EP0548252A1 - Vaccin contre la coccidiose mis au point par genie genetique - Google Patents

Vaccin contre la coccidiose mis au point par genie genetique

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Publication number
EP0548252A1
EP0548252A1 EP91917491A EP91917491A EP0548252A1 EP 0548252 A1 EP0548252 A1 EP 0548252A1 EP 91917491 A EP91917491 A EP 91917491A EP 91917491 A EP91917491 A EP 91917491A EP 0548252 A1 EP0548252 A1 EP 0548252A1
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European Patent Office
Prior art keywords
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glu
gly
sequence
pro
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EP91917491A
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German (de)
English (en)
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EP0548252A4 (en
Inventor
James W. Jacobson
Robert L. Strausberg
Susan D. Wilson
Sharon H. Pope
Susan Lee Strausberg
Wolfgang Raether
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Hoechst AG
Enzon Labs Inc
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Hoechst AG
Genex Corp
Enzon Labs Inc
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Publication of EP0548252A1 publication Critical patent/EP0548252A1/fr
Publication of EP0548252A4 publication Critical patent/EP0548252A4/en
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/44—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from protozoa
    • C07K14/455—Eimeria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P33/00—Antiparasitic agents
    • A61P33/02—Antiprotozoals, e.g. for leishmaniasis, trichomoniasis, toxoplasmosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies

Definitions

  • This invention is in the field of avian coccidiosis and is directed to recombinant antigenic proteins of avian coccidia and to the genes that encode the proteins. These antigenic proteins may be used in a vaccine against avian coccidia.
  • Coccidiosis is a disease of both invertebrates and vertebrates, including man, caused by intracellular parasitic protozoa which generally invade the epithelial cells lining the alimentary tract and the cells of associated glands.
  • the crowded conditions under which many domestic animals are raised have contributed to increased incidence of the disease.
  • Virtually every domestic animal is susceptible to infection, and distribution of the parasite is world-wide.
  • Coccidiosis is therefore the cause of signifi ⁇ cant economic loss throughout the world.
  • Eimeria contains the most economically important species. Various species of Eimeria infect a wide range of hosts, including mammals, but nine species have been recognized as being pathogenic to varying degrees in chickens: Eimeria acervulina. E. mivati. E. mitis. E. praecox. E. hagani, E. necatrix-, E. maxima, E. brunetti and E. tenella.
  • Eimeria are highly host specific, their life cycles are similar.
  • the developmental stages of the avian coccidia can be illustrated by the species Eimeria tenella. which proliferates in the cecum of the chicken.
  • the life cycle of the Eimeria species begins when the host ingests previously sporulated oocysts during ground feeding or by inhalation of dust Mechanical and chemical action in the gizzard and intestinal tract of the chicken ruptures the sporulated oocyst, liberating eight sporozoites.
  • the sporozoites are carried in the digestive contents and infect various portions of the intestinal tract by penetration of epithelial cells.
  • E. tenella has received the most attention.
  • E. tenella is an extremely pathogenic species, with death often occurring on the fifth or sixth day of infection.
  • chemotherapeutic agents poultry producers' attempts to control coccidiosis were limited to various management programs. These programs were directed toward attempts at sanitation through disinfection, or by mechanical removal of litter. Despite these efforts, sufficient oocysts usually remained to transmit the disease.
  • Another means of combating coccidia is drug treatment after the poultry is infected.
  • One drug that has been used is sulfanilamide which has shown anticoccidial activity against six species of coccidia.
  • medication may be started too late to be effective.
  • the best method for combating coccidia is preventive medication. Since the advent of the use of sulfonamide drugs, over forty compounds have been marketed for preventive medication against coccidia. There have been many problems with the use of such drugs, including anticoccidial contamination of layer flock feeds, inclusion of excessive anticoccidial drugs in the feed causing toxicity in the birds and omission of the anticoccidial from the feed resulting in coccidiosis outbreaks. A particularly frustrating problem has been the development of drug-resistant strains of coccidia.
  • the process involves the insertion of DNA (derived either from enzymatic digestion of cellular DNA or by reverse transcription of mRNA) into an expression vector.
  • DNA derived either from enzymatic digestion of cellular DNA or by reverse transcription of mRNA
  • expression vectors are derived from either plasmids or bacteriophage and contain: (1) an origin of replication functional in a microbial host cell; (2) genes encoding selectable markers, and (3) regulatory sequences including a promoter, operator, and a ribosome binding site which are functional in a microbial host cell and which direct the transcription and translation of foreign
  • eukaryotic proteins are often produced in prokaryotic cells as a fusion with sequences from the amino-terminus of a prokaryotic protein.
  • ⁇ -Galactosidase or the product of one of the E. coli tryptophan operon genes have been used successfully in this manner.
  • Expression vectors have also been developed for expression of foreign proteins in eukaryotic host cells, e.g., yeast and Chinese hamster ovary tissue culture cells.
  • Host cells transformed with expression vectors carrying foreign genes are grown in culture under conditions known to stimulate production of the foreign protein in the particular vector.
  • Such host cell/expression vector systems are often engineered so that expression of the foreign protein may be regulated by chemical or temperature induction.
  • Proteins which are secreted may be isolated from the growth media, while intracellular proteins may be isolated by harvesting and lysing the cells and separating the intracellular components. In this manner, it is possible to produce comparatively large amounts of proteins that are otherwise difficult to purify from native sources.
