AU620366B2 - Cloned genes coding for avian coccidiosis antigens which induce a cell-mediated response and method of producing the same - Google Patents

Description

WO 89/07650 PCT/US88/04172 -1- 1 CLONED GENES CODING FOR AVIAN COCCIDIOSIS 2 ANTIGENS WHICH INDUCE A CELL-MEDIATED RESPONSE 3 AND METHOD OF PRODUCING THE SAME 4 BACKGROUND OF THE INVENTION Coccidiosis, an intestinal disorder of poultry, 6 causes an assortment of problems in the infected host.

7 These problems range from poor feed conversion ratios in 8 light infections to acute death in heavier infections.

9 The disease has been estimated to cost U.S. broiler producers S300 million per year, due in part to 11 unrealized weight gains, loss of skin pigmentation, and 12 poor feed utilization, and in part to the cost of anti- 13 coccidial drugs.

14 Coccidiosis is caused by protozoans belonging to the genus Eimeria. The members of this genus have a 16 complicated life cycle which consists of both asexual and 17 sexual stages. The cycle is initiated when birds ingest 18 sporulated oocysts (generally associated with fecal 19 material). These oocysts contain invasive asexual sporozoites which are released into the bird's digestive 21 tract. The sporozoites invade epithelial cells and 22 develop into multinucleate structures called schizonts.

23 Each schizont matures and releases numerous invasive 24 asexual structures, known as merozoites, into the bird's digestive tract, where they in turn invade other 1 J WO 89/07650 PCT/US88/04172 2 1 epithelial cells. The sexual stage of the coccidiosis 2 life cycle is initiated when merozoites differentiate 3 into gametocytes. The developing asexual and/or sexual 4 stages produce the pathological digestive tract lesions characteristic of coccidiosis. Gametocytes then fuse 6 and the fertilization products, called oocysts, are 7 released in the feces. The formation of oocysts 8 completes the parasite's life cycle.

9 Infections by protozoans of the genus Eimeria can be alleviated, and even prevented, by the administration 11 of anti-coccidial agents. However, drug-resistant 12 strains arise at a frequent rate and the cost of 13 development of anti-coccidials is quite high. Chickens 14 can be vaccinated against the disease by infection with live attenuated strains of Eimeria or with non-living 16 parasite material. However, there is an appreciable 17 disease effect using the former approach and a 18 prohibitive amount of material would be required to make 19 the latter useful on a large-scale basis. Furthermore, protection with the latter is far from complete. An 21 alternative solution would be to produce, by genetic 22 engineering, the protective antigens of Eimeria 23 parasites. Once developed, the immunogens could be 24 produced in a prokaryotic or eukaryotic culture system in an unlimited supply and used to vaccinate chickens 26 against subsequent disease.

27 Immune responses are mediated by two different 28 effector mechanisms. One mechanism, which involves the 29 production of antibodies by lymphoid tissue, is termed "humoral immunity". The other, which involves the 31 activation of white blood cells such as T-lymphocytes 32 previously sensitized to the immunogen, is termed "cell- 33 mediated immunity". See Immunology: Basic Processes, 12 34 Bellanti 2d ed., 1985).

^i l WO 89/07650 PCT/US88/04172 3 1 PCT Application Publication No. WO 86/00528 to 2 Anderson, et al., titled "Cloned Gene and Method for 3 Making and Using the Same", discloses cloned genes which 4 code for antigenic proteins of Eimeria species. The procedure taught by this application for screening 6 transformed cells to identify these DNA sequences 7 involves the use of polyvalent chicken antiserum obtained 8 from chickens previously infected with Eimeria tenella.

9 See Id. at 9, 41-43 and 44. Chicken antiserum, however, reflects only the humoral immune response of the bird to 11 the antigen to which the bird has been exposed, and does 12 not reflect the bird's cell-mediated immune response.

13 Accordingly, the Anderson patent teaches how to obtain 14 DNA sequences which code for the production of antigens which evoke a humoral immune response. While an 16 important contribution to the art, Anderson does not 17 address whether these same antigens evoke a cell-mediated 18 immune response, and if not-- how such antigens might 19 be obtained. Nor does it address the relative contribution of the cell-mediated immune response and the 21 humoral immune response to the integrated immune response 22 of birds to avian coccidiosis.

23 Accordingly, an object of the present invention is 24 to provide a means for producing DNA sequences which code for antigens which evoke a cell-mediated immune response 26 to avian coccidiosis.

27 An additional important object of the present 28 invention is to provide a means for identifying DNA 29 sequences which code for antigens which evoke a cellmediated immune response to avian coccidiosis and which 31 may not otherwise be identified by prior art procedures.

32 A further object of the present invention is to 33 provide cloned genes which code for Eimeria sporozoite or 34 merozoite cell surface antigens, particularly Eimeria acervulina cell surface antigens.

1 i-i it I i WO 89/07650 PCTI/US88/04172 4 A still further object of the present invention is to provide transformed host cells which produce antigens which evoks a cell-mediated immune response to avian coccidiosis.

Still further objects' of the present invention are to provide methods and vaccines useful for protecting birds against avian coccidiosis.

SUMMARY OF THE INVENTION 9 11 12 13 14 16 17 18 19 21 22 23 24 26 27 28 29 31 32 The present invention, in a first respect, involves a method for obtaining desired DNA sequences.

The method involves, initially, providing a multiplicity of Eimeria DNA sequences (ultimately provided as either a genomic DNA or complementary DNA library). These sequences are then inserted into DNA expression vectors to form recombinant expression vectors. Next, the recombinant expression vectors are inserted into suitable hosts to form transformants which express the DNA sequences. These transformants are then screened with contacted to) antibodies which are directed against Eimeria cell surface antigens to identify transformants containing DNA sequences which code for Eimeria cell surface antigens. After this, Eimeria cell surface antigens are produced from the DNA sequences identified as coding for such antigens. These Eimeria cell surface antigens are then contacted to white blood cells that have been sensitized to antigenic Eimeria protein (specifically, white blood cells which effect a bird's cell-mediated immune response) to thereby identify DNA sequences which code for antigens that induce a cellmediated immune response to avian coccidiosis.

It has unexpectedly been found that, by following the foregoing procedure, DNA sequences which code for I Ii IL. It& I I 2 l WO 89/07650 PCI/US88/04172 1 antigenic proteins useful as vaccines for avian 2 coccidiosis can be obtained which could not be obtained 3 by prior sequence identification processes. DNA 4 sequences provided by the foregoing procedure which are not provided by prior art procedures are identified by 6 contacting the Eimeria cell surface antigens produced by 7 the procedure described above to immune sera taken from 8 an Eimeria infected bird. Those antigens not recognized 9 by the immune sera are coded for by DNA sequences which would not have been identified by prior art procedures.

11 A second aspect of the present invention is the 12 products which may be produced by the foregoing 13 procedures. These products are DNA sequences which 14 comprise a cloned gene or fragment thereof that activate white blood cells which effect a cell-mediated immune 16 response, which white blood cells are sensitized to an 17 antigenic Eimeria protein. The antigens are preferably 18 directed against Eimeria cell surface antigens, such as 19 Eimeria sporozoite or Eimeria merozoite cell surface antigens. One group of DNA sequences of the present 21 invention code for antigenic proteins not recognized by 22 immune sera taken from Eimeria infected birds.

23 Recombinant expression vectors are also disclosed 24 herein. These vectors comprise an expression vector having a promoter and a DNA sequence as described above 26 inserted in the vector downstream of the promoter and 27 operatively associated therewith. Transformed cells s 28 disclosed herein, useful for making antigens, comprise a 29 host cell and a recombinant DNA expression vector as described above contained within the host cell. The 31 expression vector promoter is selected so as to be 32 operable in the host cell.

33 A further aspect of the present invention is the 34 antigens produced by the transformed host cells, which antigens are discussed in part above. These antigens 4 f 'V I m m -6comprise proteins or fragments thereof that activate white blood cells, which white blood cells effect a cell-mediated immune response, and which white blood cells are sensitized to an antigenic Eimeria protein.

Methods and vaccines useful for protecting birds against infection by avian coccidiosis are also disclosed herein. The method comprises administering to a bird an antigen as described above in an amount effective to immunize the bird against the avian coccidiosis. The vaccines are comprised of such antigens, in an amount effective to immunize a bird against avian coccidiosis, in combination with a pharmaceutically acceptable carrier. The term "immunize", as used herein, means any level of protection which is of some benefit in a Spopulation of birds, whether in the form of decreased mortality, decreased lesion scores, improved feed conversion ratios, or the reduction of any detrimental effect of avian 'coccidiosis, regardless of 15 whether the protection is partial or complete.

According to a first embodiment of this invention, there is provided a DNA sequence which encodes an antigenic protein upon expression, wherein said antigenic protein is recognized by monoclonal antibody or polyclonal antibodies raised in animals immunized with denatured Eimeria antigens, which Eimeria antigens activate avian T cells, but not avian B cells, derived from an avian species that has been previously infected with Eimeria parasites.

According to a second embodiment of this invention, there is provided a recombinant DNA expression vector, comprising an expression 25 vector having a promoter, and a first DNA sequence inserted in said vector downstream of said promoter and operatively associated therewith, wherein said DNA sequence encodes an antigenic protein upon expression, wherein said antigenic protein is recognized by monoclonal antibody or polyclonal antibodies raised in animals immunized with denatured Eimeria antigens, which Eimeria antigens activate avian T cells, but not avian B cells, derived from an avian species that has been previously infected with Eimeria parasites.

According to a third embodiment of this invention, there is provided a transformed host cell comprising a recombinant DNA expression vector contained within said host cell, said vector comprising a promoter operable in said host cell and a DNA sequence inserted at a site downstream of said promoter and operatively asociated therewith, wherein said DNA sequence encodes an antigenic protein upon expression, wherein said antigenic protein is recognized by monoclonal antibody or polyclonal 1/666Z Y i: 6A antibodies raised in animals immunized with denatured Elmeria antigens, which Elmeria antigens activate avian T cells, but not avian B cells, derived from an avian species that has been previously infected with Eimeria parasites.

According to a fourth embodiment of this invention, there is provided a method of identifying DNA sequences that code for antigenic proteins useful as vaccines against avian coccidiosis, said method comprising: a. providing a multiplicity of Eimeria DNA sequences; b. inserting the DNA sequences into DNA expression vectors to form recombinant expression vectors; c. inserting the recombinant expression vectors into suitable host cells to form transformants which express the DNA sequences; d. contacting the transformants with antibodies directed against 15 denatured Eimeria antigens to identify transformants containing DNA sequences which code for Eimeria antigens; S"e. expressing Eimeria antigens from the DNA sequences identified in and f. contacting the expressed Eimeria antigens with T cells, which T cells are sensitized to an antigenic Eimeria protein, to thereby S" identify DNA sequences which code for antigens that are useful in a primary immunization of avian species against infection by avian coccidia.

