AU2015202009B2 - Compositions and methods of enhancing immune responses to eimeria - Google Patents

Abstract

Abstract Vaccines comprising TRAP polypeptides and Salmonella enteritidis vectors comprising TRAP polypeptides are 5 provided. The vaccines may also include a CD 154 polypeptide capable of binding to CD4Q. Also provided are methods of enhancing an immune response against Apicomplexan parasites and methods of reducing morbidity associated with infection with Apicomplexan parasites. 6390642_1 (GHMatters) P84253.AU 1 PETERB 14/04115

Description

EDITORIAL NOTE 2015202009

There are 18 pages of Description The first page is not numbered

COMPOSITIONS AND METHODS OF ENHANCING IMMUNE RESPONSES TO 2015202009 21 Apr 2015

EIMERIA CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application Serial No. 60/984,612, filed November 1,2007. which is incorporated hei'ein by reference in its entirety.

The present application is a divisional application of Australian patent application no. 2008318357, whicli is incorporated herein by reference in its'entirety.

INTRODUCTION

Coccidiosis, an infectious disease of poultry, swine, and cattle caused by the Apicomplexan protozoal parasite Eimeria, presents problems worldwide. C.occidiosis is among the top ten infectious diseases of poultty in terms of its economic impact on the poultry indtistry. Other members O'ftlie Apic.omplexan family- also cause disea.se, including Plasmodium, Cryptosporidium and Toxoplasma which are the causative agents of malaria, cryptosporidiosis and toxoplasmosis, respectively. The vaccines currently available against Eimeria are based on controlled low dosage O'f essentially folly,' virulent but drug-sensitive Eimeria parasites. Vaccination with current Eimeria-based vaccines produc.es substantial vaccine-reaction moi-bidity and economic losses in vaccinated flocks. Tlitis an effective, low vil-ulence vaccine against Eimeria is needed. An effective vaccine for Eimeria may also piove useful as a vaccine against other Apicomplexan parasites.

SUMMARY A vaccine comprising a first polyitucleotide seqtien.ce encoding a TRAP polypeptide or an immunogenic fragment thereof is disclosed. Tlie TRAP polypeptide may comprise comprises SEQ ID ΝΟ:1, SEQ ID NO:2, or SEQ ID NO:3, or a.n immunogenic fragment thereof. The vaccines optionally -further include a second polynuc-leotide seqtience encoding a CD 154 polypeptide capable ofbinding CD40. Tlie CD154 polypeptides include fewer than 50 amino acids and comprise amino acids 140-149, or a homolog thereof.

Vaccines according to the present invention may be comprised withilt a vec.toi', such as a virus, bacterium, or liposome. In one aspect, a vaccine c.omprising a Salmonella

S39081SJ (GHIters) Ρβ4253.Α٧.1 PETERB -2- 2015202009 21Aug2017 enteritidis comprising a first polynucleotide sequence encoding a TRAP polypeptide is provided.

In still another aspect, the invention includes methods of enhancing the immune response against an Apicomplexan parasite in a subject by administering a vaccine according to the present invention.

In a still firrther aspect, the invention includes methods of reducing morbidity associated with infection with an Apicomplexan parasite in a subject by administering a vaccine according to the present invention.

The present invention as claimed herein is described in the following items 1 to 23: 1. A vaccine comprising a first polynucleotide sequence encoding a TRAP polypeptide comprising SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, an immunogenic fiagment ofSEQ ID ΝΟ:1, an immunogenic fragment ofSEQ ID NO:2, or an immunogenic fiagment ofSEQ ID NO:3, and a second polynucleotide sequence encoding a CD154 polypeptide capable of binding CD40, the CD154 polypeptide having fewer than 50 amino acids and comprising amino acids 140-149 ofSEQ ID NO:4 or a homolog thereof. 2. The vaccine of item 1, wherein the TRAP polypeptide is SEQ ID NO:2, or an immunogenic fragment of 6 or more consecutive amino acids ofSEQ ID NO:2. 3. The vaccine of item 1 or 2, wherein the CD154 polypeptide comprises SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, or SEQ ID NO:9. 4. The vaccine of any one of items 1-3, wherein the vaccine comprises more than one copy of the first polynucleotide sequence, more than one copy of the_second polynucleotide sequence, or more than one copy of the first polynucleotide sequence and more than one copy of the second polynucleotide sequence. 5. The vaccine of any one of items 1-4, wherein the first polynucleotide sequence is linked in the same reading frame to the second polynucleotide sequence. 6. The vaccine of any one of items 1-5, wherein the first polynucleotide and second polynucleotide are comprised within a vector. 7. The vaccine of item 6, wherein the vector is selected from the group consisting of a vims, a bacterium, and a liposome. 9346396-1 (GHMatters) Ρ84253.Α٧.1 2a 2015202009 21Aug2017 8. The vaccine of item 7, wherein the vector is a bacterium. 9. The vaccine of item 8, the bacterium comprising the TRAP polypeptide on its surface. 10. The vaccine of item 8 or 9, wherein the bacterium is selected from the group consisting of Salmonella species. Bacillus species, Escherichia species, and Lactobacillus species. 11. The vaccine of item 10, wherein the bacterium is Salmonella enteritidis selected from strains deposited as ATCC ΡΤΑ-7871, ATCC ΡΤΑ-7872 andATCC ΡΤΑ-7873. 12. The vaccine of any one of items 1-11, wherein the first polynucleotide and the second polynucleotide are inserted into a polynucleotide sequence encoding an external portion of a transmembrane protein. 13. A vaccine comprising a variant of Salmonella enteritidis 13Α comprising a first polynucleotide sequence encoding a TRAP polypeptide comprising SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3, and a second polynucleotide sequence encoding a CD 154 polypeptide capable of binding CD40, the CD 154 polypeptide having fewer than 50 amino acids and comprising amino acids 140-149 of SEQ ID NO: 8 or a homolog thereof 14. A method of enhancing the immune response against an Apicomplexan parasite in a subject comprising administering to the subject the vaccine of any one of items 1-13 in an amount effective to enhance the immune response of the subject to the Apicomplexan parasite. 15. The method of item 14, wherein the vaccine is administered by a method selected from the group consisting of oral, intranasal, parenteral, and in ovo. 16. The method of any one of items 14 or 15, wherein the enhanced immune response comprises an enhanced antibody response or an enhanced T cell response. 17. The method of any one of items 14-16, wherein the subject is member of a poultry species or a mammal. 18. The method of any one of items 14-17, wherein the vaccine is killed prior to administration to the subject. 19. The method of any one of items 14-18, wherein the vaccine is not capable of replicating in the subject. 20. The method of any one of items 14-19, wherein the Apicomplexan parasite is selected from Ik gup cowim؟0 ؛؟Eimeria, Plasmodium, Toxoplasma, هااًه Cryptosporidium. .6396-1 (GHMallers) PW253.AU.1 2b 2015202009 21Aug2017 21. A method of reducing morbidity associated with infection with Apicomplexan parasite in a subject comprising administering to the subject the vaccine of any one of items 1-13 in an amount effective to enhance the immune response of the subject to the Apicomplexan parasite. 22. Use of a vaccine of any one of items 1-13 in the manufacture of a medicament for enhancing the immune response against an Apicomplexan parasite in a subject. 23. Use of vaccine of any one of items 1-13 in the manufacture of a medicament for reducing morbidity associated with infection with Apicomplexan parasite in a subject. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 depicts the scheme for making site-directed mutations in Salmonella enteritidis. Figure 2 depicts the design scheme of the overlapping extension PCR method used to generate the TRAP and TRAP-CD154 insertions into loop 9 of the lamB polynucleotide.