  • Such microbially produced proteins may be characterized by many well-known methods, including the use of monoclonal antibodies, hereinafter referred to as "MAbs," which are homogeneous antibodies that react specifically with a single antigenic determinant and display a constant affinity for that determinant, or by use of polyvalent antibodies, which may be derived from infected birds or other animals that have been immunized with life forms of Eimeria or with Eimeria protein, which react with a variety of different antigens and often with multiple determinants on a single antigen.
  • MAbs monoclonal antibodies
  • vaccinia has a long history of use as a vaccine and has been employed to virtually irradicate smallpox in humans. It now has been demonstrated that vaccinia virus can be effectively genetically engineered to express foreign antigens (Smith et al.. Nature 302:490-495 (1983); Panicali et al.. Proc. Natl. Acad. Sci. USA 80:5364-5368 (1983); Mackett et al.. J.
  • Fowl pox virus is very similar to vaccinia virus and many of the methods developed for vaccinia for the creation of recombinants expressing foreign antigens can be applied to fowl pox.
  • Attenuated fowl pox virus engineered to produce avian coccidia antigens thus is another method to produce an anticoccidial vaccine.
  • Live vaccines have the advantage of being inexpensive to produce and are characterized by the production of rapid immunity development
  • a second type of live vaccine results in the presentation of antigen in the gut where coccidia normally invades.
  • This method utilizes secretion or outer surface expression of the antigen by harmless bacteria introduced into the intestinal microbial population by incorporation in feed. Secretion is obtained by fusion of an antigen gene to the gene coding for a protein which is normally secreted, leaving the necessary secretion signal sequence intact Outer surface expression is achieved by fusion of the antigen genes to the genes that code for proteins normally localized on the outer surface.
  • This type of live vaccine is especially advantageous since manufacturing costs are minimal and the immune response stimulated is of a type particularly effective against coccidia invasion of the gut
  • a third type of live vaccine is the use of live recombinant bacteria expressing Eimeria antigens, which bacteria are injected subcutaneously or by other accepted routes, and which elicit an immune response to the expressed antigen (Miller et al. Infect Immun. 57:2014-2020 (1989). When compared to purified antigen or inactivated bacterial vaccines
  • bacteria the live bacterial vaccine resulted in a more protective immune response.
  • the subunit vaccines can also be used to raise an immune response against coccidiosis in ovo using techniques described by, for example, Hebrank U.S. Patent No. 4,681,063, Hebrank, EPC Patent Application
  • This invention relates to novel recombinant antigenic proteins of avian coccidiosis, and fragments thereof containing antigenic determi ⁇ nants, and to the genes that encode the antigenic peptides. It has now been found that particular polypeptides present in avian cells infected with coccidiosis, when purified and isolated, contain an antigenic determinant or determinants which can elicit an antibody response. This invention also relates to vaccines made using the novel antigenic proteins of avian coccidiosis and to methods of immunizing chickens against avian coccidia.
  • Figure 1 shows the nucleotide sequence of the 5'-3' strand of cDNA encoding the E. maxima antigen mc-4c gene (Sequence ID No. 1).
  • Figure 2 shows the amino acid sequence of E. maxima antigen mc- 4c (Sequence ID No. 2).
  • Figure 3 shows the nucleotide sequence of the 5'-3' strand of cDNA encoding the E. maxima antigen mc-5c gene (Sequence ID No. 3).
  • Figure 4 shows the amino acid sequence of E. maxima antigen mc- 5c (Sequence ID No.4).
  • Figure 5 shows the nucleotide sequence of the 5'-3' strand of cDNA encoding the E. maxima antigen mc-30c gene (Sequence ID No.
  • Figure 6 shows the amino acid sequence of E. maxima antigen mc-
  • Figure 7 shows the nucleotide sequence of the 5'-3' strand of cDNA encoding the E. maxima clone mc-35c gene (Sequence ID No. 7).
  • Figure 8 shows the amino acid sequence of E. maxima clone mc- 35c (Sequence ID No. 8).
  • Figure 9 shows the nucleotide sequence of the 5'-3' strand of DNA encoding the E. tenella antigen tg-3e gene (Sequence ID No. 9).
  • Figure 10 shows the amino acid sequence of E. tenella antigen tg- 3e (Sequence ID No. 10).
  • Figure 11 shows the nucleotide sequence of the 5'-3' strand of cDNA encoding the E. tenella antigen tc-lle gene (Sequence ID. No. 11).
  • Figure 12 shows the amino acid sequence of E. tenella antigen tc- lle (Sequence ID No. 12).
  • Figure 13 shows the nucleotide sequence of the 5'-3' strand of cDNA encoding the E. tenella antigen tc-23g gene (Sequence ID No. 13).
  • Figure 14 shows the amino acid sequence of E. tenella antigen tc- 23g (Sequence ID No. 14).
  • Figure 15 shows the nucleotide sequence of the 5'-3' strand of cDNA encoding the E. tenella antigen tc-26h gene (Sequence ID No. 15).
  • Figure 16 shows the amino acid sequence of E. tenella antigen tc- 26h (Sequence ID No. 16).
  • Figure 17 shows the nucleotide sequence of the 5'-3' strand of cDNA encoding the E. tenella antigen tc-30c gene (Sequence ID No. 17).
