EP0717629A1 - NOUVEAUX ANTICORPS ET ANTIGENES D'$i(EIMERIA) ET PROCEDES POUR LEUR UTILISATION - Google Patents

NOUVEAUX ANTICORPS ET ANTIGENES D'$i(EIMERIA) ET PROCEDES POUR LEUR UTILISATION

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Publication number
EP0717629A1
EP0717629A1 EP94924563A EP94924563A EP0717629A1 EP 0717629 A1 EP0717629 A1 EP 0717629A1 EP 94924563 A EP94924563 A EP 94924563A EP 94924563 A EP94924563 A EP 94924563A EP 0717629 A1 EP0717629 A1 EP 0717629A1
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EP
European Patent Office
Prior art keywords
antigen
eimeria
kda
antibody
antigens
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Application number
EP94924563A
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German (de)
English (en)
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EP0717629A4 (fr
Inventor
Timothy J. Miller
George Strang
David Brake
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Pfizer Inc
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Pfizer Inc
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Publication of EP0717629A1 publication Critical patent/EP0717629A1/fr
Publication of EP0717629A4 publication Critical patent/EP0717629A4/fr
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/20—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans from protozoa
    • 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

Definitions

  • the present invention relates generally to the field of parasitology. More particularly, the invention relates to novel antibodies and antigens from Eimeria and methods of using and detecting the same.
  • Avian coccidiosis is an enteric, parasitic disease of domestic and wild bird species caused by members of the protozoan genus Eimeria.
  • the disease is spread by ingestion of sporulated oocysts from feces of an infected host.
  • the infectious process is rapid and characterized by parasite replication in host cells, causing extensive damage to the intestinal mucosa.
  • the avian eimerian life cycle is complex and occurs in two stages - the exogenous phase which takes place in the litter, and the endogenous phase, which occurs in the intestinal tract of the host.
  • stage-specific antigens become the potential targets of host protective B and T-cell immune responses.
  • active infection is able to generate a natural immune response in some animals.
  • there is no practical method for determining whether a particular animal is indeed immune to infection which can only be confirmed by visually inspecting the intestinal tract of a sacrificed bird.
  • Subunit vaccine preparations have been developed in an attempt to alleviate the problems inherent in the use of the above-described preparations.
  • EPA Publication No. 453,055 (published 23 October 1991 ) describes multicomponent vaccine compositions including mixtures of a 25 kDa E. tenella antigen, a 26 kDa E. necatrix antigen or a 55 kDa E. maxima antigen, derived from Eimeria oocysts.
  • EPA Publication No. 256,536 (published 24 February 1988) describes the isolation of E. maxima macrogametocytes and microgametocytes and vaccines comprising heterogenous protein extracts derived from the gametocytes.
  • the present invention is based on the development of novel Eimeria antibody preparations, produced locally or which traffic to, the site of parasitic infection.
  • the antibodies so produced provide for the discovery of protective extracellular and intracellular Eimeria antigens in biological samples, including in culture systems which support high levels of parasite growth and development.
  • Intracellular antigens have not previously been identifiable using conventional cell culture techniques and polyclonal or monoclonal antibody preparations.
  • the antigens and antibodies can be used in protective Eimeria vaccines. Diagnostic tests as well as bioassays to measure B- and T-cell dependent immune responses at the local site of parasite entry are also made possible by the present discoveries.
  • the invention is directed to an isolated, locally generated, Eimeria antibody preparation.
  • the antibody preparation comprises cecal lymphocyte immune products (CLIP), splenic lymphocyte immune products (SLIP), rectal antibody test (RAT) or cage dropping antibody test (CD AT).
  • CLIP cecal lymphocyte immune products
  • SLIP splenic lymphocyte immune products
  • RAT rectal antibody test
  • CD AT cage dropping antibody test
  • the invention is directed to a method for detecting the presence or absence of an Eimeria antigen in a biological sample, the method comprising:
  • the subject invention is directed to a method for detecting the presence or absence of an Eimeria tenella antigen in a biological sample, the method comprising:
  • the invention is directed to a method for diagnosing coccidiosis infection in an avian subject, the method comprising: (a) providing a biological sample from the avian subject;
  • the invention is directed to an intracellular Eimeria antigen, identifiable using a locally generated, Eimeria antibody preparation.
  • the antigen has a molecular weight of approximately 28 kDa, 35 kDa, 38 kDa, 40 kDa, 43 kDa, 55 kDa, 70 kDa, 100 kDa or 110 kDa, as determined by Western immunoblot analysis.
  • the invention is directed to a method for detecting the presence or absence of an Eimeria antibody in a biological sample, the method comprising:
  • the invention is directed to a kit for diagnosing coccidiosis in an avian subject, the kit comprising a locally generated, Eimeria antibody preparation, packaged in a suitable container.
  • the invention is directed to a kit for diagnosing coccidiosis in an avian subject, the kit comprising an Eimeria tenella monoclonal antibody reactive with an intracellular Eimeria antigen, packaged in a suitable container.
  • the invention is directed to a kit for detecting the presence or absence of antibodies to Eimeria in a biological sample, the kit comprising an intracellular Eimeria antigen, packaged in a suitable container.
  • Eimeria antigen refers to a molecule derived from an Eimeria species which contains one or more epitopes that will stimulate a host's immune system to make a secretory, humoral and/or cellular antigen-specific response.
  • antigens can be derived from any of the known Eimeria species, the choice of species being dependent on the host and coccidial disorder to be treated.
  • domestic fowl can be infected by any of E. tenella, E. necatrix, E. brunetti, E. maxima, E. acervulina and E.
  • praecox. Turkeys are susceptible to infection by E. melagrimitis, E. dispersa, E. meleag ⁇ dis, E. gallopavonis, E. adenoides, E. innocua and E. subrotunda.
  • domestic and wild ducks suffer from infections caused by E. anatis and geese (Anser) can be infected by E. anseris, E. nocens, E. parvula, E. hermani, E. striata and E.fulva.
  • Antigens of the present invention can therefore be identified and derived from any of the above species.
  • an "Eimeria antigen” includes antigens substantially homologous and functionally equivalent to the corresponding native Eimeria antigen.
  • the term “Eimeria antigen” encompasses modifications, such as deletions, additions and substitutions (generally conservative in nature), to the native sequences. Such modifications of the primary amino acid sequence may result in proteins which have enhanced or decreased activity as compared to the native sequence. These modifications may be deliberate, as through site-directed mutagenesis, or may be accidental, such as through mutations of hosts which produce the antigens. All of these modifications are included, so long as the molecule remains capable of eliciting an immunological response, as defined below, and activity is not destroyed.
  • an "Eimeria antigen” denotes a protein which may be modified by combination with other biological materials, such as lipids and saccharides, or by side chain modification, such as acetylation of amino groups, phosphorylation of hydroxyl side chains including phosphorylation of tyrosine, serine, threonine or any other side chains, whether or not these residues are normally phosphorylated in the native molecule, or oxidation of sulfhydryl groups, as well as other modifications of the encoded primary sequence.
  • side chain modification such as acetylation of amino groups, phosphorylation of hydroxyl side chains including phosphorylation of tyrosine, serine, threonine or any other side chains, whether or not these residues are normally phosphorylated in the native molecule, or oxidation of sulfhydryl groups, as well as other modifications of the encoded primary sequence.
  • glycosylated and unglycosylated forms the amino acid sequences with or without associated phosphat
  • intracellular antigen an Eimeria antigen, as described above, which is expressed during intracellular stages of parasite development, i.e., during asexual development within the villous or epithelial cells of the intestinal mucosa.
  • the intracellular stage includes intracellular sporozoite metabolism, trophozoites and asexual development into multinucleate schizonts or meronts. Accordingly, intracellular antigens produced during these events are covered by the definition.
  • the production of such intracellular antigens can be seen in cell lines adapted to support the growth of intracellular Eimeria spp. forms, including both primary and continuous cell lines. Representative cell lines are described further below.
  • intracellular antigens are also referred to herein as "tissue culture derived antigens.”
  • the term “intracellular antigen” encompasses proteins which are both secreted into the cell culture media in which the parasite is developing, as well as those antigens which are retained within the cell, such as an antigen associated with the cell membrane, endoplasmic reticulum and so forth.
  • An intracellular antigen can be either soluble or insoluble.
