding the entire polypeptide, as well as nucleic acid encoding a subsequence of the polypeptide, or a fragment that produces a protective response. For example, a nucleic acid encoding a polypeptide that is not full length but nevertheless has protective activity against Eimeria. The present invention includes not only nucleic acids that include nucleotide sequences as shown herein, but also nucleic acids that are substantially identical to, or correspond to, or are substantially complementary to the exemplified embodiments. For example, the invention includes nucleic acids comprising nucleotide sequences having at least about 70% identity to the nucleotide sequences set forth herein, more preferably at least 75%, even more preferably at least 80%, more preferably at least 85%, 85.5%, 86%, 86.5%, 87%, 87.5%, 88%, 88.5%, 89%, 89.5%, even more preferably at least 90%, 90.5%, 91%, 91.5%, 92%, 92.5%, 93%, 94.5%, 94%, 94.5%, and even more preferably at least about 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 95.5%, 100% identity (or any percentage therebetween) to the exemplified nucleotide sequences. The nucleotide sequence may be modified as described above so long as any polypeptide produced is capable of inducing the production of a protective response, including a strong serum antibody response in an animal.

[0024] Nucleic acid can be obtained using various methods known to those skilled in the art. Suitable nucleic acid (e.g., cDNA, genomic or partial sequence) can be cloned or amplified by in vitro methods, such as polymerase chain reaction (PCR), ligase chain reaction (LCR), transcription-based amplification system (TAS), or self-sustained sequence replication system (SSR), using suitable primers. A wide variety of cloning methodologies and in vitro amplification methodologies are widely known to those skilled in the art. Various examples of these techniques and instructions sufficient to guide one of skill through much of the cloning exercise are found in Berger and Kimmel, Guide to Molecular Cloning Techniques, Methods in Enzymology 152, Academic Press, Inc., San Diego, Calif. (Berger); Sambrook et al. (2001) Molecular Cloning-A Laboratory Manual (3rd ed.), Vol. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor Press, NY (Sambrook et al.); Current Protocols in Molecular Biology, edited by FMA Ausubel et al., Current Protocols, a consortium between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., (1994 Supplement) (Ausubel); Cashion et al., U.S. Pat. No. 5,017,478; and Carr, European Patent No. 0,246,864.Various examples of techniques sufficient to guide one of skill through in vitro amplification methods can be found in Berger, Sambrook, and Ausubel, as well as in Mullis et al. (1987), U.S. Pat. No. 4,683,202; PCR Protocols A Guide to Methods and Applications (Innis et al., eds.), Academic Press Inc. San Diego, Calif. (1990) (Innis); Amheim & Levinson (October 1, 1990), C&EN, 36-47; The Journal of NIH Research (1991) 3:81-94; Kwoh et al. (1989) Proc. Natl. Acad. Sci. USA, 86:1173; Guatelli et al. (1990) Proc. Natl. Acad. Sci. USA, 87, 1874; Lomell et al. (1989) J. Clin. Chem., 35:1826; Landegren et al. (1988) Science, 241:1077-1080; Van Brunt (1990) Biotechnology, 8:291-294; Wu and Wallace (1989) Gene, 4:560; and Barringer et al. (1990) Gene, 89:117. Improved methods for cloning in vitro amplified nucleic acids are described in U.S. Patent No. 5,426,039 to Wallace et al. The various nucleic acids, or subsequences of these nucleic acids, can be prepared by any suitable method, including, for example, cloning and restriction of appropriate sequences, as described above.

[0025] "Codon optimization" can be used to optimize sequences for expression, which is defined as modifying a nucleic acid sequence for enhanced expression in a recombinant host cell by replacing at least one codon, two or more codons, or a significant number of codons of the native sequence with a codon that is more frequently or most frequently used in the recombinant host. Different organisms exhibit certain biases for certain codons of specific amino acids.

[0026] As used herein, "polypeptide" generally refers to peptides and proteins. In certain embodiments, a polypeptide may be at least, or more than, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids, or any amount therebetween. A peptide is generally considered to be more than 50 amino acids. The terms "fragment", "derivative" and "homolog", when referring to a polypeptide according to the present invention, refer to a polypeptide that retains essentially the same biological function or activity as said polypeptide, i.e., acts as an antigen and / or provides treatment for and / or protection from a disease. Such fragments, derivatives and homologs can be selected based on their ability to retain one or more of the biological activities of the polypeptide, i.e., acts as an antigen and / or provides treatment for and / or protection from a pathogen. The polypeptide vaccine of the present invention may be a recombinant polypeptide, a natural polypeptide or a synthetic polypeptide, preferably a recombinant polypeptide. One of skill in the art will recognize that it is possible for protective polypeptides to be expressed by genes in host cells, and plant compositions administered to animals, and to be extracted from the plant prior to administration.

[0027] "Conservatively modified variants" applies to both amino acid and nucleic acid sequences. With respect to a particular nucleic acid sequence, conservatively modified variants refer to such nucleic acids that code for identical or essentially identical amino acid sequences, or essentially identical sequences if the nucleic acid does not code for an amino acid sequence. Due to the degeneracy of the genetic code, a large number of functionally identical nucleic acids code for any given polypeptide. For example, the codons CGU, CGC, CGA, CGG, AGA, and AGG all code for the amino acid arginine. Thus, at any position where arginine is specified by a codon, this codon can be changed to any of the corresponding codons described without changing the encoded polypeptide. Such nucleic acid variations are "silent substitutions" or "silent variations", which are a species of "conservatively modified variations". Any polynucleotide sequence described herein that codes for a polypeptide also represents any and all silent variations, unless otherwise noted. Thus, silent substitutions are an implicit feature of any nucleic acid sequence that codes for an amino acid. One of skill in the art will recognize that each codon in a nucleic acid (except AUG, which is normally the only codon for methionine) can be modified by standard techniques to obtain a functionally identical molecule. In some embodiments, the nucleotide sequence encoding the protective polypeptide is preferably optimized for expression in the particular host cell (e.g., yeast, mammalian, plant, fungus, and the like) used to produce the polypeptide or RNA.

[0028] For amino acid sequences, those skilled in the art will recognize that individual substitutions, deletions or additions to nucleic acid, peptide, polypeptide or protein sequences that change, add or delete only one amino acid or a small percentage of amino acids in the encoded sequence are "conservatively modified variants," which are referred to herein as "variants" that result from the change by replacing an amino acid with a chemically similar amino acid. A variety of conservative substitution tables providing functionally similar amino acids are well known in the art. See, for example, Davis et al., "Basic Methods in Molecular Biology," Appleton & Lange, Norwalk, Conn. (1994). Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs and alleles of the present invention.

[0029] The following eight groups each contain amino acids that are conservative substitutions for one another: 1) alanine (A), glycine (G); 2) aspartic acid (D), glutamic acid (E); 3) asparagine (N), glutamine (Q); 4) arginine (R), lysine (K); 5) isoleucine (I), leucine (L), methionine (M), valine (V); 6) phenylalanine (F), tyrosine (Y), tryptophan (W); 7) serine (S), threonine (T); and 8) cysteine ​​(C), methionine (M).

[0030] The isolated variant protein can be purified from cells that express it naturally, or can be purified from cells that have been modified to express it (recombinant), or can be synthesized using known protein synthesis methods.For example, the nucleic acid molecule encoding the variant polypeptide is cloned into an expression vector, the expression vector is introduced into a host cell, and the variant protein is expressed in the host cell.The variant protein can then be isolated from the cell by an appropriate purification scheme using standard protein purification techniques.Many of these techniques are described in detail below.In particular, the term Eimeria protein is used herein synonymously with Eimeria antigen.

[0031] The methods include those comprising amino acid sequences having at least about 70% identity to the amino acid sequences set forth herein, more preferably at least 75%, even more preferably at least 80%, more preferably at least 85%, 85.5%, 86%, 86.5%, 87%, 87.5%, 88%, 88.5%, 89%, 89.5%, even more preferably at least 90%, 90.5%, 91%, 91.5%, 92%, 92.5%, 93%, 94.5%, 94%, 94.5%, and even more preferably at least about 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 95.5%, 100% identity (or any percentage therebetween) to the exemplified nucleotide sequences. The sequence may be modified as described above, so long as the polypeptide is capable of inducing the production of a protective response.

[0032] The variant protein used in the method can be attached to a heterologous sequence to form a chimeric or fusion protein. Such chimeric and fusion proteins include a variant protein fused in frame to a heterologous protein having an amino acid sequence that is not substantially homologous to the variant protein. The heterologous protein can be fused to the N-terminus or C-terminus of the variant protein.

[0033] Chimeric or fusion proteins can be produced by standard recombinant DNA techniques. For example, DNA fragments encoding different protein sequences are linked together in frame according to conventional techniques. In another embodiment, fusion genes can be synthesized by conventional techniques, including automated DNA synthesizers. Alternatively, PCR amplification of gene fragments can be performed using anchor primers that generate complementary overhangs between two consecutive gene fragments that can be subsequently annealed and re-amplified to generate chimeric gene sequences (see Ausubel et al., Current Protocols in Molecular Biology, 1992). Moreover, many expression vectors are commercially available that already code for fusion components (e.g., GST proteins). Nucleic acids encoding variant proteins can be cloned into such expression vectors so that the fusion components are linked in frame to the variant protein.

[0034] Polypeptides sometimes contain other amino acids than the 20 amino acids commonly designated as the 20 naturally occurring amino acids. In addition, many amino acids, including terminal amino acids, can be modified by natural processes, such as processing and other post-translational modifications, or by chemical modification techniques that are well known in the art. Common modifications that occur naturally in polypeptides are described in basic texts, detailed monographs, and research literature, and are well known to those skilled in the art. Thus, the variant peptides of the present invention also include derivatives or analogs in which the substituted amino acid residue is not one encoded by the genetic code, derivatives or analogs that include a substituent, derivatives or analogs in which the mature polypeptide is fused to another compound, such as a compound for increasing the half-life of the polypeptide (e.g., polyethylene glycol), or derivatives or analogs in which additional amino acids are fused to the mature polypeptide, such as a leader sequence or secretory sequence, or a sequence for purifying the mature polypeptide or proprotein sequence.