  • Figure 18 shows the amino acid sequence of E. tenella antigen tc-
  • Figure 19 shows the nucleotide sequence of the 5'-3' strand of cDNA encoding the E. tenella clone tc-32c gene (Sequence ID No. 19).
  • Figure 20 shows the amino acid sequence of E. tenella clone tc-32c
  • Figure 21 shows the nucleotide sequence of the 5'-3' strand of DNA encoding the E. tenella antigen tc-33c gene (Sequence ID No. 21).
  • Figure 22 shows the amino acid sequence of E. tenella antigen tc- 33c (Sequence ID No. 22).
  • Figure 23 shows the nucleotide sequence of the 5'-3' strand of cDNA encoding the E. tenella antigen tc-35c gene (Sequence ID. No. 23).
  • Figure 24 shows the amino acid sequence of E. tenella antigen tc-
  • Figure 25 shows the nucleotide sequence of the 5'-3' strand of cDNA encoding the E. maxima antigen mc-37c gene (Sequence ID. No. 25).
  • Figure 26 shows the amino acid sequence of E. maxima antigen mc-37c (Sequence ID No. 26).
  • T is thymidyl
  • GLY is glycine
  • ALA is alanine
  • VAL valine
  • LEU leucine ILE is isoleucine
  • THR is threonine
  • PHE is phenylalanine
  • TRP is tyryptophan
  • CYS is cysteine
  • GLU glutamic acid
  • LYS is lysine
  • ARG is arginine
  • HIS histidine
  • GLN glutamine
  • ASN is asparagine.
  • the present invention relates to recombinant antigenic proteins, and fragments thereof containing antigenic determinants, that can elicit an antibody response against avian coccidiosis, and to the cloned genes that encode the antigenic proteins and fragments.
  • These antigenic proteins, and the fragments thereof containing antigenic determinants will bind with a specific monoclonal antibody or with polyvalent antibodies from infected chickens, or from other animals that have been immunized with life forms of Eimeria or Eimeria proteins, directed against an antige ⁇ nic protein of avian coccidia.
  • the antigenic proteins of this invention may be used for several applications: (1) the protein(s) can be used in an avian coccidia assay to detect antibodies against the coccidia; (2) antibodies may be prepared from the antigenic protein(s); (3) the protein(s) can be used for preparing vaccines against avian coccidiosis.
  • Antibodies directed against coccidial-antigens are used to identify, by immunological methods, transformed cells containing DNA encoding coccidial antigens.
  • the MAbs are used as a tool for identifying cells containing DNA sequences encoding coccidial antigens that are either species specific or common to all nine species.
  • Screening transformants with polyvalent chicken antiserum or chicken bile is used to identify DNA sequences encoding a wide spectrum of coccidial proteins which are antigenic in chickens upon infection.
  • Screening transformants with poly- valent rat antiserum is used to identify DNA sequences encoding coccidia proteins, which are antigenic when injected subcutaneously in rats and which may be antigenic in chickens. DNA sequences from the trans ⁇ formants thus identified then may be incorporated into a microorganism for large scale protein production.
  • the antigenic proteins, as native proteins or as hybrids with other proteins, may be used as vaccines to immunize birds to protect them from subsequent infection.
  • DNA sequences comprising the genes that encode antigenic proteins and fragments thereof may be used as DNA probes. These probes have a variety of uses, including screening a DNA library for additional genes that may encode antigenic determinants.
  • the DNA probe may be labeled with a detectable group.
  • detectable group can be any material having a detectable physical or chemical property. Such materials have been well-developed in the field of immunoassays and in general almost any label useful in such methods can be applied to the present invention.
  • Particularly useful are enzymatically active groups, such as enzymes (see Clin. Chem. 22:1243 (1976)), enzyme substrates (see British Pat. Spec. 1,548,741), coenzymes (see U.S. Pat Nos. 4,230,797 and 4,238,565) and enzyme inhibitors (see U.S. Pat No. 4,134,792); fluorescers (see Clin. Chem.
  • chromophores such as chemiluminescers and bioluminescers (see Clin. Chem. 25:512 (1979)); specifically bindable ligands; proximal interacting pairs; and radioisotopes such as 3 H, 35 S, 32 P, 125 I and 14 C.
  • labels and labeling pairs are detected on the basis of their own physical properties (e.g., fluorescers, chromophores and radioisotopes) or their reactive or binding properties (e.g., enzymes, substrates, coenzymes and inhibitors).
  • a cofactor-labeled probe can be detected by adding the enzyme for which the label is a cofactor and a substrate for the enzyme.
  • an enzyme which acts upon a substrate to generate a product with a measurable physical property e.g., one can use an enzyme which acts upon a substrate to generate a product with a measurable physical property.
  • Examples of the latter include, but are not limited to, beta-galactosidase, alkaline phosphatase and peroxidase.
  • the term "antigenic” or “antigenic determinant” is meant immunologically cross-reactive antigenic determinants with which a given antibody will react Therefore, the antigenic peptides of this invention will include chemically synthesized peptides, peptides made by recombinant DNA techniques, and antibodies or fragments thereof which are anti-idiotypic towards the determinant of the peptides of this invention.
  • Several procedures may be used to construct a microorganism that produces an antigenic protein that binds with a monoclonal or polyvalent antibody that is directed against an antigenic protein of avian coccidia.
  • mRNA messenger RNA
  • cDNA complementary DNA
  • This route is referred to as the mRNA route.
  • the advantage to this route is that only "expressed" genes are cloned, reducing the number of individual transformants required to represent the entire population of genes.
