MXPA98002800A - Vacu - Google Patents

Abstract

Compositions comprising Eimeria proteins, or variants / fragments of said proteins are described and can be used to produce a coccidiosis vaccine. The proteins are present in the hydrophilic phase of an extract of Trito X-114 from Eimeria sporozoites and have molecular masses of 26-30 kDañ5 kDa when determined through SDS-PAGE under reducing conditions.

Description

VACCINES DESCRIPTION OF THE INVENTION The present invention relates, inter alia, to coccidiosis vaccines. The parasitic protozoa belonging to the genus Eimeria are the causative agents of intestinal coccidiosis, an enteritis which affects birds. This causes a significant economic loss, especially in the poultry industry. (For the purposes of the present application, the term "poultry" means birds that serve as sources of eggs or meat The term includes, among others, chickens, turkeys, ducks, geese, guinea fowl, pheasants, pigeons and Peacock) . Currently, coccidiosis is mainly controlled through the use of antibiotics in food. The rapid emergence of drug-resistant strains (Chapman 1993) and the prohibitive development and registration costs of a novel drug have led to an increased interest in the development of an alternative method of control. The development of effective vaccines, therefore, has been desirable for many years. However, only partial success has been obtained. Vaccination strategies currently available consist of controlled infections with either virulent or live attenuated parasites (Shirley 1993). For safety and cost reasons, the most desirable method of immunoprophylaxis against coccidiosis appears to be the use of a subunit vaccine. Although many attempts have been made to immunize chickens against coccidiosis with fractions of parasite material (Murray et al., 1986, McKenzie &Long 1986) or recombinant Eimeria proteins (Danforth et al., 1989, Jenkins et al., 1991) only has been able to achieve limited protection against attack infection. Parasite stages responsible for the induction of protective immunity are generally thought to be stages of asexual development (Jenkins et al., 1991). Initially, the selection of candidate antigens was performed using antibodies from immune chickens but, in view of the fundamental role of cell-mediated responses in protective immunity (reviewed by Lillehoj &Trout 1993, Rose) r attention has now been placed on antigens. of classification for its ability to stimulate specific T-cell responses (Dunn et al., 1995). It has been previously reported that about 8 days after an E. tenella primary infection in chickens with the parasite, specific T cells circulate in the blood periphery (Breed et al., 1996). These cells appear to be initiated to proliferate and / or produce IFN-α. in response to activate total sporozoite antigens (Breed et al., 1997). In accordance with the present invention, an intact Eimeria parasite-free composition is provided, which comprises one or more proteins or fragments or variants thereof; wherein said proteins: (a) are present in the hydrophilic phase of an extract of Triton X-114 from Eimeria sporozoites, (b) have molecular masses of 26-30 kDa + 5 kDa (ie, 21-35 kDa) , when determined by SDS-PAGE under reducing conditions. The inventors of the present have found that said composition can be used to provide a vaccine, which provides a significant degree of protection to birds (preferably poultry) against disorders mediated by Eimeria. For example, protection against the formation of cecal lesions can be achieved in birds immunized with said vaccine, when they are subjected to a subsequent attack with Eimeria parasites. This finding could not be predicted from previous studies. Although several groups have described proteins derived from Eimeria, which, by chance, may have molecular masses within the scale described above, 26-30 kDa +. 5 kDa, these proteins are quite different from the proteins of the present invention. For example, a surface protein is described in EP-A-0231537 (Newman et al.). However, under reducing conditions, it is divided to form two bands, in SDS-PAGE, of about 17 and about 8 kDa, while the proteins of the present invention had relative molecular masses of at least 21 kDa when operated under reduction conditions. Bouvier et al (J. Biol. Chem. (1985) 260 (29); p. 15504-15509) teach that the use of Triton X1 14 extraction only detects amphiphilic proteins (associated membrane) in the detergent phase and not in the hydrophilic phase. US-A-4710377 (Schenkel et al) discloses antigens with molecular masses of approximately 28 and 26 kDa. However, these are amphiphilic outer membrane components and, therefore, can not be present in the hydrophilic phase of an extract of Triton X-1 14, which can be used to prepare proteins of the present invention. Eimeria proteins that are amphiphilic are also described in WO 92/04461 (Jacobson et al), EP-A-0324648 (Liberator et al.), AU-A-28542/49 (Turner et al.), EP- A-0344808 (Alternburger et al.) And EP-A-0167443 (Murray et al.). It is also important to keep in mind that there seems to be no direct correlation between the ability of proteins to stimulate T cell responses after a primary Eimeria infection and the ability of proteins to provide a protective response in chickens in vaccination studies. A composition of the present invention will normally be free of one or more Eimeria proteins, which occur in Eimeria by nature. Preferably, it will be substantially free of Eimeria proteins, other than the Eimeria proteins that are present in the hydrophilic phase of a Triton X-1 14 extract of Eimeria sporozoites. Therefore, proteins that are membrane-bound in Eimeria may be absent. A substantial part of the proteinaceous material present in the composition (for example, at least 50% w / w, at least 70% w / w at least 90% w / w of total protein present) can cst of one or more proteins of the present invention or their fragments or variants. For example, a plurality of proteins (or variants or fragments thereof) may be present in a composition. Two or three of these proteins (or variants or fragments thereof) may be present. Alternatively, only one of these proteins (or a variant or fragment thereof) can be provided. It can be provided substantially in pure form, ie, in a form which is essentially free of any other Eimeria protein. Purification can be achieved through techniques known to those skilled in the art. These techniques are described in general terms below. A suitable purification procedure for providing the proteins of the present invention at a sufficient level of purity for sequencing is as follows: The proteins of the composition can work on 12-18% (preferably 1 5%) of acrylamide separation gel, in a suitable pH regulating system in the presence of SDS. After staining the proteins (eg, 0.1% Coomassie Brilliant Blue in 45% methanol, 10% acetic acid), the bands containing the individual proteins can be cut and digested in situ using trypsin. After extraction of the tryptic peptides and subsequent purification by HPLC, the peptides can be sequenced using an automatic gas phase amino acid sequencer. Using this method, an internal amino acid sequence of as little as 20-50 pmoles of protein can be obtained, which can be provided with sporozoites using the described methods. Variants of the proteins of the present invention can be provided. The term "proteins" is used herein to mean any molecule comprising a plurality of amino acids linked together through peptide bonds. Therefore, they include peptides and polypeptides within their scope. They also include, within their scope, polypeptide chains, which can be separated from larger proteins under reducing conditidue to cleavage of disulfide bonds. The term "variants" is used to encompass molecules having one or more amino acid deleti insertiand / or substitutirelative to a protein of the present invention, but which retain one or more immunogenic determinants of an Eimeria antigen. The variants will have one or more epitopes, preferably complete antigens, that are capable of producing an immune resp in a host animal (if necessary when combined with a larger molecule). It will be understood that, for the proteins of the present invention, there may be natural variatiin the amino acid sequence between parasites or strains of individual Eimeria. Said natural variants are within the scope of the present invention, since they are variants that do not exist by nature. Amino acid substituti which do not substantially alter the biological and immunological activities, have been described, for example, by Neurath et al. "The Proteins" ("The Proteins"), Academic Press, New York (1979). Amino acid replacements among amino acids or related replacements, which have frequently existed in evolution are, among others, Ser / Ala, Ser / Gly, Asp / Gly, Asp / Asn, lie / Val (see Dayhof, M.C., Atlas of protein sequence an structure, Nati. Biomed. Res. Found, Washington D. C, 1978, volume 5, suppl. 3) . Other amino acid substitutions include Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn / Ala / Val, Thr / Phe, Ala / Pro, Lys / Arg, Leu / lie, Leu / Val and Ala / Glu. Based on this information, Lipman and Pearson have developed a method for rapid and sensitive protein comparison (Science, 227, 1435-1441) and determination of functional similarity between homologous proteins. Amino acid substitutions are within the scope of the invention. As indicated above, the variants are not limited to substitutions, but may also / alternatively include deletions and / or insertions, which do not remove the immunological activity. However, deletions or insertions that significantly affect the structural configuration of a protein will generally only be present in variants of the present invention if they do not result in the loss of immunologically important Eimeria epitopes or antigens. Preferred variants of the proteins of the present invention have a high degree of sequence identity with said proteins, for example, at least 50%, at least 70%, at least 90% or at least 95% identity of sequence. One way to determine the amino acid sequence identity is to align a given amino acid sequence with another amino acid sequence in a form which achieves the maximum number of amino acid sets over the length of the given amino acid sequence. The percent sequence identity will then be (m / t) x 100, where m is the number of sets between the two sequences aligned over the length of the given amino acid sequence, and t is the total number of amino acids present in the Amino acid sequence given. Any way to determine the sequence identity allows the introduction of gaps when two amino acid sequences coincide (0.1.2.3, or even more gaps may be allowed). This can be done, for