# Patent

## Title
Nucleic acids encoding recombinant 56 and 82 kDa antigens from gametocytes of Eimeria maxima and their uses

## Abstract

The present invention provides the recombinant cloning and sequencing of two of the major Eimeria maxima gametocyte antigens having molecular weights of 56 and 82 kDa and the expression of these recombinant antigens in an E. coli expression system using the plasmid pTrcHis. The subject invention also provides a vaccine against coccidiosis comprising the recombinant 56 kDa or 82 kDa antigen. The subject invention also provides two 30 kDa proteins and three 14 kDa proteins from Eimeria maxima gametocytes having at the N-terminal end the amino acid sequence described herein. The subject invention also provides a vaccine against coccidiosis comprising the recombinant 56 kDa or 82 kDa antigen and any of the aforementioned proteins. Additionally, the subject invention also provides a method of immunizing a subject against infection by Eimeria tenella, Eimeria maxima, Eimeria acervulina, Eimeria necatrix, Eimeria praecox, Eimeria mitis or Eimeria brunetti, or a microorganism expressing an immunologically cross-reactive antigen, comprising the step of administering to the subject any of the aforementioned vaccines.

## Background
This application is a §371 national stage of PCT International Application No. PCT/US02/21233, filed Jul. 3, 2002, designating the United States of America, which claims priority of U.S. provisional Application No. 60/303,699, filed Jul. 6, 2001, the contents of which are hereby incorporated by reference.
This application claims the benefit of U.S. Provisional Application No. 60/303,699, filed Jul. 6, 2001, the contents of which are hereby incorporated by reference into this application.


Throughout this application various publications are referenced in parenthesis. Full citations for these publications may be found listed in alphabetical order at the end of the specification immediately preceding the claims. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this invention pertains.
BACKGROUND OF THE INVENTION
The organisms which cause the disease known as “coccidiosis” in chickens belong to the phylum Apicomplexa, class Sporozoa, subclass Coccidia, order Eucoccidia, suborder Eimeriorina, family Eimeriidae, genus Eimeria. Within the Eimerian genus there are many species, several of which are pathogenic in chickens. The species of major concern to the chicken industry are Eimeria tenella, Eimeria maxima, Eimeria acervulina, Eimeria necatrix and Eimeria brunetti. 
Coccidiosis has become a major economic problem in the chicken industry over the past several decades, mainly due to the overcrowding of chicken houses and the development of drug resistance by the parasite. The rearing of chickens under crowded conditions on a litter floor provides optimal conditions for the growth and spread of Eimeria parasites. Under such circumstances, sanitary control is impossible and the farmer must rely on the effectiveness of coccidiostat drugs. However, drugs must be kept in the feed at all times, shuttle programs must be used to avoid the appearance of drug resistance strains of Eimeria, and certain drugs have costly side effects. Furthermore, these coccidiostats also have antibacterial effects and therefore are considered to be in-feed antibiotics. Recently the European Union has decided to ban the use of all in-feed antibiotics in the chicken industry including anticoccidial drugs. Thus, the only viable approach to the control of coccidiosis in the future is by vaccine development.
The Eimeria parasite undergoes a complex life cycle in the mucosa of the intestinal tract. This life cycle is very similar to that of the other hemosporidian parasites (i.e. plasmodium, babesia, etc.) except for the lack of an arthropod vector. Oocysts sporulate on the litter floor producing four sporocysts, each containing two sporozoites (thus belonging to the class sporozoa). The oocysts are ingested by the chicken, and the sporocysts are released by the mechanical grinding of the gizzard. The sporozoites are then released from the sporocysts due to the digestion of the sporocyst wall by proteolytic enzymes in the intestine. Mobile sporozoites then invade lymphocytes and go on to invade epithelial cells where the asexual cycle begins. The parasite goes through 2-4 cycles of replication and division (each species having a defined number of divisions) leading to the production of large numbers of daughter merozoites. After the final cycle of merozoite production the sexual cycle begins with the production of the macrogametocyte (female) and microgametocyte (male). The macrogametocyte is characterized by the production of wall forming bodies, while microgametocytes contain the components involved in the formation of microgametes, which bud off from the surface of the intracellular parasite. Microgametes are flagellated and are responsible for the fertilization of the macrogamete. A zygote is formed which matures into the oocyst by fusion of the wall forming bodies and condensation of the nucleus. Oocysts are secreted in the feces, thus completing the cycle.
Over the past several years, native antigens from the sexual (gametocyte) stages of Eimeria maxima have been used to immunize laying hens. Offspring chicks were consequently vaccinated via maternal immunity (protective maternal antibody). Three major protective antigens have previously been identified in E. maxima gametocytes having molecular weights of 250, 82 and 56 kDa (EP Patent No. 0 256 536, U.S. Pat. Nos. 5,496,550, and 5,932,225). EP Patent No. 0 256 536, U.S. Pat. Nos. 5,496,550, and 5,932,225 are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this invention pertains. It was shown that these antigens are well conserved amongst Eimeria species (Wallach 1995) and can cross protect against the 3 major species that cause coccidiosis in broiler chickens, E. maxima, E. tenella and E. acervulina. More recently, it was shown that in floor pen trials, chicks from hens vaccinated with these native gametocyte antigens were protected against Eimeria under field conditions (Wallach 1996). This protection acts to lower the peak in oocyst shedding to a level which does not cause any damaging effect on the performance of the broiler chicken. Based on the above results it was concluded that these antigens are effective against coccidiosis in chickens and also have the potential for use against coccidiosis in other domestic animals including turkeys, geese, sheep, cattle, pigs and fish.
These three antigens were also characterized at the molecular level. Cell free translation experiments were carried out to identify the RNA molecules that encode them (Mencher er al.). cDNA molecules that encode these antigens were cloned by immunoscreening of a cDNA library made in the expression vector lambda zap (4, U.S. Pat. No. 5,932,225). By this approach, the gene encoding the 250 kDa antigen was cloned and sequenced. The clone pEM 250/14 was partially sequenced in U.S. Pat. Nos. 5,932,225 and 5,496,550. FIG. 13a of the subject application reproduces FIG. 11 of U.S. Pat. Nos. 5,932,225 and 5,496,550, which portrays the DNA sequence of the first 293 nucelotides of clone pEM 250/14. FIG. 13b of the subject application reproduces FIG. 12 of U.S. Pat. Nos. 5,932,225 and 5,496,550, which shows the DNA sequence of the last 196 nucelotides of clone pEM 250/14. Also, in in U.S. Pat. Nos. 5,932,225 and 5,496,550, the putative genes encoding the 56 and 82 kDa antigens were cloned and sequenced.
Subsequently, Fried et al. sequenced the entire pEM 250/14 clone and found that the antigen had a molecular weight of 230 kDa rather than 250 kDa as had been previously thought. Fried et al. found that the 230 kDa gene contains highly repetitive motifs and that these repeats are contained throughout the entire gene (Fried et al.). This clone was expressed in bacteria using the pATH plasmid vector and it was shown that it is recognized by convalescent chicken sera taken 14 days post infection with E. maxima. Finally, it was shown that this gene is expressed only in the macrogametocyte stage and by immunofluorescence was found to be located in the wall forming bodies of the macrogamete (Fried et al.).
cDNA clones encoding the 56 and 82 kDa antigens were also obtained by screening the library with polyclonal antibodies as well as a monoclonal antibody against the 56 kDa antigen. This monoclonal antibody was previously shown to provide passive immunity to naive chicks (Wallach 1990). A few clones were obtained and analyzed. One of the clones was found to encode a small 10 kDa antigen and therefore was not the desired clone. Another clone was found to contain only a small part of the open reading frame (ORF) and by northern blotting was shown to hybridize with two mRNAs of about the expected size for the 56 and 82 kDa antigens. It was therefore concluded that this was the desired clone. Genomic libraries were then screened to obtain the full length clone. However, due to the highly repetitive GCA motifs in this clone, it was not possible to specifically isolate the full length clone. Attempts to clone the full length cDNA molecule were also not successful due to these repeats. Finally, attempts to express the partial cDNA clones in bacteria failed as well probably due to their unusual sequences and a reasonable level of gene expression was not obtained. It has previously been shown that the 56 and 82 kDa antigens are glycosylated (U.S. Pat. No. 5,932,225). This is based on their strong reactivity with Soybean lectin. Therefore, glycosylation may be required in order to obtain good expression of these genes and for proper conformation of the gene products.
In addition to the 56, 82 and 230 kDa antigens, a 14 kDa antigen obtained from highly purified fractions of oocyst walls has been proposed as a possible candidate for vaccines against coccidiosis (Eschenbacher et al.). However, this hypothesis has not been explored.
Several laboratories have been working on a subunit vaccine against coccidiosis. Most of these researchers have focused their efforts on the extracellular asexual stages of the life cycle, in particular the sporozoite and merozoite stages which are considered to be the most vulnerable to immune attack. In a previous study it was found that sporozoite extracts from E. tenella could induce in broilers protection against challenge infections against this parasite for up to 7 weeks of age (Karkhanis et al.). Work carried out using monoclonal antibodies against antigens from sporozoites of E. tenella led to the identification of a 25,000 molecular weight antigen which was cloned and sequenced (Eur. Patent publication No. 0 164 176, Dec. 11, 1985). Several other sporozoite genes were identified and their recombinant antigens or the transformed bacteria themselves were tested for protective immunity (Danforth et al.). The results indicated that these recombinants were only able to provide a relatively low level of protection against challenge infection with Eimeria and did not always prevent the appearance of significant lesions.
A vaccine using antigens from the merozoite stage has also been tested (European patent publication No. 0 135 073). Using these antigens to immunize young broiler chicks, it was once again found that the protection afforded was relatively low (Danforth et al.).
In 1993, it was found that there was a correlation between protective maternal immunity with the appearance of maternal antibodies against a 230 kDa merozoite (as opposed to gametocyte) antigen of Eimeria maxima (Smith et al.). This protection was often over 90% and was found to occur even when the maternal antibody level was relatively low (although reactivity with the 230 kDa protein remained strong). It was also found that a very small quantity of the native 230 kDa merozoite antigen cut out of an SDS-PAGE gel could induce a significant (60%) level of protective maternal immunity against infection with E. maxima in offspring chicks. Furthermore, Western blotting showed that this protein was expressed in both merozoites and sporozoites of E. maxima and is also well conserved between Eimeria species.
SUMMARY OF THE INVENTION
The present invention provides the nucleic acid encoding two of the major Eimeria maxima gametocyte antigens having molecular weights of 56 and 82 kDa.
The subject invention also provides a 30 kDa protein from Eimeria maxima gametocytes having at the N-terminal end the amino acid sequence shown by SEQ. ID NO. 35.
The subject invention also provides a 30 kDa protein from Eimeria maxima gametocytes having at the N-terminal end the amino acid sequence shown by SEQ. ID NO. 42.
The subject invention also provides a 14 kDa protein from Eimeria maxima gametocytes having at the N-terminal end the amino acid sequence shown by SEQ. ID NO. 37.
The subject invention also provides a 14 kDa protein from Eimeria maxima gametocytes having at the N-terminal end the amino acid sequence shown by SEQ. ID NO. 39.
The subject invention also provides a 14 kDa protein from Eimeria maxima gametocytes having at the N-terminal end the amino acid sequence shown by SEQ. ID NO. 41.
The subject invention also provides a vaccine against coccidiosis comprising the recombinant 56 kDa antigen alone or in combination with any of the aforementioned proteins.
The subject invention also provides a vaccine against coccidiosis comprising the recombinant 82 kDa antigen alone or in combination with any of the aforementioned proteins.
The subject invention also provides a method of immunizing an subject against infection by Eimeria tenella, Eimeria maxima, Eimeria acervulina, Eimeria necatrix, Eimeria praecox, Eimeria mitis or Eimeria brunetti, or a microorganism expressing an immunologically cross-reactive antigen, comprising the step of administering to the subject any of the aforementioned vaccines.



