MXPA98006900A - Vaccine against neosp - Google Patents

Abstract

The present invention relates to a homogenate prepared from Neospora cells and vaccines against neosporosis prepared from them and which are useful for the prevention of clinical disease and animal abortion.

Description

VACUMA AGAINST IMEOSPQRA FIELD OF THE INVENTION The present invention relates to a vaccine against the pathogenic protozoan Neospora > This vaccine is useful for the prevention of clinical disease and abortion in mammals. The vaccine of the invention comprises a homogenate prepared from cells of a Neospora species.

BACKGROUND OF THE INVENTION Neosporosis in mammals is caused by infection with a pathogenic strain of the protozoan parasite Neospora and has been identified as the main cause of miscarriage »neonatal death» congenital infection and encephalitis. Dubey and Lindsay »199S» Vet. Parasite!. Today 9: 452-453. Neospora caninum infects dogs and congenitally infects puppies, often causing paralysis. N. caninum tachyzoites have been isolated from naturally infected puppies. Lindsay and Dubey »1989» J. Parasite !. 75: 163-165. The infection with Neospora is also one of the main causes of abortion in dairy cows. Cases of neosporosis have also been reported in goats »sheep and horses. Although N. cam'nuro is super cially similar to the pathogen Toxoplasma gondii, N. caninum and T. gondii, it is possible to distinguish between antigenic and ultrastructural. Dubey and Lindsay »1993» above. It was also found that protozoan parasites similar to Neospora isolated from the brains of aborted bovine fetuses and grown in continuous vitro were antigenically and ultrastructurally distinct from T. gondi and Haromondia hammondi and more similar to N. caninum. Conrad et al. 1993 »Parasi toi ogy 106: 239-249. Likewise »the analysis of the ribosomal RNA genes of the small nuclear subunit revealed that there were no nucleotide differences between Neospora strains isolated from cows and dogs» but there were consistent differences when compared to T. gondi i 'thus conferring the distinction between pathogens. Marsh et al. »1995, J. Paras toi. 81: 530-535. The etiological role of a Neospora bovine isolate in bovine abortions and congenital disease has been confirmed. Barr et al. "1994, J. Vet. Diag. Invest. 6: 207-215. A rodent model of neosporosis in the central nervous system has been developed using Balb / c inbred mice infected with N. caninum. Lindsay et al. »1995» J. Parasitol. 81: 313-315. In addition, 3e models have been described to study transplacental transmission of N. caninum in pregnant exogamic and inbred female mice, Colé et al., 1995, J Parasitol. Bi: 730-732 and Long et al., 1996 »J. Parasitol. 82: 608-611 »respectively. Likewise, an experimental model of N. can num has been developed in the # pygmy goat very similar to the abortion of the cows induced by Neospora naturally acquired. Lindsay et al., 1995. Am. J. Vet. Res. 5S: 1176-1180. In WO 9525541 a culture is described biologically pure bovine Neospora, the methods for detecting anti-Neospora antibodies and the specific Neospora nucleic acids "and a composition containing a Neospora bovine antigen and a vehicle for use as a vaccine. 10. Previously, an effective vaccine against neosporosis containing a homogenate prepared from Neospora cells had not been described.

BRIEF DESCRIPTION OF THE INVENTION In a first aspect, the present invention provides a homogenate prepared from Neospora cells, which is capable of inducing a protective response against neosporosis in a mammal. In a second aspect, the present invention provides a vaccine for protecting a mammal against neosporosis, which contains an immunologically effective amount of a homogenate prepared from Neospora cells, the homogenate being capable of inducing a response protective against neosporosis in a mammal, and a veterinarily acceptable vehicle. The vaccine of the present invention may also contain one or more additional immunomodulatory components, eg, an adjuvant or cytokine. In a third aspect, the present invention provides a method for the preparation of a vaccine that protects a mammal against neosporosis., which comprises the homogenization of the Neospora cells to form a homogenate capable of inducing a protective response against neosporosis in a mammal and the combination of an immunogenic amount. Logically effective! homogenized with a veterinarily acceptable carrier in a form suitable for administration to the mammal. In a fourth aspect, the present invention provides a method for the protection of a mammal against neosporosis, comprising the mammalian administration of a vaccine containing an immunologically effective amount of a homogenate prepared from Neospora cells, this homogenate being capable of inducing a protective response against neosporosis in a mammal and a veterinarily acceptable vehicle. The vaccine of the present invention can be administered to any species of mammal susceptible to infection and disease caused by Neospora, including but not limited to dogs, goats, sheep and horses. In a fifth aspect, the present invention provides a combination vaccine for the protection of a mammal against neosporosis and »optionally, one or more pathological diseases or disorders which may affect the mammal» whose combination vaccine contains an immunologically effective amount of a first composition containing a homogenate prepared from Neospora cells, which is capable of inducing a protective response against neosporosis in a mammal, an immunologically effective amount of a second composition capable of inducing a protective response against a disease or pathological disorder which affects the mammal, and a veterinarily acceptable vehicle. The second composition of the combination vaccine is selected on the basis of its ability to produce a protective response against neosporosis or another pathological disease or disorder affecting members of mammalian species as is known in the art. The combination vaccine of the present invention may further contain one or more additional immunomodulatory components including, for example, an adjuvant or cytokine, among others. In a sixth aspect, the present invention provides a kit for vaccinating a mammal against neosporosis "containing a first package having an immunologically effective amount of a homogenate prepared from Neospora cells" being this homogenate capable of inducing a protective response against neosporosis in a mammal, and a second container having a veterinarily acceptable vehicle or diluent suitable for mixing with e! content of the first container. In a seventh aspect, the present invention provides antibodies specific for one or more antigenic components present in a homogenate prepared from Neospora cells.