  • An "extracellular antigen” is an Eimeria antigen which is produced during the extracellular stages of parasite development, i.e., the exogenous phase of the Eimeria lifecycle which occurs in the litter and the endogenous phases where the organism has not yet invaded and entered host cells.
  • antigens include the extracellular sporozoite and merozoite forms of the organism.
  • antigens of Eimeria spp. are sometimes identified below with reference to their molecular mass in kilodaltons (kDa).
  • kDa molecular mass in kilodaltons
  • an antigen having a molecular mass of about 35 kDa is identified herein as P35; an antigen of about 40 kDa in molecular mass is identified as P40, and so on.
  • a "locally generated" antibody preparation is a composition containing one or more antibodies which are produced at, or traffic to, the local site of Eimeria infection.
  • Examples of locally generated antibody preparations include antibody preparations derived from immune lymphocyte populations found within the intestinal tract of a previously or currently infected avian subject, such as those derived from any portion of the cecum or intestine and termed cecal lymphocyte immune products ("CLIP”) herein; splenic lymphocyte immune products (“SLIP”), produced by trafficking memory splenic lymphocytes in infected avian subjects; coprantibody preparations (i.e., antibodies derived from fecal material) isolated from fecal material either directly from the intestinal tract or externally, of a previously infected or currently infected avian subject, such as rectal antibody test
  • RAT which is isolated from the intestinal digesta of the rectum of infected avian subjects
  • CDAT cage dropping antibody test
  • an “isolated” antigen or antibody an antigen or antibody which is separate and discrete from a whole organism (live or killed) with which the protein sequence is normally associated in nature.
  • an antigen contained in a cell free extract would constitute an “isolated” antigen, as would an antigen synthetically or recombinantly produced.
  • an “isolated” antibody preparation includes antibodies present in crude mixtures, blood, serum, etc., so long as the mixture is separate and discrete from the organism with which the antibodies are normally found.
  • isolated encompasses both polyclonal and monoclonal antibody preparations. Additionally, the term “isolated” with respect to both antigens and antibodies, is not meant to imply a particular degree of purity.
  • a crude extract is encompassed by the term, as is a highly purified preparation.
  • polypeptide and protein are used interchangeably and refer to any polymer of amino acids (dipeptide or greater) linked through peptide bonds.
  • polypeptide and protein include oligopeptides, protein fragments, analogs, muteins, fusion proteins and the like.
  • epitope refers to the site on an antigen or hapten to which specific B cells and T cells respond.
  • the term is also used interchangeably with "antigenic determinant” or “antigenic determinant site.”
  • An "immunological response" to a composition or vaccine is the development in the host of a secretory, cellular and/ or antibody-mediated immune response to the composition or vaccine of interest.
  • such a response includes but is not limited to one or more of the following effects; the production of antibodies from any of the immunological classes, such as immunoglobulins A, D, E, G or M; B cells; helper T cells; suppressor T cells; and or cytotoxic T cells and/or ⁇ T cells, directed specifically to an antigen or antigens included in the composition or vaccine of interest.
  • immunological classes such as immunoglobulins A, D, E, G or M
  • B cells helper T cells; suppressor T cells; and or cytotoxic T cells and/or ⁇ T cells
  • IgA immunoglobulin A
  • GALT gut-associated lymphoid tissue
  • BALT bronchial-associated lymphoid tissue
  • SIgA secretory IgA
  • immunological antigen or protein refers to an antigen or protein having an amino acid sequence which elicits an immunological response as described above.
  • immunogenic fragment is meant a fragment of a polypeptide which includes one or more epitopes and thus elicits an immunological response, as defined above. Such fragments can be identified by, e.g., concurrently synthesizing large numbers of peptides on solid supports, the peptides corresponding to portions of the Eimeria protein molecule, and reacting the peptides with antibodies while the peptides are still attached to the supports.
  • Such techniques are known in the art and described in, e.g., U.S. Patent No. 4,708,871; Geysen, H.M. et al. (1984) Proc. Natl. Acad. Sci. USA £:3998-4002; Geysen, H.M. et al. (1986) Molec. Immunol. 22:709- 715, all incorporated herein by reference in their entireties.
  • Such fragments will usually be at least about 2 amino acids in length, more preferably about 5 amino acids in length, and most preferably at least about 10 to 15 amino acids in length.
  • fragments there is no critical upper limit to the length of the fragment, which could comprise nearly the full length of the protein sequence, or even a fusion protein comprising fragments of two or more of the Eimeria antigens or one or more of the Eimeria antigens fused to, e.g., a bacterial, fungal, viral or protozoal protein.
  • Two polypeptide sequences are "substantially homologous" when at least about 65% (preferably at least about 80% to 90%, and most preferably at least about 95%) of the amino acids match over a defined length of the molecule.
  • substantially homologous also refers to sequences showing identity to the specified polypeptide sequence.
  • avian subject domestic, wild and game birds, including animals belonging to the order Galliformes, such as chickens, turkeys, pheasants, partridges, quail, grouse, guinea fowl and peacocks, as well as birds of the order Anseriformes, such as ducks and geese.
  • the definition encompasses birds of all ages, including subjects in ovo.
  • a "biological sample” refers to a sample of tissue or fluid isolated from an avian subject, including but not limited to, for example, blood, plasma, serum, fecal matter, urine, bone marrow, bile, spinal fluid, lymph fluid, samples of the skin, respiratory, intestinal, and genitourinary tracts, blood cells, organs, egg constituents and also samples of in vitro cell culture constituents
  • a "biological sample” also refers to a sample taken from any of the various developmental stages of Eimeria spp., including samples of sporulated oocysts, infectious sporozoites, intracellular sporozoites, merozoites, gametocytes, and the like.
  • treatment refers to both (i) the prevention of infection or reinfection (prophylaxis), and (ii) the reduction or elimination of symptoms (therapy) of coccidiosis.
  • the antibodies can be used in a variety of assays, including assays to identify antigens produced during the intracellular and extracellular phases of the eimerian life cycle.
  • the antibodies also allow the detection and characterization of antigens appearing at particular time periods during the infective process as well as at particular sites of infection.
  • the antibodies can be used as diagnostic reagents, to detect coccidiosis infection, as well as to determine host immunity levels; to immunopurify Eimeria antigens; and as screening agents to detect the presence of homologous genes in other medically important species.
  • the identified antigens can be isolated, characterized and used in subunit vaccine compositions, thus avoiding problems inherent in prior attenuated and killed vaccine preparations.
  • the identified antigens can also be used as diagnostic tools, for detecting Eimeria infection in biological samples and for determining the level of host immunity to the Eimeria species of interest.
  • CIP cecal lymphocyte immune products
  • SLIP splenic lymphocyte immune products
  • RAT rectal antibody test
  • CDAT cage dropping antibody test
  • CLIP and SLIP antibody preparations can be derived from splenic and cecal lymphocytes isolated from an appropriate immune bird subject. Particularly useful subjects are inbred bird lines which have been treated to simulate natural immunity to coccidiosis infection, as described in the examples.
  • Splenic and cecal lymphocytes can be isolated from the spleens and cecal pouches, respectively, of the bird subject and cultured in vitro in single cell suspension, using cell isolation and cultivation techniques known to those of skill in the art. In general, lymphocytes are cultivated for approximately five days after which time culture supernatants containing secreted Eimeria spp.-specific antibodies are removed, clarified by centrifugation, filtered, and stored at -20°C or lower. Antibody-containing supernatants can also be generated from in vitro cultivation of defined immune T- and B-cell lymphocyte subsets, or combinations thereof, using known techniques.
  • RAT and CD AT slgA-containing coprantibodies are isolated from the rectum digesta and from bird fecal droppings, respectively, of Eimeria spp.- immune birds. Exemplified herein is the isolation of RAT and CD AT from an inbred natural avian immune model. Avian slgA coprantibody samples may also be obtained from naturally exposed Eimeria spp. outbred broilers raised in wire batteries, floor pens, or broiler houses.
  • wet fecal material is resuspended in any suitable physiological balance solution or media, such as phosphate-buffered saline (PBS), followed by agitation, the addition of protease inhibitors, and two or more successive centrifugations at low and moderate speeds.
  • PBS phosphate-buffered saline
  • the antibody-containing solution is adjusted to physiological pH, filtered and stored at at least -20°C. Further purification of slgA can be achieved using ammonium sulfate precipitation, size and affinity chromatography or other biophysical methods readily known to those of skill in the art.