BRIEF DESCRIPTION OF THE FIGURES
FIG. 1 depicts a Coomassie stained SDS PAGE gel of affinity purified native gametocyte antigens. Arrows point to the 56 and 82 kDa antigens. Molecular weight marker proteins are indicated.
FIG. 2 depicts a two-dimensional (2D) SDS-PAGE gel of affinity purified native gametocyte antigens after immunoblotting and silver staining. Molecular weight marker proteins are indicated.
FIG. 3 depicts a Coomassie stained PVDF filter from a two-dimensional SDS PAGE gel and the identification of the spots that were cut out for sequence analysis. Arrows point to the 56 and 82 kDa native antigens.
FIGS. 4A-4C depicts the complete DNA sequence of the 56 kDa gametocyte antigen. The amino terminus as well as internal tryptic peptide fragments are designated. In addition, the predicted initiator methionine and signal peptide cleavage site are shown. The coding sequence, its complement and amino acid sequences are shown (SEQ. ID. NOs. 1-3).
FIGS. 5A-5D depicts the complete DNA sequence of the 82 kDa gametocyte antigen. The amino terminus as well as internal tryptic peptide fragments are designated. In addition, the predicted initiator methionine and signal peptide cleavage site are shown. The coding sequence, its complement and amino acid sequences are shown (SEQ. ID. NOs. 4-6).
FIG. 6 depicts a Southern blot of gametocyte and control chicken DNA probed with the cDNA clone for the 56 kDa antigen. The restriction enzymes used for digestion of the DNA and the marker band sizes in kilobases are indicated.
FIG. 7 depicts a Southern blot of gametocyte and control chicken DNA probed with the cDNA clone for the 82 kDa antigen. The restriction enzymes used for digestion of the DNA and the marker band sizes are indicated.
FIG. 8 depicts a northern blot of gametocyte (G) and control (C) chicken total RNA probed with the 82 kDa cDNA clone. The sizes of the marker bands in kilobases are indicated on the left.
FIG. 9 depicts an immunoblot showing reactivity of the anti polyhistidine antibody and chicken anti-APGA with proteins expressed by IPTG induced and non-induced (control) bacteria containing the 56 kDa cDNA clone in pTrcHisB. As a further negative control, bacteria that were transformed with the pTrcHisB plasmid containing no insert were tested. Finally, native APGA was used as a positive control for the blot with the anti APGA antiserum. The sizes of the protein marker bands are indicated. Arrows show the positions of the 41 kDa recombinant and 56 and 82 kDa native proteins.
FIG. 10 depicts a Coomassie stained gel and immunoblot of proteins from bacteria containing pTrcHisB-82 kDa cDNA cloned plasmids. The immunoblot shows reactivity of the anti polyhistidine, chicken anti-APGA as well as uninfected chicken (negative control) sera with the 82 kDa recombinant protein under IPTG induced and non-induced conditions at various times after induction. As a negative control, the experiment was also performed using bacteria transformed with the same plasmid without an insert. As a positive control, native APGA is also run. The arrow shows the position of the 82 kDa recombinant protein. The sizes of the protein marker bands are indicated on the left.
FIG. 11 depicts an immunoblot of a whole lysate of unsporulated E. maxima oocysts separated by 2 dimensional SDS PAGE. The gel was blotted onto a membrane filter and probed with an antiserum raised against APGA. The strongly reacting 30 kDa spot is shown with an arrow. This was the spot that was cut out of the gel and used for sequence analysis.
FIGS. 12A-12K depicts DNA sequence alignment of the 230 kDa cDNA E. maxima clone with a homologous DNA sequence from patent WO 90/00403 showing 60% homology (SEQ. ID. NOs. 26-27).
FIG. 13a depicts the DNA sequence of the first 293 nucelotides of clone pEM 250/14. The coding sequence and its amino acid sequences are shown (SEQ. ID. NOs. 28-29). FIG. 13b depicts the DNA sequence of the last 196 nucelotides of clone pEM 250/14. The coding sequence and its amino acid sequences are shown (SEQ. ID. NOs. 30-31).
FIGS. 14A & B ELISA results for chicken immunogenicity trial of the recombinant form of the 56 kDa and 82 kDa gametocyte antigen. All serum samples were tested at 1:1000 dilution. A) Coating antigen: APGA to test sera against APGA; r56 purified to test sera taken from chickens immunized with PBS, FIA and the two doses of r56. B) Coating antigen: APGA to test sera against APGA; r82 purified protein to test sera taken from chickens immunized with PBS, FIA and the two doses of r82.
FIG. 15 DNA and encoded amino acid sequence of the expressed protein fragment from the 250 kDa asexual stage protein (SEQ. ID NOS. 32-33).
FIG. 16 Mouse immunogenicity trial of the recombinant fragment of the 250 kDa asexual stage protein. The average of each group for the three consecutive bleeds is shown, with standard error bars indicated. All serum samples were tested at 1:1000 dilution. Coating antigen was 100 ng of APGA for sera from the positive control APGA group, or 100 ng of the recombinant protein for the negative control PBS and PBS/FIA groups and the two recombinant protein doses (r0.5 μg and r5 μg)
FIG. 17 Chicken immunogenicity trial of the recombinant fragment of the 250 kDa asexual stage protein. The average of each group for the three consecutive bleeds is shown, with standard error bars indicated. All serum samples were tested at 1:1000 dilution. Coating antigen was 100 ng of APGA for sera from the positive control APGA group, or 100 ng of the recombinant protein for the negative control PBS and PBS/FIA groups and the two recombinant protein doses (1 82 g and r10 82 g).
FIG. 18 Anti-r56 recognition of gametocyte and wall antigens in Eimeria maxima. 
FIG. 19 Anti-r82 recognition of gametocyte and wall antigens in Eimeria maxima. 
FIG. 20 Alignment of the N-terminus sequence of the oocyst wall proteins to the 56 kDa and 82 kDa gametocyte antigens (SEQ. ID NOS. 34-42).