[0035] Known modifications include, but are not limited to, acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of a flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cystine, formation of pyroglutamate, formylation, gamma carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins (such as, for example, arginylation), and ubiquitination.

[0036] The methods of the present invention further provide functional fragments of the nucleic acid molecules and polypeptides (including variant proteins of the polypeptides), in addition to proteins and peptides comprising, and consisting of, such fragments, provided that such fragments act as antigens and / or provide treatment for and / or protection from Eimeria.

[0037] As used herein, the term "subunit" refers to a portion of a microorganism that confers protection and that may itself be antigenic, i.e., capable of inducing an immune response in an animal. The term should be interpreted to include subunits obtained by both recombinant and biochemical methods.

[0038] In one embodiment, a method is provided for identifying a protective sequence of a virus or nucleic acid that induces protection. This method also includes fragments, derivatives, or homologs of the nucleic acid molecule. In one aspect, the method includes administering such a sequence to a test animal. The test and control animals are then challenged with an infectious dose of a disease-causing microorganism. Various methods and techniques are known to those skilled in the art for determining whether protection is provided, including, but not limited to, observing differences in disease symptoms between test and control animals. A reduction in any of the symptoms observed in the test animal compared to the control animal indicates that the protective molecule provides some degree of protection against the disease. An increase in any of the similar symptoms or symptoms observed in the test animal compared to the symptoms observed in the control animal indicates that the protective molecule does not provide protection.

[0039] In another embodiment, determining whether a molecule provided protection against Eimeria involves determining the presence or absence of challenge disease in a test animal by electron microscopy or antibodies, or assays such as the fluorescent focus neutralization (FFN) test or Western blot assays can be used. PCR methods may be used to determine whether a protective molecule is present. Northern blotting can detect the presence of diagnostic sequences. In another embodiment, ELISA or similar assays, such as hemagglutinin inhibition assays, are a number of different types of assays that can determine whether a protective molecule is effective. ELISA or enzyme-linked immunoassays have been known since 1971. In general, antigens solubilized in a buffer are coated onto a plastic surface. Upon addition of serum, antibodies can bind to the antigen on the solid phase. The presence or absence of these antibodies can be demonstrated when conjugated to an enzyme. Addition of an appropriate substrate detects the amount of bound conjugate, which can be quantified. A common ELISA assay uses a biotinylated anti-(protein) polyclonal antibody and an alkaline phosphatase conjugate. For example, the ELISA used to quantify protein levels can be an antibody sandwich assay that utilizes commercially available polyclonal rabbit antibodies. The antibodies are conjugated to alkaline phosphatase for detection. In another example, the ELISA assay for detecting trypsin or trypsinogen uses biotinylated anti-trypsin or anti-trypsinogen polyclonal antibodies and streptavidin-alkaline phosphatase conjugates.

[0040] Clearly, many such methods are available to one of skill in the art to ascertain whether a molecule confers protection, and whether it does so at the levels administered to an animal.

[0041] The nucleic acid molecule, polypeptide or fragment thereof, when administered to a subject animal, produces a protective response against Eimeria. A protective response is induced in the animal. The subject animal may or may not produce antibodies in response, but as further described herein, the animal will experience reduced morbidity or mortality due to administration of the vaccine. As used herein, the terms "protect", "protection", "protective immunity" or "protective immune response" are intended to mean that the host subject animal mounts an active immune response to the vaccine or polypeptide of the present invention, such that upon exposure to a disease challenge, the subject animal is able to fight infection. Thus, a protective immune response reduces the incidence of morbidity and mortality due to exposure to a microorganism between the host animal. The subject animal is protected from subsequent exposure to the pathogen. In one embodiment, the animal may be protected by treating an animal already exposed to a pathogen by administering the vaccine or polypeptide after such exposure. In such instances, reduced morbidity and mortality are also shown. Those skilled in the art will understand that in a commercial animal environment, the generation of protective immune response is evaluated by evaluating the effect of vaccination on the entire population, and for example, a small number of vaccinated animals may still experience morbidity and mortality. Furthermore, protection also includes a reduction in the severity of gross or histopathological changes and / or symptoms of disease compared to the changes or symptoms typically caused by the isolate in similar animals that are not protected (i.e., against appropriate controls). Thus, a protective immune response reduces the symptoms of disease compared to control animals.

[0042] A "construct" is a package of genetic material that is inserted into the genome of a cell through various techniques. A "vector" is a means for the transfer of nucleic acid into a host cell by whatever means. A vector can be a replicon to which another DNA segment can be attached to effect replication of the attached segment. A "replicon" is any genetic element (e.g., plasmid, phage, cosmid, chromosome, virus) that functions as an autonomous unit of DNA or RNA replication in vivo, i.e., capable of replication under its own control. The term "vector" includes both viral and non-viral means for introducing nucleic acid into a cell in vitro, ex vivo, or in vivo. Viral vectors include alphavirus vectors, retrovirus vectors, adeno-associated virus vectors, pox vectors, baculovirus vectors, vaccinia vectors, herpes simplex vectors, Epstein-Barr vectors, rabies virus vectors, vesicular stomatitis virus vectors, and adenovirus vectors. Non-viral vectors include, but are not limited to, plasmids, liposomes, charged lipids (cytofectins), DNA-protein complexes or RNA-protein complexes, and biopolymers. In addition to the nucleic acid, the vector may also contain one or more regulatory regions and / or selectable markers that are useful in selecting, measuring, and monitoring the results of nucleic acid transfer (such as which tissues are transferred, duration of expression, etc.).

[0043] "Cassette" refers to a segment of DNA that can be inserted into a vector at particular restriction sites. The segment of DNA encodes a polypeptide of interest or produces RNA, and the cassette and restriction sites are designed to ensure insertion of the cassette in the correct reading frame for transcription and translation.

[0044] A nucleic acid molecule is introduced into a cell when it is inserted into the cell. A cell has been "transfected" by exogenous or heterologous DNA or RNA when such DNA or RNA has been introduced inside the cell.

[0045] Once a gene has been engineered to contain the desired characteristics (e.g., a desired subcellular localization sequence), the gene can then be placed into an expression vector by standard methods. Selection of an appropriate expression vector will depend on the method for introducing the expression vector into a host cell. A typical expression vector contains a prokaryotic DNA element that codes for a bacterial origin of replication, as well as an antibiotic resistance gene to provide for growth in a bacterial host and selection of the expression vector; a cloning site for insertion of an exogenous DNA sequence; a eukaryotic DNA element that controls initiation of transcription of the exogenous gene; and DNA elements that control processing of the transcript, such as transcription termination / polyadenylation sequences. Expression vectors can also contain such sequences as are required for the eventual integration of the vector into the host chromosome. By "promoter" is meant a regulatory region of DNA that is capable of regulating the transcription of a sequence linked to it. This regulatory region usually contains a TATA box that is capable of directing RNA polymerase II to initiate RNA synthesis at the appropriate transcription start site for a particular coding sequence. A promoter is a minimal sequence that is sufficient to direct transcription in a desired manner. The term "regulatory region" is also used to denote sequences which are capable of initiating transcription in a desired manner.

[0046] The nucleic acid molecule may be used in conjunction with its own promoter or another promoter. In one embodiment, the selection marker, the nucleic acid molecule of interest may be operably linked to the same promoter. In another embodiment, they may be operably linked to different promoters. In yet a third and fourth embodiment, the expression vector may contain two or more genes of interest that may be operably linked to the same promoter or different promoters. For example, one promoter may be used to drive the nucleic acid molecule of interest and the selection marker, or different promoters may be used for one or each. These other promoter elements may be constitutive or sufficient to make promoter-dependent gene expression controllable as cell type-specific, tissue-specific, or time- or developmental stage-specific, or inducible by an external signal or agent. Such elements may be located in the 5' or 3' region of the gene. Although the additional promoter may be the endogenous promoter of the structural gene of interest, the promoter may also be an exogenous regulatory sequence. The promoter elements used to control the expression of the product protein and the selection gene may be any host-compatible promoter.These can be plant gene promoters, such as the ubiquitin promoter (European Patent Application No. 0342926); the promoter for the small subunit of ribulose-1,5-bis-phosphate carboxylase (ssRUBISCO) (Coruzzi et al., 1984; Broglie et al., 1984); or promoters from tumor-inducing plasmids derived from Agrobacterium tumefaciens, such as the plant-active nopaline synthase, octopine synthase and mannopine synthase promoters (Velten and Schell, 1985); or viral promoters, such as the 19S and 35S promoters of the cauliflower mosaic virus (CaMV) (Guilley et al., 1982; Odell et al., 1985), the Figwort mosaic virus FLt promoter (Maiti et al., 1997), or the coat protein promoter of TMV (Grdzelishvili et al., 2000). Alternatively, plant promoters can be used, such as heat shock promoters (e.g., soybean hsp17.5-E (Gurley et al., 1986)) or ethanol-inducible promoters (Caddick et al., 1998). For a review of exemplary plant promoters that may be suitably used, see International Patent Application Publication No. WO 91 / 19806.

[0047] A promoter may additionally contain other recognition sequences (referred to as upstream promoter elements) that affect the rate of transcription initiation, typically located upstream, i.e., 5', of the TATA box. It is recognized that since the nucleotide sequences for promoter regions have been identified, it is within the state of the art to isolate and identify additional regulatory elements in the 5' region upstream of the specific promoter regions identified herein. Thus, promoter regions are generally further defined by including upstream regulatory elements, such as regulatory elements responsible for tissue and temporal expression of coding sequences, enhancers, and the like.