  • the antibody preparations produced above can be further characterized and used for a variety of purposes.
  • eimerian SLIP, CLIP, RAT, and CD AT reagents can be characterized in anti-parasite assays, such as a parasite neutralization assay (PN) and in vitro parasite inhibition assays (PI).
  • PN parasite neutralization assay
  • PI in vitro parasite inhibition assays
  • Total, isotype- specific and Eimeria spp.-spcci c antibodies, present in SLIP, CLIP, RAT, and CD AT can be quantified using conventional ELISAs, known to those of skill in the art and described further in the examples.
  • Eimeria antigens can be identified in a variety of biological samples, including in samples containing sporulated oocysts, extracellular sporozoites and merozoites, intracellular sporozoites and merozoites, and the like.
  • tissue and fecal samples e.g., samples from the spleen, cecum, rectum and from bird droppings
  • Intracellular antigens can be identified in media from cell culture systems that support the growth of the intracellular stages of the parasitic life cycle.
  • the present invention makes it possible to discriminate between antigens having similar molecular weights but produced during different stages of parasitic infection and/or at different sites of infection.
  • intracellular and extracellular antigens that specifically appear in the spleen, ceca, intestinal digesta and feces, after challenge in immune versus naive birds, can be identified.
  • Intracellular antigens have not heretofore been recognized by conventional antibody probes such as by sera produced in Eimeria immune chickens, nor have they been identified using polyclonal antibodies produced in common animal species, such as rabbits, or by monoclonal antibodies from mice immunized with Eimeria sporozoites and merozoites.
  • the identification of Eimeria intracellular antigens is now possible using the antibody preparations of the invention to assay culture media from avian Eimeria spp. grown in continuous cell lines able to support growth oi Eimeria during its entire life cycle, from sporozoite to oocyst, particularly during the intracellular phases of development.
  • Such cell lines are described in International Publication No. WO 93/01276 (published 21 January 1993) and include cell clones SB-CEV- 1/P (ATCC Accession
  • Eimeria spp. grown in cell line SB-CEV-1/F7 (ATCC Accession No. CRL10495), using the above antibodies.
  • This cell line is optimally cultured in Medium 199 (Gibco Laboratories, Grand Island, NY) under incubation conditions of 5% CO 2 and 40.5°C.
  • Fetal bovine serum, antibiotics and antifungal agents can also be added at proportions readily determined by one of skill in the art. Culturing conditions for SB-CEV- 1/F7 are detailed further in International Publication No. WO 93/01276 (published 21 January 1993).
  • intracellular antigens including antigens having molecular masses of 28 kDa, 35 kDa, 38 kDa, 40 kDa, 43 kDa, 55 kDa, 70 kDa, 100 kDa and 110 kDa, respectively, have been identified in E. tenella-de ⁇ vcd ammonium sulfate-treated tissue culture supernatants from SB-CEV- 1/F7 using the antibodies of the present invention, as well as using hyperimmune sera prepared from naturally exposed, inbred chickens.
  • the 35, 38, 43, 55 and 70 kDa antigens are recognized by SLIP, CLIP, RAT and immune sera; the 40 kDa antigen by CLIP, RAT and immune sera; the 100 kDa antigen by SLIP, RAT and immune sera; the 100 kDa antigen by SLIP and RAT; the 28 kDa antigen by CLIP and RAT; and the 55 and 70 kDa antigens by SLIP, CLIP and RAT.
  • the antigens appear at different times during the infective process.
  • the 35, 38, 40, 43 and 70 kDa antigens are present in both E. tenella and E.
  • Eimeria antigens can be identified in a biological sample using the above-described antibodies and any of several standard identification techniques.
  • the antibodies can be used in immunoassays, such as competition, direct reaction, or sandwich type assays, for identifying the presence or absence of the proteins by forming complexes therewith.
  • immunoassays include, but are not limited to, Western blots, agglutination tests, enzyme-labeled and mediated immunoassays, such as ⁇ LISAs, biotin/avidin type assays, radioimmunoassays, immunoelectrophoresis, immunoprecipitation, etc.
  • the reactions generally include revealing labels such as fluorescent, chemiluminescent, radioactive, or enzymatic labels or dye molecules, or other methods for detecting the formation of a complex between the antigen present in the biological sample and the antibody or antibodies reacted therewith.
  • an immunoassay for detecting one or more of the Eimeria proteins will involve selecting and preparing the test sample and then reacting it with one or more of SLIP, CLIP, RAT, and CD AT, under conditions that allow protein-antibody conjugates to form.
  • Solid supports can be used such as nitrocellulose, in membrane or microtiter well form; polyvinylchloride, in sheets or microtiter wells; polystyrene latex, in beads or microtiter plates; polyvinylidine fluoride; diazotized paper; nylon membranes; activated beads, and the like.
  • the solid support is first reacted with the biological sample, washed and then the antibodies applied.
  • a sandwich type format such as a sandwich ELISA assay
  • a commercially available anti-immunoglobulin i.e. anti- rabbit immunoglobulin conjugated to a detectable label, such as horseradish peroxidase, alkaline phosphatase or urease, can be added.
  • a detectable label such as horseradish peroxidase, alkaline phosphatase or urease.
  • An appropriate substrate is then used to develop a color reaction.
  • a particularly convenient method for identifying and characterizing antigens using the antibodies of the invention involves immunoblot analysis. Briefly, parasite antigens are prepared and separated on SDS polyacrylamide preparative minigels. Separated proteins are electroblotted onto membranes, cut into strips, and residual protein-binding sites on the membrane are blocked with an appropriate agent, such as non-fat milk, bovine serum albumin (BSA), or heat-inactivated normal bovine serum (NBS).
  • BSA bovine serum albumin
  • NBS normal bovine serum
  • the test sample can be applied neat, or more often, it can be diluted, usually in a buffered solution which contains a small amount of protein, such as milk, BSA, or NBS.
  • the membrane After incubating for a sufficient length of time to allow specific binding to occur, the membrane is washed to remove unbound sample and then incubated with a combination of conjugated anti-chicken immunoglobulins (total antibody)(ie., IgA + IgG + IgM) or a single labeled anti- chicken immunoglobulin (isotype antibody)(ie., IgA). Sufficient time is allowed for specific binding to occur again, the membrane is washed to remove unbound conjugate, and the substrate for the enzyme is added. Color is allowed to develop and the reaction stopped by rinsing in appropriate solution.
  • total antibody ie., IgA + IgG + IgM
  • isotype antibody ie., IgA
  • a "two antibody sandwich” assay can be used to detect the proteins of the present invention.
  • the solid support is reacted first with one or more of the antibodies of the present invention, washed and then exposed to the test sample.
  • Antibodies are again added and the reaction visualized using either a direct color reaction or using a labeled second antibody, such as an anti-immunoglobulin labeled with horseradish peroxidase, alkaline phosphatase or urease.
  • Assays can also be conducted in solution, such that the eimerian proteins and antibodies thereto form complexes under precipitating conditions.
  • the precipitated complexes can then be separated from the test sample, for example, by centrifugation.
  • the antigens can be further purified using any of a variety of conventional methods including liquid chromatography, both normal or reverse phase, HPLC, FPLC and the like; affinity chromatography; size exclusion chromatography; immobilized metal chelate chromatography; gel electorphoresis; etc.
  • the amino acid sequences of the purified antigens can be determined using techniques well known in the art such as repetitive cycles of Edman degradation, followed by amino acid analysis.
  • the purified antigens can be immunologically characterized using standard techniques such as MHC-restricted response profiles in genetically inbred animals. These measurements include, without limitation, Western blot, molecular weight determinations using standard techniques such as SDS-PAGE/staining, T-cell recognition assays, and assays to infer immune protection or immune pathology by adoptive transfer of cells, proteins or antibodies.
  • Genes encoding the subject antigens can be identified by constructing gene libraries, using the resulting clones to transform a suitable host cell and pooling and screening individual colonies using the antibodies of the present invention, polyclonal serum or monoclonal antibodies to the desired antigen.