example, using the "Gap" program, which is available from Genetics Computer Group as part of "The Wisconsin Package". This program is based on an algorithm provided by Smith and Waterman (Advances in Applied Mathematics, 482-489 (1981)). Any of these ways to determine sequence identity can be used with respect to the present invention, although it is preferred for one or more gaps. When high degrees of sequence identity are present, there may be relatively some differences in the amino acid sequence. Thus, for example, there may be less than 20, less than 10, or even less than 5 differences. In any case, it should be appreciated that a large number of variants are within the scope of the present invention. For example, fusion proteins can be provided, whereby one protein of the present invention binds to another portion (eg, in order to increase stability or aid in purification). Said fusion proteins are described in greater detail below. Other variants within the scope of the present invention are any Eimeria protein comprising disulfide bonds from which the proteins of the present invention can be derived through the cleavage of said disulfide bonds under reducing conditions. The protein fragments of the present invention are also included within the scope of the present invention. Said fragments preferably have a length of 10 amino acids, most preferably a length of at least 50 or at least 100 amino acids. The proteins of the present invention, or fragments or variants thereof, are useful in therapy. These may be provided as a pharmaceutical composition in combination with one or more pharmaceutically acceptable carriers. (For the purposes of the present invention, a "pharmaceutically "acceptable composition" is a composition suitable for use in veterinary medicine.The term "pharmaceutically acceptable carrier" and similar terms should be constructed in the same way.) The pharmaceutical composition can be provided in unit dosage form.It can be provided in a sealed container. or will normally be in sterile form .. Preferred pharmaceutical compositions of the present invention are vaccines (especially coccidiosis vaccines), said vaccines are discussed in more detail below The present invention is not limited to compositions comprising proteins, fragments or variants thereof of the present invention. Nucleic acid molecules are also provided. The nucleic acid molecules encoding proteins of the present invention can be provided since the amino acid sequences encoding proteins or fragments or variants thereof can be determined through standard methodology. A person skilled in the art can then use the genetic code to identify a large number of nucleic acid molecules, which can encode said amino acid sequences. Then, the "long probe" or "short probe" aspect can be used to isolate and clone specific nucleic acid molecules from Eimeria. The cDNA molecules can be derived from an appropriate mRNA source. Alternatively, the molecules can be synthesized de novo using chemical synthesis techniques. Once the nucleic molecules have been obtained, they can be amplified by normal techniques (for example using PCR). A nucleic acid molecule according to the present invention can be linked to one or more heterologous nucleic acid molecules with which it is not naturally associated, resulting in a so-called "recombinant vector". This can be used for the transformation of a suitable host. Useful recombinant vectors are preferably derived from plasmids, bacteriophages, cosmids or viruses. Specific vectors or cloning vehicles that can be used to clone nucleic acid sequences according to the present invention are known in the art and include, among others, plasmid vectors such as pBR322, the various pUC plasmids, pGEM and Bluescript; bacteriophages, for example, lambda gt-ES, Charon 28 and phage derived from M 13, or viral vectors such as SV40, adenovirus or polyoma virus (see also, Rodríguez, R. L. and DT Denhardt, ed., Vectors: A survey of molecular cloning vectors and their uses, Butterworths, 1988; Lenstra, J. A. et al., Arch, Virol., 1 10, 1-24, 1990). The methods that will be used for the construction of a recombinant vector according to the invention are known to those skilled in the art and are established, inter alia, in Maniatis, T. et al. (Molecular Cloning, A Laboratory Manual, second edition, Cold Spring Harbor Laboratory, 1989). For example, the insertion of a nucleic acid sequence according to the present invention into a cloning vector can be easily achieved when both the genes and the desired cloning vehicle have been cut with the same enzyme (s). ) of restriction, since the DNA terms complementary in this way are produced and can be annealed and then linked together. Alternatively, it may be necessary to modify the restriction sites that are produced to provide shaved ends by digesting the individual chain structure DNA or by filling the terms of the individual chain structure with an appropriate DNA polymerase. Subsequently, ligation of the shaved end with an enzyme such as T4 DNA ligase can be performed. If desired, any restriction site can be produced by ligating adapters on the DNA terms. Said adapters may comprise specific oligonucleotide sequences that encode the restriction site sequences. The restriction enzyme cleavage vector and the nucleic acid sequence can also be modified through homopolymer prolongation. "Transformation", as used herein, refers to the introduction of a nucleic acid sequence homologous to a host cell, regardless of the method used, for example, through direct consumption or through transduction. The heterologous nucleic acid sequence can be maintained through autonomous replication or, alternatively, can be integrated into the host genome. If desired, the recombinant vectors are provided with appropriate control sequences compatible with the desired host. These sequences can regulate the expression of the inserted nucleic acid sequence. In addition to microorganisms, cultures derived from multicellular organisms can also be used as hosts. The vectors of the present invention preferably contain one or more markers that can be used to select for desired transformants, such as ampicillin and tetracycline resistance in pBR322. A suitable host cell is a microorganism or cell that can be transformed through a nucleic acid sequence encoding a protein of the present invention or a variant or fragment thereof, or through a recombinant vector comprising said acid sequence nucleic. If desired, it can be used to express said protein (or a variant or fragment thereof). The host cell may be of prokaryotic origin, for example, a bacterium such as a species of Escherichia coli, Bacillus subtilis, or Pseudomonas; or of eukaryotic origin, such as yeasts, for example, Sacharomyces cerevisiae or higher eukaryotic cells, for example, insect, plant or mammalian cells, including HeLa cells and Chinese hamster ovary cells (CHO). Insect cells include the Sf9 cell line of Spodopter frugiperda (Luckow et al., Biotechnology 6, 47-55, 1988). Parasites can still be used (for example, Leishmania, Toxoplasma or Eimeria). Information regarding cloning and expression in eukaryotic cloning systems can be found in Esser, K. et al. , (Plasmids of Eucaryotes, Springer-Verlag, 1986). In general, prokaryotes are preferred for the construction of recombinant vectors useful in the present invention. Strains of E. coli K12 are particularly useful, especially strains DH5a or MC 1061. For expression, the nucleic acid molecules of the present invention can be introduced into an appropriate expression vector, wherein said molecules are operably linked to expression control regions. Said control regions may comprise promoters, enhancers, operators, inductors, ribosome binding sites, etc. Therefore, the present invention provides a recombinant vector comprising a nucleic acid sequence encoding an Eimeria protein of the present invention, or a fragment or variant thereof, operably linked to one or more expression control sequences. Of course, it should be understood that nucleotide sequences inserted at the selected site of the cloning vector may include nucleotides, which are not part of the actual structural gene for a desired expression product, or may include only a fragment of the complete structural gene for a product of <; desired expression, since the transformed host will produce an expression product having at least one or more determinants (epitopes) of an Eimeria protein antigen. When the host cells are bacteria, useful expression control sequences, which may be used, include the Trp promoter and operator (Goeddel, et al., Nucí, Acids Res., 8, 4057, 1980); the promoter and operator Lac (Change, et al., Nature 275 65 1978); the outer membrane protein promoter (Nakamura, K. and Inouge, M., EMBO J. 1, 771-775, 1982); promoters and operators of lambda bacteriophage (Remaut, E., et al., Nucí Acids Res., 1 1, 4677-4688, 1983); and other expression enhancing and control sequences compatible with the selected host cell. When the host cells are yeast, useful, illustrative expression control sequences include, for example, matching factors. For insect cells, polyhydrin or baculovirus p10 promoters can be used (Smith, G. E. et al., Mol Cell. Biol. 3, 2156-65, 1983). When the host cell is of mammalian origin, illustrative useful expression control sequences include the SV-40 promoter (Berman, PW et al., Science, 222, 524-527, 1983), the metabolism promoter (Brinster, RL , Nature, 296, 39-42, 1982) or a heat shock promoter (Voellmy et al., Proc, Nati, Acad. Sci. USA, 82, 4949-53, 1985). Alternatively, the expression control sequences present in Eimeria can also be provided. To maximize gene expression, see also Roberts and Lauer (Methods in Enzymology, 68, 473, 1979). Therefore, the invention further comprises non-bird hosts that contain a nucleic acid sequence or a recombinant nucleic acid molecule or a recombinant vector described above, capable of producing an Eimeria protein through the expression of an encoded protein by said nucleic acid sequence. Immunization of birds against an Eimeria infection can be achieved by administering to the birds a protein (or a variant or fragment thereof) according to the present invention as a so-called subunit vaccine. The subunit vaccine according to the invention may comprise a protein (or variant or fragment thereof) in a substantially pure form, optionally in the presence of a pharmaceutically acceptable carrier. The protein (or variant or fragment thereof) can be covalently linked to a heterologous protein (or variant or fragment thereof), which may be advantageous in purification. Examples of said heterologous protein are β-galactosidase, protein A, pro-chymosin, blood coagulation factor Xa, etc.