Figure 3 is a bar graph showing the percent mortality at five days post-infection with Eimeria maxima after inoculation with a Salmonella vector expressing the indicated Eimeria TRAP sequence.

DETAILED DESCRIPTION

Recombinant DNA technologies enable relatively easy manipulation of many bacterial and viral species. Some bacteria and vimses are mildly pathogenic or non-pathogenic, but are capable of generating a robust immune response. These bacteria and virnses make attractive vaccines for eliciting an immune response to antigens. Bacterial or viral vaccines may mimic a natural infection and produce robust and long lasting mucosal immunity. Vaccines are often relatively inexpensive to produce and administer. In addition, such vectors can often carry more than one antigen and may provide protection against multiple infectious agents. 9346396-1 (GHMatters) Ρ^253.Α٧.1 - 2c - 2015202009 27 Mar 2017

In one aspect, a vaccine comprising a first polynucleotide sequence encoding a TRAP polypeptide or an immunogenic fragment thereof is provided. The TRAP polypeptide may comprise SEQ ID NO: 11 or an immunogenic fragment of SEQ ID NO: 11. A vaccine includes any composition comprising a polynucleotide encoding an antigenic polypeptide that is capable of eliciting an immune response to the polypeptide. In another aspect, the use of vectors, such as bacteria] vectors, for vaccination and generation of immune responses 87^789-1 (GHMallers) P84253.AU! 2015202009 21 Apr 2015 -3- against Eirneria or other Apicomplexan parasites such as Plasmodium (the causative agent of malaria). Toxoplasma and Cryptosporidium is disclosed. Salmonella strains make suitable vectors because bacterial genes may be mutated or attenuated to create bacteria with low to no pathogenesis to the infected or immunized subject, while maintaining immunogenicity. A high molecular mass, asexual stage antigen from Eirneria maxima (EmTFP250) was demonstrated to be a target for maternal antibodies produced by breeding hens infected with this protozoan parasite. Analysis of the amino acid sequence of the antigen revealed a novel member of the TRAP (thrombospondin-related anonymous protein) family, containing 16 thrombospondin type-1 repeats and 31 epidermal growth factor-like calcium binding domains. EmTFP250 or TRAP also contains two low complex, Irydrophilic regions ricli in. glutamic acid and glycine residues, and a ttansmembrane domain/cytosolic tail associated with parasite gliding motility that is liighly conserved within apicomplexan microneme proteins. Several potential epitopes were selected and are identified in SEQ ID NO:l-3 and 11. Due to the conserved nature of this antigen, expression of these epitopes by a vector may induce protective immunity against multiple Apicomplexan parasites.

Salmonella may provide a usefirl vector because it can survive tire gastrointestinal tract; of the host and give rise to a mucosal immune response. Oral vaccines using a Salmonella vector produce a robust mucosal immune response and are relatively easy to administer to both animals and hum airs. However, many of the curcent Salmonella vaccine strains are not as effective in generating a strong protective immune I'esponse as compared to their more vinilent counterparts. Virulent strains provide a robust immune response but may also cause significant morbidity to the vaccinated subject. A Salmonella strain tlrat could be used for effective mucosal, e.g., oral, vaccination would provide a vector that could be used to t'eadily vaccinate a subject against one or more pathogenic agents, such as Apicomplexan parasites. A Salmonella enteritidis strain usefill as a vector, and various recombinant vectors made using this strain, are described. Specifically, a Salmonella enteritidis 13Α (SE13A) capable of expressing an exogenous TRAT polypeptide is provided. In addition, a vaccine and methods of enhancing an immune response in a subject by administering the vaccine comprising a TRAP polynucleotide sequence encoding a TRAP polypeptide and a CD154 polynucleotide sequence encoding a polypeptide of CD154 or a homolog thereof that is capable of binding to CD40 are disclosed. The vaccines may be used to enhance an imnrune response against Eirneria or anodrer Apicomplexan parasite, such as -4-

Toxoplasma or Cryptosporidium, or may be used to reduce the morbidity associated with an infection cattsed by an Apicomplexan parasite. A wild-type isolate of Salmonella, Salmonella enteritidis 13Α (SE13A) (deposited with the American Type Culture Collection (ATCC) on Septenrber 13, 2006, deposit number ΡΤΑ-7871), was selected based upon its unusual ability to cause mucosal colonization and sub-mucosal translocation in chickens, permitting robust presentation of associated antigens or epitopes in commercial clrickens. Importantly, this wild-type Salmonella isolate causes no clinically detectable disease or loss of performance in commercial chickens, indicating little disease-causing potential of the wild-type Salmonella in vertebrate animals.

The SE13A isolate may be further attenuated by inactivating at least one gene necessary for sustained replication of the bacteria outside of laboratory or manufacturing conditions. Attenuated or variant Salmonella strains that can be used as vectors are described below. SE13A was used to generate attenuated Salmonella strains to develop vaccines and generate enhanced immune responses. SE13A is invasive, non-pathogenic for poultry and causes no measurable morbidity. These featitres result in an enhanced immune response as compared to non-invasive bacterial vectors. Attenuation of SE13A by mutation of genes that limit the ability of the bacterium to spread may increase the safety of the vaccine. SE13A strains with mutations in aroA or htrA retain the ability to generate an immune I'esponse, but have limited replication in the host. Thus, the attenuation increases the safety of the vectoi-without compromising the immunogenicity.