DETAILED DESCRIPTION OF THE INVENTION
The subject invention provides an isolated nucleic acid comprising a nucleotide sequence encoding a 56 kDa polypeptide from Gametocytes of Eimeria maxima, or encoding a homolog of the polypeptide, or a complement of the nucleic acid.
In one embodiment, the polypeptide has the amino acid sequence shown in FIG. 4 (SEQ. ID. NO. 3).
In another embodiment, the homolog of the polypeptide has at least 50% identity to the polypeptide having the sequence shown as SEQ. ID. NO. 3.
In an additional embodiment, the homolog of the polypeptide has at least 60% identity to the polypeptide having the sequence shown as SEQ. ID. NO. 3.
In a further embodiment, the homolog of the polypeptide has at least 70% identity to the polypeptide having the sequence shown as SEQ. ID. NO. 3.
In an added embodiment, the homolog of the polypeptide has at least 75% identity to the polypeptide having the sequence shown as SEQ. ID. NO. 3.
In yet another embodiment, the homolog of the polypeptide has at least 80% identity to the polypeptide having the sequence shown as SEQ. ID. NO. 3.
In a further embodiment, the homolog of the polypeptide has at least 85% identity to the polypeptide having the sequence shown as SEQ. ID. NO. 3.
In one embodiment, the homolog of the polypeptide has at least 90% identity to the polypeptide having the sequence shown as SEQ. ID. NO. 3.
In another embodiment, the homolog of the polypeptide has at least 93% identity to the polypeptide having the sequence shown as SEQ. ID. NO. 3.
In an additional embodiment, the homolog of the polypeptide has at least 95% identity to the polypeptide having the sequence shown as SEQ. ID. NO. 3.
In a further embodiment, the homolog of the polypeptide has at least 97% identity to the polypeptide having the sequence shown as SEQ. ID. NO. 3.
In yet another embodiment, the homolog of the polypeptide has at least 99% identity to the polypeptide having the sequence shown as SEQ. ID. NO. 3.
In an additional embodiment, the nucleotide sequence has at least 50% identity to the nucleotide sequence starting at nucleotide No. 103 and ending at nucleotide No. 1529 shown in FIG. 4 (SEQ. ID. NO. 1.)
In another embodiment, the nucleotide sequence has at least 60% identity to the nucleotide sequence starting at nucleotide No. 103 and ending at nucleotide No. 1529 shown as SEQ. ID. NO. 1.
In a further embodiment, the nucleotide sequence has at least 70% identity to the nucleotide sequence starting at nucleotide No. 103 and ending at nucleotide No. 1529 shown as SEQ. ID. NO. 1.
In one embodiment, the nucleotide sequence has at least 75% identity to the nucleotide sequence starting at nucleotide No. 103 and ending at nucleotide No. 1529 shown as SEQ. ID. NO. 1.
In another embodiment, the nucleotide sequence has at least 80% identity to the nucleotide sequence starting at nucleotide No. 103 and ending at nucleotide No. 1529 shown as SEQ. ID. NO. 1.
In an added embodiment, the nucleotide sequence has at least 85% identity to the nucleotide sequence starting at nucleotide No. 103 and ending at nucleotide No. 1529 shown as SEQ. ID. NO. 1.
In one embodiment, the nucleotide sequence has at least 90% identity to the nucleotide sequence starting at nucleotide No. 103 and ending at nucleotide No. 1529 shown as SEQ. ID. NO. 1.
In a further embodiment, the nucleotide sequence has at least 93% identity to the nucleotide sequence starting at nucleotide No. 103 and ending at nucleotide No. 1529 shown as SEQ. ID. NO. 1.
In another embodiment, the nucleotide sequence has at least 95% identity to the nucleotide sequence starting at nucleotide No. 103 and ending at nucleotide No. 1529 shown as SEQ. ID. NO. 1.
In an added embodiment, the nucleotide sequence has at least 97% identity to the nucleotide sequence starting at nucleotide No. 103 and ending at nucleotide No. 1529 shown as SEQ. ID. NO. 1.
In one embodiment, the nucleotide sequence has at least 99% identity to the nucleotide sequence starting at nucleotide No. 103 and ending at nucleotide No. 1529 shown as SEQ. ID. NO. 1.
In a further embodiment, the nucleic acid is a DNA molecule.
In yet another embodiment, the DNA molecule is a cDNA molecule.
In an added embodiment, the nucleic acid has the nucleotide sequence starting at nucleotide No. 103 and ending at nucleotide No. 1529 shown in FIG. 4(SEQ. ID. NO. 1).
In another embodiment, the nucleic acid is an RNA molecule.
In one embodiment, the isolated nucleic acid is operatively linked to a promoter of RNA transcription.
The subject invention also includes a vector comprising an isolated nucleic acid comprising a nucleotide sequence encoding a 56 kDa polypeptide from Gametocytes of Eimeria maxima, or encoding a homolog of the polypeptide, or a complement of the nucleic acid.
In one embodiment, the vector comprises the nucleic acid having the nucleotide sequence starting at nucleotide No. 103 and ending at nucleotide No. 1529 shown in FIG. 4 (SEQ. ID. NO. 1).
In another embodiment, the vector is a plasmid.
In a further embodiment, the plasmid comprises the nucleic acid having the nucleotide sequence starting at nucleotide No. 103 and ending at nucleotide No. 1529 shown in FIG. 4 (SEQ. ID. NO. 1).
In an additional embodiment, the plasmid comprises an isolated nucleic acid comprising a nucleotide sequence encoding a 56 kDa polypeptide from Gametocytes of Eimeria maxima, or encoding a homolog of the polypeptide, or a complement of the nucleic acid.
In yet another embodiment, the plasmid is the plasmid designated 56TRCHisb1 plasmid (Australian Government Analytical Laboratories Accession No. NM01/22400).
The subject invention also encompasses a host cell comprising a vector which comprises an isolated nucleic acid comprising a nucleotide sequence encoding a 56 kDa polypeptide from Gametocytes of Eimeria maxima, or encoding a homolog of the polypeptide, or a complement of the nucleic acid.
In one embodiment, the host cell comprises a vector comprising a nucleic acid having the nucleotide sequence starting at nucleotide No. 103 and ending at nucleotide No. 1529 shown in FIG. 4 (SEQ. ID. NO. 1).
In another embodiment, the host cell is selected from the group consisting of a bacterial cell; a plant cell; an insect cell; and a mammalian cell.
The subject invention additionally presents a transformed cell comprising an isolated nucleic acid comprising a nucleotide sequence encoding a 56 kDa polypeptide from Gametocytes of Eimeria maxima, or encoding a homolog of the polypeptide, or a complement of the nucleic acid.
In one embodiment, the transformed cell is the transformed cell designated clone 56TRCHisb1 in bacteria (Australian Government Analytical Laboratories Accession No. NM01/22401).
A plasmid encoding the 56 kDa antigen was deposited with the Australian Government Analytical Laboratories, Pymble, Australia, on Jun. 26, 2001, under Accession No. NM01/22400. The bacterial cell transformed with the 56 kDa antigen was deposited with the Australian Government Analytical Laboratories, Pymble, Australia, on Jun. 26, 2001, under Accession No. NM01/22401. Both deposits were made according to the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure.
In an added embodiment, the transformed cell further comprises a 56 kDa polypeptide from Gametocytes of Eimeria maxima, or a homolog of the polypeptide.
The subject invention further contains a method of producing a recombinant 56 kDa polypeptide from Gametocytes of Eimeria maxima comprising culturing a transformed cell comprising an isolated nucleic acid comprising a nucleotide sequence encoding a 56 kDa polypeptide from Gametocytes of Eimeria maxima, or encoding a homolog of the polypeptide, or a complement of the nucleic acid and isolating the recombinant 56 kDa polypeptide from Gametocytes of Eimeria maxima. The recombinant polypeptide produced by this method is also encompassed by the subject invention.
The subject invention also provides a vaccine against Eimeria tenella, Eimeria maxima, Eimeria acervulina, Eimeria necatrix or Eimeria brunetti, Eimeria praecox, Eimeria mitis or a microorganism expressing an immunologically cross-reactive antigen, comprising the isolated nucleic acid comprising a nucleotide sequence encoding a 56 kDa polypeptide from Gametocytes of Eimeria maxima, or encoding a homolog of the polypeptide, or a complement of the nucleic acid.
In one embodiment of the vaccine, the isolated nucleic acid is a plasmid.
In addition, the subject invention presents a vaccine against Eimeria tenella, Eimeria maxima, Eimeria acervulina, Eimeria necatrix or Eimeria brunetti, Eimeria praecox, Eimeria mitis or a microorganism expressing an immunologically cross-reactive antigen, comprising a recombinant 56 kDa polypeptide from Gametocytes of Eimeria maxima produced by culturing a transformed cell comprising an isolated nucleic acid comprising a nucleotide sequence encoding a 56 kDa polypeptide from Gametocytes of Eimeria maxima, or encoding a homolog of the polypeptide, or a complement of the nucleic acid and isolating the recombinant 56 kDa polypeptide from Gametocytes of Eimeria maxima. 
In another embodiment, the vaccine is comprised of a mixture of the isolated nucleic acid of the subject invention and the recombinant polypeptide of the subject invention.
In another embodiment, the vaccine is comprised of a mixture of the isolated nucleic acid of the subject invention, the recombinant polypeptide of the subject invention and a plasmid comprising the isolated nucleic acid comprising a nucleotide sequence encoding a 56 kDa polypeptide from Gametocytes of Eimeria maxima. 
In an added embodiment, the vaccine further comprises a second antigen.
In one embodiment, the second antigen is selected from the group consisting of a nucleic acid coding for an antigen from Eimeria maxima, a plasmid comprising such a nucleic acid, and a polypeptide coded by such a nucleic acid.
In another embodiment, the second antigen is selected from the group consisting of a 30 kDa protein from Eimeria maxima gametocytes having at the N-terminal end the amino acid sequence shown as SEQ. ID NO. 35, or SEQ. ID NO. 42 or a 14 kDa protein from Eimeria maxima gametocytes having at the N-terminal end the amino acid sequence shown as SEQ. ID NO. 37, SEQ. ID NO. 39 or SEQ. ID NO. 41.
In yet another embodiment, the second antigen is a nucleic acid having the nucleotide sequence shown in Seq. ID. No. 4, a plasmid comprising the nucleic acid or a polypeptide coded by the nucleic acid.
In a further embodiment, the vaccine further comprises a third antigen.
The subject invention also provides a vaccine wherein the third antigen is a 230 kDa sporozoite/merozoite antigen from E. maxima. 
The 230 kDa antigen was isolated from purified E. maxima sporozoites which are present in sporulated oocysts (see life cycle above). The isolation procedure involved extraction of proteins from the sporulated oocysts and separation of the extracted proteins on a DEAE-sephacel anion-exchange column. This was followed by SDS-PAGE of the peak fractions and Western blotting to identify the 230 kDa antigen. Furthermore, protective maternal antisera both from vaccinated hens and offspring chicks were used to confirm the identity of the purified antigen. Finally, the 230 kDa protein was isolated from a PVDF membrane filter for carrying out protein sequencing and cloning.
The amino terminal and tryptic peptide digest products of the 230 kDa antigen were sequenced. The sequences from the tryptic digest were used to design degenerate PCR oligonucleotide primers. The primers were used in RACE (rapid amplification of cDNA ends) PCR to amplify partial gene products. From the sequences of these products, gene specific primers were designed and used in RACE PCR to define the 3′ and 5′ ends of the mRNA. A full length 7 kilobase cDNA clone encoding the antigen was then amplified by PCR using gene specific primers designed to the 5′ and 3′ ends. This clone was fully sequenced and shown to contain the correct DNA sequence at its 5′ end when compared to the amino acid sequence of the N-terminus of the native protein. Thus, this nucleic acid sequence encoded the protective 230 kDa sporozoite/merozoite antigen and could now be used to produce recombinant antigen for vaccination of chickens against coccidiosis.
A plasmid encoding the 230 kDa antigen was deposited with the Australian Government Analytical Laboratories, Pymble, Australia, on Jun. 26, 2001, under Accession No. NM01/22396. The bacterial cell transformed with the 230 kDa antigen was deposited with the Australian Government Analytical Laboratories, Pymble, Australia, on Jun. 26, 2001, under Accession No. NM01/22397. Both deposits were made according to the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure.
It was previously thought that the antigen from the sporozoites/merozoites of E. maxima was a 230 kDa antigen. However, our subsequent studies have revealed that the antigen actually is a 250 kDa antigen of the sporozoites/merozoites of E. maxima. 
In an additional embodiment, the third antigen is a nucleic acid having the nucleotide sequence shown in FIG. 12 (SEQ. ID. NO. 26), a plasmid comprising the nucleic acid, or a polypeptide coded by the nucleic acid.
In still another embodiment, the vaccine further comprises a fourth antigen.
In one embodiment, the fourth antigen is a polypeptide from Gametocytes of Eimeria maxima having a molecular weight from 230 kDa to 270 kDa, a nucleotide sequence encoding the polypeptide, or a plasmid comprising the nucleotide sequence.
In a further embodiment, the antigen comprises a polypetide having the amino acid sequence shown in FIG. 13a (SEQ. ID. NO. 29) at its 5′ end or the amino acid sequence shown in FIG. 13b (SEQ. ID. NO. 31) at its 3′ end.
The subject invention also provides a method of immunizing a subject against infection by Eimeria tenella, Eimeria maxima, Eimeria acervulina, Eimeria necatrix, Eimeria praecox, Eimeria mitis or Eimeria brunetti, or a microorganism expressing an immunologically cross-reactive antigen, comprising the step of administering to the subject the vaccine of the subject invention.
In one embodiment, the subject is a species selected from the group consisting of cattle, sheep, pigs and fish.
In another embodiment, the subject is an avian species.
In a further embodiment, the avian species is selected from the group consisting of chickens, turkeys, geese, ducks, bantams, quail and pigeons.
In an additional embodiment, the avian species is chickens.
In one embodiment, the administering step comprises spraying the vaccine into the nostrils of the subject.
In a further embodiment, the administration comprises intravenous, intramuscular or intraperitoneal injection.
In another embodiment, the administration is performed in ovo.
In a further embodiment, the administration is to the air sac of an egg, thus contacting an embryo with the vaccine.
The subject invention also contains a fertilized egg from an avian species having an air sac which is inoculated with the vaccine of the subject invention, which vaccine is capable of inducing before or immediately after hatching an immune response in the embryo against a virulent form of Eimeria tenella, Eimeria maxima, Eimeria acervulina, Eimeria necatrix, Eimeria praecox, Eimeria mitis or Eimeria brunetti, or a microorganism expressing an immunologically cross-reactive antigen.
In one embodiment, the avian species is selected from the group consisting of chickens, ducks, turkeys, geese, bantams, quail and pigeons.
In another embodiment, the avian species is chickens.
The subject invention additionally provides an isolated nucleic acid comprising a nucleotide sequence encoding a 82 kDa polypeptide from Gametocytes of Eimeria maxima, or encoding a homolog of the polypeptide, or a complement of the nucleic acid.
In one embodiment, the polypeptide has the amino acid sequence shown in FIG. 5 (SEQ. ID. NO. 6).
In another embodiment, the homolog of the polypeptide has at least 50% identity to the polypeptide having the sequence shown as SEQ. ID. NO. 6.
In an additional embodiment, the homolog of the polypeptide has at least 60% identity to the polypeptide having the sequence shown as SEQ. ID. NO. 6.
In a further embodiment, the homolog of the polypeptide has at least 70% identity to the polypeptide having the sequence shown as SEQ. ID. NO. 6.
In another embodiment, the homolog of the polypeptide has at least 75% identity to the polypeptide having the sequence shown as SEQ. ID. NO. 6.
In yet another embodiment, the homolog of the polypeptide has at least 80% identity to the polypeptide having the sequence shown as SEQ. ID. NO. 6.
In an added embodiment, the homolog of the polypeptide has at least 85% identity to the polypeptide having the sequence shown as SEQ. ID. NO. 6.
In one embodiment, the homolog of the polypeptide has at least 90% identity to the polypeptide having the sequence shown as SEQ. ID. NO. 6.
In a further embodiment, the homolog of the polypeptide has at least 93% identity to the polypeptide having the sequence shown as SEQ. ID. NO. 6.