In some cases, the ability to increase protective immunity using these proteins (or variants or fragments thereof), per se, may be low. Therefore, the fragments (sometimes referred to as "haptens") can be conjugated to carrier molecules in order to increase their immunogenicity. The vehicles suitable for this purpose are macromolecules. These include natural polymers (e.g., proteins such as key limpet hemocyanin and albumin, toxins), synthetic polymers such as polyamino acids (polylysine, polyalanine), or micelles of amphiphilic compounds such as saponins. Alternatively, these fragments can be provided as their polymers, preferably linear polymers. If required, the proteins (or variants or fragments thereof), according to the invention, which are to be used in a vaccine, can be modified in vitro or in vivo, for example, through glycosylation, amidation , carboxylation or phosphorylation. An alternative to subunit vaccines is live vaccines. A nucleic acid molecule according to the invention can be introduced through recombinant DNA techniques to a microorganism (eg, a bacterium or virus) in such a way that the recombinant microorganism is still capable of replicating, thus expressing a polypeptide encoded by the inserted nucleic acid sequence and producing an immune response in the infected host bird. M. A. Barry et al., Nature (1995), 377; p. 632-635, teach how to prepare vaccines using nucleic acid molecules. A preferred embodiment of the present invention is a recombinant vector virus comprising a heterologous nucleic acid sequence described above, capable of expressing the DNA sequence in (a) host cell (s) or a host bird infected with the vector virus recombinant. The term "heterologous" indicates that the nucleic acid sequence, according to the invention, is not normally present by nature in the vector virus. In addition, the invention also comprises, (a) host cell (s) or a cell culture infected with the recombinant vector virus, capable of producing the Eimeria protein (or variant or fragment thereof) through the expression of the nucleic acid sequence. For example, the well-known technique of homologous recombination in vivo can be used to introduce a heterologous nucleic acid sequence according to the invention, to the genome of the vector virus. First, a DNA fragment corresponding to an insertion region of the vector genome, i.e., a region which can be used for incorporation of a heterologous sequence into the essential functions of vector disruption, such as those necessary for infection or replication, is inserted into a cloning vector according to normal recombinant DNA techniques. The insertion regions have been reported for a large number of microorganisms (e.g., EP 80,806, EP 1 10, 385, EP 83,286, EP 314, 569, WO 88/02022, WO 88/07088, US 4,769,330 and US 4,722, 848). Second, if desired, a deletion can be introduced to the insertion region present in the recombinant vector molecule obtained in the first step. This can be achieved, for example, through the exonuclease III digestion or the appropriate restriction enzyme treatment of the recombinant vector molecule of the first step. Third, the heterologous nucleic acid sequence is inserted into the insertion region present in the recombinant vector of the first step or in place of the DNA removed from said recombinant vector. The insertion region DNA sequence must be of appropriate length to allow homologous recombination to occur with the vector genome. Then, suitable cells can be infected with the wild type vector virus or transformed with the genomic DNA of the vector in the presence of the recombinant vector containing the insert flanked by the appropriate vector DNA sequences., so that recombination occurs between the corresponding regions in the recombinant vector and the genome of the vector. The progeny of the recombinant vector can now be produced in cell culture and can be selected, for example, genotypically or phenotypically, for example, through hybridization, detection of enzyme activity encoded by a co-integrated gene together with the sequence of heterologous nucleic acid, or detection of the heterologous antigenic protein, expressed, immunologically, by the recombinant vector. Next, these recombinant microorganisms can be administered to birds for immunization. It can then be maintained by itself for a certain time, or even replicated in the body of the inoculated animal, expressing, in vivo, a protein encoded by the inserted nucleic acid sequence according to the invention, resulting in stimulation of the immune system. of the inoculated animal. Suitable vectors for the incorporation of a nucleic acid sequence, according to the invention, can be derived from viruses such as virus from rashes, for example vaccine virus (EP 1 10, 385; EP 83,286, US 4,769, 330 and US 4,722,848) or poultry pustulation virus (WO 88/02022), herpes virus, such as HVT (WO 88/07088 or Marek's disease virus, adenovirus or virus of the influenza, or bacteria such as E. coli or the specific species Salmonella.With recombinant microorganisms of this type, the protein synthesized in the host animal can be exposed as a surface antigen.In this context, the fusion of the protein with proteins of OM P (or variants or fragments thereof), or "pilus" proteins (or variants or fragments thereof) of, for example, E. coli or the synthetic provision of signal and anchor sequences, which are recognized by It is also possible that the Eimeria protein, if desired as part of a larger whole, is released into the animal to be immunized.In all these cases, it is also possible that one or more immunogenic products find expression. , the c ual generates protection against several pathogens and / or against several antigens of a given pathogen. A vector vaccine according to the invention can be prepared by culturing a recombinant bacterium or a host cell infected with a recombinant vector comprising a nucleic acid sequence according to the invention, then the recombinant bacterium or the cells containing the vector and / or the recombinant vector viruses developed in the cells can be harvested, optionally in a pure form, and optionally formed into a vaccine in a pure form, and optionally formed into a vaccine in a lyophilized form. The host cells transformed with a recombinant vector, according to the invention, can also be cultured under conditions, which are favorable for the expression of a protein encoded by said nucleic acid sequence. The vaccines can be prepared using crude culture samples, host cell lysates or host cell extracts, although in other embodiments, more purified proteins according to the invention are formed to a vaccine, depending on the intended use. In order to purify the proteins produced, the host cells transformed with a recombinant vector, according to the invention, are cultured in an appropriate medium and the proteins produced are isolated from said cells, or from the medium, if the protein (or variant or fragment of it) is excreted. The proteins excreted in the medium can be isolated and purified by normal techniques, for example, salt fractionation, centrifugation, ultrafiltration, chromatography, gel filtration, or immuno affinity chromatography, while intracellular proteins can be isolated by first collecting said cells, breaking the cells, for example, through sound application or through other mechanically cutting means such as the French press, followed by the separation of the proteins from the other intracellular components and the formation of the proteins to a vaccine . Cell disruption can also be achieved through chemical means (for example, using EDTA or using detergents, such as Triton X1 14) or through enzymatic means, such as digestion with lysozyme. Antibodies or derivatives thereof (for example, fragments such as Fab, F (ab ') 2 or Fv fragments), which are directed against a protein according to the invention have potential uses in passive immunotherapy, immunoassays of diagnosis and in the generation of anti-idiotypic antibodies. Preferably these are specific for the Eimeria proteins of the present invention or variants / fragments thereof. Serum comprising antibodies or derivatives thereof can also be provided.