Mutations may be made in a variety of other Salmonella genes including, but not limited to, cya, crp, asd, cdt, phoP, phoQ, ompR, outer membrane proteins, dam, htrA or other stress related genes, aro, pur and gua. As shown in the Examples, mutations in aroA and htrA were found to attenuate SE13A. The aro genes are enzymes involved in the shikimate biosyndiesis pathway or the aromatase pathway and aro mutants are auxotrophic for the aromatic amino acids tryptophan, tyrosine and phenylalanine. htrA is a stress response gene that encodes a periplasmic protease that degrades aberrant proteins. Mutants in htrA are also attenuated and display increased sensitivity to ltydrogen peroxide.

The mutations in aroA and htrA described in the Examples are deletion mutations, but the mutations can be made in a variety of ways. Suitably, the mutations are non-reverting mutations that cannot be repaired in a single step. Suitable mutations include deletions, inversions, insertions and substitutions. A vector may include more than one mutation, for -<؟- example a vector may contain mutations in both aroA and htrA. Methods of making such mutations are well known in the art.

Polynucleotides encoding TRAP polypeptide antigens and other antigens from any number of pathogenic organisms may be inserted into the vector (e.g.5 SE13A) and expressed by the bacteria. The expression of these polynucleotides by the vector will allow generation of antigeiric polypeptides following immunization of the subject. The polynucleotides may be inserted into the chromosome of the bacteria or encoded on plasmids or other extrachromosomal DNA. Those of skill in the art will appreciate that nunrerous methodologies exist for obtaining expression of polynucleotides in vectors such as Salmonella. The polynucleotides may be operably connected to a promoter (e.g., a constitutive promoter, an inducible promoter, etc.) by nrethods known to tlrose of skill in the art. Suitably, polynucleotides encoding TRAP antigens are inserted into a bacterial polynucleotide that is expressed. Suitably, the bacterial polynucleotide encodes a transmembrane protein, and the polynucleotide encoding the TRAP antigen is inserted into the bacterial polynucleotide sequence to allow expression of the TRAP antigen on the surface of the bacteria. For example, the polynucleotide encoding TRAP may be inserted in frame into the bacterial polynucleotide in a region encoding an external loop region of a transmembrane protein such that the bacterial polynucleotide sequence remains in frame. See Example 1.

Alternatively, the first polynucleotide encoding TRAP antigen may be inserted into a polynucleotide encoding a secreted polypeptide. Those of skill in the art will appreciate that the polynucleotide encoding the TRAP antigen could be inseited in a wide variety of bacterial polynucleotides to provide expression and presentation of the TRAP antigen to the immune cells of a subject treated with the vaccine. In the Examples, a first polynucleotide encoding the TRAP polypeptide was inserted into loop 9 of the lamB gene of SE13A. The polynucleotide encoding the TRAP antigen nray be included in a single copy or more than one copy. A bacterial vector containing multiple copies of the TRAP antigen inserted into loop 9 of وئ may also be generated. Alternatively, multiple copies of an epitope may be inserted into the bacterial vector at more than one location.

Suitably the first polynucleotide encodes a portion of the TRAP polypeptide or the entire TRAP polypeptide, file polynucleotide may be inserted into the vector. In the Examples, three polypeptides (SEQ ID ΝΟΤ-3) were incorporated into SE13A. Suitably, the portion of the TRAP polypeptide inserted into the vector is an inrmunogenic fi-agment. An immunogenic fragment is a peptide or polypeptide capable of eliciting a cellular or humoral immune response. Suitably, an immunogenic fragment of TRAP may be 6 or more consecutive amino acids, 10 or more amino acids, 15 or more amino acids or 20 or more anrino acids of tire frill-length protein sequence.

Other suitable epitopes for inclusion in a vaccine having TRAP comprised within a vector include, but are not limited to, polynucleotides encoding other Eimeria-related polypeptides. One of skill in the art will appreciate that a variety of sequences may be used in combination with any other antigen and may also be used in conjunction with polypeptides encoding immune stimulatory peptides suclr as a polypeptide of CD154.

As described in more detail below, a vaccine including a vector may include a CD154 polypeptide that is capable of Irinding CD40 in the subject and stimulating the subject to respond to the vector and its associated antigen. Involvement of dendritic cells (DCs) is essential for the iiritiation of a powerful immune response as they possess the unique ability to activate naive T cells, causing T cell expansion and differentiation into effector cells. It is the role of the DC, which is an antigen presenting cell (APC) found in viltually all tissues of the body, to capture mtigens, transport them to associated lymphoid tissue, and then present tliem to naive T cells. Upon activation by DCs, T cells expand, differentiate into effector cells, leave the secondary immune organs, arrd enter peripheral tissues. Activated cytotoxic T cells (CTLs) are able to destroy virus-infected cells, tumor cells or even APCs infected with intracellular parasites (e.g.. Salmonella) and have been shown to be critical in the protection against viral infection. CD40 is a member of the TNF-receptor family of molecules and is expressed on a variety of cell types, including professional antigen-presenting cells (APCs), such as DCs and B cells. Interaction of CD40 with its ligand CD154 is extremely important and stimulatory for both humoral and cellular immunity. Stimulation of DCs via CD40, expressed on tire surface of DCs, can be simulated by anti-CD40 antibodies. In the body, however, this occurs by interaction with the natural ligand for CD40 (i.e. CD154) expressed on the surface of activated T-cells. Interestingly, the CD40-binding regions of CD154 have been identified. Tire CD40-binding region of CD154 may be expressed on tire surface of a vector, such as a Salmonella vector, and results in an enhanced immune response against a co.presented peptide sequence.

As described above, polynucleotides encoding CD154 polypeptides may be inserted into the chromosome ofthe vector or maintained extrachromosomally. A CDl 54 polypeptide may be a portion of CD154 frill-length protein or the entire CDl 54 protein. Suitably, the ٠٦- CD154 polypeptide is capable of binding CD40. One of skill in the art will appreciate that these polynucleotides can be inserted in frame in a variety of polynucleotides and expressed in different paits of the vector or may be secreted. The polynucleotide encoding a CD154 polypeptide capable of enhancing the immune response to TRAP may also encode the TRAP antigen. The polynucleotide encoding a CD154 polynucleotide encoding the TRAP antigen, such tliat in the vector, the CD154 polypeptide and the TRAP antigen are present on the same polypeptide. In the Examples, a polynucleotide encoding a polypeptide of CD154 that is capable of binding to CD40 also encodes the TRAP antigen. See SEQ ID NOS: 1,2,3 and 11 in the attached sequence listing. In the Examples, the polynucleotides (SEQ ID ΝΟΤ3-15) encoding the TRAP antigen and the polynucleotide encoding the CD154 polypeptide are botlr inserted in loop 9 of the latnB gene. Those of skill in the art will appreciate that bacterial polynucleotides encoding other transmembrane proteins and other loops 0'fthe lamB gene may also be used.