  • oligonucleotide probes which contain codons for a portion of the determined amino acid sequences can be prepared and used to screen DNA libraries for genes encoding the subject proteins. See, e.g., DNA Cloning: Vol. I, supra; Nucleic Acid Hybridization, supra; Oligonucleotide Synthesis, supra; T. Maniatis et al., supra. Synthetic DNA sequences, encoding the proteins of interest, can also be prepared, based on the determined sequence, using known techniques. See, e.g., Edge (1981) Nature 222:756; Nambair et al. (1984) Science 222:1299; Jay et al. (1984) /. Biol. Chem. 252:6311.
  • the coding sequences can be cloned into any suitable vector or replicon. Numerous cloning vectors are known to those of skill in the art, and the selection of an appropriate cloning vector is a matter of choice. See, generally, DNA Cloning: Vols. I & II, supra; T. Maniatis et al, supra; B. Perbal, supra.
  • the gene can be placed under the control of a promoter, ribosome binding site (for bacterial expression) and, optionally, an operator (collectively referred to herein as "control" elements), so that the DNA sequence encoding the desired protein is transcribed into RNA in the host cell transformed by a vector containing this construct.
  • the coding sequence may or may not contain a signal peptide or leader sequence. If so, the proteins can be expressed with or without the native sequences. Alternatively, heterologous signal sequences can be used. Leader sequences can be removed by the bacterial host in post-translational processing. See, e.g., U.S. Patent Nos. 4,431,739; 4,425,437; 4,338,397.
  • regulatory sequences may also be desirable, which allow for regulation of the expression of the protein sequences relative to the growth of the host cell. Regulatory sequences are known to those of skill in the art, and exarnples include those which cause the expression of a gene to be turned on or off in response to a chemical or physical stimulus, including the presence of a regulatory compound. Other types of regulatory elements may also be present in the vector, for example, enhancer sequences.
  • control sequences and other regulatory sequences may be ligated to the coding sequence prior to insertion into a vector, such as the cloning vectors described above.
  • a vector such as the cloning vectors described above.
  • the coding sequence can be cloned directly into an expression vector which already contains the control sequences and an appropriate restriction site.
  • Mutants or analogs may be prepared by the deletion of a portion of the sequence encoding the antigen, by insertion of a sequence, and/or by substitution of one or more nucleotides within the sequence. Techniques for modifying nucleotide sequences, such as site-directed mutagenesis, are described in, e.g., Sambrook et al., supra; DNA Cloning, Vols. I and II, supra; Nucleic Acid Hybridization, supra.
  • the expression vector is then used to transform an appropriate host cell.
  • the transformed host cells are cultured under conditions providing for expression of the antigen of interest.
  • the antigen is then isolated from the host cells and purified. If the expression system secretes the protein into growth media, the protein can be purified directly from the media. If the protein is not secreted, it is isolated from cell lysates.
  • the selection of the appropriate growth conditions and recovery methods are within the skill of the art.
  • the antigens of the present invention may also be produced by chemical synthesis, such as solid phase peptide synthesis, using known amino acid sequences or amino acid sequences derived from the DNA sequence of the genes of interest. Such methods are known to those skilled in the art. Chemical synthesis of peptides may be preferable if a small fragment of the antigen in question is capable of raising an immunological response in the subject of interest.
  • the isolated, recombinantly or synthetically produced Eimeria antigens can be used in immunoassays, such as the competition, direct reaction or sandwich-type assays described above, to detect the presence of Eimeria antibodies in biological samples. In this way, not only can the diagnosis of coccidiosis be made, but the host level of immunity can be determined.
  • immunoassays such as the competition, direct reaction or sandwich-type assays described above.
  • naturally occurring nn ⁇ -Eimeria spp. antibodies are produced by the infected chicken in its fecal material. The presence of these antibodies can be determined by reacting a sample of fecal material with one or more of the Eimeria antigens of the present invention. Antibodies present in the fecal sample will form an antibody- antigen complex with the antigen.
  • the reaction mixture can be analyzed to determine the presence or absence of these antibody-antigen complexes using any of a number of standard methods, such as those immunodiagnostic methods described above.
  • the isolated antigens can be conjugated to a solid support, such as any of the above-described supports, a fecal sample is then incubated with the conjugate, and the reaction mixture analyzed to determine the presence of the antibodies.
  • the filter cup and dipstick include the filter cup and dipstick.
  • the antigen of this invention is fixed to a sinter glass filter to the opening of a small cap.
  • the fecal sample is resuspended in diluent and then passed through the filter. If the antibody is present, it will bind to the filter which is then visualized through a second antibody detector.
  • the dipstick assays involves fixing an antigen to a filter, which is then dipped in the resuspended fecal sample, dried and screened with a detector molecule.
  • the Eimeria proteins of the present invention or their fragments can also be used to produce antibodies, both polyclonal and monoclonal.
  • polyclonal antibodies are desired, a selected mammal, (e.g., mouse, rabbit, goat, horse, etc.) is immunized with an antigen of the present invention, or its fragment, or a mutated antigen. Serum from the immunized animal is collected and treated according to known procedures. If serum containing polyclonal antibodies is used, the polyclonal antibodies can be purified by immunoaffinity chromatography, using known procedures. Monoclonal antibodies to the Eimeria antigens of the present invention, and to fragments thereof, can also be readily produced by one skilled in the art. The production of several monoclonal antibodies raised against E.
  • te «e// ⁇ -infected SB- C ⁇ V/F7 tissue culture supernatants is described in the examples.
  • MAb 1-2-6 which reacts with the 40 kDa intracellular protein which has been identified as being protective herein.
  • the Eimeria monoclonal antibodies are produced by using hybridoma technology.
  • immortal antibody-producing cell lines can be created by cell fusion, as well as by other techniques such as direct transformation of B lymphocytes with oncogenic DNA, or transfection with Epstein-Barr virus. See, e.g., M.
  • Monoclonal antibodies are useful in purification, using immunoaffinity techniques, of the individual antigens against which they are directed.
  • the antibodies are also useful in diagnosis of coccidiosis infection, and can be used e.g., in immunoassays such as those described above, as well as in therapeutic compositions for the passive immunization of avian subjects.
  • kits with suitable instructions and other necessary reagents, in order to conduct immunoassays as described above.
  • the antibodies, eimerian antigens, or both can be provided in a diagnostic immunoassay test kit to provide for the detection of coccidiosis infection or to test the state of immunity to coccidiosis of an avian subject.
  • the kit can also contain, depending on the particular immunoassay used, suitable labels and other packaged reagents and materials (i.e. wash buffers and the like). Standard immunoassays, such as those described above, can be conducted using these kits.
  • the antigens can also be formulated into subunit vaccine compositions to provide immunity to coccidiosis.
  • the antigens and antibodies of the present invention can be used either alone or in combination with other antigens and antibodies, from the same or different species of Eimeria.
  • intracellular Eimeria antigens may be combined with extracellular Eimeria proteins, such as those present in extracellular sporozoites and merozoites.
  • the antigens may be provided in the form of a fusion protein or a larger, multimeric protein.
  • fusion proteins or multimeric proteins may be produced recombinantly, as described in, e.g., U.S. Patent No. 4,366,246, or may be synthesized chemically.
  • antigens of this invention may be employed in combination with antigens from other avian pathogens, to provide broad spectrum protection against a variety of avian diseases.
  • crude mixtures of the antigens such as partially purified mixtures of intracellular antigens derived from ammonium sulfate precipitation of culture media which supports the growth of intracellular forms of Eimeria spp., can be used in vaccine compositions without further purification. See, e.g., the examples, where such crude extracts are shown to be protective against E. tenella challenge.
  • the vaccine compositions are generally formulated with a pharmaceutically acceptable vehicle or excipient.
  • Suitable vehicles are, for example, water, saline, dextrose, glycerol, ethanol, or the like, and combinations thereof.
  • the vehicle may contain minor amounts of auxiliary substances such as wetting or emulsifying agents and pH buffering agents.
  • Preservatives known in the art such as thimerosal, phenol and other phenolic compounds, as well as antibiotics, can also be added to the vaccine compositions of the present invention.
  • Suitable vaccine vehicles and additives are known, or will be apparent, to those skilled in the art. See, e.g., Remington's Pharmaceutical Sciences, Mack Publishing Company, ⁇ aston, Pennsylvania, 18th edition, 1990.
  • Adjuvants which enhance the effectiveness of the vaccine may also be added to the formulation.
  • Adjuvants may include for example, muramyl dipeptides, avridine, aluminum hydroxide, oils, oil in water emulsions, saponins, cytokines, and other substances known in the art.