[0048] Tissue-preferred promoters can be utilized to target enhanced transcription and / or expression within a particular tissue. When referring to preferential expression, what is meant is expression at a higher level in a particular tissue than in other tissues. Examples of these types of promoters include embryo-preferred expression, such as that mediated by the phaseolin promoter (Bustos et al. (1989), The Plant Cell, Vol. 1, 839-853). For dicotyledonous plants, embryo-preferred promoters include, but are not limited to, pea β-phaseolin, napin, β-conglycinin, soybean lectin, cruciferin, and the like. For monocotyledonous plants, embryo-preferred promoters include, but are not limited to, the 15 kD zein, 22 kD zein, 27 kD zein, gamma-zein, waxy, shrunken1, shrunken2 genes of maize, the Ltp1 gene (see, e.g., U.S. Pat. No. 7,550,579), the Ltp2 gene (Opsahl-Sorteberg, H.G. et al. (2004), Gene, 341:49-58, and U.S. Pat. No. 5,525,716), and the oleosin gene. See also International Publication WO 00 / 12733, which discloses embryo-preferred promoters from the end1 and end2 genes.Seed-preferred promoters also include promoters that direct gene expression primarily to specific tissues within a seed, such as the endosperm-preferred promoter of gamma-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 maize (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, a correction to Plant Cell 1997, 1:109), the globulin-1 promoter from maize (Belanger and Kriz (1991) "Molecular basis for Allelic Polymorphism of the maize Globulin-1 gene" Genetics 129:863-972, and GenBank Accession No. L22344), a promoter that directs expression to the seed coat or seed husk of the corn kernel, such as the pericarp-specific glutamine synthetase promoter (Muhitch et al., (2002) "Isolation of a Promoter Sequence From the Glutamine Synthetase. 1-2Gene Capable of Conferring Tissue-Specific Gene Expression in Transgenic Maize," Plant Science, 163:865-872, and GenBank Accession No. AF359511), and promoters that direct expression to the embryo (germ), such as the promoters disclosed in U.S. Pat. No. 7,169,967. When referring to a seed-preferred or embryo-preferred promoter, it is meant that the promoter causes an operably linked sequence to be expressed to a greater extent in seed or embryo tissue than in other plant tissues. A seed-preferred or embryo-preferred promoter may be expressed during seed or embryo development along with expression at other stages, and may be expressed strongly during seed or embryo development and to a much lesser extent at other times. The globulin promoters shown herein are preferentially expressed in plant embryos. Globulin-1 is the most abundant protein in maize embryos and is a vicilin-like storage protein encoded by the globulin-1 gene. See, e.g., Liu et al. (1992) MNL Vol. 22:108-109. As described in Belanger et al. (1991), globulins are storage proteins recognized to constitute 10-20% of maize embryo protein, with globulin 1 being one of the most abundant proteins encoded by the globulin 1 gene. Belanger, FC and Kriz, AL (1991) “Molecular basis for allelic polymorphism of the maize globulin-1 gene” Genetics 129, 863-872. The two most abundant proteins in maize embryos are saline-soluble, water-insoluble globulins, one a 63,000 Da molecular weight protein encoded by the globulin-1 gene and the other a 45,000 Da molecular weight protein encoded by the globulin-2 gene. See, e.g., Kriz (1989) Biochem Genet. 27(3-4):238-51.If a null allele is present, no globulin 1 protein is produced. Belanger et al. (1991), supra. Belanger et al. note that the protein is easily detected in Coomassie stained gels of protein extracts from embryos, and several alleles have been recognized. Belanger et al. (1991), p. 865. Those skilled in the art will appreciate that nucleic acid molecules encoding globulin 1 proteins are well known and easily identified using techniques available to those skilled in the art, including, by way of example and not limitation, comparison to known sequences, library preparation and screening with probes, antibody binding using Northern, Southern or Western blots, among many other means available, as discussed herein. The promoter of the gene encoding globulin-1 can be used in plants to express an operably linked nucleic acid molecule in the plant. Examples of globulin promoters, without intending to be limiting, include the 1.45 kb maize globulin-1 promoter and the untranslated leader described in Belanger and Kriz, 1991, supra, and GenBank accession L22344.

[0049] The range of promoters that can be used includes inducible promoters. An inducible regulatory element is a regulatory element that can directly or indirectly activate the transcription of one or more DNA sequences or genes in response to an inducer. In the absence of an inducer, the DNA sequence or gene will not be transcribed. Typically, the protein factor that specifically binds to the inducible regulatory element and activates the transcription is present in an inactive form that is subsequently converted directly or indirectly into an active form by the inducer. The inducer can be a chemical agent, such as a protein, a metabolite, a growth regulator, a herbicide or a phenolic compound, or a physiological stress, imposed directly by heat, cold, salt or a toxic element, or indirectly through the action of a pathogen or disease vector (such as a virus). Cells containing an inducible regulatory element can be exposed to the inducer by applying the inducer externally to the cell or plant, such as by spraying, watering, heating or similar methods.

[0050] Any inducible promoter can be used. See Ward et al., Plant Mol. Biol. 22:361-366 (1993). Exemplary inducible promoters include the ecdysone receptor promoter, U.S. Pat. No. 6,504,082; the promoter from the ACE1 system, which responds to copper (Mett et al., PNAS 90:4567-4571 (1993)); the In2-1 and In2-2 genes from maize, which respond to benzenesulfonamide herbicide safeners (U.S. Pat. No. 5,364,780; Hershey et al., Mol. Gen. Genetics, 227:229-237 (1991), and Gatz et al., Mol. Gen. Genetics, 243:32-38 (1994)), the Tet repressor from Tn10 (Gatz et al., Mol. Gen. Genet. 227:229- 237 (1991), or the Tet repressor from a steroid hormone gene, the transcriptional activity of which is induced by glucocorticosteroid hormones. Schena et al., Proc. Natl. Acad. Sci. USA 88:10421 (1991); the maize GST promoter, which is activated by hydrophobic electrophilic compounds used as pre-emergence herbicides; and the tobacco PR-1a promoter, which is activated by salicylic acid. Other chemically regulated promoters of interest include steroid-responsive promoters (e.g., Schena et al. (1991), ... 88:10421-10425, and McNellis et al., (1998) Plant J. 14(2):247-257), as well as tetracycline-inducible and tetracycline-repressible promoters (see, e.g., Gatz et al., (1991) Mol. Gen. Genet. 227:229-237, and U.S. Pat. Nos. 5,814,618 and 5,789,156).

[0051] Other components of the vector may be included, again depending on the intended use of the gene. Examples include selection markers, targeting or regulatory sequences, stabilizing or leader sequences, introns, etc. A variety of general descriptions and examples of plant expression vectors and reporter genes can be found in Gruber et al., "Vectors for Plant Transformation", Method in Plant Molecular Biology and Biotechnology (Glick et al., eds., CRC Press, pp. 89-119 (1993)). The selection of an appropriate expression vector will depend on the host and the method of introducing the expression vector into the host. The expression cassette will also include transcriptional and translational termination regions that are functional in plants at the 3' end of the heterologous nucleotide sequence of interest.

[0052] The expression vector can also optionally contain a signal sequence located between the promoter and the gene of interest and / or after the gene of interest. A signal sequence is a nucleotide sequence that is translated to provide an amino acid sequence that is used by the cell to direct the protein or polypeptide of interest to a specific location inside or outside the eukaryotic cell. Many signal sequences are known in the art. See, for example, Becker et al. (1992) Plant Mol. Biol. 20:49; Knox, C. et al., "Structure and Organization of Two Divergent Alpha-Amylase Genes from Barley" Plant Mol. Biol. 9:3-17 (1987); Lerner et al. (1989) Plant Physiol. 91:124-129; Fontes et al. (1991) Plant Cell 3:483-496; Matsuoka et al. (1991) Proc. Natl. Acad. Sci. 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. The use of a signal sequence is required when targeting a protein to the cell wall and / or apoplast. An example is the barley alpha-amylase signal sequence. Rogers, JC (1985) "Two barley alpha-amylase gene families are regulated differently in aleurone cells" J. Biol. Chem. 260:3731-3738.

[0053] In those cases where it is desirable to target the expression product of the heterologous nucleotide sequence to a particular organelle, particularly the plastid, amyloplast, or endoplasmic reticulum, or to secrete it to the cell surface or outside the cell, the expression cassette can further include a coding sequence for a transit peptide. A variety of such transit peptides are well known in the art, including, but not limited to, transit peptides for acyl carrier protein, the small subunit of RUBISCO, plant EPSP synthase, maize Brittle-1 chloroplast transit peptide (Nelson et al., Plant Physiol 117(4):1235-1252(1998); Sullivan et al., Plant Cell 3(12):1337-48; Sullivan et al., Planta(1995), 196(3):477-84; Sullivan et al., J.Biol.Chem.(1992)267(26):18999-9004), and the like. Those skilled in the art will readily appreciate the many options available in expressing a product to a particular organelle. The use of transport peptides is widely known (see, e.g., U.S. Pat. Nos. 5,717,084 and 5,728,925). Proteins can be directed to the endoplasmic reticulum of plant cells. This can be achieved by the use of localization sequences, such as KDEL. This sequence (Lys-Asp-Glu-Leu) contains a binding site for a receptor in the endoplasmic reticulum. (Munro et al., (1987) "A C-terminal signal prevents secretion of luminal ER proteins." Cell. 48:899-907. A wide variety of endoplasmic reticulum retention signal sequences are available to those skilled in the art, the KDEL sequence being one example. Another example is HDEL (His-Asp-Glu-Leu). See, e.g., Kumar et al., which discusses methods for producing various 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.