The Eimeria proteins (or variants or fragments thereof), as characterized above, can be used to produce antibodies, which can be polyclonal, monospecific or monoclonal (or derivatives thereof). If polyclonal antibodies are desired, techniques for producing and processing polyclonal sera are known in the art (eg, Mayer and Walter, eds., Immunochemical Methods in Cell and Molecular Biology, Academic Press, London, 1987). Monospecific antibodies to an immunogen can be affinity purified from polyspecific antisera through modification of the method of Hall et al (Nature, 31 1, 379-387, 1994). In monospecific antibody, as used herein, it is defined as a single antibody species or multiple antibody species with homogeneous binding characteristics for the relevant antigen. Homogeneous binding, as used herein, refers to the ability of the antibody species to bind to a specific antigen or epitope. Monoclonal antibodies, reactive against Eimeria proteins (or variants or fragments thereof) according to the present invention, can be prepared by immunizing innate mice through techniques known in the art (Kohler and Milstein, Nature, 256, 495 -497, 1975). Hybridoma cells are selected through growth on hypoxanthine, thymidine and aminopterin in an appropriate culture medium, such as Dulbecco's modified Eagle's medium. Antibody production hybridomas were cloned, preferably using the MacPherson soft agar technique (Soft Agar Techniques, Tissue Culture Methods and Applications, Kruse and Paterson, eds., Academic Press, 276, 1973). Discrete colonies were transferred to individual wells of culture dishes for cultivation in an appropriate culture medium. The antibody production cells were identified by classifying with the appropriate immunogen. Immunogen positive hybridoma cells are maintained by techniques known in the art. Specific anti-monoclonal antibodies are produced by culturing the hybridomas in vitro or by preparing ascites fluid in mice after injection of the hybridoma by methods known in the art. The anti-idiotypic antibodies are immunoglobulins, which carry an "internal image" of the antigen of the pathogen against which protection is desired, and can be used as an immunogen in a vaccine (Dreesman et al., J. Infecí Disease, 151 , 761, 1985). Techniques for increasing anti-idiotypic antibodies are known in the art (MacNamara et al., Science 226, 1325, 1984). The vaccine according to the invention can be administered in a conventional active immunization scheme: the individual or repeated administration in a manner compatible with the dose formulation, and in such amount that it will be prophylactically effective, i.e., the amount of antigen of immunization or recombinant microorganism capable of expressing said antigen that will induce immunity in birds (especially poultry) against attack by virulent Eimeria parasites. Immunity is defined as the induction of a significant level of protection in a population of birds after vaccination, compared to an unvaccinated group. A vaccine comprising the protein of the invention can reduce the number of oocysts disseminated by the infected animals. Normally, the disseminated oocysts will infect other animals in the group. After a reduction in the number of disseminated oocysts will also give a reduction in the number of animals, which are subsequently infected and also a reduction in the number of disseminated oocysts will give rise to a lower infection burden. In addition, even without the effect on the same parasite, a vaccine can reduce the incidence of diseases. This is special when the symptoms of the disease are caused by products released by the parasite. Vaccines directed against these products alleviate the symptoms with the attack of the parasite. In any case it is preferred that a vaccine of the present invention be capable of reducing the number of caecal lesions in a bird when attacked with a subsequent Eimeria infection. For live viral vector vaccines, the dose regime per chicken can vary from 103 to 10 8 pfu (but still <; 1000 pfu may be sufficient, for example for HVT). A typical subunit vaccine comprises from 0.1 to 100 μg of the protein (or variant or fragment thereof) according to the present invention. Preferably, at least 5 μg will be present. Said vaccines can be administered intradermally, subcutaneously, intramuscularly, intraperitoneally, intravenously, orally or intranasally. In addition, the vaccine may also obtain an aqueous medium or a suspension containing water, usually mixed with other constituents in order to increase the activity and / or shelf life. These constituents may be salts, pH regulators, stabilizers (such as skim milk or casein hydrolyzate), emulsifiers, adjuvants for improving the immune response (eg, muramyl dipeptide, aluminum hydroxide, saponin, polyanions and amphipathic substances) and / or conservatives. A preferred auxiliary is Quil A. This may be administered at a level of about 150 μg / dose (eg, a vaccine comprising a protein, variant or fragment of the present invention may also comprise other E. tenella immunogenic proteins or Immunogenic proteins from other Eimeria species Said combination vaccine can reduce the parasite burden in a group of birds and can increase the level of protection against coccidiosis A vaccine according to the present invention can also contain immunogens related to other bird pathogens , or may contain nucleic acid sequences encoding these immunogens, such as the antigens of Marek's disease virus (MDV), Newcastle disease virus (N DV), infectious bronchitis virus (IBV), chicken anemia agent (CAA), Reo virus, bird retrovirus, poultry adenovirus, turkey rhinotraqueatis virus, or E. coli. provide a multivalent vaccine. The invention also relates to an "immunochemical reagent", which comprises a protein (or variant or fragment thereof) according to the invention. The term "immunochemical reagent" means that the protein (or a variant or fragment thereof) according to the present invention is attached to a suitable support or is provided with a branded substance. The supports that can be used are, for example, the internal wall of a micro-test cavity or a cuvette, a tube or capillary, a membrane, a filter, a test strip or the surface of a particle such as, for example, a latex particle, an erythrocyte, a gel sol, a metal sol or a metal compound such as sun particles. (Sol is a suspension of microparticles, which as such are not soluble in water, such as metals). The brand substances, which may be used, are, among others, a radioactive isotope, a fluorescent compound, an enzyme, a dye sol, a metal sol or metal compounds such as sol particles. A nucleic acid molecule according to the present invention can also be used to provide specific probes for hybridization to allow the detection of Eimeria related to nucleic acids in a sample. Preferably, said hybridization occurs under severe conditions (for example at a temperature of about 35 to about 65 ° C using a salt solution which is about 0.9 molar) However, the skilled person will be able to various such parameters as appropriate for take into account variables such as probe length, base composition, type of ions present, etc.). The present invention also comprises a test kit comprising nucleic acid molecules of the present invention useful in the diagnosis of an Eimeria infection. These can be provided as probes or initiators. For example, primers can be used in PCR. The invention also comprises a kit useful in an immunoassay. This test equipment may contain at least one reagent Nanochemical in accordance with the invention. The immunochemical reaction, which is presented using such test equipment, is preferably a sandwich reaction, an agglutination reaction, a competition reaction or an inhibition reaction. To perform a sandwich reaction, the test kit may comprise, for example, a protein, variant or fragment thereof according to the present invention attached to a solid support, for example, the inner wall of a micro-test well. . Alternatively, it may comprise a labeled protein according to the present invention. This test equipment, however, does not comprise the aforementioned components. It may comprise one or more antibodies or derivatives thereof. The present invention will now be described by way of example only with reference to the accompanying drawings.