In yet another embodiment, the homolog of the polypeptide has at least 95% identity to the polypeptide having the sequence shown as SEQ. ID. NO. 6.
In one embodiment, the homolog of the polypeptide has at least 97% identity to the polypeptide having the sequence shown as SEQ. ID. NO. 6.
In an additional embodiment, the homolog of the polypeptide has at least 99% identity to the polypeptide having the sequence shown as SEQ. ID. NO. 6.
In an additional embodiment, the nucleotide sequence has greater than 50% identity to the nucleotide sequence starting at nucleotide No. 100 and ending at nucleotide No. 1886 shown in FIG. 5 (SEQ. ID. NO. 4).
In another embodiment, the nucleotide sequence has greater than 60% identity to the nucleotide sequence starting at nucleotide No. 100 and ending at nucleotide No. 1886 shown as SEQ. ID. NO. 4.
In a further embodiment, the nucleotide sequence has at least 70% identity to the nucleotide sequence starting at nucleotide No. 100 and ending at nucleotide No. 1886 shown as SEQ. ID. NO. 4.
In an additional embodiment, the nucleotide sequence has at least 75% identity to the nucleotide sequence starting at nucleotide No. 100 and ending at nucleotide No. 1886 shown as SEQ. ID. NO. 4.
In another embodiment, the nucleotide sequence has at least 80% identity to the nucleotide sequence starting at nucleotide No. 100 and ending at nucleotide No. 1886 shown as SEQ. ID. NO. 4.
In yet another embodiment, the nucleotide sequence has at least 85% identity to the nucleotide sequence starting at nucleotide No. 100 and ending at nucleotide No. 1886 shown as SEQ. ID. NO. 4.
In one embodiment, the nucleotide sequence has at least 90% identity to the nucleotide sequence starting at nucleotide No. 100 and ending at nucleotide No. 1886 shown as SEQ. ID. NO. 4.
In an additional embodiment, the nucleotide sequence has at least 93% identity to the nucleotide sequence starting at nucleotide No. 100 and ending at nucleotide No. 1886 shown as SEQ. ID. NO. 4.
In another embodiment, the nucleotide sequence has at least 95% identity to the nucleotide sequence starting at nucleotide No. 100 and ending at nucleotide No. 1886 shown as SEQ. ID. NO. 4.
In a further embodiment, the nucleotide sequence has at least 97% identity to the nucleotide sequence starting at nucleotide No. 100 and ending at nucleotide No. 1886 shown as SEQ. ID. NO. 4.
In one embodiment, the nucleotide sequence has at least 99% identity to the nucleotide sequence starting at nucleotide No. 100 and ending at nucleotide No. 1886 shown as SEQ. ID. NO. 4.
In a further embodiment, the nucleic acid is a DNA molecule.
In yet another embodiment, the DNA molecule is a cDNA molecule.
In an added embodiment, the nucleic acid has the nucleotide sequence starting at nucleotide No. 100 and ending at nucleotide No. 1886 shown as SEQ. ID. NO. 4.
In another embodiment, the nucleic acid is an RNA molecule.
In one embodiment, the isolated nucleic acid is operatively linked to a promoter of RNA transcription.
The subject invention also includes a vector comprising an isolated nucleic acid comprising a nucleotide sequence encoding a 82 kDa polypeptide from Gametocytes of Eimeria maxima, or encoding a homolog of the polypeptide, or a complement of the nucleic acid.
In one embodiment, the vector comprises the nucleic acid having the nucleotide sequence starting at nucleotide No. 100 and ending at nucleotide No. 1886 shown in FIG. 5 (SEQ. ID. NO. 4).
In another embodiment, the vector is a plasmid.
In a further embodiment, the plasmid comprises the nucleic acid having the nucleotide sequence starting at nucleotide No. 100 and ending at nucleotide No. 1886 shown in FIG. 5 (SEQ. ID. NO. 4).
In an additional embodiment, the plasmid comprises an isolated nucleic acid comprising a nucleotide sequence encoding a 82 kDa polypeptide from Gametocytes of Eimeria maxima, or encoding a homolog of the polypeptide, or a complement of the nucleic acid.
In yet another embodiment, the plasmid is the plasmid designated 82TRCHisb8 plasmid (Australian Government Analytical Laboratories Accession No. NM01/22398).
The subject invention also encompasses a host cell comprising a vector which comprises an isolated nucleic acid comprising a nucleotide sequence encoding a 82 kDa polypeptide from Gametocytes of Eimeria maxima, or encoding a homolog of the polypeptide, or a complement of the nucleic acid.
In one embodiment, the host cell comprises a vector comprising a nucleic acid having the nucleotide sequence starting at nucleotide No. 100 and ending at nucleotide No. 1886 shown in FIG. 5 (SEQ. ID. NO. 4).
In another embodiment, the host cell is selected from the group consisting of a bacterial cell; a plant cell; an insect cell; and a mammalian cell.
The subject invention additionally presents a transformed cell comprising an isolated nucleic acid comprising a nucleotide sequence encoding a 82 kDa polypeptide from Gametocytes of Eimeria maxima, or encoding a homolog of the polypeptide, or a complement of the nucleic acid.
In one embodiment, the transformed cell is the transformed cell designated clone 82TRCHisb8 in bacteria (Australian Government Analytical Laboratories Accession No. NM01/22399).
A plasmid encoding the 82 kDa antigen was deposited with the Australian Government Analytical Laboratories, Pymble, Australia, on Jun. 26, 2001, under Accession No. NM01/22398. The bacterial cell transformed with the 82 kDa antigen was deposited with the Australian Government Analytical Laboratories, Pymble, Australia, on Jun. 26, 2001, under Accession No. NM01/22399. Both deposits were made according to the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure.
In an added embodiment, the transformed cell further comprises a 82 kDa polypeptide from Gametocytes of Eimeria maxima, or a homolog of the polypeptide.
The subject invention further contains a method of producing a recombinant 82 kDa polypeptide from Gametocytes of Eimeria maxima comprising culturing a transformed cell comprising an isolated nucleic acid comprising a nucleotide sequence encoding a 82 kDa polypeptide from Gametocytes of Eimeria maxima, or encoding a homolog of the polypeptide, or a complement of the nucleic acid and isolating the recombinant 82 kDa polypeptide from Gametocytes of Eimeria maxima. The recombinant polypeptide produced by this method is also encompassed by the subject invention.
The subject invention also provides a vaccine against Eimeria tenella, Eimeria maxima, Eimeria acervulina, Eimeria necatrix, Eimeria praecox, Eimeria mitis or Eimeria brunetti, or a microorganism expressing an immunologically cross-reactive antigen, comprising the isolated nucleic acid comprising a nucleotide sequence encoding a 82 kDa polypeptide from Gametocytes of Eimeria maxima, or encoding a homolog of the polypeptide, or a complement of the nucleic acid.
In one embodiment of the vaccine, the isolated nucleic acid is a plasmid.
In addition, the subject invention presents a vaccine against Eimeria tenella, Eimeria maxima, Eimeria acervulina, Eimeria necatrix, Eimeria praecox, Eimeria mitis or Eimeria brunetti, or a microorganism expressing an immunologically cross-reactive antigen, comprising a recombinant 82 kDa polypeptide from Gametocytes of Eimeria maxima produced by culturing a transformed cell comprising an isolated nucleic acid comprising a nucleotide sequence encoding a 82 kDa polypeptide from Gametocytes of Eimeria maxima, or encoding a homolog of the polypeptide, or a complement of the nucleic acid and isolating the recombinant 82 kDa polypeptide from Gametocytes of Eimeria maxima. 
In another embodiment, the vaccine is comprised of a mixture of the isolated nucleic acid of the subject invention and the recombinant polypeptide of the subject invention.
In another embodiment, the vaccine is comprised of a mixture of the isolated nucleic acid of the subject invention, the recombinant polypeptide of the subject invention and a plasmid comprising the isolated nucleic acid comprising a nucleotide sequence encoding a 82 kDa polypeptide from Gametocytes of Eimeria maxima. 
In an added embodiment, the vaccine further comprises a second antigen.
In one embodiment, the second antigen is selected from the group consisting of a nucleic acid coding for an antigen from Eimeria maxima, a plasmid comprising such a nucleic acid, and a polypeptide coded by such a nucleic acid.
In another embodiment, the second antigen is selected from the group consisting of a 30 kDa protein from Eimeria maxima gametocytes having at the N-terminal end the amino acid sequence shown as SEQ. ID NO. 35, or SEQ. ID NO. 42 or a 14 kDa protein from Eimeria maxima gametocytes having at the N-terminal end the amino acid sequence shown as SEQ. ID NO. 37, SEQ. ID NO. 39 or SEQ. ID NO. 41.
The subject invention also provides a method of immunizing a subject against infection by Eimeria tenella, Eimeria maxima, Eimeria acervulina, Eimeria necatrix, Eimeria praecox, Eimeria mitis or Eimeria brunetti, or a microorganism expressing an immunologically cross-reactive antigen, comprising the step of administering to the subject the vaccine of the subject invention.
In one embodiment, the subject is a species selected from the group consisting of cattle, sheep, pigs and fish.
In another embodiment, the subject is an avian species.
In a further embodiment, the avian species is selected from the group consisting of chickens, turkeys, geese, ducks, bantams, quail and pigeons.
In an additional embodiment, the avian species is chickens.
In a further embodiment, the administration comprises intravenous, intramuscular or intraperitoneal injection.
In one embodiment, the administering step comprises spraying the vaccine into the nostrils of the subject.
In another embodiment, the administration is performed in ovo.
In a further embodiment, the administration is to the air sac of an egg, thus contacting an embryo with the vaccine.
The subject invention also contains a fertilized egg from an avian species having an air sac which is inoculated with the vaccine of the subject invention, which vaccine is capable of inducing before or immediately after hatching an immune response in the embryo against a virulent form of Eimeria tenella, Eimeria maxima, Eimeria acervulina, Eimeria necatrix, Eimeria praecox, Eimeria mitis or Eimeria brunetti, or a microorganism expressing an immunologically cross-reactive antigen.
In one embodiment, the avian species is selected from the group consisting of chickens, ducks, turkeys, geese, bantams, quail and pigeons.
In another embodiment, the avian species is chickens.
The subject invention also provides a recombinant polypeptide, wherein the amino acid sequence is shown as SEQ. ID NO. 3.
The subject invention also provides a recombinant polypeptide, wherein the amino acid sequence is shown as SEQ. ID NO. 6.
The subject invention also provides a 30 kDa protein from Eimeria maxima gametocytes having at the N-terminal end the amino acid sequence shown as SEQ. ID NO. 35
The subject invention also provides a 30 kDa protein from Eimeria maxima gametocytes having at the N-terminal end the amino acid sequence shown as SEQ. ID NO. 42.
The subject invention also provides a 14 kDa protein from Eimeria maxima gametocytes having at the N-terminal end the amino acid sequence shown as SEQ. ID NO. 37.
The subject invention also provides a 14 kDa protein from Eimeria maxima gametocytes having at the N-terminal end the amino acid sequence shown as SEQ. ID NO. 39.
The subject invention also provides a 14 kDa protein from Eimeria maxima gametocytes having at the N-terminal end the amino acid sequence shown as SEQ. ID NO. 41.
The aforementioned proteins and their corresponding nucleotide sequences can be used in the same manner as described above for the 56 kDa and 82 kDa proteins, including being used to immunize a subject, and to incorporate a plasmid containing a nucleotide sequence encoding the protein into a host cell.
The subject invention also provides a method of conferring upon a newborn subject of an avian species maternal immunity (antibodies) against infection by Eimeria tenella, Eimeria maxima, Eimeria acervulina, Eimeria necatrix, Eimeria praecox, Eimeria mitis or Eimeria brunetti, or a microorganism expressing an immunologically cross-reactive antigen, comprising the step of administering to the mother of the subject at a suitable time prior to the laying of a fertilized egg the vaccine of the subject invention in order to thereby confer protection via maternal immunity against infection by Eimeria tenella, Eimeria maxima, Eimeria acervulina, Eimeria necatrix, Eimeria praecox, Eimeria mitis or Eimeria brunetti, or a microorganism expressing an immunologically cross-reactive antigen, in the newborn subject.
In a further embodiment, the avian species is selected from the group consisting of chickens, turkeys, geese, ducks, bantams, quail and pigeons.
In a further embodiment, the administration comprises intravenous, intramuscular or intraperitoneal injection.
The subject invention also provides a method of reducing the output of Eimeria oocysts in feces from a newborn subject of an avian species which comprises the step of administering to the mother of the subject at a suitable time prior to the laying of a fertilized egg the vaccine of the subject invention in order induce an immune response and transmit maternal antibodies to the newborn so that the output of oocysts from the newborn is reduced.
In a further embodiment, the avian species is selected from the group consisting of chickens, turkeys, geese, ducks, bantams, quail and pigeons.
In a further embodiment, the administration comprises intravenous, intramuscular or intraperitoneal injection.
The subject invention also provides a method of immunizing a subject against infection by Eimeria tenella, Eimeria maxima, Eimeria acervulina, Eimeria necatrix, Eimeria praecox, Eimeria mitis or Eimeria brunetti, or a microorganism expressing an immunologically cross-reactive antigen, comprising the step of administering to the subject a live vaccine comprising a living non-virulent micro-organism or live virus that expresses a 56 kDa or 82 kDa polypeptide from the gametocytes of Eimeria maxima. 
In one embodiment, the live virus is the pox virus.
In one embodiment, the subject is a species selected from the group consisting of cattle, sheep, pigs and fish.
In another embodiment, the subject is an avian species.
In a further embodiment, the avian species is selected from the group consisting of chickens, turkeys, geese, ducks, bantams, quail and pigeons.
In a further embodiment, the administration comprises intravenous, intramuscular or intraperitoneal injection.
The subject invention also provides a method of immunizing a subject against infection by Eimeria tenella, Eimeria maxima, Eimeria acervulina, Eimeria necatrix, Eimeria praecox, Eimeria mitis or Eimeria brunetti, or a microorganism expressing an immunologically cross-reactive antigen, comprising the step of feeding to the subject a plant whose cells express a 56 kDa or 82 kDa polypeptide from the gametocytes of Eimeria maxima. 
In one embodiment, the plant is wheat.
In another embodiment, the plant is corn.
In one embodiment, the subject is a species selected from the group consisting of cattle, sheep, pigs and fish.
In another embodiment, the subject is an avian species.
In a further embodiment, the avian species is selected from the group consisting of chickens, turkeys, geese, ducks, bantams, quail and pigeons.
The subject invention also provides a method of immunizing a subject against infection by Eimeria tenella, Eimeria maxima, Eimeria acervulina, Eimeria necatrix, Eimeria praecox, Eimeria mitis or Eimeria brunetti, or a microorganism expressing an immunologically cross-reactive antigen, comprising the step of administering to the subject a plasmid comprising an isolated nucleic acid comprising a nucleotide sequence encoding a 56 kDa or 82 kDa polypeptide from the gametocytes of Eimeria maxima, or encoding a homolog of the polypeptide, or a complement of the nucleic acid.
In one embodiment, the subject is a species selected from the group consisting of cattle, sheep, pigs and fish.
In another embodiment, the subject is an avian species.
In a further embodiment, the avian species is selected from the group consisting of chickens, turkeys, geese, ducks, bantams, quail and pigeons.
In a further embodiment, the administration comprises intravenous, intramuscular or intraperitoneal injection.