  • the cloned DNA sequence is advantageously transferred to a suitable expression vector/host cell system for large scale production of the antigenic protein.
  • the DNA sequence that is to be isolated encodes an antigenic protein that will elicit an immune response when administered to chickens which will protect them from subsequent infections. It is not necessary to isolate a complete coccidial gene encoding such a protein, since those portions of the protein termed antigenic determinants are sufficient for triggering a protective immune response (Lerner, supra). This antigenic determinant should be on the surface of the folded microbially-produced protein to trigger the response (Lerner, supra).
  • the sequence may be isolated from the sporo- zoite life stage of the parasite. It has been demonstrated that part of the protective immune response in chickens is directed against the sporozoite.
  • Antigenic proteins isolated from other life stages also may be effective as vaccines.
  • MAbs or polyvalent antibodies which bind to various sporozoite proteins can be used to identify cloned DNA sequences encoding those proteins. Such proteins can be isolated and used to elicit a protective immune response in chickens.
  • Sporozoites can be obtained from oocysts by excystation using the method of Doran and Vetterling, Proc. Helminthol Soc. Wash. 34:59-65 (1967), and purified by the leucopak filter technique of Bontemps and Yvore, Ann. Rech. Vet 5:109-113 (1974). Although the method of
  • sporozoite mRNA may be isolated from intact sporulated oocysts, which contain the sporozoites.
  • Isolation of mRNA coding for the antigenic proteins of interest is advantageously accomplished by lysis of intact sporulated oocysts under conditions which minimize nuclease activity. This is accomplished using a modification of the procedure described by Pasternak et al. Molec.
  • RNA may be isolated by grinding the oocysts with glass beads in a solution containing guanadine thiocyanate, Sarkosyl, and Tris Buffer pH 8.0. Oocyst proteins are removed by phenol chloroform extraction. The total cellular RNA is separated from DNA by precipitation with lithium chloride. Oligo
  • (dT)-cellulose chromatography then can be used to isolate mRNA from the total RNA population.
  • Synthesis of cDNA may be accomplished using either a kit from
  • Moloney Leukemia virus reverse transcriptase and RNase H The kits are used according to the instructions provided by the manufacturer.
  • the poly r(A) tail of mRNA permits oligo(dT) (of about 12-18 nucleotides) to be used as a primer for cDNA synthesis or alternatively DNA oligonucleotides of random sequence can be used as a primer for cDNA synthesis.
  • the ds-cDNA prepared as described above is generally inserted into a suitable cloning vector, which is used for transforming appropriate host cells.
  • suitable cloning vectors include various plasmids and phages, but a bacteriophage lambda is preferred.
  • a cloning vector For a cloning vector to be useful for the expression of foreign proteins which are to be detected with antibodies, it should have several useful properties. Most importantly, it should have a cloning site within a gene which is expressed in the host being used. There should also be a means of controlling expression of the gene. The vector should be able to accept DNA of the size required for synthesis of the desired protein product and replicate normally. It is also useful to have a selectable property which allows identification of vectors carrying inserts. A cloning vector having such properties is the bacteriophage ⁇ gtll (ATCC 37194) (Young and Davis. Proc. Nat'l Acad. Sci. USA 80:1194-1198 (1983)).
  • This vector has a unique EcoRI site near the end of the bacterial gene coding for /3-galactosidase. That site can be used for insertion of foreign DNA to make hybrid proteins made up of /3-galactosidase and the foreign gene product
  • the expression of /3-galactosidase is under control of the lac promoter and can be induced by the addition of isopropyl- -D- thiogalactopyranoside (IPTG).
  • IPTG isopropyl- -D- thiogalactopyranoside
  • the ⁇ gtll phage contains 43.7 kb of DNA which is considerably smaller than wild type ⁇ . This allows insertion of pieces of DNA up to 8.3 kb in length, before the DNA becomes too large to fit inside the phage head.
  • the ds-cDNA can be conveniently inserted into the phage by addition of linkers containing an EcoRI restriction site and any convenient second restriction enzyme recognition site to the DNA and ligation into the EcoRI-cut ⁇ gtll DNA.
  • linkers containing an EcoRI restriction site and any convenient second restriction enzyme recognition site to the DNA and ligation into the EcoRI-cut ⁇ gtll DNA.
  • the DNA is packaged, in vitro, into ⁇ phage heads (Enquist and Sternberg, Methods in Enzvmology 68:281-298 (1979) and those phages are used to infect a suitable ⁇ -sensitive host With the proper choice of host, the phage may be screened as plaques or lysogens
  • yeast Old and Primrose, supra. pp. 62-68
  • filamentous fungi insect cells
  • mammalian cells U.S. 4,745,051 and 4,879,2366
  • the DNA described herein may be inserted into the above vectors by various techniques including homopolymeric tailing, blunt-end ligation or by use of linker molecules (Old and Primrose, supra, at p. 92).
  • the recombinant bacteriophages can be used to infect a suitable E. coli host which allows the formation of phage plaques on agar (or agarose) plates.
  • the plaques can be transferred to nitrocellulose membranes while being induced with IPTG.
  • the proper antibodies are then reacted with the filters.
  • the positive reactions are detected by reaction with either [ 125 I] Staphylococcus aureus Protein A or a second antibody conjugated with horseradish peroxidase, alkaline phosphatase, or vitamin B12.