MATERIALS AND METHODS Experimental Design Sporozoite protein fractions from E. tenella were tested for their ability to stimulate T cells from chickens infected with E. tenella, in vitro, as measured by lymphocyte proliferation and MAF activity in their supernatants. Then, the chickens were orally inoculated with 1000 sporulated E. tenella oocysts, 8 days after PBL was isolated for in vitro stimulation. Sporozoite fractions were used that stimulated T cells as vaccine preparations and were tested for their immunogenicity related to the T cell and for its efficacy. The efficacy of vaccination was determined through the reduction in cecal lesions after a vigorous attack with E. tenella.

Chickens White Leghorn chickens without sexuality, of mixed breed, developed under specific pathogen-free conditions, were kept in isolation with free access to food and water. The feces were checked weekly to ensure that the animals were free of unwanted coccidial infections. For chickens with infection they were used at an age of 5-7 weeks. Three weeks old chickens were used for the vaccination.

Parasites and Purification of Sporozoites Weybridge strain of E. tenella was used (Shirley 1986). Parasites were passed through at regular intervals through chickens free of coccidia. The management of the oocysts, sporocysts and sporozoite release from sporulated oocysts were performed as described previously (Long et al., 1976), using 0.4% taurocholate (Sigma, St. Louis, MO, USA) instead of bile salts (Tomaya &Kitano, 1983). The sporozoites were also purified through a passage of nylon wool (Larsen et al., 1984) and stored as pellets at -70 ° C.