A homolog of the nucleic acid of the invention is a nucleic acid that codes for a polypeptide which has substantially the same biological activity as the polypeptide encoded by the nucleic acid. The term “homology”, as used herein, refers to a degree of complementarity. There may be partial homology or complete homology (i.e., identity). A partially complementary sequence is one that at least partially inhibits an identical sequence from hybridizing to a target nucleic acid; it is referred to using the functional term “substantially homologous.” The inhibition of hybridization of the completely complementary sequence to the target sequence may be examined using a hybridization assay (Southern or northern blot, solution hybridization and the like) under conditions of low stringency. A substantially homologous sequence or probe will compete for and inhibit the binding (i.e., the hybridization) of a completely homologous sequence or probe to the target sequence under conditions of low stringency. This is not to say that conditions of low stringency are such that non-specific binding is permitted; low stringency conditions require that the binding of two sequences to one another be a specific (i.e., selective) interaction. The absence of non-specific binding may be tested by the use of a second target sequence which lacks even a partial degree of complementarity (e.g., less than about 30% identity); in the absence of non-specific binding, the probe will not hybridize to the second non-complementary target sequence.
As known in the art, numerous equivalent conditions may be employed to comprise either low or high stringency conditions. Factors such as the length and nature (DNA, RNA, base composition) of the sequence, nature of the target (DNA, RNA, base composition, presence in solution or immobilization, etc.), and the concentration of the salts and other components (e.g., the presence or absence of formamide, dextran sulfate and/or polyethylene glycol) are considered and the hybridization solution may be varied to generate conditions of either low or high stringency different from, but equivalent to, the above listed conditions.
It is an object of the present invention to provide nucleotide sequences encoding the 56 and 82 kDa antigens from Gametocytes of Eimeria maxima and the deduced amino acid sequence therefor. Specifically exemplified coding sequences are given in FIGS. 4 and 5, together with the deduced amino acid sequence. All synonymous coding sequences for the exemplified amino acid sequences are within the scope of the present invention.
It is a further object of the present invention to provide functionally equivalent coding and protein sequences, including equivalent sequences from other Eimeria species. Functionally equivalent 56 and 82 kDA antigens from Gametocytes of Eimeria maxima coding sequences are desirably from about 50% to about 80% nucleotide sequence homology (identity) to the specifically identified coding sequence, from about 80% to about 95%, and desirably from about 95% to about 100% identical in coding sequence to the specifically exemplified coding sequence.
Hybridization conditions of particular stringency provide for the identification of homologs of the coding sequence from other species and the identification of variant sequences, where those homologs and/or variant sequences have at least (inclusively) 50 to 85%, 85 to 100% nucleotide sequence identity, 90 to 100%, or 95 to 100% nucleotide sequence identity. Each integer and each subset of each specified range is intended within the context of the present invention.
The coding sequence and methods of the present invention include the homologous coding sequences in species other than Eimeria maxima. Methods can be employed to isolate the corresponding coding sequences (for example, from cDNA) from other organisms, including but not limited to other species such as Eimeria tenella, Eimeria acervulina, Eimeria necatrix, Eimeria praecox, Eimeria mitis and Eimeria brunetti useful in the methods of this invention using the sequences disclosed herein and experimental techniques well known to the art.
Specifically included in this invention are sequences from other species than those exemplified herein, which sequences hybridize to the sequences disclosed under stringent conditions. Stringent conditions refer to conditions understood in the art for a given probe length and nucleotide composition and capable of hybridizing under stringent conditions means annealing to a subject nucleotide sequence, or its complementary strand, under standard conditions (i.e., high temperature and/or low salt content) which tend to disfavor annealing of unrelated sequences.
“Conditions of high stringency” means hybridization and wash conditions of 65°-68° C., 0.1×SSC and 0.1% SDS (indicating about 95-100% nucleotide sequence identity/similarity). Hybridization assays and conditions are further described in Sambrook et al. (1989) Molecular Cloning, Second Edition, Cold Spring Harbor Laboratory, Plainview, N.Y. As used herein, conditions of moderate (medium) stringency are those with hybridization and wash conditions if 50-65° C., 1×SSC and 0.1% SDS (where a positive hybridization result reflects about 80-95% nucleotide sequence identity). Conditions of low stringency are typically those with hybridization and wash conditions of 40-50° C., 6×.SSC and 0.1% SDS (reflecting about 50-80% nucleotide sequence identity).
A homolog of the polypeptide of the invention is a polypeptide which has substantially the same amino acid sequence and biological activity as the polypeptide. Thus, a homolog may differ from the polypeptide of the invention by the addition, deletion, or substitution of one or more non-essential amino acid residues, provided that the resulting polypeptide retains the biological activity of the polypeptide. Persons skilled in the art can readily determine which amino acids residues may be added, deleted, or substituted (including with which amino acids such substitutions may be made) using established and well known procedures, including, for example, conventional methods for the design and manufacture of DNA sequences coding for bacterial expression of polypeptide homologs of the subject polypeptide, the modification of cDNA and genomic sequences by site-directed mutagenesis techniques, the construction of recombinant polypeptides and expression vectors, the bacterial expression of the polypeptides, and the measurement of the biochemical activity of the polypeptides by means of conventional biochemical assays.
Examples of homologs are deletion homologs containing less than all the residues specified in the subject polypeptide, substitution homologs wherein one or more residues specified are replaced by other residues, and addition homologs wherein one or more amino acids residues are added to the polypeptide. All such homologs share the biological activity of the polypeptide of the invention.
“Substantially the same polypeptide” is herein defined as encompassing the deletion, addition or substitution of fewer than four amino acids at the N-terminus of the amino acid sequence of the polypeptide. Furthermore, there may be deletions, additions or substitutions in the sequence which do not eliminate the biological activity of the polypeptide. Such modifications are known to those skilled in the art. For example, substitutions may encompass up to 10 residues in accordance with the homologous or equivalent groups described by e.g. Lehninger, Biochemistry, 2nd ed. Worth Pub., New York. (1975); Creighton, Protein Structure, a Practical Approach, IRL Press at Oxford Univ. Press, Oxford, England (1989); and Dayhoff, Atlas of Protein Sequence and Structure 1972, National Biomedical Research Foundation, Maryland (1972).
The term “biologically active”, as used herein, refers to a polypeptide having structural, regulatory, or biochemical functions of a naturally occurring molecule. Likewise, “immunologically active” refers to the capability of the natural, recombinant, or synthetic polypeptide, or any oligopeptide portion thereof, to induce a specific immune response in an animal or cells and to bind with specific antibodies.
“Substantially the same biological activity” refers to biological activity the same as that of the naturally occurring molecule possibly differing slightly in degree or level which would still be known by the skilled artisan to be the same biological activity.
The term “portion”, as used herein, in connection with a polypeptide (as in “a portion of a given polypeptide”) refers to fragments of that polypeptide. The fragments may range in size from four (4) amino acid residues to the entire amino acid sequence minus one amino acid. The term “portion”, as used herein, in connection with a nucleic acid (as in “a portion of a given nucleic acid”) refers to fragments of that nucleic acid. The fragments may range in size from twelve (12) nucleotide residues to the entire nucleic acid sequence minus one nucleotide.
A “deletion”, as used herein, refers to a change in either amino acid or nucleotide sequence in which one or more amino acid or nucleotide residues, respectively, are absent.
An “insertion” or “addition”, as used herein, refers to a change in an amino acid or nucleotide sequence resulting in the addition of one or more amino acid or nucleotide residues, respectively, as compared to the naturally occurring molecule.
A “substitution”, as used herein, refers to the replacement of one or more amino acids or nucleotides by different amino acids or nucleotides, respectively.
The present invention provides the recombinant cloning and sequencing of two of the major Eimeria maxima gametocyte antigens having molecular weights of 56 and 82 kDa.
The present invention also provides the expression of these recombinant antigens in an E. coli expression system using the plasmid pTrcHis.
The subject invention also provides a vaccine against coccidiosis comprising the recombinant 56 kDa antigen. In addition, the present invention provides a vaccine against coccidiosis comprising the recombinant 82 kDa antigen.
The present invention provides the cloning and sequencing of two of the major Eimeria maxima gametocyte antigens having molecular weights of 56 and 82 kDa.
The production of gametocytes was scaled-up in order to isolate enough gametocyte antigen to carry out amino acid sequencing (i.e. milligram quantities of the specific antigens) on the 56 and 82 kDa glycoproteins themselves. This scale up production was in itself a very difficult task, and required infecting several thousand chickens in order to provide enough material for carrying out sequence analyses. After achieving this goal, it was possible to produce enough affinity purified gametocyte antigen (APGA) to start isolating the two glycoproteins on a large scale.
The purified gametocyte antigenic glycoproteins were separated by two-dimensional, SDS polyacrylamide gel electrophoresis. After analysis of the two-dimensional gels by staining, the position of the 56 and 82 kDa antigens was determined by transfer to a PVDF membrane filter and immunodetection using antisera to APGA. After identification and removal of the 56 and 82 kDa antigens from the filter, amino acid sequencing of both their N-termini as well as internal protein sequences obtained from tryptic peptides was performed. These peptide sequences were used to predict the DNA sequences, based on which small, specific oligonucleotide probes were synthesized.
The specific oligonucleotide probes were used in RACE PCR (rapid amplification of cDNA ends) to prepare cDNA molecules from the gametocyte RNA that encodes the 56 and 82 kDa antigens. This method allowed for the production of full length cDNA molecules that are specifically amplified from mRNA molecules that contain within them the RNA sequences that encode the desired peptides. This cDNA product was then fully sequenced and the presence of the various peptides sequenced above was confirmed. Surprisingly, we found that the cDNA clones we obtained were not related to those described in Wallach et al., U.S. Pat. No. 5,932,225. Therefore, it appears that in Wallach et al., artifacts occurred when screening the cDNA library with antibodies and the clones thought to encode the 56 and 82 kDa antigens which were isolated did not in fact encode these antigens.
Finally the two new cDNA clones were used as a probe in Southern and northern blotting experiments to identify the specific gene(s) and mRNA molecule(s) that encode for the 56 and 82 kDa antigens. Whereas previously no clear banding patterns could be obtained on blots (U.S. Pat. No. 5,932,225), the number and size of gene fragments and mRNA transcripts that encode for the two antigens were clearly discerned.
The present invention further provides a method for cloning the 56 and 82 kDa antigens into a bacterial expression vector, pTrcHis, containing a poly his tag (to aid in the purification of the recombinant antigens). The two genes are then expressed in E. coli by adding a specific inducer molecule (isopropyl-α-D-thiogalactopyranoside), followed by the identification of the recombinant 56 and 82 kDa antigenic proteins by western blotting. The results of these blots showed that the 56 and 82 kDa recombinant antigens had the correct size based on measurements by mass spectrometry, and were recognized by antibodies to the his tag as well as protective antisera raised against the native 56 and 82 kDa gametocyte antigens. These results confirmed the identity of the clones and showed that these recombinant antigens can be used to replace the native antigens in the maternally based vaccine against coccidiosis in chickens.
The present invention further describes the relationship between the 56 kDa gametocyte antigen with a 30 kDa oocyst protein. This oocyst protein was shown, by immunoblotting, to strongly react with antiserum against the 56 and 82 kDa gametocyte antigens. By sequencing the amino terminus of the 30 kDa oocyst protein, we found that there was a precise match with the amino terminus of the 56 kDa antigen. It was therefore concluded that the 56 kDa antigen is processed during the development of oocysts from gametocytes into the 30 kDa protein.
The invention is further illustrated by the following examples which in no way should be construed as being further limiting. One skilled in the art will readily appreciate that the specific methods and results discussed are merely illustrative of the invention as described more fully in the claims which follow thereafter.
EXPERIMENTAL DETAILS
Example 1
Purification of Eimeria maxima Gametocytes on a Large Scale
In order to produce very large quantities of gametocytes, 4,000 heavy breed chickens were infected with 10,000 sporulated E. maxima oocysts, and were then sacrificed on day six (about 134 hours) post infection. The chicken intestines were removed, washed with PBS and cut open longitudinally. They were then cut into 1 cm long pieces and placed in a SAC buffered solution (170 mM NaCl, 10 mM Tris pH 7, 10 mM glucose, 5 mM CaCl2, 1% powdered milk) containing 0.5 mg/ml hyaluronidase (Type III from Sigma, 700 units/mg). The intestinal pieces were incubated at 37° C. for 20 minutes after which they were placed on top of a gauze filter. The pieces were rinsed with large quantities of SAC buffer and the resulting filtrate was collected. This was then filtered through a 17 micron polymon filter (Swiss Silk Bolting Cloth Mfg. Co. Ltd., Zurich, Switzerland) and the resulting filtrate was then filtered through a 10 micron filter. The gametocytes were collected from the top of the 10 micron filter, examined and counted microscopically, and placed in centrifuge bottles, which were spun at 800×g for 10 minutes. The gametocytes were then washed twice with SAC buffer, and frozen at −70° C.
Example 2
Purification of the 56, 82 and 230 kDa Gametocyte Antigens
The frozen gametocytes were thawed at room temperature and the proteins were extracted as described previously (Wallach 1995). The 56 and 82 kDa gametocyte antigens were isolated from the protein extract by running it over a Sepharose 4B column containing the monoclonal antibody 1E11-11 raised against the 56 kDa antigen. A complex of the gametocyte antigens were allowed to bind to the monoclonal antibody attached to the resin, the non-specific material was washed off using buffer, and the affinity purified gametocyte antigens (APGA) were eluted from the column. The purified APGA was lyophilized. A small sample of APGA was analyzed by SDS-PAGE where the 56 and 82 kDa native antigens were clearly visualized (FIG. 1).
Example 3
Two Dimensional Gel Electrophoresis of APGA and Isolation of the Major 56 and 82 kDa Antigens
The 56 and 82 kDa gametocyte antigens were isolated from APGA. Lyophilized APGA was prepared as described in Example 2, and was solubilized in water. The proteins were then separated by two-dimensional SDS-PAGE (FIG. 2), and identified by immunoblotting using a polyclonal chicken anti-APGA antibody, which recognizes both the 56 and 82 kDa proteins. Once identified and their location established on two-dimensional SDS-PAGE gels, the proteins were then transferred to a PVDF membrane filter, and stained with Coomassie Blue (FIG. 3). Immunoblotting was carried out at the same time, and the two blots were compared to clearly identify the 56 and 82 kDa proteins. The spots corresponding to the 56 and 82 kDa gametocyte antigens were cut out of the membranes and the amino-terminus of each antigen was sequenced.
Example 4
Amino Acid Sequencing of the Amino-Terminus as Well as Internal Tryptic Peptides from the 56 and 82 kDa Antigens
The amino-termini of the 56 and 82 kDa proteins were sequenced:










amino-terminus of the 56 kDa protein:












VPSTTPVENQVHPY-EM
(SEQ. ID. NO. 7)




 










amino-terminus of the 82 kDa protein:












-PTVLDTTTG-QVEDT
(SEQ. ID. NO. 8)







In order to determine the protein sequence of internal tryptic fragments of the 56 and 82 kDa proteins, the APGA preparation was first separated by one dimensional SDS-PAGE and stained with Coomassie Blue. The proteins were then excised from gels and digested with trypsin and sequenced.
Several tryptic peptide sequences were obtained from both proteins and the results are summarized in Table 1.








TABLE 1







Amino acid sequences of tryptic peptides isolated



from the 56 and 82 kDa antigens.


 










Peptide
56 kDa Antigen
82 kDa antigen
SEQ. ID. NO.














A
VQDV(L/I)VDA(L/I)WAS(L/I)R
ATGFSEEEVMR
 9, 10



 


B
VTEMMDM(L/I)SNR
TGGLFDQACNDAPPSR
11, 12


 


C
Q(L/I)Q(L/I)QDQMMR
TGP(L/I)STTGATGATTGPVAA(L/I)R
13, 14


 


D
AAEEF(L/I)HR
P(L/I)THVE
15, 16


 


E

R(L/I)AAVPGTTAGT
17


 


F
D(L/I)QEY(L/I)STAFNWA-
(L/I)AEGAEPRPVMPPAAATAAANLR
18, 19


 



ENQSTAYTR


 


G
RQTAAWMDRTA(L/I)EQEETT

20


 


H
MNAAMDSSNE(L/I)MTT

21


 


I
KFPET(L/I)F

22









The amino acid sequences obtained did not show any homology to any other known protein.
Example 5
RACE PCR Cloning and Sequencing of the Genes Encoding the 56 and 82 kDa Antigens
The genes for the 56 and 82 kDa proteins were amplified from gametocyte cDNA using SMART RACE PCR technology (Clonetech). RNA was isolated from E. maxima gametocytes and mRNA was purified using Dynal beads (Dynal). SMART ready cDNA was synthesized following the protocols according to the manufacturer's instructions using the reverse transcriptase Powerscript (Clonetech). Amplifications of both the 5′ and 3′ ends were carried out using the protocols described in the SMART RACE PCR manual, and the DNA polymerase, Advantage Taq (Clonetech), a high fidelity enzyme mixture.
The gametocytes had been isolated from chicken intestines, filtered a number of times and washed thoroughly as described in Example 1. There was a concern that residual chicken intestinal material was still present in this preparation. Consequently, PCRs carried out using degenerate primers designed to the amino-terminus of the 56 and 82 kDa genes and degenerate primers designed to internal tryptic peptide fragments gave rise to bands in both cDNA samples prepared from purified gametocytes and uninfected chicken cells. In this situation PCR bands, which stained intensely with ethidium bromide on agarose gels, were purified, cloned into pGEMT-Easy (Promega) and sequenced (SUPAMAC sequencing service, Sydney, Australia). In some cases, when rearrangements were observed or the cloned fragment was difficult to sequence, sequence was obtained directly from the PCR product. If the DNA sequence data from the PCR product translated to any of the amino acid sequences of the tryptic peptides, the PCR product was of parasitic origin and sequencing continued.
The full length sequence of the 56 and 82 kDa proteins are shown in FIGS. 4 and 5, respectively. The full length sequence of the 230 kDa protein is presented in FIG. 12.
Amino acid sequence of the tryptic peptides and N-terminus of the 56 gametocyte antigen matched the deduced amino acid sequence arising from the corresponding cloned DNA.
Nine tryptic peptides were sequenced for the 56 kDa protein (Table 1). All peptides but one, sb56i, could be mapped to the cloned gene corresponding to the 56 kDa protein (FIG. 4). This tryptic fragment may correspond to a contaminating band present in the sample. In detail:

    
    
        Tryptic peptides sb56a, sb56b, sb56c, sb56d, sb56f and sb56h matched precisely to the deduced amino acid sequence predicted by the cloned DNA.
        Tryptic peptide sb56g did not match precisely to the deduced amino acid sequence predicted by the cloned DNA. The sequence of the tryptic fragment was reanalysed, and the new sequence matched more closely with the predicted sequence derived from the cloned DNA.

Tryptic fragment sb56g original sequence:

    
    













RQ--TAAWMDR-TA[L/I]EQEETT
(SEQ. ID. NO. 23)







Reanalysed sb56g sequence:











RGVQTAAWMDGVTA I EKEETT
(SEQ. ID. NO. 24)







Deduced aa sequence from DNA:











RGVQTAAWMNGVTA I EKEETT
(SEQ. ID. NO. 25)







A discrepancy still remains in this peptide at amino acid 10, where the protein sequence reveals a D and the DNA sequence predicts a N. This segment of DNA was sequenced 4 times, and each time predicted an N.
Amino acid sequence of the tryptic peptides and N-terminus of the 82 gametocyte antigen match the deduced amino acid sequence arising from the corresponding cloned DNA.
Seven tryptic peptides were sequenced for the 82 kDa protein (Table 1). All peptides but two, sb82d and sb82e could be mapped to the cloned gene corresponding to the 82 kDa protein. This tryptic fragment may correspond to a contaminating band present in the sample. In detail:

    
    
        Tryptic peptides sb82a, sb82b, sb82c, sb56d and sb82f matched precisely to the deduced amino acid sequence predicted by the cloned DNA.
        Tryptic peptides sb82d and sb82e did not match to the deduced amino acid sequence predicted by the cloned DNA.
    