  • the protein may be linked to a carrier in order to increase the immunogenicity thereof.
  • Suitable carriers include large, slowly metabolized macro- molecules such as proteins, including serum albumins, keyhole limpet hemocyanin, immunoglobulin molecules, thyroglobulin, ovalbumin, and other proteins well known to those skilled in the art; polysaccharides, such as sepharose, agarose, cellulose, cellulose beads and the like; polymeric amino acids such as polyglutamic acid, polylysine, and the like; amino acid copolymers; and inactive virus particles.
  • the Eimeria antigens may be used in their native forms or functional groups modified for attachment to these carriers.
  • avian species have a mucosal immune network consisting of gut-associated lymphoid tissue termed GALT or Peyer's patches), bronchial-associated lymphoid tissue (BALT), and the Harder gland, located ventrally and posteriomedially to the eyeball. Presentation of an antigen to these tissues triggers proliferation and dissemination of committed B- cells to the secretory tissues and glands in the body, with the ultimate production of secretory IgA (slgA). SlgA serves to block the colonization and invasion of specific surface antigens that colonize on, and pass through, a mucosal surface. It appears that the intestinal slgA system plays an essential role in the protective immune response to Eimeria. Davis, PJ. et al. Immunology (1978) 24:879-888. Accordingly, the
  • Eimeria antigens of the present invention may also be administered using avirulent carrier microbes, able to invade and proliferate in the cells of the GALT and BALT.
  • avirulent carrier microbes able to invade and proliferate in the cells of the GALT and BALT.
  • Such delivery allows for a generalized secretory immune response as well as humoral and cellular immune responses.
  • recombinant plasmids containing genes for the Eimeria antigens can be introduced into one of several avirulent strains of bacteria, designed for delivering antigens to avian subjects.
  • Such avirulent organisms generally contain mutations in genes necessary for long- term survival and include mutant derivatives oi Salmonella, E. coli and E. coli- Salmonella hybrids. Such mutants are described in e.g., Curtiss, R. Ill, et al. Infect. Immun. (1987) 55:3035-3043 and U.S. Patent Nos. 4,968,619; 4,
  • the proteins may be formulated into vaccine compositions in either neutral or salt forms.
  • Pharmaceutically acceptable salts include the acid addi ⁇ tion salts (formed with the free amino groups of the active polypeptides) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed from free carboxyl groups may also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine, and the like.
  • Vaccine formulations are prepared by combining an effective amount of one or more antigens described above, the exact amount being readily determined by one skilled in the art.
  • an "effective amount" of an antigenic vaccine component will be that amount required to generate an amount of circulating antibody sufficient to prevent or reduce coccidiosis disease symptoms.
  • An effective amount of an Eimeria antigen will vary, depending on the mode of administration, the particular species oi Eimeria targeted, the degree of protection desired and the age and health of the subject to be treated.
  • compositions to be delivered parenterally generally between about 10 ⁇ g to about 1 mg, more preferably about 25 ⁇ g to about 200 ⁇ g, and most preferably about 50 ⁇ g to about 100 ⁇ g, in about 0.5 to about 10 ml, preferably about 1 ml to 3 ml, will constitute an effective amount of antigen.
  • the vaccine compositions of the present invention can be administered parenterally, e.g., by intramuscular, subcutaneous, intravenous or intraperitoneal injection. It may also be desirable to introduce the vaccine composition directly into the gut or bronchus, to stimulate a preferred response of the GALT or BALT, such as by oral administration, intranasal administration, gastric intubation or aerosol administration, as well as air sac and intratracheal inoculation.
  • the vaccines can be conveniently placed directly into water given to the avian subjects.
  • Other suitable methods of administering the vaccines of the invention are, e.g., via the conjunctiva to reach the Harder gland or in ovo administration, by inoculating avian eggs before they hatch.
  • the initial inoculation might involve parenteral administration while subsequent boosters might be given orally.
  • the avian subject is immunized by administration of the vaccine formulation, in at least one dose, and preferably two or more doses. However, the animal may be administered as many doses as is required to maintain a state of immunity against coccidiosis.
  • boosters can be given at regular intervals, i.e., at six months or yearly, in order to sustain immunity at an effective level.
  • the antigens of Eimeria spp. are sometimes identified in the examples with reference to their molecular mass in kilodaltons (kDa). Thus, an antigen having a molecular mass of about 35 kDa is identified below as P35; an antigen of about 40 kDa in molecular mass is identified as P40, and so on.
  • P35 molecular mass of about 35 kDa
  • P40 antigen of about 40 kDa in molecular mass
  • the following acronyms are used in the examples and are defined as follows: UI/UC, unimmunized, unchallenged (naive) UI/C, unimmunized, challenged
  • NE/C naturally exposed, challenged SLIP, splenic lymphocyte immune product CLIP, cecal lymphocyte immune product RAT, rectal antibody test CD AT, cage dropping antibody test
  • MAb monoclonal antibody spz, extracellular sporozoite mrz, extracellular merozoite
  • NE Inbred Natural Exposure
  • Chicks were fed a nonmedicated starter/grower diet and water ad libitum.
  • Groups (NE/C) of one-day chicks were immunized with 500 E. tenella oocysts from a strain designated L.S. 65 (provided by D. Strout, Univ. of New Hampshire) per os for five consecutive days. Unless specifically noted, all trickle immunizations and challenge infections were performed with E.
  • tenella L.S. 65 Control groups (UI/C) were similarly immunized with distilled water. At either 6, 8, 11 or 15 days following the last day of parasite exposure, birds were weighed and challenged with 3.5 x IO 4 E. tenella oocysts. Mean body weight gain and cecal lesions were determined at 6 days post- challenge. The duration of immunity to homologous challenge in trickle- immunized chicks was found to be at least 2 weeks. NE/C birds from all three haplotypes showed significantly higher weight gains and lower lesions than their
  • Example 2 Preparation of SLIP and CLIP Samples from Inbred NE Model For preparation of SLIP samples, spleens were removed (typically 4- 6/group) at the desired timepoint and mononuclear cells isolated using standard Histopaque 1077 centrifugation (Sigma, St. Louis, MO.).
  • Viable cells were counted using trypan blue and a hemacytometer and cultivated at a density of 5 x IO 6 cells/ml in serum-free modified LM-Hahn medium (LMH) (Calnek, et. al. Infect. Immun. (1981) 24:483-491.) for 5 days (40.5°C, 5% CO 2 ). Supernatant was harvested, spun (800 x g, 10 min., 4°C), and cell-free supernatant collected. Sodium azide was added to 0.1% (v/v) final concentration and samples filtered (0.2 ⁇ M). SLIP samples were stored at 4°C for up to 2 weeks or aliquots prepared and frozen at -20°C until use.
  • LMH serum-free modified LM-Hahn medium
  • both cecal pouches per bird were removed (typically 4-6 birds/group) at the desired timepoint and intestinal lymphocytes isolated according to standard procedures. Briefly, cecal pouches were cut at the ileocecal junction, the distal end excised, and cecal contents expressed out. Tissue was placed into a tube containing cHBSS (Ca 2+ and Mg 2+ -free HBSS containing 2X antibiotic/antimycotic, 25 mM HEPES, pH 7.4) and placed on ice until further manipulations. Tubes were shaken vigorously for 10 sec. to remove additional gut contents, and S/N discarded following gravity sedimentation of tissue. Cecal tissue was placed into a petri dish and each cecum opened longitudinally.
  • cHBSS Ca 2+ and Mg 2+ -free HBSS containing 2X antibiotic/antimycotic, 25 mM HEPES, pH 7.4
  • the mucosal surface was gently scraped to remove any residual clumps of fecal material and tissue minced into small 1-2 cm fragments.
  • Tissue fragments were placed in a 50 ml tube containing 10-20 ml cHBSS, and samples hand shaken vigorously for 10-20 sec, followed by a low speed spin (70 x g, 1 min. R.T.). S/N was discarded and 10- 20 fresh cHBSS added to tissue. The shake/spin procedure was repeated two additional times, followed by the addition of 10-20 ml cHBSS/DTT (lOmM DTT, IX genticin and polymyxin B sulfate) per sample.
  • 10-20 ml cHBSS/DTT lOmM DTT, IX genticin and polymyxin B sulfate
  • cHBSS/ES containing 1 mg/ml collagenase (Clostridiopeptidase A, Type VII, Sigma) was added and minced tissue solution transferred to a glass flask containing a stir bar.