DEFINTIONS

SThe following terms are employed herein: 25 Cloning. The selection and propagation of genetic material from a single individual, a vector containing one gene or gene fragment, or a single organism containing one such gene or gene fragment.

Cloning Vector. A plasmid, virus, retrovirus, bacteriophage or nucleic acid sequence which is able to replicate in a host cell, characterized by one or a small number of restriction endonuclease recognition sites at which the sequence may be cut in a predetermined SLMM/666 LMM/666Z I i:i WO 89/07650 PCT/US88/04172 -7- 1 fashion, and which contains a marker suitable for use in 2 the identification of transformed cells, e.g., 3 tetracycline resistance or ampicillin resistance. A 4 cloning vector may or may not possess the features necessary for it to operate as an expression vector.

6 Codon. A DNA sequence of three nucleotides 7 (a triplet) which codes (through mRNA) for an amino acid, 8 a translational start signal or a translational 9 termination signal. For example, the nucleotide triplets TTA, TTG, CTT, CTC, CTA and CTG encode for the amino acid 11 leucine TAG, TAA and TGA are translational stop 12 signals, and ATC is a translational start signal.

13 DNA Sequence. A linear series of 14 nucleotides connected one to the other by phosphodiester bonds between the 3' and 5' carbons of adjacent pentoses.

16 Expression. The process undergone by a 17 structural gene to produce a polypeptide. Expression 18 requires both transcription of DNA and translation of 19 RNA.

Expression Vector. A plasmid, virus, 21 retrovirus, bacteriophage or nucleic acid sequence which 22 is able to replicate in a host cell, characterized by a 23 restriction endonuclease recognition site at which the 24 sequence may be cut in a predetermined fashion for the insertion of a heterologous DNA sequence. An expression 26 vector has a promoter positioned upstream of the site at 27 which the sequence is cut for the insertion of the 28 heterologous DNA sequence, the recognition site being 29 selected so that the promoter will be operatively associated with the heterologous DNA sequence.

31 Fusion Protein. A protein produced when two 32 heterologous genes or fragments thereof coding for two 33 different proteins not found fused together in nature are 34 fused together in an expression vector. For the fusion protein to correspond to the separate proteins, the WO 89/07650 PCT/US88/04172 8 1 separate DNA sequences must be fused together in correct 2 translational reading frame.

3 Genome. The entire DNA of an organism. It 4 includes, among other things, the structural genes encoding for the polypeptides of the substance, as well 6 as operator, promoter and ribosome binding and 7 interaction sequences including sequences such as the 8 Shine-Dalgarno sequences.

9 Heterologous DNA. A DNA sequence inserted within or connected to another DNA sequence which codes 11 for polypeptides not coded for in nature by the DNA 12 sequence to which it is joined.

13 Nucleotide. A monomeric unit of DNA or RNA 14 consisting of a sugar moiety (pentose), a phosphate, and nitrogenous heterocyclic base. The base is lined to the 16 sugar moiety via the glycosidic carbon carbon of the 17 pentose) and that combination of base and sugar is a 18 nucleoside. The base characterizes the nucleotide.

19 The four DNA basis are adenine guanine cytosine and thymine The four RNA bases 21 are A, G, C and uracil 22 Phage or Bacteriophage. Bacterial virus 23 many of which include DNA sequences encapsidated in a 24 protein envelope or coat ("capsid"). In a unicellular organism, a phage may be introduced by a process called 26 transfection.

27 Plasmid. A non-chromosomal double-stranded 28 DNA sequence comprising an intact "replicon" such that 29 the plasmid is replicated in a host cell. When the plasmid is placed within a unicellular organism, the 31 characteristics of that organism may be changed or 32 transformed as the result of the DNA of the plasmid. For 33 example, a plasmid carrying the gene for tetracycline 34 resistance (TetR) transforms a cell previously sensitive to tetracycline into one which is resistant to

H!

I,

WO 89/07650 PCT/US88/04172 -9- 1 it. A cell transformed by a plasmid is called a 2 "transformant".

3 Polypeptide. A linear series of amino acids 4 connected one to the other by peptide bonds between the alpha-amino and carboxy groups of adjacent amino acids.

6 Promoter. A DNA sequence within a larger 7 DNA sequence defining a site to which RNA pjlymerase may 8 bind and initiate transcription.

9 Reading Frame. The grouping of codons during translation of mRNA into amino acid sequences.

11 During translation the proper reading frame must be 12 maintained. For example, the DNA sequence GCTGGTTGTAAG 13 may be translated via mRNA into three reading frames, 14 each of which affords a different amino acid sequence: GCT GGT TGT AAG Ala-Gly-Cys-Lys 16 G CTG GTT GGTA AG Leu-Val-Val 17 GC TGG TTG TAA A Trp-Leu-(STOP) 18 Recombinant DNA Molecule. A hybrid DNA 19 sequence comprising at least two DNA sequences-, the first sequence not normally being found together in nature with 21 the second.

22 Ribosomal Binding Site. A nucleotide 23 sequence of mRNA, coded for by a DNA sequence, to which 24 ribosomes bind so that translation may be initiated. A ribosomal binding site is required for efficient 26 translation to occur. The DNA sequence coding for 27 ribosomal binding site is positioned on a larger DNA 28 sequence downstream of a promoter and upstream from a 29 translational start sequence. Shine-Dalgarno sequences are prokaryotic ribosomal binding sites.

31 Start Codon. Also called the initiation 32 codon, is the first mRNA triplet to be translated during 33 protein or peptide synthesis and immediately precedes the I, 1 1 1 WO 89/07650 PCT/US88/04172 10 1 structural gene being translated. The start codon is 2 usually AUG, but may sometimes also be GUG.

3 Structural Gene. A DNA sequence which 4 encodes through its template or messenger RNA ("mRNA") a sequence of amino acids characteristic of a specific 6 polypeptide that is an integral constituent of a cellular 7 organelle (such as a cell membrane).

8 Transcription. The process of producing 9 mRNA from a structural gene.

Translation. The process of producing a 11 polypeptide from mRNA.

12 DETAILED DESCRIPTION OF THE INVENTION 13 In practicing the present invention, any protozoan 14 species in the genus Eimeria may be employed. These include Eimeria acervulina, E. mivati, E. mitis, E.

16 praecox, E. hagaini, E. necatrix, E. maxima, E. brunetti 17 and E. tenella. These protozoa are known and available 18 to those skilled in the art, as avian coccidiosis is 19 found on poultry farms throughout the world. See, e.g., M. Wisher, Molec. Biochem. Parasitol., 21:7 (1986); P.

21 Murray, et al., European Patent Application Publication 22 No. 0223710; R. Schenkel, et al., European Patent 23 Application Serial No. 0135073. Particularly preferred 24 for practicing the present invention is E. acervulina.

The present invention may be practiced with any 26 bird susceptible to avian coccidiosis, including 27 chickens, turkeys, ducks, geese, quail, partridge, and 28 pheasant. A particularly preferred bird with which to 29 practice the present invention is the chicken.

A multiplicity of DNA sequences obtained from an 31 Eimeria species for use in practicing the present 32 invention may be generated by conventional techniques.

:jI -E 14. A transformed host cell comprising a recombinant DNA expression vector contained within said host cell, said actor comprising a promoter operable in said host cell and a DNA sequence t,-dA ed at a S. /2 WO 89/07650 PCT/US88/04172 11 1 One approach is to digest the genomic DNA of an Eimeria 2 species, with the ultimate goal being the preparation of 3 a genomic DNA library. See generally, R. Old and S.

4 Primrose, Principles of Gene Manipulation, 102-109 (3d ed., 1985). A more preferable approach is to isolate 6 mRNA from an Eimeria species and generate cDNA sequences 7 therefrom, with the ultimate goal being the preparation 8 of a cDNA library. See generally, R. Old and S.

9 Primrose, supra at 109-111; T. Maniatis, E. Fritsch and J. Sambrook, Molecular Cloning: A Laboratory Manual, 11 187-246 (1982). DNA sequences in a cDNA library do not 12 contain introns, as these have been removed by the 13 splicing of the mRNA from which the cDNA sequences are 14 prepared. If the DNA sequences are to expressed in a bacterial host, they should preferably be cDNA sequences, 16 as eukaryotic introns are not spliced out by bacteria.

17 A variety of vector-host combinations may be 18 employed in practicing the present invention. Host cells 19 may be either prokaryotic or eukaryotic cells, and, when the host cells are bacterial cells, they may be either 21 gram negative or gram positive bacteria. Useful hosts 22 include Escherichia coli (including, for example, E. coli 23 X1776, E. coli X2282, E. coli HB101 and E. coli MRC1), 24 species of Salmonella (including, for example, S.

typhimurium, S. enteriditis, and S. dublin) species of 26 Pseudomonas (including, for example, P. aeruginosa and P.

27 putida), Bacillus subtilis, yeasts and other fungi (for S28 example, Saccharomyces cerevisiae), plant cells such as 29 plant cells in culture (including, for example, both angiosperms and gymnosperms) and animal cells such as 31 animal cells in culture.

32 Vectors used in practicing the present invention 33 are selected to be operable as cloning vectors or 34 expression vectors in the selected host cell. The vectors may, for example, be bacteriophage, plasmids, o WO 89/07650 PCT/US88/04172 12- 1 2 3 4 6! 7 8.

9.

11 12 13 14 16 17 18 19 21 22 23 24 26 27 287 29 31 32 33 34 viruses, or hybrids thereof. Vectors useful in E. coli including plasmids (for example, pSC101, ColEl, RSF2124, pBR322, pBR324, pBR325, pAT153, pUC-6 and pUC-8), bacteriophage lambda, cosmids, phasmids, and filamentous coliphages. Salmonella species may be transformed, for example, with plasmids such as pJC217, pBRDOO1, and pBRD026. Vectors useful in gram negative bacteria generally include plasmids of incompatibility groups P, Q or W, which have broad host ranges (for example, Sa, RP4, and RSF1010), and Transposons such as TnT. Bacillus subtilis, a gram positive bacteria, can be transformed with S. auerus plasmids (for example, pC194, pE194, pSa0501, pUB110, and pT127). Yeast host vectors include yeast integrating plasmids (such as pYeLeu 10), yeast episomal plasmids (such as pJDB219 and pJDB248), yeast replicating plasmids (which contain an autonomously replicating sequence, or "ars", derived from a yeast chromosome), and yeast centromere plasmids (which contain centromeres functional in yeast). Plant cell vectors include Geminiviruses, Caulimoviruses (such as CaMV, CERV, DaMV, FMB, MMV, CVBFV and ThMV) and Agrobacterium tumefaciens containing Ti plasmids. Mammalian cell vectors include viruses (such as SV40), retroviruses, and adenoviruses.

Within each specific vector various sites may be selected for insertion of the isolated DNA sequence.