As discussed above, a CD154 polynucleotide encoding a CD154 polypeptide that is capable of enhancing the immune response to the antigen may be included in the vaccine. Suitably, the CD154 polypeptide is fewer than 50 amino acids long, more suitably fewer than 40, fewer than 30 or fewei' than 20 amino acids in length. The polypeptide may be between 10 and 15 amino acids, between 10 and 20 amino acids or between 10 and 25 amino acids in length. The CD154 sequence and CD40 binding region are not highly conserved among tire various species. The CD154 sequences of chicken and human are provided in SEQ ID NO: 10 and SEQ ID NO:4, respectively.

The CD40 binding regions of CD154 have been determined for a number of spec-ies, including human, chicken, duck, mouse and cattle and are shoi in SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9, respectively. Although there is variability in the sequences in the CD40 binding region bet'ween species, the human CD154 polypeptide was able to enhance the immune response in chickens. Therefore, one may practice the invention using species specific CD154 polypeptides or a heterologous CD 154 polypeptide.

In the Examples, several SE13A recombinant bacteria were generated. In eaclr of the SE13A strains containing botlr the TRAP and CD154 polynucleotides, the TRAP polypepetide and the CD154 polypeptide were encoded on tire same polynucleotide aird were in frame witlr eaclr other and with tire Salmonella lamB polynucleotide in which they were inserted. In alternative embodiments, tire CD154 polypeptide and the TRAP polypeptide may be encoded by distinct polynucleotides. SE13A aroA htrA TRAP contains a deletion in aroA and htrA and encodes both the TRAP epitope (SEQ ID NO:l-3) and optionally the CD154 polypeptide (SEQ ID NO :4) inserted into loop 9 of lamB.

Compositions comprising an attenuated Salmonella strain and a pharmaceutically acceptable carcier are also provided. A phamaceutically acceptable carrier is any carrier suitable for in vivo administration. Examples of phannaceutically acceptable carriers suitable for use in the composition include, but are not limited to, water, buffered solutions, gluc-ose solutions or bacterial culture fluids. Additional components of the compositions may suita-bly include, for example, excipients such as stabilizers, preservatives, diluents, emulsifiers and lubricants. Examples of pharmaceutically acceptable carriers or diluents include stabilizes such as carbohydrates (e.g., sorbitol, mannitol, starch, sucrose, glucose, dextran), proteins such as albumin or casein, protein-containing agents such as bovine serum or skimmed milk and buffere (e.g.١ phosphate buffer). Especially when such stabilizers are added to the compositions, the composition is suitable for freeze-drying or spray-drying.

Methods of enhancing immune responses in a subject by administering a vaccine containing a TRAP polypeptide and a CD154 polypeptide capable of binding to CD40 and activating CD40 are also provided. The vaccine comprising the polynucleotide encoding a CD154 polypeptide capable of binding to CD40 is administered to a subject in an amount effective to enhance the immune response of the subject to the vaccine. Suitably, the vaccine contains a polynucleotide encoding a polypeptide including amino acids 140-149 of the human CD154 polypeptide (SEQ ID NO:4) or a homolog thereof Tlrerefore, a homologue of amino acid 140-149 derived from one species may be used to stimulate an immune response in a distinct species.

Several suitable polypeptides are identified herein. Suitably, the polynucleotide encodes a CD154 polypeptide from tire same species as the subject. Suitably, a polynucleotide encoding the polypeptide of SEQ ID NO:5 is used in human subjects, a polynucleotide encoding the polypeptide of SEQ ID NO:6 is used in clrickens, a polynucleotide encoding the polypeptide of SEQ ID NO:7 is used in ducks, a polynucleotide encoding the polypeptide of SEQ ID NO:8 is used in mic.e, and a polynucleotide encoding the polypeptide of SEQ ID NO:9 is used in cows. In the Examples, the human CD154 polypeptide (SEQ ID NO:5) was used in a chicken vaccine and was demonstrated to enhance the inrmune response to a foreign antigen. Thus other heterologous combinations of CD154 polypeptides and subjects may be usefill in the methods of the invention. The CD154 -9- polypeptide may be used to enhance the immune response in the subject to any foreign antigen or antigenic polypeptide present in the vaccine in addition to the TRAP polypeptide. One of skill in tire art will appreciate that the CD154 polypeptide could be used to enhance the immune response to more than one antigenic polypeptide present in a vaccine.

The polypeptide from CD154 stimulates an immune response at least in part by binding to its receptor, CD40. The Examples used a polypeptide homologous to the CD154 polypeptide which is expressed on immune cells of the subject and which is capable of binding to the CD40 receptor on macrophages and other antigen presenting cells. Binding of this ligand-receptor complex stimulates macrophage (and macrophage lineage cells suclr as dendritic cells) to enhance phagocjdosis and antigen presentation rvhile increasing cytokine secretions known to activate other local immune cells (such as B-lymphocytes). As such, molecules associated with the CD154 peptide are preferentially targeted for immune response and expanded antibody production.

Potential vectors for use in the methods include, but are not linrited to. Salmonella (Salmonella enteritidis), Shigella, Escherichia (E. coli). Yersinia, Bordetella, Lactococcus, Lactobacillus, Bacillus, Streptococcus, Vibrio (Vibrio cholerae). Listeria, έιν'η, poxvirus, herpesvirus, alphavirus, and adeno-associated virus.