Fractionation of Sporozoite Protein Extraction of Triton X-114 Extraction of Triton X-114 was performed to isolate the hydrophilic phase of total sporozoite (HPS) proteins (Bordier 1981). Then, 2 x 109 purified E. tenella sporozoites (1 x 109 / ml) were suspended in 10 mM Tris. HCl, 150 mM NaCl, pH 7.4 (TBS), supplemented with protease inhibitors; 1 mM phenylmethylsulfonyl fluoride (PMSF, Serva, Heidelberg, Germany), 5 μg / ml Aprotinin, 1 μg / ml Leupeptin and 1 μg / ml Pepstatin A and sound was applied three times to 20 seconds in position 7 , on ice (using a sound applicator from Branson, Soest, The Netherlands). Pre-condensed Triton X-114 (Serva) was added to the sporozoite suspension at a final concentration of 10% (v / v) and mixed well to dissolve the proteins (total volume of 10 ml). The non-solubilized material was pelleted through centrifugation (20 minutes, 12000 g at 4 ° C). The recovered supernatant was layered on a sucrose cushion and incubated for 15 minutes at 40 ° C (phase separation) and rotated 10 minutes at 400 g at room temperature (RT). The extraction of the hydrophilic fraction was repeated once more in 10% (v / v) and subsequently in 20% (v / v) of Triton X-114 precondensed. The total protein concentration was determined using the bichinchonic acid (BCA) assay (Pierce Chemicals, Rockford, Illinois, USA). The hydrophilic phase was stored at -70 ° C until further used.

Fractionation of cell preparation All procedures were performed at 4 ° C. Prior to fractionation, HPS was concentrated through acetone precipitation. After centrifugation, at 10 minutes, 15000 g at 4 ° C and air drying, the pellets were dissolved to reduce the pH regulator of the sample (Laemmli 1970) containing 30 mg / ml of dithiothreitol (DTT) and boiled for 3 hours. minutes at 100 ° C. Hydrophilic proteins were fractionated using 12% (w / v) gel separation (7 cm) of polyacrylamide (PAA) and 4% of a stacking gel of PAA (w / v) in a 37 mm tube of the apparatus Prepcell Bio-Rad (Bio-Rad Labs, Richmond, CA), according to the manufacturer's protocol. The Prepcell was operated at 40mA, 500V max. The fractions were collected (+3 ml) overnight and stored at -85 ° C. The samples of the fractions were diluted once in 2 x pH regulator sample of resistance reduction and were taken for analysis with sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) using a PAA gel 12% (p / v) (Laemmli 1970). The gels were stained with silver according to Wray et al. (1981). Fifty fractions were combined in 9 fractions based on their relative molecular mass and dialyzed against 0.01M of pH regulated saline with phosphate (PBS) pH 7.3. The total protein concentration in the background fractions was determined using the BCA assay.

Preparation of sporozoite fractions for the lymphocyte proliferation assay The background fractions and HPS were treated with BioBeads (Calbiochem, La Jolla, CA) to remove the residual detergent. The beads were first washed 5 times with PBS (0.04 M, pH 7.3, 280-300 mOsm / kg) and incubated for 30 minutes with RPMI 1640 medium (Dutch Modification) supplemented with 2 mM L-glutamine, 1 mM Na pyruvate, 200 μU / ml penicillin, 200 μg / ml streptomycin (RPMI-GPPS), and 2% normal chicken serum (GI BCO, Life Technologies Paisley, UK). The samples were incubated with the beads for 1 hour at room temperature on a rotating platform and subsequently dialysed against PBS at 4 ° C. After precipitation of acetone, the protein pellets were washed with 96% ethanol, air dried and dissolved in RPMI-GPPS.

Lymphocyte Proliferation Assay The PBL proliferation responses were measured through the incorporation of [3 H] thymidine as described (Breed et al., 1996). In summary, PBL of infected, vaccinated or control chickens (1 x 106 / cavity) were cultured in 96-well round-bottom microtiter plates (Nunc, Kamstrumpo, Denmark) in the presence of either E. tenella sporozoite antigen. total (equivalent to 3 x 105 of sporozoites per cavity), H PS (10-15 μg / well) or bottom fractions of these (50 μl / well) or with medium only in a total volume of 150 μl of RPMI-GPPS supplemented with 1% (v / v) of normal chicken serum (GI BCO, Life Technologies, Paisley, United Kingdom). After 64 hours of incubation, the plates were centrifuged, a culture fluid of 80 μl was removed and analyzed for the presence of MAF activity (see nitrite assay). A fresh medium of RPM I-GPPS was added and the cells were pulsed during the last 8 hours with 18.5 kBq of [3 H] thymidine per cavity (specific gravity 185 GBq / mmole). The incorporation of [Hjtimidine using a Betaplate 1205 (Wallac, Turku, Finland) was measured. Proliferation responses were expressed as stimulation indices (SI), which represent the relationship of the mean proliferation in counts per minute (CPM) after stimulation for the value of the controls in the medium.

Nitrite assay The production of nitrite oxide through chicken macrophages of H D1 1 (Beug et al., 1979) was used as a measure of MAF activity, most likely due to I NF-? (Breed et al., 1997). Nitric oxide was quantified through the accumulation of nitrite in the culture medium as described by Breed et al. (1997). In summary, 100 μl of HD 1 1 cells (1.5 x 106 / ml of RPMI-GPPS + 20% (v / v) solution of fetal calf serum and 2% (v / v) of L-arginine) were cultured in 96-well round bottom plates (Nunc) in the presence of 100 μl of lymphocyte supernatants. After 24 hours of incubation, the amount of NO2"in the culture medium was determined through the Griess reaction.The supernatant of chicken spleen cells activated in Con A was included for reference.The data are expressed as μM of N0 71.5 x 106 cells / 24 hours (MAF activity) The nitrite index (NI) represents the ratio of the average amount of nitrite measured using supernatants of stimulated cultures to the value of unstimulated cultures (controls of the medium ).

Vaccination experiments The selected antigen preparations were thawed and the volumes were adjusted to obtain 5-10 μg protein / dose (0.5 ml), unless otherwise indicated. At each dose, 150 μg / dose of Quil A (Superfos Biosector, Vedbaek, Denmark) was added as auxiliary. The different vaccine preparations were injected subcutaneously in groups of + 10 chickens. The control group was injected with adjuvant in PBS. After +. 3 weeks, the chickens were reinforced with the same preparation, which was freshly prepared from the frozen antigen material. Eleven days after vaccination, heparinized blood samples (5 ml) were taken through cardiac puncture and analyzed for proliferation and production of MAF in response to sporozoite antigen of E. tenella total (equivalent to 3 x 105 sporozoites per cavity) or the medium only. From 3 to 4 days later, all the chickens were orally inoculated with 4500 sporulated oocysts of E. tenella. One week after this attack, the chickens were analyzed for the presence of cecal lesions. The severity of the cecal lesions were classified on a scale of 0-4, as described (Johnson &Reid, 1970).

Statistical analysis The statistical analysis of the lymphocyte proliferation data was performed using the Student's t test on stimulation indices transformed with Log (bennett &Riley, 1992). A similar transformation was performed for the nitrite analysis. The differences in the group means were considered significant values of P < 0.05. The differences between the mean group values of the injury classifications were analyzed using the Student's t test.