[0054] Another example is to keep proteins in the vacuole. Various signal sequences are widely known to achieve this. For example, Raikhel's U.S. Patent No. 5,360,726 shows a vacuolar signal sequence as Warren et al.'s U.S. Patent No. 5,889,174 shows. Vacuolar targeting signals can be present in the amino-terminal portion (Holwerda et al., (1992) The Plant Cell, 4:307-318; Nakamura et al., (1993) Plant Physiol., 101:1-5), or in the carboxy-terminal portion, or in the internal sequence of targeting protein (Tague et al., (1992) The Plant Cell, 4:307-318; Saalbach et al., (1991) The Plant Cell, 3:695-708). In addition, the amino-terminal sequence, in conjunction with the carboxy-terminal sequence, is responsible for vacuolar targeting of the gene product (Shinshi et al. (1990) Plant Molec. Biol. 14:357-368).

[0055] The termination region may be native with the DNA sequence of interest or may be derived from another source. A variety of convenient termination regions are available from the Ti plasmid of A. tumefaciens, such as the termination region of octopine synthase (MacDonald et al., (1991) Nuc. Acids Res. 19(20)5575-5581) and the termination region of nopaline synthase (Depicker et al., (1982) Mol. and Appl. Genet. 1:561-573; and Shaw et al., (1984) Nucleic Acids Research Vol. 12, No. 20 pp7831-7846 (nos)). Examples of various other terminators include the pinII 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 Acid Res. 15:9627-9639.

[0056] Numerous variations on promoters, selectable markers, signal sequences, leader sequences, termination sequences, introns, enhancers, and other components of vectors are available to those of skill in the art.

[0057] Plants are used broadly herein to include any plant at any stage of development, including plant cuttings, plant cell cultures, plant organs, plant seeds and plantlets. In other words, the term plant refers to whole plants or plant materials or plant parts or plant tissues or plant cells, including collections of plant cells. For example, the various parts of a plant seed include the pericarp or kernel, the embryo or germ, and the endoplasm. A plant cell is the structural and physiological unit of a plant, including a protoplast and a cell wall. A plant cell can be in the form of an isolated single cell or cell aggregate (e.g., friable callus, etc.), or cultured cells, or can be part of a more highly organized unit (e.g., a plant tissue, a plant organ, or a plant). Thus, a plant cell can be a protoplast, a gamete-producing cell, or a cell or collection of cells that can regenerate into a complete plant. The plant tissue or plant organ may be a seed, protoplast, callus, or any group of plant cells organized into a structural or functional unit. Particularly useful parts of a plant include harvestable parts and parts useful for propagation of offspring plants. Harvestable parts of a plant may be any useful part of a plant, such as flowers, pollen, seedlings, tubers, leaves, stems, fruits, seeds, roots, and the like. Plant parts useful for propagation include, for example, seeds, fruits, cuttings, seedlings, tubers, rhizomes, and the like. In one embodiment, the tissue culture will preferably be capable of regenerating a plant. Preferably, the regenerative cells in such tissue culture will be embryos, protoplasts, meristematic cells, callus, pollen, leaves, anthers, roots, root tips, silk, flowers, kernels, ears, cobs, husks, or stems. Still further, plants may be regenerated from tissue culture.

[0058] A variety of plant species may be used, including any plant species, whether monocotyledonous or dicotyledonous, including maize (Zea mays), canola (Brassica napus, Brassica rapa ssp.), alfalfa (Medicago sativa), rice (Oryza sativa), rye (Secale cereale), sorghum (Sorghum bicolor, Sorghum vulgare), sunflower (Helianthus annuus), wheat (Triticum aestivum), soybean (Glycine max), tobacco (Nicotiana tabacum), potato (Solanum tuberosum), peanut (Arachis hypogaea), cotton (Gossypium hirsutum), sweet potato (Ipomoea batatus), cassava (Manihot esculenta), coffee (Cofea spp.), coconut (Cocos nucifera), pineapple (Ananas comosus), citrus trees (Citrus spp.), cocoa (Theobroma cacao), tea (Camellia sinensis), banana (Musa spp.), avocado (Persea americana), fig (Ficus casica), guava (Psidium guajava), mango (Mangifera indica), olive (Olea europaea), papaya (Carica papaya), cashew (Anacardium occidentale), macadamia (Macadamia integrifolia), almond (Prunus amygdalus), sugar beet (Beta vulgaris), oats (Avena), barley (Hordeum), vegetables, ornamentals and conifers.Vegetables include tomatoes (Lycopersicon esculentum), lettuce (e.g., Lactuca sativa), beans (Phaseolus vulgaris), lima beans (Phaseolus limensis), peas (Lathyrus spp.), and members of the cucumber genus, such as cucumber (C. sativus), cantalupensis, and muskmelon (C. melo). Ornamental plants include azaleas (Rhododendron spp.), hydrangeas (Macrophylla hydrangea), hibiscus (Hibiscus rosasanensis), roses (Rosa spp.), tulips (Tulipa spp.), daffodils (Narcissus spp.), petunias (Petunia hybrida), carnations (Dianthus caryophyllus), poansettia (Euphorbia pulcherrima), and chrysanthemums. Conifers that may be used in the practice of the present invention include, for example, pines such as Pinus taeda, Pinus elliotii, Pinus ponderosa, Pinus contotta, and Pinus radiata; Douglas fir (Pseudotsuga menziesii); Tsuga canadensis; Picea glauca; Sequoia sempervirens; firs such as Abies amabilis and Abies balsamea; cedars such as Thuja plicata and Chamaecyparis nootkatensis, algae or Lemnoideae (also known as duckweed), and the like. One embodiment provides that the plant is maize.

[0059] Various methods of transformation or transfection are currently available. Newer methods are available for transforming crops or other host cells, so they can be directly applied. Thus, a wide variety of methods have been developed to insert DNA sequences into the genome of a host cell to obtain transcription or transcripts and translation of the sequences to produce phenotypic changes in the organism. Thus, any method that provides efficient transformation / transfection can be used. In particular, the transformation vector that contains the sequence operably linked to the heterologous nucleotide sequence in the expression cassette can also contain at least one additional nucleotide sequence for the gene that is cotransformed into the organism. Alternatively, the additional sequence can be provided in another transformation vector.

[0060] There are various methods for introducing expression vectors into plant tissues available to those skilled in the art, and these methods will depend on the plant selected. Procedures for transforming a wide variety of plant species are widely known and described throughout the literature. (See, for example, Miki and McHugh (2004) Biotechnol. 107, 193-232; Klein et al., (1992) Biotechnology (NY) 10, 286-291; and Weising et al., (1988) Annu. Rev. Genet. 22, 421-477). For example, DNA constructs can be introduced into the genomic DNA of plant cells using techniques such as microprojectile-mediated delivery (Klein et al., 1992, supra), electroporation (Fromm et al., 1985 Proc. Natl. Acad. Sci. USA 82, 5824-5828), polyethylene glycol (PEG) precipitation (Mathur and Koncz, 1998 Methods Mol. Biol. 82, 267-276), direct gene transfer (International Publication WO 85 / 01856 and European Patent Application Publication No. 275069), in vitro protoplast transformation (U.S. Patent No. 4,684,611), and microinjection of plant cell protoplasts or embryogenic callus (Crossway, A. (1985) Mol. Gen. Genet. 202, 179-185). The Agrobacterium transformation method of Ishida et al. (1996), as well as that described in U.S. Patent No. 5,591,616, is yet another option. Co-cultivating plant tissue with Agrobacterium tumefaciens is a variation in which the DNA construct is placed into a binary vector system (Ishida et al., 1996, Nat. Biotechnol. 14, 745-750). The virulence functions of the Agrobacterium tumefaciens host will direct the insertion of the construct into the plant cell DNA when the cells are infected by the bacteria. See, for example, Fraley et al. (1983), Proc. Natl. Acad. Sci. USA, 80, 4803-4807. Agrobacterium is primarily used in dicotyledonous plants, but monocotyledonous plants, including maize, can be transformed by Agrobacterium.See, e.g., U.S. Patent No. 5,550,318. In one of many variations on this method, Agrobacterium infection of maize can be used in conjunction with heat shock treatment of immature embryos (Wilson et al., U.S. Patent No. 6,420,630) or with antibiotic selection of Type II callus (Wilson et al., U.S. Patent No. 6,919,494).

[0061] Transformation of rice is described by Hiei et al. (1994) Plant J. 6, 271-282, and Lee et al. (1991) Proc. Nat. Acad. Sci. USA 88, 6389-6393. Standard methods for transforming canola are described by Moloney et al. (1989) Plant Cell Reports, 8, 238-242. Transformation of maize is described by Fromm et al. (1990) Biotechnology (NY) 8, 833-839, and Gordon-Kamm et al. (1990) supra. Wheat can be transformed by techniques similar to those used to transform maize or rice. Transformation of sorghum is described by Casas et al. (Casas et al. (1993) Transgenic sorghum plants via microprojectile bombardment. Proc. Natl. Acad. Sci. USA 90, 11212-11216). Transformation of barley is described by Wan and Lemaux (Wan and Lemaux (1994) Generation of large numbers of independently transformed fertile barley plants. Plant Physiol. 104, 37-48). Transformation of soybean is described in numerous publications, including U.S. Pat. No. 5,015,580.

[0062] In one method, the Agrobacterium transformation method of Ishida et al. (1996), as well as that described in U.S. Patent No. 5,591,616, is largely mimicked, with modifications that we have found improve the number of transformants obtained. In Ishida's method, the A188 variety of corn is used, which produces type I callus in culture. In one embodiment, the HiII corn line is used, which gives rise to type II embryogenic callus in culture (Armstrong et al., 1991).