    


In addition to the sequence information described above:
1) The predicted size of the ORF encoding the mature form of the 82 kDa protein is 64,275 Da, which corresponded to the true size of the native protein of 62,236 Da, as determined by mass spectrometry.
2) The predicted size of the ORF encoding the mature form of the 56 kDa protein is 51,407 Da which corresponded to the true size of the native protein of 52,450 Da, as determined by mass spectrometry.
Finally, the two protein and DNA sequences did not show any homology to any other known gene or protein.
Example 6
Southern and Northern Blotting Using the 56 and 82 kDa cDNA Cloned Probes.
Southern blotting using E. maxima and chicken DNA was carried out by first cutting the DNA with a variety of restriction enzymes and separating the resulting DNA fragments on an agarose gel. This is followed by transferring the DNA to nitrocellulose paper, probing with a P32 labeled cDNA probe for the 56 (FIG. 6) or 82 (FIG. 7) kDa antigens and performing autoradiography. The results showed that for both the 56 and 82 kDa antigens there appear to be two different, single copy genes, which encodes the two proteins.
Northern blotting using E. maxima and chicken RNA was carried out by separating the RNA molecules on an agarose gel, transferring it to a nitrocellulose filter and probing with the P32 labelled 56 and 82 cDNA clones. The results showed that the 56 kDa mRNA has a molecular weight of about 1.9 KB and the 82 kDa mRNA had a molecular weight of about 2.4 KB (FIG. 8). These sizes are very similar to those predicted from the DNA sequences.
Example 7
Expression of the Recombinant 56 and 82 kDa Antigens using the pTrcHis Vector in E. coli and Their Analysis using Sera Against Native APGA.
The full length gene encoding the 82 kDa protein was amplified from E. maxima gametocyte cDNA using gene specific primers carrying terminal restriction sites to facilitate directional cloning into the expression vector pTRCHisb (Invitrogen). The full length gene included the coding region of the amino-terminus of the mature protein and sequence up to, but not including, the stop codon (575 aa). A partial fragment of the gene encoding the 56 kDa protein was amplified from E. maxima gametocyte cDNA using gene specific primers carrying terminal restriction sites. This included the amino-terminus of the protein and a further 323 amino acids of sequence, 133 amino acids shorter than the full length mature protein. Both genes were cloned into the commercially available vector pTrcHisb (Invitrogen).
1) Expression of the 56 kDa Gene in pTrcHis B
Transformed bacteria were induced with 1 mM IPTG, and bacterial lysates were analyzed by 1D-SDS PAGE and immunoblotting (FIG. 9). A commercially available anti-His antibody to the His fusion tag of the recombinant protein recognized a band of the predicted size of 40 kDa (this clone lacks the coding region for 133 amino acids) under inducing conditions. Under non-induced conditions there was also a low level of reactivity with this band indicating that there is some degree of leakiness of the gene expression. Recognition of the recombinant 56 kDa protein was then assessed by immunoblotting with the chicken polyclonal anti-APGA antibody. The immunoblot showed that the anti-APGA antibody recognized the native form of the protein by one dimensional SDS-PAGE, as well as the recombinant protein, clearly demonstrating that the cloned gene product indeed codes for the 56 kDa protein.
2) Expression of the 82 kDa Gene in pTrcHis B
Transformed bacteria were induced with 1 mM IPTG, and bacterial lysates were analysed by one dimensional SDS PAGE and immunoblotting (FIG. 10). A commercially available anti-His antibody to the His fusion tag of the recombinant protein recognized a band of the predicted size of 82 kDa under inducing and non-inducing conditions. Recognition of the recombinant 82 kDa protein was then assessed by immunoblotting with the chicken polyclonal anti-APGA antibody. This antibody was produced by immunizing chickens with native APGA isolated from purified gametocytes. The immunoblot showed that the anti-APGA antibody recognized the native form of the protein by 1D SDS-PAGE, as well as the recombinant protein, clearly demonstrating that the cloned gene product indeed codes for the 82 kDa protein.
Based on the above results together with the sequence analyses described in Example 5, we concluded that the two cDNA clones described above are the authentic genes encoding for the 56 and 82 kDa antigens. In addition, the strong reactivity with the antisera raised against the native antigens shows that these recombinant proteins can now be used to replace APGA for the immunization of chickens against coccidiosis.
Example 8
Homology of the 56 kDa Antigen With a 30 kDa Antigen from E. maxima Oocysts.
Antibodies to APGA were used to detect homologous proteins on a two-dimensional blot of oocyst antigens. We found that there was very strong reactivity with a protein of 30 kDa (FIG. 11). This spot was cut out of the membrane filter and the N-terminus of the protein was sequenced. The resulting amino acid sequence corresponded precisely to the N-terminal sequence of the gametocyte 56 kDa antigen. Based on this finding we concluded that the gametocyte 56 kDa antigen is processed into the 30 kDa protein of the oocyst stage of development.
Example 9
Expression of gam 56, a 56 kDa Gametocyte Antigen from Eimeria maxima 


    Native protein: Mr 56,000 (size determined by molecular sizing (MS): 52,450 Da)
    Source: Parasitic: Eimeria maxima 
    Life cycle stage: macrogametocyte
    Gene: 1,754 base pairs sequenced presented over 5 polymerase chain reaction (PCR) fragments, all of which are cloned into pGEMT-Easy, except for the last  ̃600 bp of the gene, which includes  ̃400 bp of the coding region.
    5′UTR(1-102 bp)
    ORF (103-1,533 bp)
    3′UTR (1,534-1,731 bp)
    polyA tail(1,732-1,754 bp)
    pI: 4.8 predicted from sequence (by 2D SDS-PAGE, the protein migrates towards the acidic end of the gel)

Expression Constructs

    Expression vectors used: pTRCHisb, pET25b
    Expression construct: given p56TRCHisb1
    Gene fragment that was cloned into the expression vector: gam 56 was amplified from cDNA using the following primer pairs: SB74/SB75 (172-1137 bp) for directional cloning into the BamHI/EcorI site of TRCHisb. The amplified region contains the sequence encoding the amino terminus of the mature protein, excluding the initiator methionine and leader sequence. It contains a tyrosine-serine rich region and excludes a proline-methionine rich region.


Amino acid composition of cloned gam 56 fragment:

    2 cysteines present
    amino acid composition of gene fragment cloned into pTRCHisb:


















S(12.7%) 
Y(11.5%) 
A(8.7%)
T(8.4%)
P(7.2%)


R(6.6%)
E(6.0%)
M(5.5%)
L(5.5%)
V(4.6%)


Q(4.3%)
N(4.3%)
G(3.8%)
D(3.5%)
W(1.7%)


F(1.4%)
K(1.4%)
I(1.4%)
H(0.9%)
C(0.6%)









    Predicted protein size: 41 kDa
    Yield: 0.9-1.4 μg/ml (nickel agarose purified protein/ml culture) Difficult to see induced protein in crude bacteria lysate on a Commassie Blue stained gel.

Expression Conditions:



The promoter is leaky, therefore we can get expression in the absence of IPTG.
Used baffled flasks, 37° C., 4 h induction, with 1 mM IPTG, 0.1 mg/ml ampicillin in 0.01 M Mg2+SOB(SOB better than LB).
Normally, one predominate band at  ̃42 kDa is obtained after purification and detection with silver staining. Often some higher molecular weight bands, which may be aggregates, are obtained after purification as well as the main  ̃42 kDa band. The protein seems to aggregate at −20° C. and 4° C.; after purification we desalt and add stabilisers (3% lactose, 1% monosodium glutamate).
Example 10
Immunization and challenge trial of the recombinant 56 kDa (r56) and 82 kDa (r82) gametocyte antigens, and the 250 kDa (r250) asexual stare antigen in chickens
Immunization
Animals


    Chickens:
    
        84 day old ( ̃12 weeks) Australorp cockerels
        kept on medicated (robenidene) food
        all chickens were individually tagged and recorded

Antigens

    
    


Recombinant proteins in the pTRCHisb expression system were grown at 37° C. in 0.1 mg/ml ampicillin in 0.01M Mg2+SOB and induced for 4 hours with 1 mM IPTG. Proteins were purified on a Ni-agarose column, concentrated, desalted, and lyophilized with stabilizers (3% lactose, 1% monosodium glutamate). Protein concentrations used for all antigens were measured using the Bradford assay. Affinity Purified Gametocyte Antigen (APGA) preparations provided by M. Wallach was used as a positive control for the trial.
Groups and Doses


    
    
        9 chickens used per group; 9 groups in total; 81 chickens used in total.
        Chickens were immunized with 0.5 ml antigen/Freunds Incomplete Antigen (FIA) cocktail (0.25 ml antigen/0.25 ml FIA) per bird, intra-muscularly, on one side only of the chicken, with the following antigens:
    
    


















Group 1
PBS only



Group 2
Adjuvant (FIA)/PBS



Group 3
APGA (2.5 g)



Group 4
r250 protein (1.0 g)



Group 5
r250 protein (10.0 g)



Group 6
r56 protein (0.5 g)



Group 7
r56 protein (5.0 g)



Group 8
r82 protein (0.5 g)



Group 9
r82 protein (5.0 g)










Immnization Schedule

















Immunization 1:
week 1



Immunization 2:
week 3



Bleed:
week 6



Bleed:
week 8



Bleed/Kill:
week 9










Analyzes

    
    
        Bleeds were taken ( ̃1.5-2 ml/bird), sera separated and tested by ELISA and immunoblotting

Results

    
    


Results of the bleeds are shown in FIG. 14.
Challenge
Animals and Parasites


    
    
        5 chickens (148 days old;  ̃4.5 months) from each group which had the highest antibody titre based on the ELISA results of bleed 1 were used; in the case of the PBS and FIA controls, chickens with the lowest antibody titres were used
        E. maxima (strain Houghton);
        robenidene was removed from the feed one week prior to challenge

Groups

    
    


The following groups and chickens were taken from the immunization trial described above, and used in the challenge experiments














Group 1
PBS only
chicken numbers 2, 3, 4,




6, 8


Group 2
Adjuvant (FIA)/PBS
chicken numbers 12-16


Group 3
APGA (2.5 g)
chicken numbers 20, 22,




23, 25, 27


Group 5
r250 protein (10.0 g)
chicken numbers 37, 39, 41,




44, 45


Group 7
r56 protein (5.0 g)
chicken numbers 57, 59, 60,




61, 63


Group 9
r82 protein (5.0 g)
chicken numbers 74, 75, 76,




79, 80









Challenge Schedule

Robenidene removed
Challenged with 100 sporulated oocysts per bird Day 6
Oocyst Harvest and Count Schedule


    Day 0 post-infection
    Day 1 post-infection
    Day 2 post-infection
    Day 3 post-infection
    Day 4 post-infection


Checked oocyst output for contamination of another species
Replaced plastic sheet to start collections.

    Day 5 post-infection Feces collected, and oocysts counted
    Day 6 post-infection Feces collected, and oocysts counted
    Day 7 post-infection Feces collected, and oocysts counted
    Day 8 post-infection Feces collected, and oocysts counted
    Day 9 post-infection Feces collected, and oocysts counted
    Day 10 post-infection Feces collected, and oocysts counted









TABLE 2







Immunization and Challenge Trial I










Groups/
Cumulative oocyst counts (×106)
Output (%)
% inhibition






















Day p.i.
6
7
8
9
10
6
7
8
9
10
6
7
8
9
10

























1. PBS
6.67
17.00
26.40
27.33
27.43
100
100
100 
100 
100
0
0
0
0
0


only



2. FIA only
3.20
14.40
17.30
17.50
17.50
48
85
66
64
64
52
15
34
36
36








(100)
(100)
(100)
(100)
(100)
(0)
(0)
(0)
(0)
(0)


3. APGA
2.77
9.35
13.48
13.58
13.61
42
55
51
50
50
58
45
49
50
50


(2.5 μg)





(87)
(65)
(78)
(78)
(78)
(13)
(35)
(22)
(22)
(22)


5. r250
0.83
8.35
13.72
14.72
14.72
12
49
52
54
54
88
51
48
46
46


(10 μg)





(26)
(58)
(79)
(84)
(84)
(74)
(42)
(21)
(16)
(16)


7. r56
0.33
4.53
7.20
8.16
8.53
5
27
27
30
31
95
73
73
70
69


(5 μg)





(10)
(32)
(42)
(47)
(49)
(90)
(68)
(58)
(53)
(51)


9. r82
4.23
10.33
14.73
14.93
15.06
63
61
56
55
55
37
39
44
45
45


(5 μg)





(132)
(72)
(85)
(85)
(86)
(0)
(28)
(15)
(15)
(14)