In addition, methods of enhancing an immune response against an Apicomplexan parasite and methods of reducing morbidity associated with subsequent infection with an Apicomplexan parasite are disclosed. Briefly, tire methods conrprise administering to a subject a vaccine comprising a first polynrrcleotide sequence encoding a TRAP polypeptide in an effective amount. The TRAP polypeptides may include SEQ ID NO:l-3 and 11. The insertion of the TRAP polypeptides into the vector may be accomplislred in a variety of ways known to those of skill in the art, including but not linrited to tire scarless site-directed mutation system described in BMC Biotechnol. 2007 Sept, 17: 7(1): 59, Scarless and Site-directed Mutagenesis in Salmonella enteritidis chromosome, wlriclr is incorporated herein by reference in its entirety. The vector may also be engineered to express the TRAP polypeptides in conjunction with other polypeptides capable of enhancing the immune response as discussed above, such as in SEQ ID NO:4 and SEQ ID NO:10. In particular, a polypeptide of CD154 capalrle of binding CD40 nray be expressed by the vector to enhance the immtme response of the subject to tire TRAP polypeptide. Optionally, the vector is a bacterium, suclr as Salmonella enteritidis. -10-

The usefirl dosage of he vaccine to be administered will vary depending on the age, weight and species of the subject, the mode and route of administration and the type of pathogen against which an immune response is sought. The composition may be administered in any dose sufficient to evoke an immune response. For bacterial vaccines, it is envisioned that doses ranging from 10ة to 10٥؛ bacteria, from 1٥4 to 1,0. bacteria, or from 10و to 10? bacteria are suitable. The composition may be administered only once or may be administered two or more tinres to increase the immune response. For example, the composition may be administered two or more times separated by one week, two weeks, or by three or more weeks. The bacteria are suitably viable prior to administtation, but in some embodiments tire bacteria may be killed prior to administration. In some embodiments, the bacteria may be able to replicate in the subject, while in other embodiments the bacteria may not be capable of replicating in the subject.

For administeation to animals or humans, the compositions may be administered by a variet'y of means including, but not limited to, intranasally, mucosally, by spraying, intradermally, parenterally, subcutaneously, orally, by aerosol or intramuscularly. Eye-drop administration or addition to drinking water or food are additionally suitable. For chickens, the compositions may be administered in ovo.

Some embodiments of the invention provide methods of enhancing immune responses in a subject. Suitable subjects may include, but are not limited to, vertebrates, suitably mammals, suitably a lruman, and birds, suitably poultry, suclr as chickens. Other animal models of infection may also be used. Enhancing an immune response includes, but is not limited to, inducing a therapeutic or prophylactic effect that is mediated by tire immmre system of the subject. Specifically, enhancing an immune response may include, but is not limited to, enhanced production of antibodies, enhanced class switching of antibody heavy chains, maturatioir of antigen presenting cells, stimulation of helper T cells, stimulation of c^olytic T cells or induction ofT and B cell memory.

It is envisioned that several epitopes or antigens from, the same or different patlrogens may be administered in combination in a single vaccine to generate an enhanced immune response against multiple antigens. Recombinant vaccines may encode antigens from nrultiple pathog'enic microorganisms, viirrses or tumor associated antigens. Administration of vaccine capable of expressing multiple antigens Iras the. advantage of induciirg immunity against two or more diseases at the sanre time. For example, live attenuated bacteria, suclr as 2015202009 21 Apr 2015 -11.

Salmonella enteritidis 13Α, provide a suitable vector for eliciting an immune response against multiple antigens.

Bacterial vaccines may be constructed using exogenous polynucleotides encoding arrtigens which may be inserted in,to the bacterial genome at any non-essential site or alternatively may be carried on a plasmid using methods well known in the art. One suitable site for insertion of polynucleotides is within external portions of transmembrane proteins or coupled to sequences that: target the exogenous polyrrucleotide for secretory pathways. One example of a suitable transnrembrane protein for insertion of polyrrucleotides is the lamB gene. In the Examples, TRAP and CD154 polynucleotides were inserted into loop 9 of the lamB sequence.

Exogenous polynucleotides include, but are not limited to, polynucleotides encodirrg antigens selected from pathogetric microorganisms or viruses and include polynucleotides that are expressed in such a way that an effective immune response is generated. Such polynucleotides may be derived from pathogenic viruses such as influenza (eg, M2e, hemagglutinin, or neuraminidase), herpesviruses (eg, the genes encoding the structural proteins of herpesviruses), retrovirases (e.g., the gplbo envelope protein), adenoviruses, paramyxoviruses, coronaviruses and the like. Exogenous polynucleotides can also be obtained from pathogenic bacteria, e.g., genes encoding bacterial pr-oteins such as toxins, and outer membrane proteins. Further, exogenous polynucleotides from parasites, such as other Apicomplexan parasites are attractive candidates for use of a vector vaccine.

Polynucleotides encoding polypeptides involved in ttiggering the immune system may also be included irr a vector, such as a live attenuated Salmonella vaccine. The polynucleotides may encode immune system molecules known for their stimulatory effects, such as an interleukin. Tumor Necrosis Factor, an interferon, or another polynucleotide involved in immune-regulation. The vaccine may also include polynucleotides encoding peptide-s known to stimulate an immune response, such as the CD154 polypeptide described herein.

The folloWng examples are meant only to be illustrative and are not meant as limitations on the scope of the invention or of the appended claims. -12-

EXAMPLES

Example 1. Construction of TRAP and TRAP/C.154 inserts.

Strains and Culture Conditions

All plasmids were first maintained in TOP 10 E. coli cells (Invitrogen, Carlsbad, CA, USA) unless described otherwise. Salmonella enteritidis 13Α was used for introduction of mutations. Salmonella enleritidis strain 13Α was a field isolate available from USDA/APPIISMVSL and deposited with the ATCC as deposit number ΡΤΑ-7871. Bacteria carrying plasmid pKD46 were grown at 30.C. Other bacteria were groi at 37.C. Plasmid curing was conducted at 37.C. I'Uria-Bertani (LB) media was used for routine growth of cells, and soc nredia (Invitrogen, Carlsbad, CA, USA) was used for phenotypic expression after electroporation. When appropriate, the following antibiotics were added to the media: ampicillin (Amp) at lOOpg/ml, kanamycin (Km) at 50pg/ml, and chloramphenicol (Cm) at 25pg/ml.

Plasmids

Plasmids ρΚ٠46, pKD13, and pBC-I-Scel were described previously (Datsenko and Wanner, PNAS 2000, 97:6640-6645 and Kang et al., ل Bacteriol 2004, 186:4921-4930, both of which are incorporated herein by reference in their entireties). Plasmid pKD46 eircodes Red recombinase enzyjnes which mediate homologous recombination of incomiirg linear DNA with chromosomal DNA. This plasjnid also contains the Ampicillin resistance gene and is temperature-sensitive so that it requires 30.C for maintenance in the cell. Plasmid pKD13 served as a template for amplification of the Km resistance (Km1) gene used in overlapping PCR. Plasmid pBC-I-Scel, which is maintained in the cell at 37.C, produces the 1-SceI enzyme, which cleaves the following 18 base pair, rare recognition sequerrce: 5'-TAGGGATAACAGGGTAAT-3' (SEQ ID NO: 16). Plasmid pBC-I-Scel also coirtains the chloramphenicol resistance (Cm٢) gene.