These sites are usually designated by the restriction enzyme or endonuclease that cuts them. For example, in pBR322 the Pst I site is located in the gene for penicillinase between the nucleQtide triplets that code for amino acids 181 and 182 of the penicillinase protein.

The particular site chosen for insertion of the selected DNA fragment into the vector to form a recombinant vector is determined by a variety of factors. These include size and structure of the ll p 11 WO 89/07650 PCT/US88/04172 13 1 polypeptide to be expressed, susceptibility of the 2 desired polypeptide to enzymatic degradation by the host 3 cell components and contamination by its proteins, 4 expression characteristics such as the location of start and stop codons, and other factors recognized by those of 6 skill in the art. None of these factors alone absolutely 7 controls the choice of insertion site for a particular 8 polypeptide. Rather, the site chosen reflects a balance 9 of these factors, and not all sites may be equally effective for a given protein.

11 Eimeria DNA sequences may be inserted into the 12 desired vector by known techniques. If, however, the 13 vector is to serve as an expression vector, the vector 14 should have a promoter, and the DNA sequence should be inserted in the vector downstream of the promoter and 16 operationally associated therewith. The vector should 17 be selected so as to have a promoter operable in the host 18 cell into which the vector is to be inserted (that is, 19 the promoter should be recognized by the RNA polymerase of the host cell). In addition, the vector should have 21 a region which does for a ribosome binding site 22 positioned between the promoter and the site at which the 23 DNA sequence is inserted so as to be operatively 24 associated with the Eimeria DNA sequence once inserted (preferably, in correct translational reading frame 26 therewith). The vector should be selected to provide a 27 region which codes for a ribosomal binding site 4 28 recognized by the ribosomes of the host cell into which 29 the vector is to be inserted. For example, if the host cell is to be a prokaryotic cell such as E. coli, then 31 the region which codes for a ribosomal binding site may 32 code for a Shine-Dalgarno sequence.

33 A preferred method for carrying out the present 34 invention is with vectors which produce a fusion protein. Such vectors include, in order from upstream to j ~I V WO 89/07650 PCT/US88/04172 -14 1 downstream, a promoter, a region which codes for 2 ribosomal binding site, a translational start codon, a 3 sequence which codes for a first protein or fragment 4 thereof immediately following the translational start codon, and a site at which the second Eimeria DNA 6 sequence is inserted. The Eimeria DNA sequence (which 7 codes for a protein or fragment thereof different from 8 the first DNA sequence, beta-galactosidase) is 9 inserted in the vector so that it is spliced directly to the first sequence and is in correct translational 11 reading frame therewith. The first and second DNA 12 sequence thereby together code for a fusion protein.

13 In the present invention, transformed host cells 14 are screened with antibodies (monoclonal or polyclonal) directed against Eimeria cell surface antigens prior to 16 the step of screening for white blood cell activation.

17 Prior art procedures employ immune sera taken from 18 Eimeria infected birds. Such sera is not directed 19 specifically to cell surface antigens. In the present invention, polyclonal antibodies taken from an animal 21 which has been administered denatured Eimeria cell 22 surface antigens are employed. Alternatively, monoclonal 23 antibodies directed against specific Eimeria cell surface 24 antigens may be employed.

One unique development disclosed herein involves 26 the use of monoclonal antibodies directed to the Eimeria 27 refractile body. Through the use of such antibodies, DNA 28 sequences which code for a refractile body protein or 29 fragment thereof are identified. These DNA sequences may also be employed in practicing the present invention.

31 Antibodies directed against Eimeria cell surface 32 antigens may be obtained by procedures known in the art.

33 See, Timmins, et al., Gene, 39:89 (1985); H.

34 Danforth and P. Augustine, Poultry Sci., 62:2145 (1983); see generally, G. Kohler and C. Milstein, Nature, 256:495 f 3 atbde drce gistseii ieracl ufc 9V :L n i WO 89/07650 PCT/US88/04172 15 1 (1975). These antibodies may be of any origin, but are 2 preferably rabbit or murine (mouse or rat) antibodies.

3 They may be of any class of immunoglobulin, including 4 IgG, IgA, IgD, IgE, and IgM. Preferred is IgG, such as IgGl, IgG2, IgG3 and IgG4.

6 For screening against white blood cells, the 7 antigens coded for by the DNA sequence of interest may be 8 produced by any conventional means. It may be exprested 9 from the same vector and host cell in which it was originally cloned, transferred to a different host for 11 expression, or transferred to a different vector and host 12 for expression. The antigenic expression product is then 13 extracted from the host cell culture and used in the 14 white blood cell screening step.

White blood cells useful for practicing the 16 present invention include any white blood cells which 17 effect a cell-mediated, as opposed to humoral, immune 18 response. Such cells include, for example, Granulocytes 19 (such as Basophils and mast cells, Eosinophils, and neutrophils), T-lymphocytes, Macrophages, K cells and NK 21 cells. The white blood cells should be white blood 22 cells sensitized to an Eimeria antigen. Particularly 23 preferred for practicing the present invention are T- 24 lymphocytes. Procedures for determining whether such cells are activated by an antigen are either known to 26 those skilled in the art or will be apparent to those 27 skilled in the art through the present disclosure, and S 28 include both in vitro and in vivo tests. For example, 29 the antigen could be topically applied to an Eimeria sensitized subject and the region to which the antigen 31 was applied examined for a wheal and flare reaction.

32 Suitable subjects for such a test would include rabbits, 33 rats and mice. Alternatively, the antigen could be 34 injected subcutaneously into the wattle of an Eimeria sensitized chicken and the chicken's wattle examined for i acervulina cell surface antigens.

WO 89/07650 PCT/US88/04172 -16 1 swelling. The most quantifiable procedure, and therefore 2 most preferable, is to incubate white blood cells taken 3 from an Eimeria sensitized bird in an aqueous solution 4 containing the antigen. See also H. Lillehoj, "Immune Response during Coccidiosis in SC and FP Chickens I. In 6 vitro assessment of T cell proliferation to stage 7 specific parasite antigens, Vet. Immunol. Immunopathol., 8 13:321 (1986); see also, M. Beaven, et al., J. Pharm.

9 Exp. Ther., 224:620 (1983); M. Beaven, et al., Clin.

Chem. Acta., 37:91 (1972). Preferably, the white blood 11 cells are obtained (removed from) a bird during the 12 bird's second exposure to Eimeria. At such time, the 13 bird's white blood cells, which have previously been 14 sensitized, are increasing in number.

Numerous issued U.S. patents are available which 16 disclose information useful to those skilled in the art 17 in practicing the present invention. U.S. Patent No.

18 4,710,463 to Murray discloses recombinant DNA expression 19 vectors incorporating DNA sequences coding for Hepatitis B virus antigens. U.S. Patent No. 4,601,980 to Goeddel 21 and Heyneker discloses the expression of a gene coding 22 for human growth hormone in a pBR322/E. coli system.

23 U.S. Patent No. 4,590,163 to Helinski and Ditta di-'closes 24. RK2 plasmids useful for gene cloning in gram-negative bacteria such as E. coli. U.S. Patent No. 4,237,224 to 2.6 Cohen and Boyer discloses methods for producing 27 recombinant DNA expression vectors. U.S. Patent No.

28, 4,332,897 to Nakano, et al. discloses lambdoid 29 bacteriophage vectors useful for transforming E. coli.

U.S. Patent No. 4,332,901 to Goldstein discloses a P4 31 derivative bacteriophage cloning vector. U.S. Patent 32 No. 4,704,362 to Itakura and Riggs and U.S. Patent No.

33 4,356,270 to Itakura disclose recombinant plasmid vectors 34 useful for transforming microbial hosts. U.S. Patent No.

4,273,875 to Manis discloses a plasmid designated pUC6 24; RK2___ p~la uocit

I

WO 89/07650 PCT/US88/04172 17 1 useful as a cloning vector for transforming microbial 2 hosts. U.S. Patent No. 4,349,629 to Carey, et al.

3 discloses plasmid vectors employing the trp bacterial 4 promoter useful as recombinant DNA expression vectors.

U.S. Patent No. 4,362,817 to Reusser discloses the 6 plasmid pUC1060, which contains a tet gene promoter, 7 useful as an expression vector. U.S. Patent No.

8 4,599,308 to Hamer, et al. discloses SV40 expression 9 vectors which can be introduced into eukaryotic cells.

U.S. Patents Nos. 4,693,976 to Schilperoort, et al., 11 4,536,475 to Anderson, and 4,459,355 to Cello and Olsen 12 all concern the transformation of plant cells with the Ti 13 plasmid of Agrobacterium tumefaciens. The disclosures 14 of all U.S. patent references cited herein are to be incorporated herein by reference.

16 In the examples below, three specific DNA 17 sequences coding for Eimeria cell surface antigens are 18 disclosed. Clone MA1 is disclosed in Table 1, clone 19 cMZ-8 is disclosed in Table 2, and clone MC17 is disclosed in Table 3. The sequences are shown in their 21 5' to 3' orientation. Those skilled in the art can make 22 numerous uses of this information. First, working from 23 the same Eimeria acervulina starting materials, they can 24 reisolate the same sequences. Second, they can generate oligonucleotide probes homologous to the sequences shown 26 in the Tables and isolate DNA sequences which hybridize 27 to the probes. Such probes are preferably at least 16, 28 and more preferably at least 20, nucleotides in length.

29 Still greater binding selectivity is achieved with probes 30 nucleotides or more in length, but the advantages of 31 probes having lengths much greater than this tend to be 32 offset by the disadvantage of the decreased yields of 33 longer probes which can be synthesized. Third, in view 34 of the known degeneracy of the genetic code (more than one codon codes for the same amino acid), they can i

A

i WO 89/07650 PCT/US88/04172 18 18 1 produce different DNA sequences which can code on 2 expression for the same polypeptides coded for an 3 expression by any of the foregoing DNA sequences.

4 The antigens of the present invention may be administered by any convenient route, such as 6 subcutaneously, intraperitoneally, or intramuscularly, in 7 the presence of physiologically acceptable diluent. The 8 antigens may be administered in a single dose or in a 9 plurality of doses. The antigens of the present invention may be administered, if desired, in combination 11 with vaccine stabilizers and vaccine adjuvants. Typical 12 stabilizers are, for example, sucrose, an alkali metal 13 hydrogen phosphate salt, glutamate, serum albumin, 14 gelatin, or casein. The stabilizer may be any one or more of the foregoing. The adjuvant may be, for 16 example, alum or a composition containing a vegetable 17 oil, isomannide monooleate and aluminum monostearate.

18 The antigens of the present invention may be stored under 19 refrigeration or in frozen or lyophilized form.

Antigens may be administered to birds via 21 biological routes. A transformed host cell which 22 expresses a DNA sequence coding for an antigen may be 23 administered to a bird so that the host cell expresses 24 the antigen in the bird. For example, a transformed Salmonella host may be orally administered to the bird.