  • fecal material typically 4-6 birds/group was collected from the ileocecaljunction down to the cloaca and placed in a preweighed 50 ml conical tube.
  • CD AT fresh cage droppings were collected from litter pans and placed in a preweighed 50 ml conical tube. The net wet weight of samples were determined, and 3 mis of Dulbecco's phosphate buffered saline (DPBS) were added per gram feces. Samples were vortexed at moderate speed for 15-20 sec. to resuspend fecal material. Samples were spun (2750 x g,4o s C, 15 min.) and supernatant above fecal pellet transferred to 30 ml Sorvall GSA-600 rotor tubes. Samples were spun
  • Example 4 In Vitro Parasite Neutralization Assay
  • control and trickle-immunized B 24 B 24 and B 30 B 30 birds were prepared as described in Example 1.
  • Thirteen days following the last parasite exposure birds were challenged with 3.5 x IO 4 oocysts and CLIP samples prepared at days 1, 3 and 5 post-challenge as described in Example 2.
  • One ml samples were incubated with an equal volume of freshly excysted L.S. 65 E. tenella sporozoites (5 x lOVml) on a rocker platform (1 hr, 40.5°C).
  • the mixture was then directly added in quadruplicate to microtiter wells containing SB-CEV/F7 cells (ATCC Accession No. CRL10495) plated (in medium 199/5% FBS) at 1 x 10 5 cells/well 24 hrs previously. After 2 hrs (40.5°C, 5% CO 2 ) extracellular sporozoites were removed by washing, and one ⁇ Ci per well of tritiated uracil (Amersham, 5 mCi/mmol) added.
  • SB-CEV/F7 cells ATCC Accession No. CRL10495
  • Example 5 In Vitro Parasite Inhibition Assay
  • the same CLIP samples obtained from Ul/C and NE/C B 24 B 24 and B 30 B 30 birds in Example 4 were used for the assay.
  • the peak inhibitory activity of these NE/C T-cell derived cytokines parallels the peak inhibitory activity of the NE/C B-cell derived antibodies obtained in Example 4.
  • antigen-specific T H 2cells located in the cecal lining elaborate specific cytokines, most likely IL5 and IL6, which enhance antibody production and extracellular sporozoite neutralization.
  • Example 6 Ouantitation of total and sporozoite-specific IgA in RAT
  • a murine anti-chicken IgA MAb (MAb 6.2.3-1 purchased as ascites from Dr. S. Naqi, Cornell University, Ithaca, NY) or MAb Jl 26.189.96 (Janssen Biochemica) was diluted 1 :500 in 50 mM sodium borate, pH 9.5.
  • Test RAT samples were initially diluted 1:100 in PBST/5% milk and serial two-fold dilutions added in duplicate to wells (100 ⁇ l/well). Serial two-fold dilutions of reference serum containing IgA (Bethyl Labs, RS10-102-l)(initial concentration 4.0 ⁇ g/ml) were similarly prepared for each plate. Plates were incubated at 4°C overnight. Primary RAT antibody incubation was performed at 4°C to decrease endogenous protease activity in the samples.
  • Plates were washed 3X with PBST and then 100 ⁇ l/well of a 1:500 dilution (PBST/5% milk) of horseradish peroxidase conjugated goat anti-chicken IgA (Bethyl Labs, A30-103P- 3) added for lhr (40°C). Plates were washed 3X with PBST and then developed by the addition of 100 ⁇ l/well TMB peroxidase substrate/peroxidase solution
  • Example 7 Immunoblots The following procedure was used for several of the examples to follow. Immunoblots were modified from previously published procedures (J.T. Roehrig et al. Virology (1985) 142:347-356 and H. Towbin et al. Proc. Natl. Acad. Sci. USA (1979) 76:4350-4354). E. tenella (LS65) oocysts were produced and maintained by passage in chickens. Pure oocysts and sporozoites were obtained essentially as previously described by Schultz, D.M. et al. J. Protozol. (1984) 3-1: 181-183.
  • Sporozoite and merozoite antigens were obtained by resuspending sporozoites and in vitro merozoites in PBS containing 0.5 mM phenylmethyl sulfonyl fluoride (Calbiochem-Behring, La Jolla, CA). The solution was freeze-thawed three times on dry ice and sonicated (Heat Systems Ultrasonics, model W-380) on ice for one min using a one second pulse, 80% duty cycle. After five cycles, each one min long, samples were transferred to microcentrifuge tubes and spun at 10,000 x g, 10 min at 4°C. Soluble material above the pellet was collected and protein concentrations determined using standard procedures. Sonicated parasite preparations were adjusted to 1 mg/ml in serum-free media 199, aliquoted and stored at -20°C for further use.
  • Proteins were immunoblotted (overnight, 40 mA, 4°C) onto Immobilon-P membranes (0.45 ⁇ M, Millipore Corp., Bedford, MA.) If necessary, membranes were cut into desired size strips, prior to subsequent manipulations. Membranes or membrane strips were washed three times in TTBS (wash buffer, Tris-buffered saline/0.01% Tween 20). Membranes were rinsed in wash buffer between all subsequent incubation steps.
  • TTBS wash buffer, Tris-buffered saline/0.01% Tween 20
  • Blots to be used for SLIP, CLIP, and sera antibody incubations were blocked in TTBS/2% skim milk/1% gelatin for a minimum of 2 hrs (R.T.); blots to be used for RAT incubations were blocked in TTBS/3% BSA for a minimum of 6 hrs. In some instances, blots were blocked overnight (R.T.).
  • samples were diluted 1/2, 1/2, and 1/500 respectively, in TTBS/1% gelatin/0J% NaN 3 ; for primary RAT incubations, samples were diluted 1/5 in TTBS/3% BSA/10% FBS. All primary antibody incubations were carried out overnight (R.T.).
  • Example 8 Determination of Total and Sporozoite-specific IgA Levels in RAT Samples Prepared from Inbred NE Model and Correlation to Protection against Disease RAT samples were prepared from NE/UC and NE/C B 19 B 19 birds following different periods of rest. Day-old chicks were trickle immunized with 500 E. tenella oocysts for 5 consecutive days. Then 10, 17 and 24 days after the last parasite exposure, 10 birds/group were weighed and either mock challenged or challenged with 3.5 x 10" homologous oocysts. Groups of age-matched naive birds were also weighed and challenged (UI/C). At day 2 post-challenge, RAT samples from 5 birds per group were prepared.
  • the spz-specific IgA results are similar in that IgA concentrations increased following parasite challenge after 10 and 17, but not 24, days rest.
  • Significant protection against weight loss in NE/C groups was observed after 10 and 17 days rest, but not after 24 days rest.
  • concentrations of both total and spz-specific IgA in RAT in birds can be determined.
  • the results can be used to determine the minimum total and spz-specific IgA titers required for protection against homologous parasite challenge. Results can also be used to better evaluate flock immunity.
  • RAT samples were prepared (2-3 birds/group) from all groups and assayed for total and sporozoite-specific IgA concentrations.
  • the data indicate that it is possible to measure total and spz-specific IgA levels in outbred broilers using a RAT ELISA, and that the levels of RAT may correlate to the immune status.
  • the highest levels of spz-specific IgA were detected in immune birds at day 6 post-challenge (groups 8 and 9) and these values were considerably higher than values obtained from naive birds at day 6 post- challenge (groups 2 and 3). results also indicated that birds immune to one strain of E.
  • results obtained can be used to determine the minimum total and spz-specific IgA titers required for protection against homologous or heterologous species challenge. Results can also be used to better evaluate flock immunity.
  • Age-matched control birds (U C) were immunized and boosted with adjuvanted tissue culture media obtained from uninfected F7 cells. At 10 days of age, all birds were weighed and challenged with 3.5 x IO 4 sporulated E. tenella oocysts per os. At 16 days of age, final bird weights were measured. In trial 1, birds were vaccinated with approximately 50 ⁇ g total protein per dose and in trials 2 and 3, birds were vaccinated with approximately 100 ⁇ g total protein per dose. A group of unimmunized, unchallenged birds (UI/UC) was also used in all 3 trials. Statistical comparisons of weight gains were performed using least square analysis and values compared to UI/C controls.