PCR

All primers used for PCR are listed in Table 1. Typically, PCR was performed using approximately o.lpg of purified genomic, plasmid or PCR-generated DNA (Qiagen, Valencia, CA, USA), lx cloned Pfu polymerase buffer, 5٧ Pfu polymerase (Stratagene La Jolla, CA, USA), ImM dNTPs (GE Healthcare Bio-Sciences Coqx, Piscataway, NJ), and 1.2μΜ of each primer in a total volume of 50 pL. The DNA engine thermal cycler (Bio-Rad, 2015202009 21 Apr 2015 .13.

Hercules, CA, USA) was used with the following amplification conditions: 94.C for 2 minutes; 30 cycles of 94.C sec for 30 sec, 58.C for 60 sec-, 72.C for 90 sec per 1 kb; and 72.C for 10 minutes for final extension. Each PCR product was gel purified (Qiagen, Valencia, CA, flSA) and either eluted in 25pL EB buffer foi- preparation of templates used in 5 overlapping extension PCR or in 50pL EB buffer, ethanol precipitated and suspended in 5pL of ddHO for electroporation into s. enteritidis.

Table 1. Primer sequences

Primer Amplified region Primer sequence lam-up-f loop 9 up S'TGTACAAGTGGACGCCAATC 3' (SEQ I٥NO:17) Jam-up-r (<؟.١\(AAC لأ١ SEQ) اد S'GTTATCGCCGTCTTTGATATAGCC lamdn-f loop 9 dn 5'ATTTCCCGmTGCCGCAGC 3' (SEQ ID NO:19) lam-dn-r 5'GTTAAACAGAGGGCGACGAG 3' (SEQ IDNO:2٥) Km-f S'GCTATATCAAAGACGGCGATAACrAACTATAACGGTCCTAAGGT Ι-Scel/Km' gene AGCGAATTTCCGGGGATCCGTCGA 3' (SEQ ID NO:21) Km.r S'GCTGCGGCATAACGGGAA.-rnGTACiGCTGGAGCTGCTTCG 3' (SEQ ID NO:22) Kan4f inside Km' gene: 5CAAAAGCGCTCTGAAGTTCC 3' (SEQ ID NO:23) Kan4r sequencing 5'GCGTGAGGGGATCTTGAAGT 3' (SEQ ID 10:24) SEQl 54 up؛hCD SEQl hCD154/ loop 9 up ١"؛2'0؛Q٠Y٥Y,؟GAG1TATCGCCGTC٢TTGATATAGCOMS[٢ reverse SEQlhCD 154 down SEQlhCD154/l00p 9 down أه^م؟8؟٠ت؛ةيل:هةا?ذجئ؟ي;ءحححلوح؟ل؟:؟ةج7٠ه٠ةححة forward ΝΟ:26) SEQ2 hCD154up reverse SEQ2-hCD154/ loop 9 up nATCGCCGTCrrTGATATAGCadSEQlONOai) SEQ2 hCD154 up SEQ2-hCD154/ loop 9 down reverse ID NO:28) SEQ3 Hcdl54up reverse SEQ3 hCD 154/loop 9 up GAGlTCGCCGTCnTGATATAGCC؛’lS£٠Q.'\٥!>\2١؟ SEQ3 hCD154up SEQ3-hCD154/ loop 9 down لج:ءءت؟حية reverse ID NO:3٥) lam 3f outer regions of loop 5'GCCATCTCGCTTGGTGATAA 3' (SEQ ID NO:31) lam3r 9: sequencing 5'CGCTGGTATnTGCGGTACA 3' (SEQ ID NO:32)

In Table 1, italicized nucleotides are complementary to either side of the lamB gene loop 9 10 insertion site, which corresponds to nucleotide 1257 using 5. typhimurium as an annotated reference genome. Bold font nucleotides represent the 1-SceI recognition site in the Km-f primer. 14-

Electroporation

Transformation of pKD46 into ت٠. enteritidis was the first step carried out so that Red recombinase enzymes could be used for mediating recombination of subsequent mutations. Plasmid pKD46 was harvested from £. coli BW25H3 (Datsenko and Wanner, PNAS 2000, 97:6640-6645) using a plasmid preparation kit (Qiagen Valencia, CA, USA). Then 0.5pL of pKD46 DNA was used for transformation irrto 5. enteritidis 13Α which had been prepared for electroporation. (Datsenko and Wanner. PNAS 2000, 97:6640-6645). Briefly, cells were inoculated into 10-15rnL of 2Χ YT broth and grown at 37٥c overnight. Then 100pL of overnight culture was re-inoculated into lOmL fresh 2Χ YT broth at 37٥c for 3-4 hours. Cells to be transformed with pKD46 plasmid were heated at 50٥c for 25 minutes to help inactivate host restriction. Cells were washed five times in ddRO watei- and resuspended in 60pL of 10% glycerol. Cells were then pulsed at 2400-2450kV for l-6ms, incubated in soc for 2-3 hours at 30.C and plated on LB media with appropriate antibiotics, s, enteritidis transformants with pKD46 were maintained at 30.C. When these transfomants were prepared for additional electroporation reactions, all steps were the same except that 15% arabinose was added to induce Red recombinase enzymes one hour prior to washing, and cells did not undergo the 50.C heat step.

Loop 9 up. 1-SceI/ Km٢٠ Loop 9 down Construct

Introduction ofl-Scel enzyme recognition site along with the Km' gene into loop 9 of the lamB gene was done by combining the Red recombi'nase system (Datsenko and Wanner, PNAS 2000, 97:6640-6645, which is incorporated herein by reference in its entirety) and overlapping PCR (Horton et al.) BioTechniques 1990, 8:528-535, which is incorporated herein by reference in ite entirety). The insertion site corresponds to nucleotide 1257 of the lamB gene using Salmonella typhimurium LT2 (S. typhimnrium) as an annotated reference genome. First, the upstream and downstream regions immediately flanking the loop 9 insertion site (loop 9 up and loop 9 down, respectively) were amplified separately. Primers used were lam-up-f and lam-up-r for loop 9 up and lam-dn-f and lam-dn-Γ for loop 9 down. Then the Km' gene from pKD13 plasmid was amplified using primers Km-fand Km-r. Here, the 1-SceI enzyme site was synthetically added to the 5' end of Km-f primer then preceded by a regioir complimentary to the Ιοορ-up-r primer. Likewise, a region complimentary to the Ιοορ-dn-f primer was added to the 5’ end of Km-r primer. The complimentary regions allow all 3 PCR products to anneal when used as templates in one PCR reaction. Figure 2a -15- represents this design scheme. PCR fragments consisting of loop 9 up- 1-SceI/ Km٢- loop 9 do^ sequence (PCR-Α) were electroporated into s, enteritidis cells) which harbored pKD46 and were induced by arabinose, and then plated on LB with Km plates. To verify the correct sequence orientation of the mutation, we performed colony PCR with primer pairs Kan4F/!am3f and Kan4R/lam3r١ where Kan4F and Kan4R are Km' gene-specific primers and lam3f and lam3r are primers located outside the lamB loop 9 region. These PCR fragments were gel purified (Qiagen, Valencia, CA, USA) and used for DNA sequencing.