[0063] Although Ishida recommends selection with phosphinothricin when the bar or pat genes are used for selection, another preferred embodiment provides for the use of bialaphos instead. Generally, as described in the '616 patent and as outlined in more detail below, dedifferentiation is obtained by culturing plant explants on a dedifferentiation-inducing medium for 7 days or more, and the dedifferentiated or dedifferentiated tissue is contacted with Agrobacterium carrying the gene of interest. The cultured tissue can be, for example, callus, somatic embryo-like tissue, or suspension cells. In this preferred embodiment, the Agrobacterium suspension is incubated for 10 min at 4° C. for 24 hours at 4° C. ... 6 ~10 11The Agrobacterium has a cell population of 10 ... In yet another embodiment, we find that EHA101 strain is preferred, since LBA4404 strain is not highly pathogenic and has limited application without the superbinary plasmid. This strain is a disarmed helper strain derived from the hypervirulent A281 strain. The cointegrated superbinary / cloning vector from the LBA4404 parent is isolated and electroporated into EHA101, selecting for spectinomycin resistance. A plasmid is isolated to ensure that EHA101 contains the plasmid. EHA101 contains a disarmed pTi that conveys resistance to kanamycin. See Hood et al. (1986).

[0064] Furthermore, the Ishida protocol as described provides for growing a fresh culture of Agrobacterium on a plate, scraping the bacteria off the plate and resuspending them in co-cultivation medium as described in the '616 patent for incubation with corn embryos. This medium contains 4.3 g MS salts, 0.5 mg nicotinic acid, 0.5 mg pyridoxine hydrochloride, 1.0 ml thiamine hydrochloride, casamino acids, 1.5 mg 2,4-D, 68.5 g sucrose and 36 g glucose per liter, all at a pH of 5.8. In a further preferred method, the bacteria are grown overnight in a 1 ml culture, and then a fresh 10 ml culture is reinoculated the next day when transformation is to take place. The bacteria grow into log phase and reach an OD 600 = 0.5, preferably between 0.2 and 0.5. The bacteria are then centrifuged to remove the medium and resuspended in co-cultivation medium. Since HiII is used, the medium preferred for HiII is used. This medium is described in some detail by Armstrong and Green (1985). The resuspension medium is the same as the medium described above. All further HiII media are as described by Armstrong and Green (1985). The result is redifferentiation of the plant cells and regeneration into plants. Redifferentiation is sometimes indicated as dedifferentiation, but the former term more accurately describes the process in which cells start out with a morphology and identity and are placed on a medium where the cells lose that identity and are "reprogrammed" to have a new identity. Thus, the scutellum cells become embryogenic callus.

[0065] Transgenic plants can be produced that contain an introduced nucleic acid molecule encoding a polypeptide.

[0066] When referring to the introduction of a nucleotide sequence into a plant, it is meant to include transformation into a cell as well as crossing a plant carrying the sequence with another plant, as in conventional plant breeding techniques, so that the second plant contains the heterologous sequence. Such breeding techniques are well known to those skilled in the art. This can be accomplished by any means known in the art for breeding plants, such as cross-pollinating the transgenic plant described above with another plant and selecting for plants from subsequent generations that express the amino acid sequence. The plant breeding methods used herein are well known to those skilled in the art. For a discussion of plant breeding techniques, see Poehlman (1995) Breeding Field Crops (AVI Publication Co., Westport Conn, 4th ed.). Many crop plants useful in this method are bred through techniques that take advantage of the plant's pollination method. If the pollen from one flower is transferred to the same flower or another flower of the same plant, the plant is self-pollinating. If the pollen comes from a flower on a different plant, the plant is cross-pollinating. For example, in the Brassica genus, plants are usually self-sterile and only cross-pollination is possible unless self-compatibility is obtained through the discovery of mutants or through genetic intervention. In self-pollinating species, such as rice, oats, wheat, barley, pea, bean, soybean, tobacco and cotton, male and female plants are anatomically juxtaposed. During natural pollination, the male reproductive organ of a given flower pollinates the female reproductive organ of the same flower. Maize plants (Zea mays L.) can be bred by both self-pollination and cross-pollination techniques. Maize has male flowers located on the tassel and female flowers located on the ear on the same plant. Maize can be self-pollinating or cross-pollinating.

[0067] Pollination can be by any means, including but not limited to hand, wind or insect pollination, or mechanical contact between male fertile and male sterile plants. For commercial scale production of hybrid seeds in most plant species, wind or insect pollination is preferred. Tighter control of the pollination process can be achieved by using various methods to make one pool of plants male sterile and the other male fertile pollen donor. This can be achieved by hand detasseling, cytoplasmic male sterility, or controlling male sterility through various methods well known to experienced breeders. Examples of more sophisticated male sterility systems include those described by Brar et al., U.S. Pat. Nos. 4,654,465 and 4,727,219, and Albertsen et al., U.S. Pat. Nos. 5,859,341 and 6,013,859.

[0068] Backcrossing methods can be used to introduce genes into plants. This technique has been used for decades to introduce various traits into plants. An example of a description of this and other widely known plant breeding methodologies can be found in references, such as Neal (1988). In a typical backcrossing protocol, the original variety of interest (recurrent parent) is crossed to a second variety (non-recurrent parent) that carries only one gene of interest to be transferred. The progeny obtained from this cross is then crossed again to the recurrent parent: this process is repeated until a plant is obtained in which essentially all of the desired morphological and physiological characteristics of the recurrent parent are regained in the transformed plant in addition to the only transferred gene from the non-recurrent parent.

[0069] The selection and breeding techniques described above can result in a number of transgenic plants that are harvested in a conventional manner. The plant expressing the recombinant polypeptide, or whatever part expressing the recombinant polypeptide, can be used in a commercial process or the polypeptide can be extracted. When using the plant or part itself, the plant or part can be, for example, milled and then applied in a commercial process. Polypeptide extraction from biomass can be achieved by known methods. Downstream processing for any production system refers to all unit operations after product synthesis, in this case protein production in transgenic seeds (Kusnadi, AR, Nikolov, ZL, Howard, JA, 1997. Biotechnology and Bioengineering. 56:473-484). For example, the seeds can be processed so that either the whole seed can be ground into flour or fractionated so that the germ can be separated from the husk and endosperm. If germ is used, it is usually defatted using an extraction process and the remaining broken germ is ground into a meal or sieved flour. In some cases, the germ is used directly in the process or the protein can be extracted (see, for example, International Publication WO 98 / 39461). Extraction is generally performed in an aqueous buffer at a specific pH to enhance extraction of recombinant proteins and minimize extraction of native seed proteins. Subsequent protein concentration or purification can then follow.

[0070] The compositions and processes described herein are also for producing and administering vaccines to protect animals against Eimeria.

[0071] As used herein, the term "vaccine" refers to a pharmaceutical composition that contains at least one protective molecule that induces a protective response in a subject, and possibly, but not necessarily, one or more additional components that enhance the activity of said active ingredient. A vaccine may further contain additional components typical of pharmaceutical compositions. In another form, the immunologically active components of a vaccine may contain appropriate elements of said organism (subunit vaccine), whereby these elements are produced by disrupting the whole organism or a growing culture of such a microorganism and subsequent purification steps to obtain the desired structure, or by a synthetic process induced by appropriate manipulation of an appropriate system, such as, but not limited to, bacteria, insects, mammals or other species, and subsequent isolation and purification procedures, or by induction of said synthetic process in an animal that requires a vaccine by direct incorporation of genetic material using an appropriate pharmaceutical composition (polynucleotide vaccination). A vaccine may simultaneously contain one or more of the above elements.

[0072] The vaccine of the present invention may include pharma- ceutically acceptable carriers, excipients, carriers, stabilizers and / or diluents. Examples include, but are not limited to, wetting agents and lubricants, preservatives, lipids, stabilizers, solubilizers and emulsifiers. Carriers may be solvents or dispersion media, such as water, ethanol, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc.), suitable mixtures thereof, and vegetable oils. One possible carrier is a physiological salt solution. Examples of stabilizers include, for example, glycerol / EDTA, carbohydrates (such as sorbitol, mannitol, trehalose, starch, sucrose, dextran or glucose), proteins (such as albumin or casein) and protein degradation products (e.g., partially hydrolyzed gelatin).

[0073] It is possible to provide an adjuvant in the vaccine. Adjuvants enhance the immunogenicity of antigens, but are not necessarily immunogenic themselves. Adjuvants may act by retaining antigens locally near the site of administration, resulting in a depot effect that promotes a slow, sustained release of antigens to cells of the immune system. Adjuvants may also attract cells of the immune system to the antigen depot and stimulate such cells to elicit an immune response. Immune stimulants or adjuvants have been used for many years to improve the host immune response to, for example, vaccines. The vaccines of the present invention may be used in combination with adjuvants such as lipopolysaccharides, aluminum hydroxide and aluminum phosphate (alum), saponins complexed to membrane protein antigens (immunostimulating complexes), pluronic® polymers with mineral oil, inactivated mycobacteria in mineral oil, Freund's complete adjuvant, bacterial products such as muramyl dipeptide (MDP) and lipopolysaccharide (LPS), and lipid A, as well as liposomes. Desirable characteristics of an ideal adjuvant may include the following: (1) lack of toxicity; (2) ability to stimulate sustained immune responses; (3) ease of manufacture and stability on long-term storage; (4) ability to induce both CMI and HIR to antigens administered by various routes; (5) synergy with other adjuvants; (6) ability to selectively interact with populations of antigen-presenting cells (APCs); (7) ability to specifically induce an immune response specific to the appropriate T cell helper 1 (TH1) or TH2 cells; (8) ability to selectively increase appropriate antibody isotype levels (e.g., IgA) to an antigen. Adjuvants used with the compositions and methods of the invention need not have all of these characteristics to be used.

[0074] As used herein, an "immunogenic effective amount" refers to an amount effective to reduce, eliminate, treat, prevent, or control the symptoms of an infection, disease, disorder, or condition.

[0075] The amount administered depends on the subject being treated, including, for example, the ability of the individual's immune system to mount a protective response. The appropriate regimen for initial and booster doses is also variable, but may include an initial dose followed by subsequent doses. For example, it may be desirable to provide an initial dose of vaccine followed by a booster dose. The need to provide an effective amount of protective molecule must also be balanced against the cost of providing a larger amount of protective molecule. A cost-effective vaccine is one whose cost of producing it is less than the value that can be obtained from using it. Measuring and determining the effectiveness of any of the compositions and vaccines of the present invention can be accomplished by any of the many methods available to those skilled in the art.