Example 11
Expression of a Recombinant Fragment of the 250 kDa Asexual Stage Protein
The region of the 250 kDa protein encoding the predicted transmembrane domain/cytosolic tail and upstream hydrophilic domain was selected for expression studies (FIG. 15). The area was chosen for a number of reasons and are as follows: 1) similar 3′ hydrophilic tail regions have been identifiied in a number of apicomplexan microneme proteins and appear unique to this family of proteins; 2) such regions have been identified in other microneme proteins also recognised as immunodominant, primarily Eimeria tenella microneme protein 1 (EtMIC1) and surface antigen 5401 (EtMIC4); 3) a similar region was expressed from the E. tenella 5401 antigen (EtMIC4) and was found to afford significant protection against challenge with E. tenella (Danforth et al, 1988); 4) other regions of the protein consist primarily of the EGF-like and TSP-1-like domains. These domain types are found highly conserved within eukaryotes and therefore the possiblility of their inducing auto-immunity must be considered. Furthermore because of the prevalence of such domain types it seems unlikely that they would be responsible for inducing a strong immune response.
PCR primers EP006 (5′-TTGGATCCCGAATTGCACCCCA TTCC-3′) and EP007 (5′-TTGAATTCTGAATGTCGCCGCTGTCG-3′) were designed to amplify the selected DNA region from a cDNA clone encoding the 250 kDa protein. The primers incorporated BamHI (EP006) and EcoRI (EP007) restriction sites to facilitate cloning into the selected expression vector. The PCR product subsequently generated using the primers was gel-purified and its identity confirmed by sequencing.
The bacterial expression vector pTrcHisB (Invitrogen) was selected for expression studies. Plasmid vector DNA and gel purified cDNA insert were digested with the restriction enzymes BamHI and EcoRI, and the digested DNA fragments gel purified and ligated. The ligation mixture was transformed into E. coli strain DH5-a and following plating and incubation, resulting colonies were selected, cultured and used for plasmid preparation. The identity of the selected recombinants was confirmed by DNA sequencing.
In preparation for expression, plasmid DNA containing the expression construct was transformed into the E. coli host expression strain TOP10. Following plating and incubation, a single bacterial colony was selected and used to establish an O/N culture in LB media. A vector only negative control culture was also established. Aliquots of each culture were then transferred to fresh LB media and incubated until the cells reached mid-log phase, at which stage expression was induced with the addition of 1 mM IPTG. Samples from the expression culture and negative control culture were taken at 0, 1, 2, 5 and 24 hrs post induction, and centrifuged to pellet the bacterial cells. All pellets were subsequently resuspended in TE buffer, sonicated and centrifuged to separate the aqueous soluble fraction (supernatant) from the insoluble fraction (pellet). All fractions were analysed under reducing conditions on SDS-PAGE gels and subsequently stained with Coomassie Blue. When compared to the negative control samples, an over-expressed protein was detected in the soluble fractions, migrating at just below the 45 kDa marker. Western analyis of the soluble fractions using an antibody reactive with the 6× Histidine tag of pTrcHis expression products, detected a protein band of the same apparent molecular weight. The predicted size of the expressed protein is approximately 30 kDa, somewhat less than that observed on SDS-PAGE gels. The size difference might be explained by the high frequency of proline residues in the expressed protein, known to cause proteins to migrate with apparently high molecular weight.
In preparation for immunogenicity trials, the expressed protein was purified using Ni-NTA Agarose nickel-charged resin (QIAGEN), with minor modifications to the manufacturer's recommended protocol. Expressed proteins containing the 6× His tag bind to the resin and are displaced by an increased concentration of imidazole in the elution buffer. Briefly, cell pellets were resuspended in Lysis buffer (50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole, pH 8.0), containing 1 mg/ml lysozyme. The suspension was sonicated on ice and centrifuged to pellet insoluble material. The supernatant containing the soluble expressed protein was then mixed with Ni-NTA resin and added to a disposable elution column. The slurry was allowed to settle then washed with Wash buffer (50 mM NaH2PO4, 300 mM NaCl, 20 mM imidazole, pH 8.0), before elution with Elution buffer (50 mM NaH2PO4, 300 mM NaCl, 250 mM imidazole, pH 8.0). The purity of eluted fractions was analysed by reducing SDS-PAGE and Coomassie Blue staining.
Details for the immunogenicity trials are as for the 56 kDa and 82 kDa trials. For the mouse trial, 0.5 μg and 5 μg doses of the recombinant protein per mouse were used (6 mice/group). For the chicken trial, 1 82 g and 10 82 g doses per bird were used (9 chickens/group). ELISA results for the collected serum samples from the mouse and chicken trials are presented in FIGS. 16 and 17 respectively.
Example 12
The oocyst wall-of Eimeria is derived from precursor proteins found in the sexual stage of the parasite (macrogametocyte) which undergo processing and di-tyrosine crosslinking to form the hardened, protective barrier of the excreted form of the parasite
The genes encoding the 56 kDa and 82 kDa sexual stage, macrogametocyte antigens have been cloned and sequenced. Both genes show an unusual amino acid composition, and in particular, both have tyrosine-rich regions; the 56 kDa protein possesses one tyrosine-rich region and the 82 kDa protein possesses two tyrosine-rich regions. Proteins rich in tyrosine have been previously implicated in oocyst wall formation in E. acervulina and E. tenella. (Eschenbacher et al.) Thus, the role of the tyrosine rich region in the 56 kDa and 82 kDa sexual stage antigens in oocyst wall formation was explored in Eimeria maxima. 
Antibodies to the recombinant form of the 56 kDa protein (anti-r56) and antibodies to the recombinant form of the 82 kDa protein (anti-r82) recognize a  ̃30 kDa protein in unsporulated and sporulated oocysts, and a  ̃30 kDa protein in purified wall fragments (see FIGS. 18 and 19). They also recognize their native form counterparts in gametocyte extracts. The  ̃30 kDa protein recognized in purified oocyst wall fragments by the anti-r82 kDa antibody is not the same as the  ̃30 kDa protein recognized by the anti-r56; it is slightly smaller. The  ̃30 kDa protein recognized by the anti-r56 antibody was purified and the N-terminus sequenced. The N-terminus of the  ̃30 kDa protein corresponds exactly to the N-terminus of the 56 kDa gametocyte antigen (see FIG. 20a).
Others have shown by SDS-PAGE and coomassie blue staining that the oocyst wall of Eimeria is composed of two predominant proteins of 14 kDa and 30 kDa. Using better SDS-PAGE separation techniques, we have resolved the 14 kDa protein into 3 components of  ̃10-14 kDa, and named them 14.1, 14.2 and 14.3, where 14.1 represents the protein which has migrated the slowest on SDS-PAGE gels, and 14.3 the fastest (see FIG. 18c). We have sequenced the N-terminus of all four proteins and the results are presented in FIG. 20. In summary, the 30 kDa protein is a novel protein which does not show any similarity to any other previously characterized protein as determined through a BLAST protein search (see FIG. 20c). The N-terminus of protein 14.3 corresponds to the beginning of the tyrosine rich region in domain 1 of the 82 kDa protein (see FIG. 20b), the N-terminus of protein 14.2 corresponds to the beginning of the tyrosine rich region in domain 2 of the 82 kDa protein (see FIG. 20b), and the N-terminus of protein 14.1 corresponds to the beginning of the tyrosine rich region in the 56 kDa protein (see FIG. 20a).
Together these results show that the oocyst wall of Eimeria is derived from precursor proteins found in the wall forming bodies of the sexual stage (macrogametocyte) of the parasite. Through some signaling mechanism, they are proteolytically processed into several shorter proteins of  ̃30 kDa and  ̃14 kDa. Contrary to previous findings, our data indicates that the oocyst wall is composed of more than two proteins. Our findings suggest that the oocyst wall is composed of several proteins present at different levels in the parasite, some of which are in high abundance that they are recognized by coomassie blue staining of SDS-PAGE gels, and others that are present at low levels, only detected through the more sensitive technique of immunoblotting. The  ̃30 kDa protein seen in coomassie blue stained SDS-PAGE gels is not related to the 56 kDa and 82 kDa gametocyte antigens, however, the smaller  ̃10-14 kDa proteins are. Our most recent finding that di-tyrosine is present at detectable levels in the order of 0.00338 mmol/mol in oocysts, indicates that the small tyrosine rich proteins are probably held in the wall through a mechanism involving di-tyrosine crosslinks. However, we believe that not all the proteins are held in the wall in this way and are currently investigating this.
REFERENCES
Eschenbacher, K. H., Eggli, P., Wallach, M. and Braun, R. (1995) Characterization of a 14 kDa oocytst wall protein of Eimeria tenella and E. Acervulina, Parasitol., 112:169-176.
Fried, M., Mencher, D., Sar-Shalom, O., and Wallach, M. (1992) Developmental gene expression of a 230-kilodalton macrogamete-specific protein of the avian coccidial parasite, Eimeria maxima. Mol. & Biochem. Parasitol., 51:251-262.
Mencher, D., Pugatsch, T. and Wallach, M. (1989) Antigenic proteins of Eimeria maxima gametocytes: cell-free translation and detection with recovered chicken serum. Exp. Parasitol. 68:40-48.
Wallach, M., Pillemer, G., Yarus, S., Halabi, A., Pugatsch, T. and Mencher, D. (1990) Passive immunization of chickens against Eimeria maxima infection with a monoclonal antibody developed against a gametocyte antigen. Infection & Immunity 58:557-562.
Wallach, M., Smith, N. C., Petracca, M., Miller, C. M. D., Eckert, J. and Braun, R. (1995) Eimeria maxima gametocyte antigens: potential use in a subunit maternal vaccine against coccidiosis in chickens. Vaccine, 13:347-354.
Wallach, M. and Vermeulen, A., (1996) Progress Towards a Subunit Vaccine Against Coccidiosis. Misset's World Poultry, Supplement Coccidiosis (2), 22-24.

## Claims

1. An isolated nucleic acid sequence comprising a nucleotide sequence encoding a 56 kDa polypeptide having the amino acid sequence set forth as SEQ. ID. NO. 3 present in gametocytes of Eimeria maxima, or the full complement of the nucleic acid.

2. An isolated vector comprising an isolated nucleotide sequence encoding a 56 kDa polypeptide having the amino acid sequence set forth as SEQ. ID. NO. 3 present in gametocytes of Eimeria maxima, or the full complement of the nucleic acid.

3. An isolated host cell comprising an isolated vector comprising an isolated nucleotide sequence encoding a 56 kDa polypeptide having the amino acid sequence set forth as SEQ. ID. NO. 3 present in gametocytes of Eimeria maxima, or the full complement of the nucleic acid.

4. A method of producing a 56 kDa polypeptide present in gametocytes of Eimeria maxima comprising culturing isolated host cells comprising an isolated nucleotide sequence encoding a 56 kDa polypeptide having the amino acid sequence set forth as SEQ. ID. NO. 3 and isolating the 56 kDa polypeptide from the cells so cultured.

5. A vaccine for immunizing a subject against infection by Eimeria maxima comprising an isolated nucleic sequence encoding a 56 kDa polypeptide having the amino acid sequence set forth as SEQ. ID. NO. 3 present in gametocytes of Eimeria maxima or the full complement of the nucleic acid.

6. A method of immunizing a subject against infection by Eimeria maxima comprising administering to the subject the vaccine of claim 5.

7. The isolated nucleic acid of claim 1, wherein the nucleotide sequence is set forth as SEQ ID NO: 1.

8. The isolated nucleic acid of claim 1, wherein the isolated nucleic acid is a plasmid designated 56TRCHisbl plasmid deposited under Australian Government Analytical Laboratories Accession No. NM01/122400.

9. The nucleic acid of claim 1, wherein the nucleic acid is a DNA molecule or an RNA molecule.

10. The nucleic acid of claim 9, which is a cDNA molecule.

11. The nucleic acid of claim 1, further comprising an operatively linked promoter.

12. The vector of claim 2, wherein the vector is a plasmid.

13. The host cell of claim 3, wherein the cell is a bacterial cell, a plant cell, an insect cell, or a mammalian cell.

14. An isolated transformed cell comprising an isolated nucleotide sequence encoding a 56 kDa polypeptide having the amino acid sequence set forth as SEQ. ID. NO. 3 present in gametocytes of Eimeria maxima or the full complement of the nucleic acid.

15. The transformed cell of claim 14, wherein the cell is designated clone 56TRCHisbl bacteria deposited under Australian Government Analytical Laboratories Accession No.NM01/22401.

16. The vaccine of claim 5, wherein the subject is an avian species.

17. The vaccine of claim 16, wherein avian species is chickens, ducks, turkeys, geese, bantams, quail, or pigeons.

18. The vaccine of claim 5, wherein the vaccine is designed to be administered by intravenous, intramuscular or intraperitoneal injection; or by spraying said vaccine into the nostrils of the subject.

19. The vaccine of claim 17, wherein the vaccine is designed to be administered in ovo.

20. The vaccine of claim 17, wherein the vaccine is designed to be administered to an air sac of an egg.