  • the plaques containing cross-reactive antigens can then be detected by autoradiography, by detection of the conjugated enzyme, or, in the case of Vitamin B12 conjugates, by binding of streptavidin conjugate to one of the reporter enzymes.
  • the phages giving positive signals in the antibody-screening procedure can be shown to contain sequences coding for coccidial proteins by excision of the DNA originally inserted into the phage DNA and examination of the ability of that DNA to hybridize with coccidia mRNA or coccidia genomic DNA.
  • the nucleotide sequence of the cDNA insert is determined using the methods of Sanger et al. Proc. Natl Acad. Scl. USA 74:5463-5467 (1977); or Maxam and Gilbert, Proc. Natl. Acad.
  • Another method of cloning coccidial antigens begins with isolation of nuclear DNA from oocysts. This DNA is then broken into fragments of a size suitable for insertion into a cloning vector. To obtain such fragments, one can use mechanical shearing methods such as sonication or high-speed stirring in a blender to produce random breaks in the DNA. Intense sonication with ultrasound can reduce the fragment length to about 300 nucleotide pairs. (Old and Primrose, supra, p.
  • nuclear DNA may be partially digested with DNAsel, which gives random fragments, with restriction endonucleases, which cut at specific sites, or with mung bean nuclease in the presence of fo ⁇ namide, which has been shown with some related organisms (McCutchan, T.F., et al Science 225:625-628 (1984)) to produce DNA fragments containing intact genes.
  • nuclear DNA fragments may be inserted into any of the cloning vectors listed for the cloning of cDNA in the mRNA experimental method. If the nuclear DNA is digested with a restriction endonuclease, it can be inserted conveniently into a cloning vector digested with the same enzyme, provided the vector has only one recognition site for that enzyme. Otherwise, DNA fragments may be inserted into appropriate cloning vectors by homopolymeric tailing or by using linker molecules (Old and Primrose, supra, at p. 92).
  • genomic DNA expression libraries are constructed after digestion on nuclear DNA with any one of a number of restriction enzymes that yield blunt end DNA fragments.
  • the size of the DNA frag ⁇ ments can be controlled by reducing the time of digestion with the restriction enzyme.
  • Linkers such as an oligonucleotide encoding restriction site for EcoRI and Notl are ligated onto the blunt end DNA fragments and fragments are ligated into the EcoRI site of bacteriophage ⁇ gtll.
  • the resulting genomic expression library can be subjected to antibody screening as described for the mRNA route.
  • Plasmid DNA is isolated from transformants found to be "positive” by the above screening methods.
  • the nuclear DNA inserts of these plasmids are then subjected to DNA sequencing.
  • the cloned DNA sequence may be transferred to expression vectors engineered for high- level production of the antigenic protein.
  • the expression vectors are transformed into suitable host cells for production of the antigenic protein.
  • These host cells may include both prokaryotic and eukaryotic organisms.
  • the prokaryotic host cells include E. coli and B. subtilis.
  • the eukaryotic host cells include yeast, insect cells, and mammalian cells.
  • Coccidial antigens advantageously may be produced at high levels in E. coli as a fusion protein comprising the antigen and an amino terminal portion of the viral MS-2 polymerase. This fusion is accomplished by inserting a DNA sequence encoding a coccidial antigen into a plasmid vector, pEX32b, carrying the polymerase gene.
  • the expression of the fusion antigenic protein is highly regulated by temperature.
  • Host expression vector systems in which expression of foreign proteins is regulatable have the advantage of avoiding possible adverse effects of foreign protein accumulation as high cell densities are reached.
  • Some investigators have proposed that expression of gene fragments such as those encoding antigenic determinants may avoid the deleterious effects that expression of the entire antigenic protein would have on E. coli host cells. (Helfman et al. Proc. Natl Acad. Scl. USA 80:31-35 (1983)).
  • Coccidial antigens also may be produced in high levels as fusions at the carboxy-terminal of E. coli 3-galactosidase.
  • the coccidia antigen gene is transferred to a small plasmid such as pGX3217 which carries the gene for ampicillin resistance and which contains a /3-galactosidase gene with an EcoRI insertion site near the 3' end of the gene. Synthesis of the fused gene products is regulated by the ]ac promoter which is induced by addition of IPTG, the inducer of the lac operon.
  • An effective subunit vaccine against avian coccidiosis may consist of a mixture of antigen proteins derived from several species of Eimeria. Alternatively, production costs may be decreased by producing two or more antigen proteins as one fusion protein thus reducing the required number of fermentations and purifications.
  • Such a fusion protein would contain the amino acid sequence comprising an antigenic epitope of each antigen protein (or repetitions of those sequences) with variable amounts of surrounding nonantigenic sequence.
  • a hybrid gene designed to code for such a protein in E. coli would contain bacterial regulatory sequence (promoter/operator) and the 5' end of an E. coli gene (the ribosome binding site and codons for several amino acids) to ensure efficient translation initiation followed by the coding sequences for the antigenic epitopes all fused in the same reading frame.
  • E. coli cells transformed with the expression vector carrying a cloned coccidial antigen sequence are grown under conditions that promote expression of the antigenic polypeptide.
  • the antigenic protein is then tested for ability to elicit an immune response in chickens that will protect them from subsequent Eimeria infections.
  • the antigen protein may be presented to birds as a live E. coli expressing the protein, as killed E. coli expressing the protein or as purified protein.