Loop 9 up- TRAP -CD154 ٠ Loop 9 down Construct

The final overlapping PCR fi'agment, PCR-Β, contained the added TRAP antigen in combination with CD154 sequences flanked by loop 9 up and down regions (Figure 2b), Combination sequences consisted of TRAP polynucleotide encoding SEQ ID NO:l-3 and CD154 along with spacers suclr as Serine (Ser) residues.

To shorten the amount of steps for constmction of the next fragment, the TRAP-CD154 sequence was synthetically added to the 5’ end of the lam-dn-f primer and preceded by the complimentary region to the Ιοορ-up-r primer. The previously used PCR product for loop 9 up could be used together with the newly constructed PCR product in which the TRAP-CD154S were incorporated at the 5' end of loop 9 down to perform the final PCR reaction. However, for other insert sequences, an extra PCR step was needed, due to the longer lengths of insert sequences, to amplify loop 9 up with added nucleotides specific to insertion sequences connected to Ιοορ-up-r primer. The coding sequence for Gly (GGT) and Serine (TCC) as well as all other amino acids were chosen based on compiled data of the most frequently used codons in E. coli and Salmonella typhimurium proteins. See Table 1 for further details of primer design. 1-SceI site/ Kmr insertion mutation

The first mutation step involved designing a PCR fragment, PCR-Α, which would serve as the carrier of the Ι-Scel site/ Km' cassette to be- inserted into the lamB site. PCR-A consisted of the Ι-Scel enzyme recognition site adjacent to the Km' gene with approximately 200- 300bp of flanking DNA on each end homologous to the upstream and downstream regions of lamB loop 9 insertion site (loop 9 up and loop 9 down, respectively). The fragment was introduced into s. enteritidis cells expressing Red recombinase enzymes and Km٢ colonies were selected. After screening a few colonies by colony PCR, positive clones -16- were sequenced for the desired inserted 1-SceI site/ Km' sequence, and the identified mutant was selected and designated as SE164.

Genomic Replacement ofl-Scel/ Km' with TRAP-CD154S

The second mutation step required constructing a PCR fragmeirt, referred to as PCR-B and shown in Figure 2Β, consisting of the final insertion sequence, the , flanked by lamB homologous fragmeirts, PCR-Β amplicons have no selection marker and must be counter-selected after replacement for the previous 1-SceI site,/ Km' mutation in SE164, Plasmid pBC-I-Scel encodes the Cm' gene and the 1-SceI enzyjne, which will cut the genome at the 1-SceI site of SE164. Therefore, pBC-1-Sce! was electroporated into SE164 along with PCR-Β. After recombination of PCR-Β to replace PCR-Α, positive clones were chosen based on the ability to grow on Cm but not on Km. After DNA sequencing of mutants to confirm successful recombination ofPCR-B, the strains were designated Sequence 1, Sequence 2 and Sequence 3. Ten random clones for each of the TRAP-CD154 insertions were used for PCR with lam 3f and lam 3r then digested using unique restriction enzymes sites for each insertion sequence and 100% of clones tested by digestion were positive for the desired mutation sequence. Sequencing results demonstrated that the insertion of TRAP-CD154 was exactly into the loop 9 region without the addition of extraneous nucleotides in each case. The inserts of the TRAP-CD154 vaccines are as follow's: TRAP-CD154 (SEQ ID NO:33); TRAP-US-CD154 (SEQ ID NO:34); TRAP-DS-CD154 (SEQ ID NO:35).

Example 2. Attenuation of TRAP-C.154 mutants/inserts.

Attenuation of SE13A was achieved by deletion mutation ofthe aroA gene and/or the hirA gene. Mutation ofthe aroA gene, a key gene in the chorismic acid pathway of bacteria, results in a severe metabolic deficiency which affects seven separate biochemical pathways. Mutation of the htrA gene reduces the cell’s ability to withstand exposure to low and high temperatures, low pH, and oxidative and DNA damaging agents and reduces the bacteria’s virulence.

To achieve deletion mutations in SE13A, the target gene sequence in the bacterial genome of s. enteritidis was replaced with the Km resistant gene sequence. This was completed using overlapping extension PCR aird electroporation of the PCR products as described above. The Km resistance gene was targeted into the genomic region containing the genes of interest (aroA or htrA) by flanking the Km resistance geire with 200-300 base pairs of sequences homologous to the genes of interest. Once Km resistant mutants were -17- obtained., the aroA and htrA deletion mutations were confirmed by DNA sequencing. Analogous aroA- and htrA-Salmonella strains were deposited with the American Type Culture Collection on September 13, 2006 (Deposit No. ΡΤΑ-7872 and Deposit No. ΡΤΑ-7873, respectively). The attenuated strains were previously tested in vivo with regards to clearance time. Both of the attenuated strains had quicker clearance times than did the wildtype 13Α strain, but both were, able to colonize the liver, spleen, and cecal tonsils of chickens after oral infection. Attenuated strains comprising the TRAP-CD154s and lacking both aroA and htrA were isolated.

Example 3. Protection of chicks from mortality after Eimeria inftction

Day-of-hatch chicks (η=280) were orally vaccinated with about 1 X 10ة cfil of the Salmonella isolates comprising the three distinct polynucleotides encoding the TRAP polypeptides of SEQ ID NO:l-3 or saline control. At 21 days of age, the chicks were orally challenged with 1.4 sporulated oocysts of Eimeria maxima. The chicks were monitored daily post challenge. As depicted in Figure 3, mortality of chicks at day 5 post challenge was reduced as compared to non-vaccinated animals irrespective of the vaccine strain given. The mortality was as follows: TRAP (SEQ ID ΝΟ:1) 7/43 (16.3%)؛ TRAP US(SEQ ID NO:2) 1/46 (2.2.%)؛ TRAP DS (SEQ ID NO:3) 6/43 (11%); Control (unvaccinated) 10/46 (21.7%). Surprisingly, the chicks vaccinated with a Salmonella comprising TRAP polypeptide of SEQ ID NO:2 demonstrated marked and significantly reduced mortality as compared to control non-vaccinated chicks (P < 0.001). Necropsy was perfomred and indicated that all mortality was related to the Eimeria maxima infection.