RESULTS Background hydrophilic fractions of E. tenella sporozoites The hydrophilic protein phase of sporozoite homogenates of E. tenella (H PS) obtained through extraction TX-14 was fractionated using gel electrophoresis preparation. The relative molecular mass of the proteins in each fraction was measured using the SDS-PAGE analysis. Nine fractions containing proteins of different relative molecular mass were obtained (Table 1). These fractions were tested for their ability to stimulate PBL from chickens infected with E. tenella.

TABLE 1 Relative Molecular Mass of Hydrophilic Fractions of Esporozoites from E. Tenella Selection of T cell stimulation antigen fractions The ability of fractions to stimulate T cell responses in PBL from infected chickens (day 8 pi) was measured through lymphocyte proliferation and MAF activity in their supernatants . The average stimulation indices are shown in Figure 1. Both control preparations (total sporozoite antigen and H PS) induced high proliferation responses in infected animals. The nine fractions induced significantly higher proliferation responses in PBL from infected animals than in PBL from uninfected controls. The highest responses were found against fractions 1, 2, 3, and 8. The stimulated PBL culture fluids were harvested and tested for the presence of MAF activity using the nitrite assay. The MAF activity, expressed as the amount of nitrite produced through macrophages, is as shown in Figure 1B. The high levels of MAF activity were measured in the PBL culture fluid stimulated with antigen preparations. control. The background levels in unstimulated cultures were low. Only fractions 1, 2 and 3 induced a significantly higher level of MAF activity in the PBL culture fluid of infected chickens than in those of uninfected chickens. The fractions that stimulated both types of responses (1, 2 and 3) and fraction 8, containing a relatively high proliferation induced capacity and reduced the activity induced by MAF, were selected to determine their vaccine potential.

Characterization of selected antigen fractions The four fractions of selected antigen were analyzed using SDS-PAGE. Figure 2 shows the different polypeptides present in the selected fractions. Fraction 1 contained polypeptides with a molecular mass of < 15 kD. Fraction 2 showed approximately 9 different polypeptides, two predominant bands of approximately 16 kD and 22 kD and minor bands ranging from 17-26 kD. Fraction 3 contained 3 polypeptides ranging from 26-30 kD (+ 5 kDa, to allow possible limitations in the measurement techniques used). Fraction 8 showed a predominant polypeptide of about 49 kD.

Determination of potential vaccine of selected antigen fractions The groups of chickens were immunized with the four selected antigen fractions. The animals received an initiation vaccination on day 0 and a booster vaccination on day 21. Eleven days after the booster vaccination (three days before the attack infection), PBL were harvested and stimulated with a total sporozoite antigen to determine the reactivity of the T lymphocyte. The lymphocytes of the four experimental groups exhibited a high reactivity after of the stimulation with the sporozoite antigen with respect to both, stimulation of lymphocyte proliferation and induction of MAF activity in their supernatants (Figures 3A and B). The lymphocytes of chickens vaccinated with fraction 3 showed the highest responses. Fourteen days after the booster vaccination, all animals were attacked with sporulated oocysts with E. tenella. Seven days later, the animals were sacrificed to determine the range of lesion in the mint. A value (present in group 3) was excluded from further analysis, since it was determined that this was the only value outside 95% of the total group reliability limit (n = 46; value> mean +1. 96 x SD). The results show that only the group of animals vaccinated with fraction 3 of the antigen had cecal lesion scales reduced as compared to the unvaccinated controls (Figure 3C). This reduction was statistically significant (P <0.05).

Dose-effect response of the protective antigen 3 fraction A dose-effect experiment was conducted to determine if the level of protection can be increased. Groups of animals were vaccinated with different amounts of antigen (0.5 and 15 μg / dose). The animals received an initiation vaccination and a booster with an interval of three weeks. Eleven days after the booster vaccination, the sensitivity of the peripheral blood lymphocyte against the total sporozoite antigen was determined. Lymphocytes from vaccinated animals reacted after stimulation with the sporozoite antigen. A clear dose-effect relationship was presented in those lymphocytes of animals that received the highest antigen dose showing higher T cell activation responses (both in the proliferation assay and in the nitrite assay, Figure 4A and B) . When it was attacked with sporulated oocysts with E. tenella four days later, it appeared that there was no dose-effect relationship between the level of protection and the dose of antigen used for vaccination; both groups of animals were protected to a similar degree with respect to the development of lesions in the mint (Figure 4C). The reduction in the mean injury scale of both groups of vaccinated animals was statistically significant, different from that of the control group (P <0.005). DISCUSSION N ueve hydrophilic fractions of sporozoite proteins, separated according to different molecular weight, were tested for their ability to stimulate T cell responses in PBL from day 8 p. i. The nine fractions induced proliferation of day 8 of P BL. Although the protein concentration varied between fractions, it was decided not to correct these concentrations for the reason that each of the fractions again contained two or more different polypeptides in unknown relationships. This means that the differences in the height of the answers, therefore, can not be completely trusted to select relevant fractions. The trial data were NOT included as a second criterion to select the fractions for vaccination-attack experiments. In addition to fractions 1, 2 and 3 that stimulated both types of T cell responses in PBL from infected animals, fraction 8 was included as containing a relatively high proliferation inducing capacity at low MAF induction activity. (probably IN F-?; Breed et al. 1 997). Although all vaccine preparations induced strong T cell responses, surprisingly only a fraction induced partial protection against oral attack infection with E. tenella oocysts. Figure 1 shows the responses of the T cell of PB L from chickens infected with E. tenella (day 8 i.p.) and of control against different hydrophobic fractions of E. tenella sporozoites, H PS or total sporozoite antigen (sporo). The fractions were tested at a concentration of 0. 1 5 x its original volume. (A) Average proliferation responses. (B) Average MAF activity through PBL supernatants. The vertical lines indicate normal errors of the mean (SEM). * Significantly different from the untreated group (* P < 0.05, ** P < 0.005, *** P < 0.0005). Figure 2 illustrates the SDS-PAGE of the hydrophilic fractions of E. tenella sporozoites used for vaccination. They were stained with silver. Each of the groups covers 3 lanes with double serial dilutions of the fraction. Figure 3 is the vaccine potential of the selected antigen fractions. PBL were taken 11 days after the booster vaccination and stimulated with E. tenella sporozoite antigen. (A) PBL responses of medium proliferation. (B) MAF activity mediated by supernatants. (C) Average cecal lesion range of vaccination groups after E. tenella attack infection. The vertical lines indicate SEM.

* Significantly different from the untreated group (* P <0.05, ** P < 0.005, *** p < 0.0005). Figure 4 is the dose-effect response of the 3 antigen fraction. PBL 1 was taken 1 day after the booster vaccination and stimulated with £ sporozoite antigen. tenella. (A) PBL responses of medium proliferation. (B) MAF activity mediated by supernatants. (C) Mean cecal lesion range of vaccinated groups after infection with E. tenella. The vertical lines indicate SEM. * Significantly different from the untreated group (* P <0.05, ** P < 0.005, *** p < 0.0005).