26 Alternatively, the antigen may be incorporated into a 27 vector capable of transforming avian cells (for example, 28 a retrovirus) and that vector administered to the subject 29 bird so that the bird's own cells serve as host cells for the vector, express the antigen, and present the antigen 31 to the subject. All of these procedures are procedures 32 for making and administering antigens of the present 33 invention to birds to protect these birds from avian 34 coccidiosis.

i S said antigenic protein Is recognized by monoclonal antibody or polyclonal M/666Z i WO 89/07650 PCT/US88/04172 19 1 The following examples are provided to more 2 completely explain the present invention. They are for 3 illustrative purposes only, and are not to be taken as 4 limiting thereof.

EXAMPLE 1 6 EXTRACTION OF RNA FROM SPOROZOITES 7 A mixture of sporulated and unsporulated Eimeria 8 acervulina occysts (2.2 x 109 total) were homogenized in 9 a Potter-Elvheim grinder in the presence of DNA/RNA extraction buffer (4 M guanidine isothiocyanate, 0.1 M 11 B-mercaptoethanol, 10mM ethylenediamine tetraacetic acid 12 (EDTA), 5mM sodium citrate, 0.5% sodium sarcosine). The 13 homogenate was diluted into 1.6 volumes cesium 14 trifluoracetate and centrifuged at 45,000 rpm for 16h at 20 0 C. The RNA band was removed by syringe needle 16 puncture of the centrifuge tube and was diluted in 2 17 vols. 100% ethanol and stored for 15 min. at -70 0 C. The 18 precipitating RNA was pelleted by centrifugation at 19 10,000 rpm for 30 min. at 4 0 C. The RNA pellet was washed twice with 70% ethanol and collected by 21 centrifugation. The RNA pellet was dried in vacuo and 22 then dissolved in H 2 0. The RNA solution was extracted 23 twice with a 1:1 solution of phenol-chloroform, twice 24 with chloroform, and then precipitated overnight with 0.1 vol. 3 M sodium acetate and 2.5 vol. ethanol at -20 0

C.

26 The RNA was collected by centrifugation, dried in vacuo, 27 and dissolved in H 2 0.

i i LMM/666Z WO 89/07650 PCT/US88/04172 1 EXAMPLE 2 2 EXTRACTION OF RNA FROM MEROZOITES 3 Merozoites of E. acervulina (2.9 x 109 total) were 4 obtained from infected chickens and purified as described in Jenkins and Dame, Molec. Biochem. Parasitol., 25:155 6 (1987). The merozoites were disrupted by pipetting in 7 the presence of DNA/RNA extraction buffer. The isolation 8 of RNA thereafter was identical to the procedure 9 described for sporozoite RNA isolation (Section 1).

EXAMPLE 3 11 SELECTION OF SUBPOPULATIONS OF RNA HAVING POLY A TAILS 12 RNA from sperozoites and merozoites were passed 13 through an oligo-dT cellulose-column (Pharmacia) to 14 enrich for poly A+ RNA mRNA). Once bound, the poly A+ RNA was eluded from the column with distilled 16 H 2 0, collected, and precipitated with ethanol and sodium 17 acetate. The mRNA was pelleted by centrifugation, 18 washed with 80% ethanol, centrifuged once again, dried in 19 vacuo, and dissolved in H 2 0.

EXAMPLE 4 21 PREPARATION OF SPOROZOITE AND MEROZOITE cDNA LIBRARIES 22 FROM THE RESPECTIVE SPOROZOITE AND MEROZOITE mRNA 23 A. Preparation of recombinant bacteriophage 24 lambda DNA. cDNA was generated from poly A+ RNA of E.

acervulina sporozoites and merozoites using established 26 techniques. See Gubler and Hoffman, Gene, 25:263-269 1 WO 89/07650 PCT/US88/04172 21 1 (1983). In brief, first strand cDNA synthesis was 2 accomplished by priming with oligo dT in the presence of 3 RNAsin, the appropriate dNTPS, and Moloney Murine 4 Leukemia Virus (MMLV) reverse transcriptase. cDNA synthesis was halted after lh at 37 0 C with EDTA and the 6 cDNA-RNA mixture was phenol-chloroform extracted and 7 precipitated with ethanol and sodium acetate, 8 centrifuged, dried in vacuo, and resuspended in H 2 0.

9 Second strand synthesis was carried out in the presence of dNTPS and RNase H by DNA polymerase I. After a lh 11 incubation at 15 0 C followed by a lh incubation at 22 0

C,

12 the reaction was halted and the double-stranded cDNA was 13 phenol-chloroform extracted, precipitated with ethanol 14 and ammonium acetate, collected by centrifugation, dried in vacuo, and resuspended in 50mM Tris, pH 7.6, ImM EDTA, 16 5mM DTT. The cDna was methylated with s-adennsyl 17 methionine by EcoRI methylase and the cDNA ends polished 18 with T4 DNA polymerase I. After phenol-chloroform 19 extractions, ethanol precipitation, centrifugation, and drying in vacuo, the methylated cDNA was tailed with 21 EcoRI linkers using T4 DNA ligase and RNA ligase. The 22 cDNA was then digested with EcoRI restriction enzyme for 23 1.5h at 37°C to produce EcoRI compatible ends, stopped 24 with EDTA, phenol-chloroform extracted, ethanol precipitated, collected by centrifugation, dried in 26 vacuo, resuspended in 10mM Tris, pH 7.5, 0.1mM EDTA, 0.4 27 M NaC1 and passed over a NACS column (Bethesda Research 28 Laboratories). The cDNA was eluted with high salt 29 buffer (2 M NaCl in TE), ethanol precipitated, collected by centrifugation, dried in vacuo, and resuspended in 31 TE. The cDNA was ligated to lambda gtll DNA containing 32 compatible EcoRI ends using T4 DNA ligase at 4 0 C for 16h.

33 B. Packaging of recombinant bacteriophage 34 lambda DNA into virions and transfection of E. coli i_ o i

I

WO 89/07650 PCT/US88/04172 -22 1 Y1090. Recombinant sporozoite cDNA- and merozoite 2 cDNA-bacteriophage DNA was packaged into intact virions 3 using a Packagene extract following the procedures 4 described by the manufacturer (Promega Biotech). After packaging, an aliquot of the recombinant bacteriophage 6 was used to transfect Escherichia coli strain Y1090 7 cells. After infection, the cells were plated on Luria 8 broth (LB) agar plates in LB agarose containing 0.2% 9 X-gal, 4mM IPTG, and 75 ug/ml ampicillin. Using this technique, greater than 95% of the bacteriophage were 11 observed to be recombinants contained cDNA 12 inserts) as judged by the percentage of white plaques of 13 the total plaques obtained. The sporozoite and merozoite 14 cDNA libraries were estimated to contain 4.1 x 106 and 2.4 x lD6 clones, respectively.

16 EXAMPLE 17 IDENTIFICATION OF RECOMBINANT PHAGES BY SCREENING 18 E. coli WITH IMMUNE SERA FROM RABBITS ADMINISTERED 19 E. acervulina SPOROZOITE OR MEROZOITE MEMBRANE FRACTIONS 21 A. Preparation of immune sera. In order to 22 screen the cDNA bacteriophage libraries, immune sera were 23 generated by immunizing rabbits with denatured membrane 24 preparations of E. acervulina sporozoites or merozoites.

Individual rabbits were immunized intramuscularly with 26 either denatured sporozoite or denatured merozoite 27 membranes emulsified in Complete Freund's Adjuvant and 28 boosted with an identical dose in Incomplete Freund's 29 Adjuvant (1 ug membrane protein) two times further at 1 week intervals. Immune sera were collected when 31 anti-sporozoite and anti-merozoite ELISA titers reached a 32 plateau and were processed using standard methods. See, i; I iP :I ~cr ylvrr~~r VL YII~I~~YI-I WO 89/07650 PCT/US88/04172 23 1 Shiigi and Slomick, 111 Selected Methods in 2 Cellular Immunology, 295-305, (Mishell and Shiigi, eds.

3 1980). Prior to screening the bacteriophage libraries, 4 immune sera were absorbed with E. coli Y1090 cellular extract to remove anti-E. coli antibodies.

6 B. Immunoscreening of Recombinant 7 Bacteriophages. Aliquots (approximately 105 clones) of 8 the SZ and MZ bacteriophage libraries were used to 9 transfect E. coli Y1090 and plated as described in Section 4B. The plates containing developing phage 11 plaques were overlaid with nitrocellulose disks which had 12 been soaked in 10mM IPTG and were incubated at 37 0 C for 13 4h to induce production of the galactosidase fusion 14 protein. The nitrocellulose disks, impregnated with the E. coli proteins containing the recombinant fusion 16 protein were removed, treated with phosphate-buffered 17 saline (PBS) .containing 0.05% Tween 20 and 1% BSA for 18 min. at room temperature to block non-specific antibody 19 (Ab) binding. The filter disks were then incubated overnight with a 10- 2 dilution of absorbed rabbit 21 anti-sporozoite membrane sera or anti-me:,ozoite membrane 22 sera. The Ab binding was developed by subsequent 23 treatment with biotinylated anti-rabbit (or anti-mouse) 24 IgG followed by avidin-peroxidase. Substrate (0.5 mg/ml 4-chloro-l-napthol and 0.1% H 2 0 2 was added and positive 26 bacteriophage cDNA plaques were identified and removed 27 from the respective culture plates with a pasteur pipet 28 into phage dilution buffer. Fifty-five positive 29 sporozoite clones and 44 positive merozoite clones were identified.

Fi til" ii WO 89/07650 PCT/US88/04172 24 1 EXAMPLE 6 2 SUBCLONING OF SELECTED BACTERIOPHAGES IN 3 E. coli Y1090 4 Once identified and removed from the respective culture plates each of the positive bacteriophage 6 identified in Example 1 above were subcloned by several 7 rounds of plating, screening, and isolation similar to 8 the steps described in Section 5B. This procedure was 9 repeated until 100% of the plaques from each individual clone produced a positive signal upon immunoscreening, 11 EXAMPLE 7 12 TRANSFER OF SELECTED BACTERIOPHAGES INTO E. coli Y1089 13 AND PRODUCTION OF FUSION PROTEINS THEREFROM 14 The titer of each clonal bacteriophage preparation prepared in Example 6 above was determined by infecting 16 E. coli Y1090 and plating on LB agar containing 17 ampicillin. Individual aliquots of an overnight culture 18 of E. coli Y1089 were infected at a M.O.I. equal to 19 with separate bacteriophage cDNA clones and grown at 32 0 C. Individual colonies were picked into microtiter 21 plates in a grid design and replica plated onto two LB 22 agar-ampicillin culture plates. One inoculated plate 23 was grown at 32 0 C, the other grown at 42 0 C. Colonies S24 growing at the lower temperature, but not at the higher, indicative of a lysogenic state, were isolated and grown 26 in bulk culture in LB broth containing ampicillin at 27 32 0 C. When the 0.D.