  • the antigen may be combined with suitable carriers and adjuvants and administered to birds in their feed or by injection.
  • the cloned antigenic proteins used in vaccines above are tested for their ability to elicit an immune response in chickens that protects the birds from subsequent infection by any of the important species of Eimeria. including E. tenella. E.
  • cloned antigenic proteins which may be useful as vaccines to protect against coccidiosis
  • another useful alternative which may be derived from cloning antigen genes is the use of small, synthetic peptides in vaccines (see Lerner, supra). After the sequence of antigenic proteins is determined, it is possible to make synthetic peptides based on that sequence. The peptides are conjugated to a carrier protein such as hemocyanin or serum albumin and the conjugate then can be used to immunize against coccidia.
  • a carrier protein such as hemocyanin or serum albumin
  • expression libraries were prepared in the lambda vector, ⁇ gtll, using cDNA prepared from poryA mRNA isolated from E. maxima oocysts. The construction of the libraries is described in Genex Patent Application, PCT/US89/02918 incorporated herein by reference.
  • the cDNA expression library was screened with rat antiserum raised against E. maxima sporozoites.
  • the rat immune serum was prepared using the following protocol. Da
  • the library to be screened was plated on a host that allows lysis and plaque formation. Following induction of the antigens encoded by the phage, the plaques were transfered to nitrocellulose filters. Positive phage were identified after screening with the rat anti-E. maxima sporozoite antiserum. The cDNA inserts from the positive clones were cloned into bacteriophage M13 and subjected to sequence analysis. Ej. maxima antigen mc-4c was identified.
  • Antigen mc-4c is an analog of E. acerv ⁇ lina antigen ac-6b and E tenella antigen GX3262 (Miller etal. Infect. Immun. 57:2014-2020 (1989); Danforth et al. Poultry Sci. 68:1643-1652 (1989)). Only the carboxy terminal sequence of mc-4c was recovered so a cDNA expression library was screened with an 18 bp oligonucleotide complementary to the 5'- sequence of the original mc-4c clone. A second clone, ⁇ mc-36c, was identified that encodes all but the six amino terminal amino acids of the antigen. The sequence of mc-4c and the 21.7 Kd translation product that it encodes are shown in Figures 1 and 2 (Sequence ID Nos.l and 2).
  • E. maxima libraries were screened with the rat anti-E. maxima sporozoite immune serum. Phage that produce antigens cross-reactive with the immune serum were identified. The cDNA inserts from the positive phage were cloned into bacteriophage
  • the carboxyl terminal sequence of antigen mc-5c is encoded by 1611 bp of open reading frame.
  • the sequence and its 59.2 Kd translation product are shown in Figures 3 and 4 (Sequence ID Nos.3 and 4).
  • a partial sequence of antigen mc-5c were expressed in pEX32b.
  • the terminal 227 amino acids encoded by an EcoRI fragment extending from the internal restriction site starting at base 928 to beyond the site of translation termination ( Figure 3) were inserted into pEX32b and expressed.
  • Antigen mc-5c is encoded in expression vector pGX5376.
  • E. maxima libraries were screened with the rat anti-E. maxima sporozoite immune serum. Phage that produce antigens cross-reactive with the immune serum were identified.
  • the cDNA inserts from the positive phage were cloned into bacteriophage M13 and subjected to sequence analysis. Following sequence analysis, E. tenella antigen mc-30c was identified.
  • Antigen mc-30c had 510 bp of open reading frame fused in frame with the /3-galactosidase gene of ⁇ gtll. Within the open reading frame are two methionine codons (double underlined) followed by 321 bp of open reading frame ending with a termination codon ( Figure 5, Sequence ID No.5). Preceding the putative methionine initiation codons are two
  • the insert contains a complete gene encoding an Eimeria antigen consisting of 109 amino acids with a molecular weight of 12.2 Kd.
  • the amino acid sequence of the mc-30c antigen encoded by the entire open reading frame of mc- 30c is shown in Figure 6 (Sequence ID No.6) with the methionines at the putative initiation site underlined.
  • Antigen mc-30c was expressed in E. coli after insertion into the plasmid expression vector pEX-32b. Antigen mc-30c is encoded in expression vector pGX5370 and is expressed as a fusion protein with 11 Kd of the MS-2 polymerase under control of the P L promoter.
  • the host strain pop2136 contains a temperature sensitive repressor of P L and expression of the fusion protein is fully induced at 42°C.
  • E. maxima libraries were screened with the rat anti-E. maxima sporozoite immune serum. Phage that produce antigens cross-reactive with the immune serum were identified.
  • the cDNA inserts from the positive phage were cloned into bacteriophage M13 and subjected to sequence analysis. Following sequence analysis, E. tenella antigen mc-35c was identified.
  • Antigen mc-35c is encoded by 1461 bp of open reading frame fused in-frame with the /3-galactosidase sequence. The 5'-sequence consists of a 24 bp sequence encoding eight amino acids which is repeated with complete fidelity a total of 24 times.
  • This repeat is linked through a single glutamic acid residue to a second, different 24 bp sequence which is repeated 16 times.
  • the repeats are followed by 501 bp of open reading frame followed by a termination codon.
  • the nucleotide sequence of mc- 35c is shown in Figure 7 (Sequence ID No.7) and the amino acid sequence of mc-35c is shown in Figure 8 (Sequence ID No.8) with the first of each of the eight amino acid repeats underlined and the connecting glutamic acid residue double underlined.