  • SLIP samples were obtained from UI/C, N ⁇ /UC, and N ⁇ /C B 19 B 19 , B ⁇ B 24 , and B 30 B 30 groups. Birds were trickle-immunized with 500 E. tenella oocysts/bird for 5 days, rested for 13 days, and then challenged orally with 3.5 x IO 4 homologous oocysts. Day 1 post-challenge, SLIP samples were prepared as described above and assayed for Western reactivity against E. tenella sonicated sporozoites and 30%(NH 4 ) 2 SO 4 44-72 hr supernatants from E. tenella SB-C ⁇ V/F7 infected cells.
  • results indicate that the spleens of all 3 UI/C haplotypes contain B cell populations capable of producing IgG reactive with the spz 40 kDa antigen (termed "P40" herein).
  • P40 spz 40 kDa antigen
  • This antigen induces an immunodominant response, since the spleens of all 3 NE/UC haplotypes contain B cell reactivity to P40 15 days after the last 500 oocyst parasite exposure.
  • this same splenic B cell population is not present at day 1 post-challenge in the B 19 B 19 and B 2 B 24 haplotypes, suggesting that this population has emigrated from the spleen.
  • CLIP samples were obtained from UI/C, NE/UC, and NE/C B 19 B 19 , B 24 B 24 , and B 30 B 30 groups day 1 and 3 post-challenge, as described in Example 10. These samples were prepared and assayed for Western reactivity against E. tenella sonicated sporozoites and 30%(NH 4 ) 2 SO 4 44-72 hr supernatants from E. tenella SB- C ⁇ V/F7 infected cells. The IgG Western reactivity profiles of the day 1 and 3 post- challenge CLIP samples are summarized in Tables 3 and 4, respectively.
  • Results show the CLIP reactivity profile within a single NE/C haplotype and group is similar but clearly different, between day 1 and day 3 post-challenge.
  • some non-MHC restricted NE/C intestinal B-cells are present at both days 1 and 3 post-challenge (e.g., P70 and P40)
  • other non-MHC restricted B-cells are not present until day 3 post-challenge (e.g., P43).
  • the differential presence of these antigen-specific B-cells is a direct result of parasite challenge, since almost identical day 1 and day 3 CLIP reactivity profiles were observed in the 3 NE/UC haplotypes.
  • RAT samples were prepared from UI/C and NE/C B 19 B 19 , B 24 B 24 , and B 30 B 30 groups. Birds were trickle-immunized with 500 E. tenella oocysts/bird for 5 days, rested for 16 days, and then challenged orally with either a predetermined low or high oocyst dose. At day 2 post-challenge, RAT samples were prepared (3 birds/group) and assayed for Western reactivity against E. tenella sonicated sporozoites and 30%(NH 4 ) 2 SO 4 44-72hr supernatants from E. tenella SB-CEV/F7 infected cells.
  • Example 14 Identification of E. tenella Antigens Using Sera Prepared from NE Model Sera samples were obtained from UI/C and NE/C B 19 B 19 groups. Birds were trickle-immunized with 500 E. tenella oocysts/bird for 5 days, rested for 14 days, and then challenged orally with 3.5 x IO 4 homologous oocysts. At day 1, 3 and 5 post-challenge, serum samples were collected (5/group) by cardiac puncture, pooled and assayed for Western reactivity against E.
  • Example 15 Purification of slgA from NE/C CLIP
  • CLIP reagent was prepared as described in Example 2.
  • Approximately 40 mL CLIP was treated with ammonium sulfate to a concentration of 35%. After stirring one hour at 4°C, the solution was centrifuged (16,800 x g), precipitate collected, dissolved in PBS and dialyzed against PBS.
  • Unbound and bound pooled fractions were subjected to SDS- PAGE (reducing) and analyzed by silver staining and Western blot. Silver stain revealed a predominant P70 species with a few minor contaminating bands. The molecular weight of the reduced alpha heavy chain is 70 kDa.
  • a portion of the positive staining fraction was applied to a Superose 6 column (1.6 x 53 cm) using a 0.5 mL/min. flow rate. Analysis of the collected fractions by SDS-PAGE silver stain identified a major P70 species. Based on the Superose 6 molecular weight standard profile, an approximate molecular weight of 170,000 daltons was assigned, indicating the presence of monomeric IgA.
  • Example 16 Purification of slgA from NE/C RAT RAT reagent was collected and prepared from the same group of birds used in Example 15 and purified in a similar manner. The eluate from the Jacalin column tested positive in the spz-specific IgA ELISA and was applied to a Superose 6 column, individual fractions collected and analyzed by SDS-PAGE silver stain. Results showed a P70 species present in both the early and late fractions.
  • a standard N ⁇ /C RAT sample lot was prepared from B 19 B 19 birds. Chicks were trickle-immunized with 500 E. tenella oocysts/bird for 5 days, rested for 15 days, challenged with 5 x IO 4 oocysts and then rechallenged 14 days later. Seven days later, RAT reagent was prepared. Polyclonal rabbit anti-E. tenella sera, designated Rb 15/16, was obtained by immunizing rabbits with freshly excysted and adjuvanted E. tenella sporozoites three times. Rb 15/16 and RAT samples were assayed for IgG and IgA reactivity, respectively, against E. tenella spz, mrz, and E.
  • tenella IgA antibodies present in RAT identified several, unique extracellular and SB-C ⁇ V/F7 intracellular E. tenella antigens not recognized by conventional rabbit antisera raised against E. tenella sporozoites. Antigens recognized by NE/C RAT represent novel vaccine candidate targets.
  • Example 19 Comparison of Anti-Eimeria Antibody Responses in Different Biological Compartments of NE/C B ⁇ B ⁇ Birds Sera, SLIP and CLIP samples were obtained from NE/C B 19 B 19 birds. Day- old chicks were trickle-immunized with 500 E.
  • a cell soluble lysis buffer (0.5% Brij-35, 300 mM NaCl, 50 mM Tris-Cl, pH 7.6 containing protease inhibitors (100 mM 1, 10 phenanthroline, 100 mM benzamidine HCL hydrate, 1 mg/ml pepstatin, 50 mM PMSF, 2 mg/ml leupeptin, 5 mg/ml soybean trypsin inhibitor, and 4 mg ml aprotinin)) for soluble membrane and cytosolic proteins, and a cell insoluble lysis buffer (0.2% sodium deoxycholate, 0.2% SDS) for insoluble material. The soluble and insoluble preparations were pooled for Western analysis.
  • protease inhibitors 100 mM 1, 10 phenanthroline, 100 mM benzamidine HCL hydrate, 1 mg/ml pepstatin, 50 mM PMSF, 2 mg/ml leupeptin, 5 mg/ml soybean trypsin inhibitor, and 4 mg
  • Antibodies produced at the local site of infection in an immune host typically recognize a more restricted set of antigens as compared to SLIP and sera. Moreover, the antigens recognized by immune CLIP and/or RAT are different than those recognized by SLIP or sera at a given timepoint, particularly early post-challenge. As stated previously, antigens in this invention are specified by MHC haplotype recognition, biological compartment, response time and immune status of the bird.
  • Chicks were trickle-immunized with 500 E. tenella L.S. 65 oocysts/bird for 5 days, rested for 15 days, boosted with 5 x IO 4 homologous oocysts, rested an additional 14 days, challenged with 5 x 10 4 oocysts, and samples prepared at day 7 post-challenge as outlined above.
  • immune RAT raised against E. tenella L.S. 65 was assayed for Western reactivity against antigens prepared from two heterologous E. tenella field strains isolated from two different geographic areas.
  • the first field strain designated GP5
  • the second field stain was isolated from an poultry farm in Arkansas in 1992 (Dr. Phil Davis, Univ. of Arkansas).
  • Oocysts from both field strains were purified and amplified in Peterson Arbor Acres broilers using standard techniques, and used to infect SB-CEV/F7 cells.
  • strain cross-reactive serum antibodies have been previously described, this is the first example in which antibodies produced at the local site of infection, i.e., immune RAT, have been shown to contain IgA antibodies which are strain cross-reactive. This strategy can be used to confirm the conservation of P43, P40 and P38 in other E. tenella and heterologous Eimeria spp. field isolates (see below).
  • Example 21 Identification of Cross-Reactive Heterologous Eimeria spp. Antigens Using RAT Prepared from E. tenella L.S. 65 NE/C Birds
  • RAT raised against E. tenella L.S. 65 was used to identify extracellular and SB-CEV/F7 intracellular antigens obtained from different Eimeria spp.: E. acervulina and E. maxima. Sporozoites obtained from pure oocyst cultures of each species were used to infect SB-C ⁇ V/F7 cells.