550 of the bulk culture reached 28 0.4-0.5, the temperature was shifted to 42 0 C and held at 29 that level for 20 min. to induce the lytic cycle. After a temperature shift to 37 0 C, the culture was induced with 31 2mM IPTG for Sr i- r WO 89/07650 PCTUS88/04172 25 1 The E. coli were then harvested by centrifugation at 2500 2 rpm fo' 15 min. at 25 0 C and the cell pellet was 3 resuspended in a volume of 0.01 M Tris, pH 7.4, 4 MgC1 2 10 ug/ml leupeptin and chymostatin, and 0.5mM TPCK and TLCK. The E. coli were lysed by freeze-thawing and 6 disrupted further by sonication for 20 sec. on ice. The 7 homogenate was treated with 10 ug/ml DNase and RNase for 8 10 min. on ice and then centrifuged at 10,000 g for 9 min. at 4 0 C and the resulting supernatant stored at -200C for further analysis.

11 EXAMPLE 8 12 PURIFICATION OF FUSION PROTEINS 13 The beta-galactosidase fusion proteins produced by 14 the procedures described in Example 7 above were purified by high performance liquid chromatography (HPLC) using a 16 GF 250 molecular sieve column (DuPont) after dilution in 17 6 M urea (due to the insoluble nature of the recombinant 18 antigens). One minute fractions (0.5 ml/fraction) were 19 collected and assayed for beta-galactosidase fusion protein by ELISA. The partially purified recombinant 21 antigens were dialyzed against several 1-1 changes of 22 DMEM.

23 EXAMPLE 9 24 SCREENING OF PURIFIED FUSION PROTEINS WITH T CELLS OBTAINED FROM E. acervulina INFECTED CHICKENS 26 Immune T lymphocytes were collected from inbred 27 strains of chickens (Hy-Line International Production 28 Center) that had been inoculated per os at 4-8 weeks of 29 age with 105 sporulated oocysts of E. acervulina and i i WO 89/07650 PCT/US88/04172 26 1 a second time with the same dose 5 weeks later. T cell 2 proliferation assays were carried out 5-10 d after the 3 second inoculation as described by Lillehoj, Vet.

4 Immunol. Immunopath., 13:321 (1986). In brief, splenic lymphocytes (3-4 x 106) enriched for T cells by passage 6 over nylon wool, see Julius, et al., Euro. J. Immunol., 7 3:645-650 (1973), were co-cultured with different 8 concentrations of sporozoites (1-2 x 106) or 9 HPLC-purified recombinant antigens prepared in Example 8 above (50-100 ng) in microtiter plates. Concanavalin A 11 (Sigma Chemical Co.) as a positive control and identical 12 fractions of lambda gtll lysogen extracts purified by 13 HPLC as a negative control were included in each assay.

14 In all assays, mitomycin C treated normal spleen cells (3.4 x 104) were used as antigen presenting cells.

16 Cultures were incubated for 3 d at 41 0 C in a humidified 17 atmosphere of 5% CO 2 in air. The cultures were pulsed 18 18h before harvesting with 1 uCl of 3 H-deoxythymidine 19 3 H-TdR, New England Nuclear). The cultures were harvested using a PHD cell harvester (Cambridge 21 Technology Inc.) and the amounts of 3 H-TdR uptake 22 quantified on a B-scintillation counter (Beckman 23 Instruments, Inc.).

24 Of the 5-10 fusion proteins screened, those designated cSZ-1 and cMZ-8 appeared to be of most 26 i interest. There was a measurable increase in the level 27 of 3 H-thymidine incorporation by immune SC T lymphocytes S 28 co-cultured with cSZ -1 compared to lambda gtll 29 controls. This increased response was similar when normal SC spleen cells or LSCT-RP9 cells were used as the 31 source of APC. There was a much greater level of 32 activation of immune SC T lymphocytes by recombinant 33 CMZ-8 antigen at the highest concentration tested. These 34 responses to cSZ-1 and cMZ-8 appear to be relevant to infection since T cells from uninfected, normal controls WO 89/07650 PCT/US88/04172 27 1 exhibited negligible stimulation in the presence of 2 either of the recombinant E. acervulina antigens 3 3 H-TdR incorporation by normal T cells in the presence of 4 recombinant antigens was less than 20% of that by immune T cells).

6 EXAMPLE 7 SUBCLONING OF RECOMBINANT cDNA INTO pUC-8, 8 TRANSFORMATION OF E. coli JM83 THEREWITH, AND 9 PRODUCTION OF CLONED GENES THEREFROM CSZ-1 and cMZ-8 were subcloned into pUC-8 to 11 facilitate determining the molecular organization and DNA 12 sequence thereof. High titer bacteriophage preparations 13 were produced. See, Maniatis, et al., (1982), and 14 concentrated by centrifugation over CsCl. Bacteriophage DNA was prepared (Maniatis, et al., 1982), digested with 16 EcoRI to release the cDNA insert, the products separated 17 by agarose electrophoresis, and the cDNA insert isolated 18 by electroelution and passage through a NACS column. The 19 cDNA insert was ligated to pUC-8 plasmid DNA which contained compatible EcoRI ends. Recombinant plasmid DNA 21 was used to transform competent JM83 cells using standard 22 procedures. See, Hanahan, J. Molec. Biol., 166: 23 557 (1983). Plasmid DNA was generated as described 24 (Maniatis, et al., 1982) and the cDNA insert isolated as outlined above.

cDAisr a iae o U- lsi N hc cotie optbeEoIeds eobnn lsi N i- WO 89/07650 PCT/US88/04172 28 1 EXAMPLE 11 2 CHARACTERIZATION OF THE cSZ-1 ANTIGEN 3 For labeling and hybridization studies, plasmid 4 DNA was digested with EcoRI, elctrophoresed on LMP agarose (FMC), and the insert DNA band was excised, 6 placed in electrophoresis buffer in dialysis bags and 7 electroeluted for 2h at 100 volts. The dialysate was 8 extracted twice with phenol, once with phenol-chloroform, 9 and once with chloroform and the insert DNA precipitated with ethanol and sodium acetate. The insert DNA was 11 labeled with 10 uCI of 32 p-alpha dCTP (3000 uCi/mMole, 12 New England Ni'clear) by nick translation, Rigby, et al., 13 J. Molec. Biol., 113:237 (1977), using DNase I and DNA 14 polymerase (BRL). Labeled DNA was separated from 32 p-dCTP by passage over a 18cm x 0.4cm Sephadex 16. column (Pharmacia). E. acervulina sporozoite and 17 merozoite DNA was purified as described by Dame and 18 McCutchan, Molec. Biochem. Parasitol., 8:263 (1983), 19 except that 2 ug/ml ethidium bromide and CsTFA instead of CsC1 was used in the centrifugation step. The purified 21 DNA (2 ug) was digested with 30 units of either EcoRI, 22 EcoRV, HindIII, or Dral restriction enzymes (BRL), 23 electrophoresed in agarose (FMC) and transferred to 24 Biodyne membrane (Pall) using Southern blotting procedures. See, Southern, J. Molec. Biol., 98:503 26 (1975). After transfer, the DNA-blotted Biodyne paper 27 was baked in vacuo at 80 0 C for 2h, prehybridized with 28 M NaCl, 0.05 M NaCitrate, pH 7.0 (6x SSC), 0.2% 29 tetra-sodium pyrophosphate, 0.2% sodium dodecylsulfate (SDS), and 50 ug/ml heparin (Sigma) for 6h at 65 0 C, and 31 hybridized with 106 cpm of 32 P-labeled probe for 16-20h 32 at 65 0 C. The blots were washed three times with 0.1X 33 SSC, 0.1% SDS at 65 0 C for 30 min. per wash and once with 34 0.05X SSC, 0.1% SDS for an additional i Il l *s -I 'i f :i .1 WO 89/07650 PCT/US88/04172 29 1 2 3 4 6 7 8 9 11 12 13 14 16 17 18 19 21 22 23 24 26 27 28 29 31 32 33 34 30 min. at 65 0 C. The blots were air dried and overlLid with photographic film (Kodak XAR) to visualize the hybridization patterns.

The lysogens were induced with 2mM IPTG as described by Young and Davis, Genetic Engineering, 7 (J.

Setlow and A. Hollander, eds. 1985), were harvested by centrifugation (2500 and lysed by freeze-thawing and sonication for 20 sec. in 0.01 M Tris-HCl, pH 7.4, 0.005 M MgCL 2 and 0.5 ug/ml chymostatin, leupeptin, 0.005 M TLCK, TPCK. After sonication, the E. coli homogenates containing the fusion protein were treated with 10 ug/ml RNase and DNase to destroy nucleic acids and were centrifuged at 7500 g for 15 min. to pellet unbroken cells and large particulate material. Protein concentrations were determined using the BCA technique (Pierce Chemical The beta-galactosidase fusion proteins were purified by immunoaffinity chromatography on an anti-beta-galactosidase column following the techniques described by the manufacturer (Promega Biotech).

Aliquots of E. coli homogenates containing the fusion protein were diluted in 2X sample buffer glycerol, 10% 2-mercaptoethanol, 4.6% SDS, 0.125 M Tris, pH 6.8) and run on a 4% stacking/7.5% resolving SDS-polyacrylamide gel as described by Laemmeli, Nature, 227:680 (1976). The SDS-page separated proteins were transferred to nitrocellulose paper (Schleicher and Schuell) as described by Towbin, et al., Proc. Nat. Acad.

Sci. USA, 76(9):4350 (1980). After Western blotting, the nitrocellulose paper was treated with 0.01 M NaHPO 4 pH 7.3, 0.01 M NaCl (PBS) containing 0.05% Tween 20 and non-fat dry milk (NFDM). Immune sera (10- 2 dilution) and monoclonal antibodies reactive with beta-galactosidase (2 x 10 4 dilution) were diluted in PBS-Tween 20-NFDM and used to probe Western blots as i Li I L ii I: i WO 89/07650 PCTI/US88/04172 30 1 2 3 4 6 7 8 9 11 12 13 14 16 17 18 19 21 22 23 24 26 27 28 29 31 32 33 34 described by Jenkins and Dame, Molec. Biochem.

Parasitol., 25:155 (1987). Polyclonal antibodies specific for the beta-galactosidase fusion proteins were purified from immune rabbit sera raised to E. acervulina sporozoite and merozoite membrane extracts using described procedures. Ozaki, et al., J. Immunol.

Methods, 89:213 (1986). E. acervulina sporozoites (108) and merozoites (108 101 0 were 125 1-surface labeled using the lodogen method. See, Markwell, Biochem., 17(22):4807 (1980). Whole cell protein extracts were prepared, separated by SDS-PAGE, transblotted to nitrocellulose paper and probed with immune sera using methods similar to those described above.

The foregoing techniques revealed that the cDNA insert cloned in cSZ-1 was 1580 bp in length and contains an internal EcoRI site which divides the sequence into a 960 bp (upper) fragment and a 620 bp (lower) fragment.