  • Antigen mc-35c was expressed in E. coli after insertion into the plasmid expression vector pEX-32b. Antigen mc-35c is encoded in expression vector pGX5367 and is expressed as a fusion protein with 11 Kd of the MS-2 polymerase under control of the P L promoter.
  • the host strain pop2136 contains a temperature sensitive repressor of P L and expression of the fusion protein is fully induced at 42°C.
  • E. tenella cDNA and genomic expression libraries in lambda phage were screened with chicken immune bile.
  • Immune bile was recovered from chickens infected with E. tenella oocysts seven days after the onset of infection. Before being used to screen the libraries, the immune bile was incubated with R coli proteins to inactivate antibodies cross-reactive with E. coli proteins. After incubation of the filters used to lift the lambda plaques from the growth plates with the immune bile, the filters were developed with goat anti-chicken IgA antibodies.
  • the positive plaques were visualized with donkey anti-goat antibodies conjugated with alkaline phosphatase. Phage that produce antigens cross-reactive with the immune bile were identified. The DNA inserts from the positive phage were cloned into bacteriophage M13 and subjected to sequence analysis.
  • Antigen tg-3e was expressed in E. coli after insertion into the plasmid expression vector pEX-32b. Antigen tg-3e is encoded in expression vector pGX5390 and is expressed as a fusion protein with 11 Kd of the MS-2 polymerase under control of the P L promoter.
  • the host strain pop2136 contains a temperature sensitive repressor of P L and expression of the fusion protein is fully induced at 42°C.
  • E. tenella cDNA libraries were screened with the chicken anti-E. tenella immune bile. Phage that produce antigens cross-reactive with the immune bile were identified. The cDNA inserts from the positive phage were cloned into bacteriophage M13 and subjected to sequence analysis. Following sequence analysis R tenella antigen tc-lle was identified.
  • Antigen tc-lle consists of a 417 bp open reading frame encoding 139 amino acids.
  • the sequence of tc-lle and the 13.9 Kd translation product that it encodes are shown in Figures 11 and 12 (Sequence ID
  • Antigen tc-lle was expressed in E. coli after insertion into the plasmid expression vector pEX-32b. Antigen tc-lle is encoded in expression vector pGX5394 and is expressed as a fusion protein with 11 Kd of the MS-2 polymerase under control of the P L promoter.
  • the host strain pop2136 contains a temperature sensitive repressor of P L and expression of the fusion protein is fully induced at 42°C.
  • the library to be screened was plated on a host that allows lysis and plaque formation. Following induction of the antigens encoded by the phage, the plaques were transferred to nitrocellulose filters using standard protocols (Berger et al. Methods in Enzvmologv, Vol 152, Academic Press, New York, NY (1987)). The filters were screened with the chicken anti-E. tenella sporozoite antiserum. Plaques recognized by chicken serum IgA antibodies were identified with goat anti-chicken IgA antibody followed by biotinylated donkey anti-goat IgG antibody. The positive plaques were visualized with an alkaline phosphatase-streptavidin conjugate using standard protocols (Berger et al.
  • Antigens tc-23g was expressed in E. coli after insertion into the plasmid expression vector pEX-32b using standard protocols (Berger et al. Methods in Enzymology. Vol. 152, Academic Press, New York, NY
  • the antigen is encoded in expression vector pGX5398 and is expressed as a fusion protein with 11 Kd of the MS-2 polymerase under control of the P L promoter.
  • the host strain pop2136 contains a tempera ⁇ ture sensitive repressor of P L and expression of the fusion protein is fully induced at 42°C.
  • Example 7 an E. tenella cDNA library was screened with chicken anti-E. tenella merozoites immune serum produced using the same injection regimen described in Example
  • a phage that produces an antigen cross-reactive with IgG antibodies in the immune serum was identified with biotinylated goat anti-chicken IgG antibody followed by visualization with an alkaline phosphatase-strep- tavidin conjugate.
  • the cDNA insert from the positive phage was cloned into bacteriophage M-13 and subjected to sequence analysis using the method of Sanger et al. (Proc. Natl. Acad. Sci. USA 74-5463-5467 (1977)). Following sequence analysis, E. tenella antigen tc-26h was identified.
  • the antigen consists of a peptide fragment which is composed of long strings of alanines and serines.
  • the nucleotide sequence of tc-26h and the 10 Kd translation product that it encodes are shown in Figures 15 and 16
  • Example 7 an E. tenella cDNA library was screened with the rat anti-E. maxima sporozoite immune serum. Phage that produce antigens cross-reactive with the immune serum were identified. The cDNA inserts from the positive phage were cloned into bacteriophage M-13 and subjected to sequence analysis using the method of Sanger et al. fProc. Natl. Acad. Sci. USA 74:5463-5467 (1977)). Following sequence analysis, E. tenella antigens to tc-30c, tc-32c, tc-33c and tc-35c were identified.
  • Antigen tc-30c consisted of 284 bp of open reading frame encoding an antigen fragment fused in-frame with the /3-galactosidase gene of ⁇ gtll. The nucleotide sequence of tc-30c and the 9.5 Kd translation product that it encodes are shown in Figures 17 and 18 (Sequence ID Nos.17 and 18).
  • Antigen tc-32c consisted of 261 bp of open reading frame encoding an antigen fragment The nucleotide sequence of tc-32c and the 8.8 Kd translation product that it encodes are shown in Figures 19 and 20 (Sequence ID Nos.19 and 20).