[0076] In one embodiment, a simple and rapid method may be to perform a Western blot analysis of a sample candidate vaccine composition to quantitate the amount of polypeptide or fragment present in the sample. In one embodiment, the amount of polypeptide may be compared to a standard known to be effective for similar polypeptides from other biotypes, and a vaccine may be prepared or tested in test animals in which the level of polypeptide produced is at least equal to or greater than this standard.

[0077] The compounds described herein can be administered to a subject in a therapeutically effective dose to prevent Eimeria-associated disease. The dosage depends on the subject receiving the vaccine and factors such as the size, weight and age of the subject.

[0078] The precise amount of the immunogenic composition of the invention to be used in the formulation will depend on the route of administration and the nature of the subject (e.g., age, size, stage / level of disease) and should be determined according to the practitioner's judgment and each subject's circumstances in accordance with standard clinical techniques. An effective immunizing amount is an amount sufficient to treat or prevent Eimeria infection in a subject, e.g., an avian subject.

[0079] The immunogenicity of a composition can be determined by monitoring the immune response of a test subject after immunization with the composition using any immunoassay known in the art. The production of humoral (antibody) responses and / or cellular immunity can be interpreted as an indication of the immune response.

[0080] The immune response of the test subject can be analyzed by various approaches, such as the reactivity of immune serum obtained against the immunogenic conjugate, as assayed by known techniques, for example, enzyme-linked immunosorbent assay (ELISA), immunoblot, immunoprecipitation, virus neutralization, etc., or by any method known in the art, such as by culturing a sample from the subject to assay the level of infectious pathogen, for example virus level, by protecting the immunized host from infection by the pathogen and / or by reducing symptoms caused by infection by the pathogen in the immunized host, or by other techniques known in the art. The level of infectious pathogen can also be determined by measuring the level of the antigen against which the immunoglobulin is directed. A reduction in the level of infectious pathogen or an improvement in the symptoms of infectious disease indicates that the composition is effective.

[0081] The therapeutic agents of the present invention can be tested in vitro for the desired therapeutic or prophylactic activity prior to in vivo use in animals. For example, in vitro assays that can be used to determine whether administration of a particular therapeutic agent is indicated include in vitro cell culture assays in which appropriate cells from a cell line, or cells cultured from a subject with a particular disease or disorder, are exposed to or otherwise administered the therapeutic agent and the effect of the therapeutic agent on the cells is observed.

[0082] Additionally, a therapeutic agent may be assayed by contacting the therapeutic agent with cells (either cells cultured from a subject or cells cultured from a cultured cell line) that are susceptible to infection by an infectious disease agent but are not infected by the infectious disease agent, exposing the cells to the infectious disease agent, and then determining whether the infection rate of cells contacted with the therapeutic agent was lower than the infection rate of cells not contacted with the therapeutic agent. Infection of cells by an infectious disease agent may be assayed by any method known in the art.

[0083] Therapeutic agents can also be evaluated by measuring the levels of the molecule against which the antibody is directed in animal models and / or human subjects before, during or at suitable time intervals after treatment. Any changes in the amount of the molecule, or the absence of changes in the amount of the molecule, can be identified and correlated with the effect of treatment on the subject. The level of the molecule can be determined by any method known in the art.

[0084] After vaccinating animals against Eimeria using the methods and compositions of the present invention, any binding assay known in the art may be used to evaluate the binding of the resulting antibodies to a particular molecule. These assays may also be performed to select antibodies that show higher affinity or specificity for a particular antigen. As one measure of vaccine efficacy, ELISA may be performed on samples taken from vaccinated individuals to determine whether antibodies against the vaccine containing the sequence, derivative, homologue or variant or fragment thereof generated anti-polypeptide antibodies. The individual's sample is measured against a reference anti-polypeptide antibody. Analysis of symptoms and measurement of animal weight gain also demonstrated a reduction in the effects of the disease in the presence of a particular dose. Fluorescence focus neutralization assay is yet another assay to detect serum neutralizing antibodies and analyze the effectiveness of the vaccine and the particular dose. When testing animals administered the vaccine, for example, measuring the antibody response is also useful in determining the effectiveness of the vaccine. As described in the examples below, serum may be collected and titers may be measured as the reciprocal of the highest dilution at which hemagglutination is inhibited. Other measurements following vaccine administration may also be used to determine efficacy, pathology assessment, pathogen isolation, symptom measurement, and the general health and weight gain of the subject.

[0085] Vaccine efficacy can also be assessed quantitatively (e.g., reduction in the proportion of diseased tissue compared to an appropriate control group) or qualitatively (e.g., isolation of virus from blood, detection of viral antigens in tissue samples by assay methods, etc.). Disease symptoms can be assessed quantitatively (e.g., body temperature / fever), semi-quantitatively (e.g., severity of pain), or qualitatively (e.g., presence or absence of one or more symptoms or reduction in the severity of one or more symptoms). Clearly, those skilled in the art have many different options available for measuring vaccine efficacy. A protective period of more than 7 days has been achieved after at least one challenge or exposure to a pathogenic microorganism, and protection of at least 2 weeks, at least 20 days, 21 days, 22 days, 23 days, 24 weeks, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 31 days, 32 days, 33 days, 34 days, 35 days, 36 days, 37 days, 38 days, 39 days, 40 days, 41 days, 42 days, 43 days, 44 days, 45 days, 46 days, 47 days, 48 ​​days, 49 days, 50 days, 51 days, 52 days, 53 days, 54 days, 55 days, 56 days, 57 days, 58 days, 59 days, 60 days or more has been achieved using the present invention. Such protective periods are also provided when the present invention is used with other animals. Protective responses are also shown herein in one embodiment to be specific to the disease as opposed to another disease, thus demonstrating specific memory.

[0086] The vaccine may be administered using standard methods, including intranasal, oral and / or parenteral (e.g., intramuscular) administration. For example, a vaccine containing an Eimeria vaccine protein may be administered intramuscularly one or more times. In another embodiment of the method, for example, the vaccine is administered orally one or more times. In an alternative embodiment, the vaccine may be administered orally after oral administration and / or at least once before administering the vaccine intramuscularly. Corn kernels may be provided to the animals as food, thereby reducing costs and antigen loss that may occur from further processing.

[0087] The following are provided by way of example without intending to limit the scope of the invention. All references cited herein are hereby incorporated by reference. EXAMPLES

[0088] Example 1 Plant-produced proteins show great promise in that they can accumulate recombinant proteins at levels much higher than microorganisms for some difficult-to-degrade proteins and are generally the cheapest protein source. 6~8 However, not all plant systems are created equal. There is wide variation in cost, scalability, substances that interfere with purification such as proteases, lignin and phenols, storage characteristics and safety with respect to allergenic, carcinogenic or toxic substances in the host. These characteristics have been reviewed elsewhere. 7 , maize has emerged as one of the leading systems leading to the recent commercialization of several recombinant proteins.

[0089] This is evidenced by the high accumulation observed in some vaccine candidates. Reports of hepatitis B surface antigen (HBsAg) accumulation in edible plant tissues (other than maize) vary dramatically, with banana fruit being one of the lowest at 0.001 μg / gram fresh weight and potato being one of the highest at 8 μg / gram fresh weight. 9、10In the maize-based system developed by the inventors, HBsAg is expressed at over 200 μg / g. This high level of antigen in the grain results in raw material costs of less than $0.01 / dose, even when accumulation is only 10 μg / g. However, purification costs can account for 90% of the product and are inversely proportional to the concentration in the biomass. Cost models indicate that levels as low as 10 μg / g may be economically feasible, but in most cases levels of >50 mg / kg are targeted to keep purification costs low. This level is about 5 times higher than has been achieved with the microorganism. The inventors have shown that the same maize-based system can be used to express Eimeria vaccine proteins at similarly high levels and low costs.

[0090] Indeed, processed corn kernels are cost-effective, heat stable, and suitable for direct feeding to animal subjects. Incorporation of subunit Eimeria vaccine proteins, such as Eimeria3-1e, Gam82, and E-1Fa, into corn kernels also allows for bioencapsulation that protects the subunit vaccine proteins from degradation in the digestive system upon oral delivery to subjects. Furthermore, Eimeria proteins bioencapsulated in corn kernels result in more effective vaccines when delivered orally. For example, the present invention is expected to provide greater than 85% protection in chickens against challenge with Eimeria species.

[0091] Therefore, when we set out to accumulate Eimeria antigens in corn to produce candidate Eimeria vaccines, we used several approaches to accumulate high levels, as described herein.

[0092] Example 2 Creating molecular constructs To develop an Eimeria vaccine, maize was transformed with four constructs encoding the Eimeria vaccine proteins 3-1e, Gam82 and / or EF-1 polypeptides (Figure 1). The four constructs included EIA (expressing 3-1e and Gam82), EIB (expressing EF-1a), EID (expressing 3-1e and EF-1a), and EIE (expressing Gam82). The sequences of 3-1e (GenBank AAD39362), Gam82 (GenBank XP_013333566) and EF-1a (GenBank XP_013234784) were optimized for maize codon usage and expressed in maize in a manner similar to our previously published work. 11 The open reading frame was cloned using the potato protease inhibitor II (pinII) termination sequence, which confers high expression in our previously published work. 12、13 is linked to an embryo-preferred promoter in a manner similar to that of 14 In particular, the terms "Eimeria vaccine protein," "Eimeria protein," and "Eimeria antigen" are used interchangeably herein. Additionally, "EF-1a" and "EF-1 alpha" are used interchangeably herein.