Each of the two fragments were subcloned into pUC-8 and lambda gtll. The respective beta-galactosidase fusion proteins in lambda gtll (upper and lower) were expressed and immunoscreened with rabbit anti-E. acervulina sporozoite serum. Identical fusion proteins were obtained from cSZ-1 and cSZ-lU, thus, immunoreactive parasite protein was associated with the large 960 bp fragment. Clones of the smaller 620 bp fragment were not immunoreactive. The 960 bp sequence from subclone pcSZ-1U in pUC-8 was cleaved from the vector by EcoRI digestion, separated by agarose electrophoresis, electroeluted, and labeled with 32 P-dCTP by nick translation for use as a probe.

The cSZ-lU probe hybridized to either one or two major bands on Southern blots of E. acervulina DNA digested with EcoRI, EcoRV, HindIII, or Dral. Although only data from EcoRI digested sporozoite and merozoite

I~

'1« i WO 89/07650 PCT/US88/04172 31 1 DNA is presented, hybridization was to the same bands in 2 DNA from both stages of the parasite for each restriction 3 enzyme. A single hybridization band was observed for 4 both HindIll- and Dral-digested E. acervulina DNA while 6 EcoRV digested genomic DNA. Except for the two bands 7 found in EcoRI digested genomic DNA, these findings can 8 be explained by restriction mapping data. The pcSZ-1U 9 insert DNA contains a single EcoRV site and no Dral or HindIII sites. It is unclear why pcSZ-lU hybridizes to 11 two EcoRI fragments of genomic DNA since no EcoRI sites 12 are presented in the insert DNA. It is possible that 13 the gene exists in more than one copy within the genome 14 or that the genomic sequence giving rise to the mRNA from which the 960 bp cDNA was prepared is interrupted with an 16 intron which contains EcoRI and EcoRV sites.

17 Lysogens of E. coli Y1089 containing the cSZ- 18 insert were treated with IPTG to induce production of the 19 cSZ-1 beta-galactosidase fusion protein. Homogenates containing the fusion protein were prepared, separated by 21 SDS-PAGE, transblotted to nitrocellulose paper, and 22 probed with mouse McAb specific for beta-galactosidase 23 and with rabbit sera raised to sporozoite membrane 24 extracts. The cSZ-1 fusion protein appears to be 130 kDa in size. The size of the fusion proteins produced by 27 bp insert encodes parasite protein. Similar to other 28 beta-galactosidase fusion proteins, Schoner, et al., 29 Biotechnology, 3:151-154 (1985), cSZ-1 forms insoluble aggregates. About 50% of the reactive p130 antigen was 31 present in thq pellet after sonication and the 7500 g 32 centrifugation. Transblots of the cSZ-l fusion protein 33 lysate were also probed with sera from chickens immune to 34 E. acervulina as a result of a previous infection with this protozoan. There did not appear to be any n iz. hesie f hefuio potin podce b L IL ~i WO 89/07650 PCT/US88/04172 -32 1 detectable recognition of the cSZ-1 fusion protein by the 2 immune chicken sera.

3 Antibodies reactive with the cSZ-1 fusion protein 4 were absorbed and purified from rabbit anti-sporozoite membrane sera and used to probe Western blots of 6 12 5 I-labeled E. acervulina sporozoite proteins. Two 7 protein bands, one at Mr 240, the other at Mr 160, were 8 recognized as antigens homologous for the cSZ-1 fusion 9 protein. Although detectable 125 I-labeling is evident, neither the p240 nor the p160 antigen appears to be a 11 major sporozoite surface protein. Immunoblotting of 12 125 I-labeled sporozoite proteins with sera from mice 13 immunized with purified cSZ-1 confirmed that the fusion 14 protein is homologous to a region of the Mr 240 and Mr 160 antigens.

16 EXAMPLE 12 17 CHARACTERIZATION OF THE cMZ-8 ANTIGEN 18 Clone cMZ-8 was characterized using the same 19 procedures as described in Example 11 above. Clone cMZ-8 contains an insert size of 800 bp which was 21 released by EcoRI digestion, purified from agarose gels 22 by electroelution, and subcloned into pUC8 plasmid giving 23 rise to clone pcMZ-8. Gel-purified pcMZ-8 insert DNA was 24 labeled by nick translation and used to probe Southern blots of restriction enzyme-digested E. acervulina 26 sporozoite and merozoite DNA. A single fragment of E.

27 acervulina genomic DNA (sporozoite or merozoite) bound 28 probe for each restriction enzyme combination (EcoRI, 29 EcoRV, HindIII, Dral) suggesting that only a single copy of the sequence may be present in the genome. Similar 31 to the pcSZ-1 hybridization results, there was no L i :n rr i WO 89/07650 PCT/US88/04172 33 1 significant difference in the banding patterns between 2 sporozoite and merozoite DNA for each restriction digest.

3 Fusion proteins of cMZ-8 were prepared as lysates 4 of the respective E. coli Y1089 lysogen, separated by SDS-PAGE, and transblotted to nitrocellulose paper as 6 above. The blots were probed with anti-beta- 7 galactosidase McAb and with rabbit sera raised to E.

8 acervulina merozoite membrane extracts. The cMZ-8 fusion 9 protein appears to be 145-150 kDA in size. The parasite encoded portion of this 150 kDA beta-galactosidase fusion 11 protein is estimated to be about 35 kDa which is similar 12 to the estimated size based on the length of the cDNA 13 insert (800 bp). Similar to the cSZ-1 fusion protein, at 14 least 50% of the cMZ-8 protein is present as insoluble aggregates. Antibodies to the cMZ-8 protein were 16 purified from rabbit anti-sera specific for merozoite 17 membranes and used to probe Western blots of 1251- 18 labeled merozoite proteins. The respective parasite 19 protein appears to be 230-250 kDa with a considerable number of breakdown products and/or cross-reactive 21 antigens between Mr 75-230 kDa. The p230-250 antigen is 22 a surface protein as indicated by 125 I-labeling. These 23 results were confirmed by probing Western blots of 24 125 I-labeled merozoite proteins with sera from mice that had been immunized with beta-galactosidase column- 26 purified cMZ-8 protein.

27 EXAMPLE 13 28 IMMUNIZATION OF CHICKENS WITH cSZ-1 and cMZ-8 29 Separate groups of one week old Sexsal chickens were immunized intramuscularly with varying doses of 31 either cSZ-l or cMZ-8 recombinant antigens or the 32 appropriate controls, boosted with an identical dose one naii WO G9/07650 PCT/US88/04172 -34- 1 week later, and challenged the following week with 103 2 sporulated oocysts of Eimeria acervulina. After 5d the 3 chickens were sacrificed and examined for signs of 4 disease intestinal lesion score). Although no differences were found for body weight gains, there was a 6 significant decrease in the lesion score from 7 cSZ-1 and cMZ-8 immunized birds compared to that observed 8 in the control groups.

9 EXAMPLE 14 IMMUNIZATION OF CHICKENS WITH cSZ-l AND cMZ-8 11 AND WITH REDUCED LIPOPOLYSACCHARIDE 12 In the previous vaccine trial, it was noted that 13 administration of the recombinant sporozoite (cSZ-1) and 14 merozoite (cMZ-8) antigens and lambda gtll control .E.

coli extracts had a depressive effect on weight gains 16 compared to unimmunized controls. To determine if 17 endotoxin lipopolysaccharide, LPS) was responsible 18 for this effect, we removed a substantial amount 19 of the LPS by treating the antigen extracts with Polymixin B agarose. Again, separate groups of one week 21 old Sexsal chickens were immunized with varying doses of 22 either LPS containing or LPS-depleted cSZ-1 or cMZ-8 23 recombinant antigens or the appropriate controls. The 24 chickens were boosted with identical doses two times thereafter, one and two weeks post-initial immunization, a 26 and challenged the following weeks with 103 oocysts of 27 Eimeria acervulina. After 5d the chickens were 28 sacrificed and examined for signs of disease. While 29 removal of LPS did improve weight gains prior to occyst challenge, there was no significant different in body 31 weight gains between groups of chickens that were 32 immunized with the recombinant antigens or those 1 WO 89/07650 PCT/US88/04172

SI

35 1 receiving the lambda gtll control. However, lesion 2 scores in the chickens that were immunized with either 3 recombinant antigen from which LPS had been removed were 4 significantly lower than lesion scores from either the lambda gtll controls or recombinant antigen preparations 6 which contained LPS.

7 EXAMPLE 8 IDENTIFICATION OF RECOMBINANT PHAGES BY SCREENING 9 E. coli WITH MONOCLONAL ANTIBODIES SPECIFIC FOR THE SPOROZOITE STAGE OF E. acervulina 11 To obtain monoclonal antibodies (McAb) reactive 12 with either stage of the parasite, BALB/C mice were 13 immunized intravenously with 106 E. acerv-,.ina 14 sporozoites or merozoites and boosted with an identical dose 2 weeks later. Spleen cells from immunized mice 16 were obtained 3d after the lest immunization and fused 17 with P 3 X myeloma cells in the presence of polyethylene 18 glycol using described procedures. See Zola and Brooks, 19 "Techniques for the Production and Characterization of Monoclonal Hybridoma Antibodies", in Hybridoma 21 Antibodies: Techniques and Applications, 1-57 (Hurrell, 22 ed. 1978). In brief overview, the hybridomas 23 were grown in Dulbecco's minimal essential medium (DMEM) 24 containing fetal bovine serum, hypoxanthine, aminopterin, and thymidine in flat bottom microtiter plates (Costar) 26 and stored at 37 0 C in a 5% CO incubator. Supernatants 27 from wells containing proliferating hybridoma colonies 28 wf.re assayed for antibodies reactive with dried E.

29 acervulina sporozoites and merozoites by immunofluorescence (IFA) using established procedures.

31 Danforth and Augustine, Poult. Sci., 62(11):2145-2151 32 (1983). Positive cultures were WO 89/07650 PCT/US88/04172

I-

36 1 subcloned by limiting dilution and tested for the 2 production a single Ig class by enzyme-linked 3 immunoabsorbent assay. Wakefield, et al., Clinica 4 Chemica Acta, 123:303-310 (1982).

Fusion of spleen cells from mice hyperimmune to E.

6 acervulina sporozoites with myeloma cells resulted in the 7 production of a hybridoma which secreted IgG2a. Although 8 no surface staining was observed, the McAb, designated 9 S 11

P

1

A

1 2 appears to recognize an 12 5 I-labeled 22 kDa surface protein as revealed by immunoblotting.

11 Subsequent experiments have shown that this 22kDa 12 antigen, called p22 is present on the parasite surface as 13 well as on the refractile body (RB) membrane. Although 14 S 11 PiA 12 is not species-specific, since it cross-reacts with E. tenella sporozoites, it is developmental 16 stage-specific since no recognition of E. acervulina 17 merozoites was observed.