REFERENCES Bennett S. & Riley E.M. (1992) The statistical analysis of data from immunoepidemiological studies. Journal of Immunological Methods 146, 229-239 Beug H., Von Kirchbach A., Doderlein G., Conscience J-F. & Graf T. (1979) Chicken hematopoietic cells transformed by seven strains of defective avian leukemia viruses display three distinct phenotypes of differentiation. Cell 18, 375-390 Bordier C. (1981) Phase separation of integral membrane proteins in Triton X-114 solution. Journal of Biological Chemistry 256, 1604-1607 Breed D.G.J., Dorrestein J. & Vermeulen A.N. (1996) Immunity to Eimeria tenella in chickens: phenotypical and functional changes in blood T-cell subsets. Avian Diseases 40, 37-48 Breed D.G.J., Dorrestein J., Schetters Th.P.M., Waart van den L., Rijke E.O. & . Vermeulen A.N. (1997) Pheripheral blood lymphocytes from E. tenella infected chickens produces gamma-interferon after stimulation in vitro. Parasite Immunology 19, (127-135) Chapman H.D. (1993) Resistance to anticoccidial drugs in fowl. Parasitology Today 9, 159-162 Danforth H.D., Augustine P.C., Ruff M.D., McCandliss R., Strausberg R.L. & Likel M. (1989) Genetically engineered antigen confers partial protection against avian coccidial parasites. Poultry Science 68, 643-1652 Dunn P.P.J., Billington K., Bumstead J.M. & Tomley F.M. (1995) Isolation and sequences of cDNA clones for cytosolic and organellar hsp70 species in Eimeria spp. Molecular and Biochemical Parasitology 70, 211-215 Jenkins M.C., Augustine P.C., Danforth H.D. & Barta J.R. (1991) X-irradiation of Eimeria tenella oocysts provides direct evidence that sporozoite invasion and early schizont development induces protective immune response (s) Infection and Immunity 59, 4042-4048 Jenkins M.C., Seferian P.G., Augustine P.C. & Danforth H.D. (1993) Protective immunity against coccidiosis elicited by radiation-attenuated Maximum Eimeria sporozoites that are incapable of asexual development. Avian Diseases 37, 74-82 Johnson, J., & Reid W.M. (1970) Anticoccidial drugs: injury scoring techniques in battery and floor-pen experiments with chickens. Experimental Parasitoloy 28, 30-36. Laemmli U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227 ', 680-685 Larsen R.A., Kyle J.E., Whitmire W.M. & Speer C.A. (1984) Effects of nylon wool porification on infectivity and antigenicity of Eimeria falciformis sporozoites and merozoites. Journal of Parasitology 70, 597-601 Lillehoj H.S. & Trout J.M. (1993) Coccidia: a review of recent advances on immunity and vaccine development. Avian Pathology 22, 3-31 Long P.L., Miliard B.J., Joyner L.P. & Norton C.C. (1976) A guide to the laboratory techniques used in the study and diagnosis of avian coccidiosis. Folia Veterinaria Latina 6, 201-217 McKenzie M. E. & Long P. L. (1986) Immunization against coccidiosis with extracts of E / mer / a-infected tissues. Poultry Science 65, 892-897 Murray P.K., Bhogal B.S. , Crane M.S.J. & MacDonald T.T. (1986) Eimeria tenella-in vivo immunization studies with sporozoite antigen.

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Claims (10)

1. CLAIMS 1 .- A complete parasite-free composition of Eimeria, which comprises one or more proteins, or fragments or variants thereof; wherein said proteins: (a) are present in the hydrophilic phase of an extract of Triton X-1 14 from Eimeria sporozoites, (b) have molecular masses of 26-30 kDa + 5 kDa (i.e. 21-35 kDa ), when determined by SDS-PAGE under reducing conditions.
2. 2. A composition according to claim 1, wherein said Eimeria sporozoite extract is an extract of sporozoites of E. tenella, E. acervulina, E. maximus, E. brunetti, E. necatrix or E. mitis.
3. 3. A composition according to claim 1 or claim 2, wherein at least 50% w / w of the proteinaceous material present is made up of one or more of said proteins, fragments and / or variants thereof.
4. 4. A composition according to any of the preceding claims, wherein a plurality of, for example two to three of said proteins, fragment or variants thereof, are present.
5. 5. A composition according to any of claims 1 to 3, wherein only one of said proteins or fragments or variants thereof is present, for example, substantially in pure form.
6. 6. A nucleic acid molecule, which: a) encodes a protein, variant or fragment thereof, as described in any of claims 1 to 5, b) is complementary to a nucleic acid molecule as described in a), c) hybridizes to a nucleic acid molecule as described in a) or b).
7. 7. A nucleic acid molecule according to claim 6, wherein it is in an isolated or recombinant form.
8. 8. A vector comprising a nucleic acid molecule according to claim 6 or claim 7.
9. 9. A host that is not a bird, comprising a vector according to claim 8 or a nucleic acid of according to claim 6 or claim 7.
10. 10. A vector according to claim 8 or a host according to claim 9, wherein it is adapted to express a protein, variant or fragment thereof as described in any of claims 1 to 5. 1 1 .- A pharmaceutically acceptable vaccine composition comprising a vector or host according to claim 10 (if it is in living form, annihilated or attenuated). 12. A pharmaceutically acceptable composition according to any of claims 1 to 5 which is in the form of a vaccine. 13. A composition according to claim 11 or claim 12, wherein said vaccine comprises an auxiliary. 14. A composition according to claim 12, wherein the auxiliary is Quil A. 15. A composition according to any of claims 12 to 14, which is in a unit dosage form. 16. - A composition according to any of claims 1 to 5 or claims 11 to 15 for use in medicine. 17. The use of a composition according to any of claims 1 to 5 in the preparation of a vaccine against a disorder mediated by Eimeria, for example, against coccidiosis. 18. An antibody or a derivative thereof that binds a protein, variant or fragment thereof as described in any of claims 1 to 5. 19. An immunological reagent comprising a protein, variant or fragment thereof. , as described in any of claims 1 to 5 attached to a support or provided with a detectable label. 20. An immunological reagent comprising a protein, variant or fragment thereof, as described in any of claims 1 to 5, which is attached to a support or provided with a branded substance. 21. A test kit for the diagnosis of Eimeria infection, comprising a nucleic acid molecule according to claim 6 or claim 7; an antibody or derivative thereof according to claim 18; or an immunological reagent according to claim 19 or claim 20.