[0093] Eimeria vaccine proteins 3-1e, EF-1α and / or Gam82 may have a targeting signal directed to the apoplast / cell wall using the well-characterized barley alpha-amylase ("BAASS") targeting sequence, or to the endoplasmic reticulum (ER) using the previously described KDEL C-terminal targeting signal sequence. 15 Both subcellular localization strategies result in high levels of expression. Indeed, the apoplast and the endoplasmic reticulum have proven to be consistent cellular locations for accumulating antigens within maize kernels.

[0094] Constructs EIA and EIB were targeted to the apoplast / cell wall, as shown in Figure 1. EID and EIE were targeted to the endoplasmic reticulum (Figure 1). A cell / organelle targeting sequence (BAASS:cell wall / apoplast) was included in all constructs, as shown in Figure 1 and Figures 5-7. A C-terminal sequence (KDEL:ER) was included in the codon-optimized EID and EIE constructs. To increase mRNA stability, the untranslated 3' region of potato protease inhibitor II (pinII) was also added to all constructs as a transcription termination element.

[0095] An embryo-preferred promoter (Pr44: seed embryo) was used to drive high gene transcription and subsequent high-yield protein production in all constructs (also referred to herein as "expression cassettes"). Maximum protein expression levels were achieved using promoter pr44 in constructs containing a single transcription unit. Notably, promoter pr44 contains a duplication of a portion of the well-characterized maize globulin-1 promoter.

[0096] Transformation After treatment, the constructs were transferred to an appropriate Agrobacterium strain for maize transformation. The constructs were transferred to Agrobacterium for maize transformation by a combination of triparental mating and electroporation as previously described. 13 Briefly, a binary vector system for Agrobacterium was used and then transferred to an Agrobacterium strain capable of transforming maize. Transformed cell lines were identified by herbicide resistance and allowed to mature into plants.

[0097] Specifically, corn was transformed by first processing at least 2000 corn embryos per construct, followed by regenerating T0 generation plants. The plants were then transferred to a greenhouse and crossed with HiII untransformed corn to obtain T1 generation seeds. The number of regenerated T1 generation plants from which seeds were obtained for each construct is listed below. In particular, the expression of individual transformation events in plants has been shown to vary greatly between independent events, and therefore, we aim to have many events per construct and many plants per event mature and set fruit. 89 plants representing EIA-18 events 50 plants representing the EIB-4 event 88 plants representing the EID-15 event 37 plants representing EIE-6 events

[0098] Protein detection A large number of single seeds of T1 generation plants were assayed for expression, yielding up to 360 samples per construct. Crude extracts were tested from ground whole seeds. Optimal results were obtained with extraction in 1x PBS + 0.05% Tween®. Robust expression of the three recombinant proteins (e.g., Eimeria vaccine protein 3-1e, EF-1 alpha and Gam82) was achieved, as shown by representative Western blots (Figures 2-4). Western blots using anti-Eimeria protein antibodies were performed on selected high-expressing lines to confirm the size of the proteins and that they were immunoreactive with Eimeria proteins.

[0099] 3-1e Figure 2 shows a Western blot of seed grain expression of 3-1e in constructs EIA and EID. 50 whole seeds were ground and extracted with 100 mg flour / 1 mL 1x PBS + 0.05% Tween®. Each sample represents seeds from one plant from an independent insertion event from either construct EIA or EID, with HiII as a non-transformed negative control. Extracts were loaded on a standard SDS PAGE gel under reducing conditions. Blots were transferred to membranes by iBlot and incubated overnight with mouse mAb and 3-1e at 1:1000. Blots were developed using alkaline phosphatase and anti-mouse IgG conjugated to BCIP / NBT. Expression was estimated at 100 mg / kg. The band migrating larger than expected in lane 1 is due to 3-1e aggregation and / or post-translational modifications.

[0100] EF-1a Figure 3 shows a Western blot of seed grain expression of EF-1a in constructs EIB and EID. 50 whole seeds were ground and extracted with 100 mg flour / 1 mL 1x PBS + 0.05% Tween® (pH 7.0). Each sample represents seeds from one plant from an independent insertion event from either construct EIB or EID, with HiII as a non-transformed negative control. Extracts were loaded on a standard SDS PAGE gel under reducing conditions. Blots were transferred to membranes by iBlot and incubated overnight with rabbit Ab against EF-1a. Blots were developed using anti-rabbit conjugated to alkaline phosphatase and BCIP / NBT. Some degradation in transport was observed in the 1 μg of positive control recombinant protein utilized. The predicted size was 51 kD, but the recombinant protein migrated larger due to post-translational modifications (Figure 3). The recombinant protein target appears at approximately 82 kD (arrowed in FIG. 3) due to the post-translational modification. Expression is estimated to be an average of 50 mg / kg. Notably, endogenous maize EF-1a is approximately 70% identical to Eimeria EF-1a, so some cross-reactivity with polyclonal antibodies may be observed. We plan further analysis using single seeds.

[0101] Gam82 Figure 4 is a Western blot of seed grain expression of Gam82 in constructs EIA and EIE. 50 whole seeds were ground and extracted with 100 mg flour / 1 mL 1x PBS + 0.05% Tween®. Each sample represents seeds from one plant from an independent insertion event from either construct EIA or EIE, with HiII as a non-transformed negative control. Extracts were loaded on a standard SDS PAGE gel under reducing conditions. Blots were transferred to membranes by iBlot and incubated overnight with rAb against Gam82. Blots were developed using anti-rabbit conjugated to alkaline phosphatase and BCIP / NBT. There is a strong signal in the control E. coli recombinant protein and some EIA strains (arrows), but they migrated larger than expected due to post-translational modifications. The predicted size is approximately 67 kD. Based on this gel and comparison with standards, expression is estimated to be up to 600 mg / kg. We contemplate further analysis using single seeds.

[0102] Quantification of expression by ELISA Expression levels of Eimeria vaccine protein 3-1e polypeptide, Gam82 polypeptide and / or EF-1 polypeptide in seeds can also be quantified by ELISA in a manner similar to that previously described. 16Briefly, individual seeds from each plant are extracted separately in PBS + 1% Triton® X-100 and the protein content in the extracts is measured by the Bradford method. 1 μg equivalent of extracted protein is then tested in an ELISA (Eimeria proteins and anti-Eimeria protein antibodies can be induced by standard methods). Purified Eimeria proteins are used as standards and control seed extracts are used as negative controls. Six individual seeds per plant can be analyzed and the expression levels in the positive seeds can be used to calculate the average antigen expression level in each plant, which can be used to calculate the average expression level of each event, which can finally be used to calculate the average expression of the various constructs. This approach can be used to select the constructs and transformation events with the highest expression. This procedure has been statistically analyzed previously and shown to be sufficient for the selection of the highest expressing lines.

[0103] Prepare materials for further testing From the constructs, we selected the strains with the highest expression levels of Eimeria vaccine protein 3-1e polypeptide, Gam82 polypeptide and / or EF-1a polypeptide. These constructs can be used to test the Eimeria vaccine proteins individually. Seeds can be propagated to select high-expressing stable lines without unintended plant alterations. Material from the next generation can be used for the studies described herein. The Eimeria vaccine protein is expressed in the high-expressing stable lines at a level of at least 1 mg / kg of seed. Kernels containing the Eimeria vaccine protein can be used to purify the antigen using an antibody affinity column as described above. 17Briefly, anti-Eimeria IgG may be coupled to cyanogen bromide-activated Sepharose beads, which may then be used to create a column. The column may be equilibrated with PBS, and the extract may be loaded onto the column, washed with PBS, and eluted with high salt at pH 3. The eluate may be run on a Western blot to confirm immunoreactivity and on a Coomassie stained gel to obtain an estimate of purity. In addition to Western blots, n-terminal sequencing and lectin binding may be used to confirm glycosylation, and spectrophotometric analysis may be used to compare plant-produced proteins to those of Eimeria proteins produced from other recombinant hosts. Kernels from the lines may be processed and used to form wafers as previously described. 18 .

[0104] Array Details (Maize codon optimized sequence) Start Methionine Stop codon Underlined: important restriction sites (NcoI, PacI, HindIII) Italics: signal sequences (barley alpha amylase (BAASS) and / or KDEL) SEQ ID NO: 1 3-1e polypeptide sequence of GenBank AAD39362 (The 3-1e polypeptide is also referred to as "3-1e," "3-1e protein," or "Eimeria acervulina 3-1e / profilin protein sequence.") SEQ ID NO: 2 BAASS 3-1e nucleotide sequence [ka] SEQ ID NO: 3 BAASS KDEL 3-1e Nucleotide Synthesis [ka] SEQ ID NO: 4 EF-1a polypeptide sequence from GenBank XP_013234784 (The EF-1a polypeptide is also referred to as "EF-1a," "EF-1a protein," or "Eimeria tenella EF-1a protein sequence.") SEQ ID NO: 5 EF-1a nucleotide sequence [ka] SEQ ID NO:6 BAASS EF-1a KDEL nucleotide synthesis [ka] SEQ ID NO: 7 Gam82 polypeptide sequence from GenBank XP_013333566 (The Gam82 polypeptide is also referred to as "Gam82", "Gam82 protein" or "Eimeria maxima Gam82 protein sequence") SEQ ID NO:8 BAASS Gam82 nucleotide synthesis [ka] SEQ ID NO:9 BAASS KDEL Gam82 Nucleotide Synthesis [ka]

[0105] Example 3 Obtaining protection from Eimeria challenge in chickens. Corn kernel preparation for feeding trials Seeds from the lines with the highest levels of Eimeria vaccine proteins 3-1e, EF-1 alpha and Gam82 were bred to obtain grain for feeding trials. The grain can be enriched for the germ fraction using a customized germ removal system. In one embodiment, the antigen is preferentially expressed in the embryo and enrichment can result in a 3-7 fold increase in antigen concentration. This approach has been used for many other proteins to increase recombinant protein concentration. The germ fraction can then be ground into flour and defatted using SFE to increase stability. The germ flour can be incorporated into standard chicken feed using a target dose of 1 mg. The material can then be tested for antigen stability in dry feed at ambient temperature. In another embodiment contemplated by the inventors, the Eimeria vaccine proteins can be delivered to the subject animal individually or in combination. For example, by mixing various lines, two or more Eimeria vaccine proteins (e.g., 3-1e, EF-1 alpha and Gam82) can be delivered to the subject simultaneously. Strains may also be crossed to provide additional means of delivering target antigens.