18 Recombinant phages prepared as described in 19 Example 4 above were screened with these monoclonal antibodies using the procedure described in Example 21 above. One positive bacteriophage was identified, which 22 was designated MA1. This bacteriophage was subcloned as 23 described in Example 6 above, and transferred into E.

24 coli Y1089 and a fusion protein produced therefrom as described in Example 6 above. The fusion protein was 26 then purified as described in Example 8 above and 27 screened with T cells from E, acervulina infected 28 chickens as described in Example 9 above. The response 29 at two different dose levels of MA1 was 4-5 times greater (P<0.05) than the activation induced by similar amount 31 of protein derived from identical fractions isolated fro 32 lambda gtll homogenate.

I

i i WO 89/07650 PCT/US88/04172 37 1 EXAMPLE 16 2 CHARACTERIZATION OF THE MAl ANTIGEN 3 The expressed beta-galactosidase fusion protein of 4 MAl was probed by immunoblotting with S 11

P

1

A

12 supernatant and appears to be about 125 kDa. This Mr 6 estimate was corroborated by immunoblotting of MAl with 7 McAb to beta-galactosidase.

8 The MAl clone was subclon. into pUC-8 and used to 9 transform E. coli JM83, as described in Example 10 above, to facilitate determining the molecular organization and 11 DNA sequence thereof.

12 Digestion of pMAl with EcoRI and subsequent 13 agarose electrophoresis of the products revealed a DNA 14 insert of about 200 bp in length. The molecular organization and expression of genes encoding the p22 16 antigen were examined by hybridization of 32 P-labeled MAl 17 insert DNA to Southern blots of restriction enzyme- 18 digested E. acervulina sporozoite DNA and Northern blots 19 of RNA from both the sporozoite and merozoite stages.

The p22 gene appears to exist as a single copy or low 21 copy number sequence since only one hybridization band 22 was observed with EcoRI (31 kbp), Dral (2.4 kbp), or 23 HindIII (12 kbp) cut sporozoite DNA. Consistent with 24 our immunological data that there is exclusive expression of the p22 antigen on sporozoites, labeled insert DNA 26 hybridized only to sporozoite RNA and not to RNA from 27 merozoites. One major hybridization band at 500 bp was 28 observed which is in the range of predicted size of the 29 mRNA (approximately 600 bp) based on the size of the p22 protein.

I

WO 89/07650 PCT/US88/04172 38 1 EXAMPLE 17 2 DNA SEQUENCING OF MA1 3 The DNA sequence of the cDNA insert MA1 was 4 determined using the dideoxy chain termination technique. Sanger, et al., Proc. Nat. Acad. Sci. USA, 6 74:5463 (1977). Purified cDNA insert was obtained in 7 accordance with Example 10 above, ligated to EcoRI 8 digested M13mpl8 DNA (BRL), and used to transfect JM101 9 cells. See Messing, Methods Enzymol., 101:20 (1983). Recombinant M13 clones (white plaque-colonies on 11 Luria broth containing X-gal and IPTG) were picked and 12 used to generate single stranded viral DNA. See Messing, 13 supra. Sequencing reactions were performed as described 14 by Williams, et al., BioTechniques, 4(2):138 (1986), with minor alterations using 30 uCi 3 5 S-dATP (NEN 500 16 Ci/mMole) and analyzed on a 6% polyacrylamide sequencing 17 gel. The complete sequence of M13 recombinant viral DNA 18 and its complement representing bcth strands of the cDNA 19 was ascertained and analyzed using the Intellegenetics DNA sequencing program. See generally, Friedman, T., 21 Intellegenetics: A Short Course in Molecular Biology 22 Software (1985) (Intellegenetics, Inc., Mountain View, 23 CA). The sequence of cDNA clone MA1, as determined by 24 these procedures, is shown in Table 1 below.

-T,

WO 89/07650 PCT/US88/04172 39 1 TABLE 1 2 DNA SEQUENCE AND PREDICTED AMINO ACID 3 SEQUENCE OF cDNA CLONE MAl 4 GTA GTC GTC GTC GTC GTC GTG GGA AGT TCG ATG CAC GTC GTG GAA 6 VAL VAL VAL VAL VAL VAL VAL GLY SER SER MET HIS VAL VAL GLU 7 8 GTT CGG TGC TTC. GGA GTC CGA AGA AGA CCA TCT ACA GAA TCA CGA 9. VAL ARG SER PHE GLY VAL ARG ARG ARG PRO SER THR GLO SER ARG 135 11 AGA AGT TCT CCTI CTG-ACL GTG TCT CCC TGC CTC TAT TCT GTT TTC 12 ARG SER SER PRO LEU THR LEU SER PRO CYS LEU TYR SER VAL PHE 13 180 14 CTC TGT CTA CTC CCC CCT GTC TCT GTA AGT TTC TGC CTT AAA AGG C LEU CYC LEU LEU PRO PRO VAL SER VAL SER PHE CYS LEU LYS ARG 16 DNA sequencing of the, pMA1 insert revealed a large 17 open reading frame in one of the two possible sequence 18 orientations. The other four reading frames are not 19 considered since cloning into the EcoRI site of the lacZ gene of lambda DNA does not destroy the reading frame, 21 but is designed to encode a viable beta-galactosidase 22 fusion protein. Quite unexpected was the finding that 23 no stop codon or long poly A tail was present in. the 24 sequence suggesting that we have cloned an internal region of the gene encoding the Mr22 protein. Consistent 26 with the hybridization results and previous mapping 27 studies no EcoRI, Dral, or HindIII sites occur within the 28 sequence.

i~ B i:1 a a !i i ij iJ

I

WO 89/07650 1 2 3 4 se th 6 se 7 8 9 PCrUS88/04172 40 EXAMPLE 18 DNA SEQUENCING OF cMZ-8 The DNA 5' portion and the quence of the cDNA insert cMZ-8 was e procedures described in Example quence is shown in Table 2 below.

3' portion of the determined using 17 above. This TABLE 2 PARTIAL DNA SEQUENCE AND PREDICTED AMINO ACID SEQUENCE OF cDNA CLONE cMZ-B 27 54 WA AAC AA G A GOA AGA T=T AGA GSA I& C AGA CCr =1 C r Tcr =T r TT AcA GIC a ACa =T =CT Pro Le Pro R~e Ser Pro Pro Ser Tr PM Val Sec Pro Ser Thr Pro Val Ser 81 108 03h a Aa TUT CAG AGA GA Wr =CTC; CAG AGaXA GSA AA 7= C= T= AM Cr-T ACA CT AC Pro Pro Ser Th Pro Val Ser Pro Pro SeC 'hi Pro Val Ser Pro Pro Ser la 135 162 GC CAA GGAAGC GSA AGU AcT C G AC c Crr G= CCr Tr AC A r C 7M CCT A a; =c Pro Val Sec Pro Pro Ser Mhr-Pro Val SeC Pro Pro Ser Mir Pro Val Ser Pro 189 216 GGh ACh TW O1 CAG AGC O A GGA ArA G3C AC zGX GSA AG.A G T= A= o GIC T r r =e =rc APk Pro Ser 'Thr Pro Val Ser Pro Pr, r Pro Val Sec Pro Pro Ser hr Pro 243 270 AGT TM ACh A o CA AGA a= CAP. GIG GC c= T M Aar ACh a Cn I r A~ O Ga i c Oc Val Ser Pro Mt Ser Mr Pro Ala Ser Pro Proer Pro Val His His Arg 297 ACT G= GC 7W CA& T7W GAA TIC CAA =G AX AG AG WA TrCA C6 A2 Ci AAC CT AC CIT GCC T7= T A= Ser Pro Pro Leau M Val Am La A Val Ala Sec Sec Sec =a Ta; CA CT -A C CIC TIC AT T CIC ACA TIC CCT AM CC A& =r Ca GAC AAC TAA CAA GAG G= AAG M GzA ACC T"IC% VT C T CC CIT C' C= CT C;A aC ACT cr arr ACr Ta AAC CC a CCC CICZ 03h M ACA =T CCz GAA Ca CA CC =r CC AC Aa; CCC VC CI CC CI C= C =P CC TIA C CM h GmC Trca; TGCI =C AM IA GM =GM% C AC I= GAG C C C dCC= GAG ChC CC

LL

I

I. iY WO 89/07650 PCT/US88/04172 41 1 The upper strand shown is believed to be the 2 coding strand, and the predicted amino acid for the 3 upper strand is shown. It is possible that sequence 4 codes in reverse from how it is shown, in which case either the upper or lower strand could be the coding 6 sequence. However, the large open reading frame 7 represented by the upper strand shown in Table 2 suggests 8 that this is the coding strand shown in proper 9 orientation.

EXAMPLE 19 11 CHARACTERIZATION AND DNA SEQUENCING OF 12 cDNA CLONE MC17 13 The cDNA clone MC17 was identified and sequenced 14 through use of essentially the same procedures as described above.

16 MC17 is a 130 kDa beta-galactosidase fusion 17 protein which was identified by immunoscreening an 18 Eimeria acervulina sporozoite cDNA library with a 19 monoclonal antibody designated S 16

P

3

A

1 MC17 represents a portion of a 58 kDa E. acervulina merozoite surface 21 protein as determined by immunoblotting of extracts of 22 125 1-labeled merozoites with S 16

P

3

A

1 This McAb, 23 Sl6P 3 Al, was prepared using the same procedures as 24 described in Example 15 above. It is of the IgG 1 subclass and recognizes only E. acervulina merozoites and S 26 not sporozoites. Probing Northern blots of E.

27 acervulina sporozoite and merozoite RNA with 32P-labeled 28 MC17 insert cDNA has shown hybridization only to nucleic 29 acid derived from the latter developmental stage.

Hybridization of this probe to Southern blots of 31 restriction enzyme-digested E. acervulina sporozoite and 32 merozoite DNA st.gests that MC17 cDNA may be interrupted j V U.UOA Id, U.C1 o1o a c uui-Li.ona 1 42 1 by an intron in the genomic sequence. This conclusion 2 is based on the observation that more than one 3 hybridization band was observed with DraI (14.0 kbp and 4 2.0 kbp) and EcoRI (23 kbp and 7.6 kbp) digested DNA and neither restriction enzyme site is present in the cDNA 6 sequence. In contrast, probing of BamHI E. acervulina 7 DNA produced only one hybridization band (21 kbp) which 8 may reflect the absence of this restriction site on the 9 proposed intron.

The DNA sequence of MC17 is shown in Table 3 11 below.