  • Antigen tc-33c consisted of 408 bp of open reading frame encoding the carboxy terminal sequence of an antigen.
  • the nucleotide sequence of tc-33c and the 12.5 Kd translation product that it encodes are shown in Figures 21 and 22 (Sequence ID Nos.21 and 22).
  • Antigen tc-35c consisted of 120 bp of open reading frame encoding the carboxy terminal sequence of an antigen. The nucleotide sequence of tc-35c and the 5.0 Kd translation product that it encodes are shown in
  • E. maxima cDNA libraries were screened with the rat anti-E. maxima sporozoite immune serum. Phage that produce antigens cross-reactive with the immune serum were identified.
  • the cDNA inserts from the positive phage were cloned into bacteriophage M-13 and subjected to sequence analysis using the method of Sanger et al. (Troc. Natl. Acad. Sci. USA 74:5463-5467 (1977)). Following sequence analysis, E. tenella antigen mc-37c was identified.
  • Antigen mc-37c consists of 442 bp of open reading frame encoding an antigen fragment that is fused in-frame with the /3-galactosidase sequence of ⁇ gtll.
  • the nucleotide sequence of mc-37c and the 16.2 Kd peptide that it encodes are shown in Figures 25 and 26 (Sequence ID Nos. 25 and 26). While certain representative embodiments and details have been shown for the purpose of illustrating the invention, it will be apparent to those skilled in the art that various changes and modifications may be made therein without departing from the scope of the invention.
  • ADDRESSEE Sterne, Kessler, Goldstein & Fox
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • AGA TTA GTA GAT TTT TGT ATA CAA GAT TTT AAG AGA AAG AAT AGA TCT 624 Arg Leu Val Asp Phe Cys He Gin Asp Phe Lys Arg Lys Asn Arg Ser 195 200 205
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • CAA CCA CCA CAC ATT CAT ACG GAG ACT CCT GCC ACA ACA CCA GTT GCA 240
  • MOLECULE TYPE DNA (genomic)
  • AGG CTC AAG CAA CAG CAA CAG CAG CAG CTT TGT CAG CAG CAG CCA GGG 384 Arg Leu Lys Gin Gin Gin Gin Gin Gin Gin Leu Cys Gin Gin Gin Pro Gly 115 120 125
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)

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Abstract

Nouvelles protéines antigéniques de recombinaison de la coccidiose aviaire, fragments desdites protéines contenant des déterminants antigéniques, et gènes codant les peptides antigéniques. On décrit aussi des vaccins préparés en utilisant les nouvelles protéines antigéniques de la coccidiose aviaire et des méthodes permettant d'immuniser les poulets contre les coccidioses aviaires.
EP91917491A 1990-09-12 1991-09-05 Genetically engineered coccidiosis vaccine Withdrawn EP0548252A4 (en)

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US5824656A (en) * 1988-01-15 1998-10-20 Merck & Co., Inc. Recombinant and native group B eimeria tenella immunogens useful as coccidiosis vaccines
US6008327A (en) 1992-03-13 1999-12-28 Akzo Nobel, N.V. Peptides and nucleic acid sequences related to the Epstein Barr virus
ZA965586B (en) 1995-07-03 1997-01-31 Akzo Nobel Nv Coccidiosis poultry vaccine
NZ500033A (en) * 1998-10-07 2001-06-29 Akzo Nobel Nv Hydrophilic eimeria polypeptides for use as coccidiosis vaccines
DE10330235A1 (de) * 2003-07-04 2005-01-20 Bayer Healthcare Ag Neues Eimeria Gen und Protein sowie deren Verwendung
EP1644408A1 (fr) * 2003-07-15 2006-04-12 Barros Research Institute Antigene eimeria tenella utilise dans l'immunotherapie de la coccidiose
CN109432006A (zh) * 2018-12-18 2019-03-08 佛山市正典生物技术有限公司 一种球虫疫苗喷滴免疫用佐剂及其应用

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EP0231537A2 (fr) * 1985-12-03 1987-08-12 Solvay Protéines antigènes et vaccins les contenant pour prévention de coccidiose
WO1988006629A1 (fr) * 1987-03-06 1988-09-07 Synergen Incorporated Polypeptides servant au diagnostic des coccidioses et procedes deproduction de ces polypeptides utilisant l'adn recombinant
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US4874705A (en) * 1985-05-16 1989-10-17 Solvay & Cie, S.A. DNA encoding an antigenic protein derived from Eimeria tenella and vaccines for prevention of coccidiosis caused by Eimeria tenella
CA1340520C (fr) * 1984-06-05 1999-05-04 Karel Z. Newman Jr. Proteines antigeniques et vaccins qui en renferment; anticorps protecteurs diriges vers eux pour prevenir la coccidiose causee par eimera tenella et eimera necatrix
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EP0231537A2 (fr) * 1985-12-03 1987-08-12 Solvay Protéines antigènes et vaccins les contenant pour prévention de coccidiose
WO1988006629A1 (fr) * 1987-03-06 1988-09-07 Synergen Incorporated Polypeptides servant au diagnostic des coccidioses et procedes deproduction de ces polypeptides utilisant l'adn recombinant
WO1990000403A1 (fr) * 1988-07-05 1990-01-25 Genex Corporation Vaccin contre la coccidiose prepare par genie genetique

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