[0106] Evaluation of an orally delivered Eimeria subunit vaccine in broiler chickens: Feeding and challenge: Feeding and dosing can be configured to achieve a dose sufficient to protect target birds at the lower end of consumption. The expected dosing is such that herd immunity can be achieved in >85% of protected birds. Based on animal models, this should be sufficient to prevent economic losses and stop the spread of the disease.

[0107] The protective efficacy of Eimeria corn-derived antigens delivered in complete diets was evaluated in a vaccination-challenge study. Three-day doses of vaccine diet were administered at 7, 8, and 9 days of age and at 14, 15, and 16 days of age. There were also non-vaccinated, non-challenged and non-vaccinated, challenged treatment groups. Details of the experimental design are summarized in Table 1 below. [Table 1]

[0108] A large dose mixed-species Eimeria challenge consisting of approximately 35,000 oocysts of E. acervulina, E. maxima and E. tenella was administered at 28 days of age (study day 21). Lesion scoring was performed 6 and 12 days after challenge. Standard statistical analyses were used to compare treatments for mean lesion score (0-4, with lesions scoring 2 resulting in a significant reduction in production), oocyst output, body weight and feed conversion.

[0109] Results of the study showed a statistically significant protective response for the 3-day high-dose vaccination regimen against E. acervulina challenge. Mean lesion scores were reduced from 1.9 in the positive control group to 1.5 in the high-dose group (Figure 9A). When a categorical analysis of birds with lesion scores above or below 2 was applied, vaccinated groups tended to have fewer birds with lesions above 2 compared to the positive controls for all Eimeria species.

[0110] Performance data demonstrated that vaccination did not adversely affect body weight or feed conversion compared to the negative control group. Vaccination did not provide statistically significant protection against post-challenge weight loss or reduced feed conversion. However, post-challenge weight gain was better in the high-dose vaccinated treatment group compared to the control group (Figure 9B).

[0111] Oocyst shedding after challenge was also examined as a measure of immunity. Reduction in oocyst shedding reduces subsequent exposure to infection within flocs and reduces carryover of oocysts between flocs. The mean number of oocysts per gram of excretion (OPG) was lower in both vaccine treatment groups at day 6 post-challenge compared to the positive control, with reductions of 85% and 75% in the vaccinated groups (Figure 9C). These results are encouraging when compared to the literature for subunit vaccines.

[0112] Overall, the orally delivered Eimeria subunit vaccine demonstrated safety and showed favorable immunogenicity results. Future studies will be performed to optimize antigen dose, timing of vaccine administration, and further characterize immune responses.

[0113] As described herein, the above examples demonstrate how oral administration of maize-produced Eimeria antigens can protect chickens against Eimeria challenge.

[0114] conclusion Disclosed herein is a vaccine platform for developing an orally delivered, low-cost, rapidly scalable Eimeria vaccine in chickens and other animals. To support this premise, we have demonstrated that: 1) plants from 43 independent transformation events were regenerated that express three apoplast or ER-targeted proteins, 3-1e, EF-1alpha, and Gam82; 2) this is used to demonstrate that oral administration of corn-produced Eimeria antigens can protect chickens from Eimeria challenge with the cost and scale-up advantages from production in plants. The initial development of an effective vaccine against avian coccidiosis may provide broad protection against some of the most common Eimeria species. Western blot analysis was performed on bulk seeds from individual events. Accumulation levels averaged 50 mg / kg whole seed for each antigen. This range is optimal for conducting animal studies without selection. Additionally, we contemplate scaling up the highest production lines so that we can enrich for germ and bulk-up kernels. The inventors are now in a position to scale up production, starting with just a single ear of corn, to billions of doses per year at a fraction of the cost of traditionally produced vaccines. A heat-stable oral vaccine also eliminates the need for a cold chain and personnel for administration, allowing an easier and faster route towards mass immunization of poultry.

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[0116] Sequence Overview SEQ ID NO: 1 3-1e polypeptide sequence of AAD39362 SEQ ID NO: 2 BAASS 3-1e nucleotide sequence SEQ ID NO: 3 BAASS KDEL 3-1e Nucleotide Synthesis SEQ ID NO: 4 EF-1a polypeptide sequence from GenBank XP_013234784 SEQ ID NO: 5 EF-1a nucleotide sequence SEQ ID NO:6 BAASS EF-1a KDEL nucleotide synthesis SEQ ID NO: 7 Gam82 polypeptide sequence from GenBank XP_013333566 SEQ ID NO:8 BAASS Gam82 nucleotide synthesis SEQ ID NO:9 BAASS KDEL Gam82 Nucleotide Synthesis

Claims

1. 1. A method for generating a protective response against Eimeria in an animal, comprising: a) orally administering to said animal a plant or plant product comprising at least one Eimeria vaccine protein, or a composition comprising a sequence having at least 90% identity thereto or a functional fragment of said at least one Eimeria vaccine protein expressed in seeds of said plant at a level of at least 10 mg / kg; b) generating a protective response in said animal against said Eimeria; A method comprising:

2. The method of claim 1 , wherein the composition comprises plant material or plant tissue.

3. The method of claim 1 , wherein the composition comprises a seed or a seed embryo.

4. The method of claim 1 , wherein the protective response comprises a serum antibody response in the animal.

5. 5. The method of claim 4, wherein the serum antibody response is at least 4 times greater than the serum antibody response in an animal that is not administered the vaccine.

6. 2. The method of claim 1, wherein the at least one Eimeria vaccine protein comprises a 3-1e polypeptide, a Gam82 polypeptide, and / or an EF-1a polypeptide.

7. 7. The method of claim 6, wherein the at least one Eimeria vaccine protein is targeted to the apoplast or the endoplasmic reticulum.

8. 1. A vaccine comprising a plant-produced polypeptide of an Eimeria vaccine protein, (a) a promoter that directs expression preferentially in plant embryonic tissue; (b) a nucleic acid molecule encoding an Eimeria vaccine protein as disclosed herein, or a sequence having at least 90% identity thereto, or a functional fragment thereof, operably linked to said promoter; (c) a nucleic acid molecule that targets expression of the polypeptide in the apoplast or endoplasmic reticulum of the plant and expresses the Eimeria vaccine protein in the plant at a level of at least 1 mg / kg of seeds of the plant. A vaccine comprising a plant or plant product comprising a construct comprising the

9. 9. The vaccine of claim 8, wherein the construct comprises at least two Eimeria vaccine proteins, the Eimeria vaccine proteins comprising a 3-1e polypeptide, a Gam82 polypeptide, or an EF-1a polypeptide.

10. 9. The vaccine of claim 8, wherein the at least one Eimeria vaccine protein is not optimized for expression in the plant.

11. 9. The vaccine of claim 8, wherein the construct comprises pr44-BAASS:3-1e pr44-BAASS:Gam82, pr44-BAASS:EF-1a, pr44-BAASS:3-1e KDEL pr44-BAASS:EF-1a KDEL, or pr44-BAASS:Gam82 KDEL.

12. 9. The vaccine of claim 8, wherein the Eimeria vaccine protein comprises a 3-1e polypeptide, said 3-1e polypeptide comprising SEQ ID NO:1 or encoded by SEQ ID NO:2 or 3.

13. 9. The vaccine of claim 8, wherein the Eimeria vaccine protein comprises an EF-1a polypeptide, said EF-1a polypeptide comprising SEQ ID NO:4 or encoded by SEQ ID NO:5 or 6.

14. 9. The vaccine of claim 8, wherein the Eimeria vaccine protein comprises a Gam82 polypeptide, said Gam82 polypeptide comprising SEQ ID NO:7 or encoded by SEQ ID NO:8 or 9.

15. 1. A method for expressing a polypeptide of an Eimeria vaccine protein or a functional fragment thereof, comprising: (a) a promoter that directs expression preferentially in plant embryonic tissue; (b) a nucleic acid molecule encoding an Eimeria vaccine protein as disclosed herein, or a sequence having at least 90% identity thereto, or a functional fragment thereof, operably linked to said promoter; (c) a nucleic acid molecule that targets expression of said polypeptide in the apoplast and / or endoplasmic reticulum of said plant and expresses said Eimeria vaccine protein in said plant at a level of at least 1 mg / kg of seeds of said plant. introducing into the plant a construct comprising:

16. The method of claim 15, wherein the construct comprises a construct EIA, a construct EIB, a construct EID and / or a construct EIE.

17. 16. The method of claim 15, wherein the Eimeria vaccine protein comprises a 3-1e polypeptide, the 3-1e polypeptide comprising SEQ ID NO:1 or encoded by SEQ ID NO:2 or 3.

18. 16. The method of claim 15, wherein the Eimeria vaccine protein comprises an EF-1a polypeptide, wherein the EF-1a polypeptide comprises SEQ ID NO:4 or is encoded by SEQ ID NO:5 or 6.

19. 16. The method of claim 15, wherein the Eimeria vaccine protein comprises a Gam82 polypeptide, wherein the Gam82 polypeptide comprises SEQ ID NO:7 or is encoded by SEQ ID NO:8 or 9.

20. 16. The method of claim 15, wherein the Eimeria vaccine protein is expressed in the plant or plant part at a level of at least 50 mg / kg.

21. 16. The method of claim 15, wherein the Eimeria vaccine protein or fragment thereof is expressed in the plant or plant product at a level of at least 10 mg / kg of whole seed.

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