12 TABLE 3 13 DNA SEQUENCE AND PREDICTED AMINO ACID O14 SEQUENCE OF cDNA CLONE MC7 15- 4245 16 GTA CGT CGT AGC GGC GCC CCG GCG GGG GTC GTC GCG TCG TCG GCA 17 HIS ALA ALA SER PRO ARG GLY ARG PRO GLN GLN ARG SER SER ARG 18 19 GTG CCC CGA CTC CCA GGT CTG TGA TGA CCT CCC CTT CGA CGA CGA 20 HIS GLY ALA GLU GLY PRO ASP THR THR GLY GLY GLU ALA ALA ALA 1 by an in35ron in the genomic sequence. This conclusion 2 GTT CAA CCT CCT CCT GCT GCA GGC CTT CTA CAC CTA GGC ACC CCC 3 hybridizGLN VAL PRO PRO PRO ALA ALA GLY LEU LEU HIS LEU GLY THR PROtion band was observed with Dral (14.0 kbp and 4 1802.0 kbp) and EcoR (23 kbp and 76 kbp) digested DNA and TGT TGG TGC TTA CGT TTG CGG TTG ACG ACA ACA ACC CCC GAA ACG 6 sequece.THR THR THR ASN ALA ASN ALA ASN CYS CYS CYS TRP GL ina LEU CYS 7 -225DNA produced only one hybridization and (21 kbp) which 8 AAC CTG ACG GAA GTT TGT GTT AAC TTT ATA CAC GAA CAC GTT TTT 9 LEU ASP CYS LEU GLN THR GLN LEU LYS TYR VAL LEU VAL GLN The DNA sequence of MC17 s shown in Table 3 11 below. TTT TTT TTT C 12 TABLE 3 13 DNA SEQUENCE AND PREDICTED AMINO ACID 14 SEQUENCE OF cDNA CLONE MC17 16 GTA CGT CGT AGC GGC GCC CCG GCG GGG GTC GTC GCG TCG TCG GCA 17 HIS ALA ALA SER PRO ARG GLY ARG PRO GLN GLN ARG SER SER ARG 18 19 GTG CCC CGA CTC CCA GGT CTG TGA TGA CCT CCC CTT CGA CGA CGA 20 HIS GLY ALA GLU GLY PRO ASP THR THR GLY GLY GLU ALA ALA ALA 21 135 j 22 GTT CAA CCT CCT CCT GCT GCA GGC CTT CTA CAC CTA GGC ACC CCC 23 GLN VAL PRO PRO PRO ALA ALA GLY LEU LEU HIS LEU GLY THR PRO 24 180 TGT TGG TGC TTA CGT TTG CGG TTG ACG ACA ACA ACC CCC GAA ACG 1 26 THR THR THR ASN ALA ASN ALA ASN CYS CYS CYS TRP GLY LEU CYS 27 -225 28 AAC CTG ACG GAA GTT TGT GTT AAC TTT ATA CAC GAA CAC GTT TTT 29 LEU ASP CYS LEU GLN THR GLN LEU LYS TYR VAL LEU VAL GLN 238 31 TTT TTT TTT TTT C i* 1

I

i WO 89/07650 PCT/US88/04172 43 1 The DNA sequence of the MC17 insert revealed a 2 long poly A tail suggesting that we have cloned the 3' 3 end of the cDNA. This sequence also appears to be 4 composed of several amphipathic alpha helices which have been associated with T lymphocyte recognition sites.

6 Consistent with this finding is the significant in vitro 7 activation of T lymphocytes obtained from E. acervulina- 8 immune chickens by the beta-galactosidase fusion protein 9 coded for by cMZ-8.

EXAMPLE 11 CHARACTERIZATION OF cDNA CLONE MA16 12 MA16 is a 140 kDa beta-galactosidase fusion 13 protein which was identified by immunoscreening an E.

14 acervulina merozoite cDNA library with an IgM subclass monoclonal antibody designated 12-07. This monoclonal 16 antibody was prepared using essentially the same 17 procedures as described in Example 15 above. MA16 18 represents a portion of a p58/p70 E. acervulina merozoite 19 surface protein as revealed by the immunoblotting of extracts of 125 I-labeled merozoites with 12-07. This 21 McAb cross-reacts with E. acervulina sporozoite surface 22 antigens which are similar in size to the merozoite 23 constituents as revealed by immunoblotting of labeled 24 sporozoit-es. The surface locale of the p58/p70 antigen has been corroborated by immunofluorescence staining of 26 E. acervulina sporozoites and merozoites with McAb 27 12-07. The relationship of p58 and p70 to each other is 28 unknown.

I t WO 89/07650 PCT/US88/04172 44 1 EXAMPLE 21 2 SCREENING OF FUSION PROTEINS WITH IMMUNE SERA 3 FROM E. ACERVULINA INFECTED CHICKENS 4 An aliquot of each fusion protein preparation (cMZ,, cSZ-1, MA1, MC17, MA 6) was separated on the 6 basis of molecular weight by SDS-polyacrylamide gel 7 electrophoresis (SDS-PAGE) and transblotted to 8 nitrocellulose paper. The Western blots were probed 9 either with monoclonal antibodies specific for the fusion protein or for beta-galactosidase or with immune sera 11 from E. acervulina infected chickens. The binding of 12 first Ab was detected with biotinylated-anti-Ig followed 13 by avidin-percxidase and developed with 0.5 mg/ml 14 4-chloro-l-napthol, 0.01% H202 Of these five fusion proteins, only cMZ-8 was 16 recognized by immune sera taken from E. acervulina 17 infected chickens.

18 The foregoing examples have been provided to 19 illustrate the present invention. They are not to be taken as limiting thereof, the scope of the invention 21 being defined by the following claims. Equivalents of 22 the claims are to be included therein.

A

i 18 Th

Claims (15)

1. 5.5 C 45 The claims defining the invention are as follows: 1. A DNA sequence which encodes an antigenic protein upon expression, wherein said antigenic protein is recognized by monoclonal antibody or polyclonal antibodies raised in animals immunized with denatured Eimeria antigens, which Eimeria antigens activate avian T cells, but not avian B cells, derived from an avian species that has been previously infected with Eimeria parasites. 2. A DNA sequence as described in Claim 1, wherein said antigenic protein binds with an antibody directed against a cell-surface antigen of an Eimeria sporozoite. 3. A DNA sequence as described in Claim 1, wherein said antigenic protein binds with an antibody directed against a cell-surface antigen of an Eimeria merozoite. 4. A DNA sequence as described in any one of Claims 1 to 3, 15 wherein said Eimeria species is Eimeria acervulina. 5. A DNA sequence as described in any one of Claims 1 to 4 and selected from clones MA1, MC17, cSZ-1 or MA16.
2. 6. A DNA sequence as described in any one of Claims 1 to 4 which codes on expression the same polypeptide coded for on expression by a 20 sequence selected from clones MA1, MC17, cSZ-1 or MA16.
3. 7. A DNA sequence as described in any one of Claims 1 to 4, wherein said sequence hybridizes to an oligonucleotide probe homologous to a segment of a clone selected from MAI, MC17, cSZ-1 or MA16.
4. 8. A DNA sequence as described in any one of Claims 1 to 4 which 25 codes on expression the same polypeptide coded for on expression by a sequence which hybridizes to an oligonucleotide probe homologous to a segment of a clone selected from MA1, MC17, cSZ-1 or MA16.
5. 9. A recombinant DNA expression vector, comprising an expression vector having a promoter, and a first DNA sequence inserted in said vector downstream of said promoter and operatively associated therewith, wherein said DNA sequence encodes an antigenic protein upon expression, wherein said antigenic protein is recognized by monoclonal antibody or polyclonal antibodies raised in animals immunized with denatured Eimeria antigens, which Eimeria antigens activate avian T cells, but not avian B cells, derived from an avian species that has been previously infected with Eimeria parasites. A recombinant DNA expression vector as described in Claim 9, Swherein said expression vector is selected from bacteriophages, plasmids, k\ viruses or hybrids thereof. M/666Z I U I 46
6. 11. A recombinant DNA expression vector as described in Claim wherein said expression vector is bacteriophage-lambda.
7. 12. A recombinant DNA expression vector as described in any one of Claims 9 to 11, further comprising a second DNA sequence spliced to said first DNA sequence in correct translational reading frame therewith, wherein said second DNA sequence codes for the production of a protein or fragment thereof different from that coded for by said first DNA sequence, whereby said first and second DNA sequences together code for the production of a fusion protein.
8. 13. A recombinant DNA expression vector as described in Claim 12, wherein said second DNA sequence codes for the production of P-galactosidase or a fragment thereof.
9. 14. A transformed host cell comprising a recombinant DNA expression vector contained within said host cell, said vector comprising 0. 15 a promoter operable in said host cell and a DNA sequence inserted at a site downstream of said promoter and operatively associated therewith, wherein said DNA sequence encodes an antigenic protein upon expression, wherein said antigenic protein is recognized by monoclonal antibody or polyclonal antibodies raised in animals immunized with denatured Eimeria antigens, which Eimeria antigens activate avian T cells, but not avian B cells, derived from an avian species that has been previously infected with Eimeria parasites. A transformed host cell as described in Claim 14, wherein said host cell is a eukaryotic cell. S• 25 16. A transformed host cell as described in Claim 14, wherein said host cell is a prokaryotic cell.
10. 17. A transformed host cell as described in Claim 14 or Claim 16, wherein said host cell is a bacterial cell.
11. 18. A transformed host cell as described in Claim 17, wherein said bacterial cell is Escherichia coli. J 19. A method of identifying DNA sequences that code for antigenic proteins useful as vaccines against avian coccidiosis, said method comprising: a. providing a multiplicity of Eimeria DNA sequences; b. inserting the DNA sequences into DNA expression vectors to form recombinant expression vectors; c. inserting the recombinant expression vectors into suitable Rhost cells to form transformants which express the DNA sequences; N A/ >LMM/666Z i j 47 d. contacting the transformants with antibodies directed against denatured Eimeria antigens to identify transformants containing DNA sequences which code for Eimeria antigens; e. expressing Eimeria antigens from the DNA sequences identified in and f. contacting the expressed Eimeria antigens with T cells, which T cells are sensitized to an antigenic Eimeria protein, to thereby identify DNA sequences which code for antigens that are useful in a primary immunization of avian species against infection by avian coccidia.
12. 20. A method according to Claim 19, wherein said expression vector is a bacteriophage expression vector and ;aid host cells are E. coli.
13. 21. A DNA sequence as defined in claim 1, which DNA sequence is substantially as hereinbefore described with reference to any one of Examples 11 or 16 to 15 22. A recombinant DNA expression vector as defined in Claim 12, which vector is substantially as hereinbefore described with reference to Example 4 or Example
14. 23. A process for preparing the recombinant DNA expression vector according to Claim 22, which process is substantially as hereinbefore described with reference to Example 4 or Example
15. 24. A host cell transformed with the vector according to Claim 22, which host cell is substantially as hereinbefore described with reference to any one of Examples 4, 6 or A method of identifying DNA sequences that code for antigenic proteins useful as vaccines against avian coccidiosis, which method is substantially as hereinbefore described with reference to Example DATED this FOURTH day of NOVEMBER 1991 The United States of America, as represented by the Secretary S/ U.S. Department of Commerce Patent Attorneys for the Applicant SPRUSON FERGUSON LMM/666Z