  • Example 22 Identification of E. tenella Antigens Using E. tenella UI/C. N ⁇ /UC and N ⁇ /C RAT Prepared from Four Different Outbred Commercial Broiler Lines and Correlation to Protection
  • RAT from each group of each line was used to screen E. tenella L.S. 65 sporozoites and 30%(NH 4 ) 2 SO 4 44-72 hr supernatants from SB-C ⁇ V/F7 infected cells.
  • the IgA Western reactivity profiles of the RAT samples are summarized in Table 11. The spz and 30% antigen reactivity profiles were dependent on both the bird line and the immune status. Results confirm several previously identified antigens, including
  • Example 23 Identification of E. tenella Antigens Using CD AT Prepared from Four Different E. tenella UI/C Outbred Broiler Lines and Correlation to Protection The same outbred commercial broiler lines used in the previous example were raised on wire and used as a source of CD AT. Birds were weighed at 14 days of age, and challenged with 5 x IO 4 oocysts (UI/C). Age-matched naive birds from each line were weighed and mock-infected (UVUC). At 36-40 hrs post-challenge, fresh cage droppings from all the groups were collected, CD AT prepared and screened by Western blot analysis using anti-IgA against E. tenella spz antigen.
  • UI/C oocysts
  • UI/C bird weights at day 6 post-challenge were determined and compared to their UI/UC counterparts. Weight performance was expressed as the percentage weight loss of the UI/C group compared to the UI/UC controls. Only antigens identified in UI/C and not their UVUC counterparts are shown in Table 12.
  • CD AT from line 1 identified Pl 10 and CD AT from line 3 recognized P55 and P35. These results indicate that in 14-day old birds acutely infected with E. tenella, RAT IgA spz- specific responses appear earliest in line 3. This result is consistent with the RAT results obtained in Example 21.
  • Example 24 Identification of E. tenella Antigens Using RAT Prepared from Commercial Poultry Field Operation Farms Infected with Eimeria spp. RAT samples were collected from 15 different commercial poultry farms (4 birds/farm, pooled) during periodic, conventional coccidiosis diagnostic screening procedures. Samples were from either broiler or roaster production lines and birds ranged in age from 2-6 weeks.
  • RAT samples were subjected to preliminary Western blot against L.S. 65 E. tenella sporozoite antigen. Two of fifteen farms showed very strong reactivity. These same two farms were subsequently confirmed by an independent laboratory to have the highest incidences of E. tenella cecal lesions in the birds examined. Five of fifteen farms tested positive for reactivity against 3 E. tenella spz antigens, P92, P40 and P38. These five RAT were then screened against 30%(NH 4 ) 2 SO 4 44-72 hr supernatant antigen from E. tenella SB-C ⁇ V/F7 infected cells. A summary of the Western reactivity is shown in Table 13, and includes the type of bird, age, and most prevalent Eimeria spp.
  • results show that the local humoral immune response, recognized by RAT, identified 3 antigens common to all five farms. These five farms differed in the most common Eimeria spp. found. Therefore, these results show that the P43, P40, and P38 antigens are cross-reactive and most likely conserved among field isolates of E. tenella, E. maxima and E. acervulina. These 3 antigens are obvious targets for inclusion in a multivalent coccidiosis vaccine designed to be protective against the most economically important Eimeria species.
  • mice were immunized i.p., at two-week intervals, with 5 x IO 6 SB-CEV/F7 culture-derived E. tenella merozoites, adjuvanted 1:1 with complete (primary immunization) or incomplete (boost) Freund's adjuvant.
  • mice were immunized both IP and IV with IO 6 unadjuvanted merozoites.
  • Spleen cells were fused with mouse myeloma cell line SP2/0 and hybridoma supernatants initially screened by ELISA against E.
  • mice were immunized and boosted as described above, with 25-30 ⁇ g of partially purified protein obtained by the biochemical separation of E. tenella infected 44-72 hr SB-CEV/F7 tissue culture supernatant. Hybridoma colonies were screened against the immunogen, and a total of three cloned MAbs were further characterized as above. Although MAb 1-2-6 recognized at least four different molecular weight proteins, it did show reactivity to P40, one of the protective proteins identified herein.
  • Example 27 Partial N-terminal Amino Acid Sequence Determination of 45%fNH 1 ' ) 2 SO J 110/100 Doublet Reactive with Rb 15/16.
  • CLIP and RAT The 45%(NH 4 ) 2 SO 4 44-72 hr SB-CEV/F7 infected supernatant was applied to a Superose 6 column equilibrated with 4 M GdSCN in PBS.
  • Western blot results using MAb 2-3 identified a strongly reactive 92 kDa species. Fractions containing MAb 2-3 reactivity were combined, dialyzed against PBS and the dialysate applied to a MAb 2-3 immunoaffinity column.
  • the sequence shows no matches to the GenEMBL database.
  • UVUC unimmunized.unchallenged
  • UVC mock immunized/challenged

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Abstract

Sont décrits de nouveaux antigènes d'Eimeria protecteurs intracellulaires et spécifiques du stade. Ces antigènes sont identifiables à l'aide de préparations d'anticorps produites localement, au niveau du site de l'infection parasitaire. Les anticorps et les antigènes sont utiles comme réactifs diagnostiques pour détecter la coccidiose ainsi que pour déterminer l'état immunitaire d'un sujet aviaire donné. Ces anticorps et ces antigènes sont également utiles dans des compositions de vaccin assurant une protection contre l'infection par Eimeria.
EP94924563A 1993-08-02 1994-08-02 NOUVEAUX ANTICORPS ET ANTIGENES D'-i(EIMERIA) ET PROCEDES POUR LEUR UTILISATION Withdrawn EP0717629A4 (fr)

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US5807551A (en) * 1996-04-01 1998-09-15 Iowa State University Research Foundation, Inc. Method to provide artificial passive immunity in birds
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US4710377A (en) * 1983-08-19 1987-12-01 American Cyanamid Company Antigens and monoclonal antibodies reactive against sporozoites of Eimeria spp.
US4650676A (en) * 1983-08-19 1987-03-17 American Cyanamid Company Antigens and monoclonal antibodies reactive against merozoites of Eimeria spp.
US5187080A (en) * 1984-06-05 1993-02-16 Solvay & Cie S.A. DNA encoding an antigenic protein derived from Eimeria tenella and vaccines for prevention of coccidiosis caused by Eimeria tenella
US5279960A (en) * 1984-07-05 1994-01-18 Enzon Corp. 25 KD coccidial antigen of eimeria tenella
US5028694A (en) * 1985-12-03 1991-07-02 Solvay & Cie, S.A. Antigenic proteins and vaccines containing them for prevention of coccidiosis caused by eimeria Eimeria necatrix and Eimeria tenella

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
DANFORTH H D: "Use of monoclonal antibodies directed against Eimeria tenella sporozoites to determine stage specificity and in vitro effect on parasite penetration and development." AMERICAN JOURNAL OF VETERINARY RESEARCH, (1983 SEP) 44 (9) 1722-7. , XP002114612 *
DAVIS P J ET AL: "A mechanism for secretory IgA-mediated inhibition of the cell penetration and intracellular development of Eimeria tenella." IMMUNOLOGY, (1979 MAR) 36 (3) 471-7. , XP002114610 *
FAYER R ET AL: "Colostrum from cows immunized with Eimeria acervulina antigens reduces parasite development in vivo and in vitro." POULTRY SCIENCE, (1992 OCT) 71 (10) 1637-45. , XP000605864 *
See also references of WO9503813A1 *
TREES A J ET AL: "Eimeria tenella: local antibodies and interactions with the sporozoite surface." JOURNAL OF PROTOZOOLOGY, (1989 JUL-AUG) 36 (4) 326-33. , XP002114611 *
ZIGTERMAN G J ET AL: "Detection of mucosal immune responses in chickens after immunization or infection." VETERINARY IMMUNOLOGY AND IMMUNOPATHOLOGY, (1993 APR) 36 (3) 281-91. , XP002114613 *

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CA2167533A1 (fr) 1995-02-09
EP0717629A4 (fr) 1999-11-03
JPH09504604A (ja) 1997-05-06

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