In a repeat experiment, mortality in the vaccinated bird (6/48) was significantly lower tharr the controls (17/50) and performance was better in the vaccinated chicks, but the difference was not significant.

In addition, seram was collected from immunized birds and an ELISA for TRAP perfomed. A robust TRAP specific antibody response was generated in the birds vaccinated withTRAP-US (SEQ IDNO:2).

Example 4. Morbidity associated with vaccination is limited

To evaluate the efficacy of TRAP US-CD154 (SEQ ID NO:34) as a potential vaccine candidate, a similar sttrdy was completed to investigate morbidity associated with vaccination. Broiler chickens were orally vaccinated with 1 X 10¾ cfir/bird of the Salmonella vaccine with TRAP US and CD 154 insert (SEQ ID NO:34) or sham vaccinated with saline. -18- 2015202009 27 Mar 2017

Coccidia challenge was performed with sporulated oocytes of Eimeria maxima (105 sporulated oocysts/bird) at three weeks post-vaccination. Body weight gain and lesions were evaluated 7 days post-challenge. Immunized birds showed a significant (ρ<0.01) improvement in performance. Immunized birds had about a 31% weight gain as compared to unvaccinated controls. Thus, vaccination with a Salmonella-based vaccine comprising a TRAP polypeptide and a CD154 polypeptide capable of binding CD40 may protect birds from morbidity and mortality associated with Eimeria infection.

In the claims which follow and in the preceding description of the invention, except where the context requires otherwise due to express language or necessary implication, the word “comprise” or variations such as “comprises” or “comprising” is used in an inclusive sense, i.e. to specify the presence of the stated features but not to preclude the presence or addition of further features in various embodiments of the invention.

It is to be understood that, if any prior art publication is referred to herein, such reference does not constitute an admission that the publication forms a part of the common genera] knowledge in the art, in Australia or any other counrty. 8796789-1 (GHMatters) Ρ84253.Α٧.1

Claims (23)

1. We claim:

   1. A vaccine comprising a first polynucleotide sequence encoding a TRAP polypeptide comprising SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, an immunogenic fragment of SEQ ID NO:1, an immunogenic fragment of SEQ ID NO:2, or an immunogenic fragment of SEQ ID NO:3, and a second polynucleotide sequence encoding a CD154 polypeptide capable of binding CD40, the CD154 polypeptide having fewer than 50 amino acids and comprising amino acids 140-149 of SEQ ID NO:4 or a homolog thereof.
2. 2. The vaccine of claim 1, wherein the TRAP polypeptide is SEQ ID NO:2, or an immunogenic fragment of 6 or more consecutive amino acids of SEQ ID NO:2.
3. 3. The vaccine of claim 1 or 2, wherein the CD154 polypeptide comprises SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, or SEQ ID NO:9.
4. 4. The vaccine of any one of claims 1-3, wherein the vaccine comprises more than one copy of the first polynucleotide sequence, more than one copy of the second polynucleotide sequence, or more than one copy of the first polynucleotide sequence and more than one copy of the second polynucleotide sequence.
5. 5. The vaccine of any one of claims 1-4, wherein the first polynucleotide sequence is linked in the same reading frame to the second polynucleotide sequence.
6. 6. The vaccine of any one of claims 1-5, wherein the first polynucleotide and second polynucleotide are comprised within a vector.
7. 7. The vaccine of claim 6, wherein the vector is selected from the group consisting of a virus, a bacterium, and a liposome.
8. 8. The vaccine of claim 7, wherein the vector is a bacterium.
9. 9. The vaccine of claim 8, the bacterium comprising the TRAP polypeptide on its surface.
10. 10. The vaccine of claim 8 or 9, wherein the bacterium is selected from the group consisting of Salmonella species, Bacillus species, Escherichia species, and Lactobacillus species.
11. 11. The vaccine of claim 10, wherein the bacterium is Salmonella enteritidis selected from strains deposited as ATCC PTA-7871, ATCC PTA-7872 and ATCC PTA-7873.
12. 12. The vaccine of any one of claims 1-11, wherein the first polynucleotide and the second polynucleotide are inserted into a polynucleotide sequence encoding an external portion of a transmembrane protein.
13. 13. A vaccine comprising a variant of Salmonella enteritidis 13A comprising a first polynucleotide sequence encoding a TRAP polypeptide comprising SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3, and a second polynucleotide sequence encoding a CD154 polypeptide capable of binding CD40, the CD154 polypeptide having fewer than 50 amino acids and comprising amino acids 140-149 of SEQ ID NO: 8 or a homolog thereof.
14. 14. A method of enhancing the immune response against an Apicomplexan parasite in a subject comprising administering to the subject the vaccine of any one of claims 1-13 in an amount effective to enhance the immune response of the subject to the Apicomplexan parasite.
15. 15. The method of claim 14, wherein the vaccine is administered by a method selected from the group consisting of oral, intranasal, parenteral, and in ovo.
16. 16. The method of any of claims 14 or 15, wherein the enhanced immune response comprises an enhanced antibody response or an enhanced T cell response.
17. 17. The method of any one of claims 14-16, wherein the subject is member of a poultry species or a mammal.
18. 18. The method of any one of claims 14-17, wherein the vaccine is killed prior to administration to the subject.
19. 19. The method of any one of claims 14-18, wherein the vaccine is not capable of replicating in the subject.
20. 20. The method of any one of claims 14-19, wherein the Apicomplexan parasite is selected from the group consisting of Eimeria, Plasmodium, Toxoplasma, and Cryptosporidium.
21. 21. A method of reducing morbidity associated with infection with Apicomplexan parasite in a subject comprising administering to the subject the vaccine of any one of claims 1-13 in an amount effective to enhance the immune response of the subject to the Apicomplexan parasite.
22. 22. Use of a vaccine of any one of claims 1-13 in the manufacture of a medicament for enhancing the immune response against an Apicomplexan parasite in a subject.
23. 23. Use of vaccine of any one of claims 1-13 in the manufacture of a medicament for reducing morbidity associated with infection with Apicomplexan parasite in a subject.