BRIEF DESCRIPTION OF THE FIGURES FISURA 1.- Western transference analysis before "Or exposure to BALB / c mice serum A whole-cell homogenate of NC-1 tachyzoites (the" NSA preparation ") was fractionated by SDS-PATE and moved to a PVDF membrane" which was then incubated. with samples of primary serum, followed by anti-mouse IgG conjugated goat with alkaline phosphatase and was developed using the chromogenic substrate BCIP / NBT. Lanes 1-3 = serum of the mice at rw which were administered adjuvant only (control) »lanes 4-6 = serum of the mice to which the NSA plus adjuvant preparation (vaccine) was administered; the patterns of molecular weights. The serum of the immunized mice contains antibodies that are reactive with the proteins of the NSA preparation having molecular weights of about 17-19 »28-30, 33, 37» 46 »48 and 56 KD. 25 FIGURE 2.- Immunorescent antibody titers 1 (IFA) before exposure (A) and after exposure (B). Serum from animals was added on day 21 post-immunization (day O before exposure) and day 21 after exposure to the wells containing tachyzoites NC-1. The wells were then incubated with anti-mouse IgG conjugated with fluorescein isothiocyanate (Fab) .A? + IgG (Kirkegaard 8-Perry »Gaithersburg» MD) »was washed and examined by epiphyseal microscopy. The antibody titer is based on the highest dilution of immune serum that produces a detectable fluorescence. The results show average titers of IFA antibodies higher in animals vaccinated before exposure (2A) and significantly higher IFA antibody titers after exposure (2B) (P <0,001) when compared to controls. In 2B »with an exposure 1045» the geometric average control title (GMT) = 2 »691; GMT of the vaccine - 25. 600. With an exposure 10"7" GMT of control = 5.382 »GMT of the vaccine = 72. 408. FIGURE 3.- Splenic-specific antigen proliferation test before exposure. 21 after the exposure »splenocytes were prepared from the mice that had been administered adjuvant alone (control) or the NSA plus adjuvant preparation (vaccine) and T cell prophylaxis assays were performed incubating the splenocytes in Presence of the NSA preparation and pulse the splenocyte cultures with C3H-ltimidi a »as described below (example 2) The results are expressed as \ cpm (mean cpm with NSA minus cpm medium with medium alone ) and demonstrate that a Neospora cell homogenate can induce a population of T cells in vivo which is able to proliferate in vitro after exposure to the NSA preparation. FIGURE 4. Production of specific spinal cytokines the antigen before the donor's exposure. On the 21st day after the exposure, the splenocytes of the mice which had been administered adjuvant alone (control) or the adjuvant NSA preparation (vaccine) were prepared and the levels of cytokine production were determined by incubation of splenocytes in presence of the NSA preparation »by collecting the cell-free supernatants and carrying out the assay for the determination of specific cytokines using specific commercial cytokine bodies» following the manufacturer's instructions (PharMingen »San Diego, CA). The results demonstrate that a Neospora cell homogenate can induce a population of T cells in vivo that is capable of producing both types of cells »of type 1 (IFN-» IL-2) and of type 2 (IL- »IL- IO> »vitro after exposure with the NSA preparation FIGURE 5. Antigen-specific splenic proliferation assay after exposure On the 21st day after the exposure, splenocytes from BALB / ca mice were prepared. they had been given only adjuvant (control) or the most adjuvant NSA preparation (vaccine) and T-cell proliferation assays were performed by pulse-testing splenocyte cultures with C3H3thymidine »as described below. exposure with 1 x 1013 (A) or 1 x 10"7 (B) of tachyzoites NC-1, significantly higher antigen-specific responses were detected (** = P <; 0. 05; **** - P < 0 »01) using splenocytes from vaccinated mice compared to control mice (n = 4 / group). '- "' FIGURE 6. Scores of lung and brain injury IO after the exhibition. Sections of lung and brain tissue were prepared on day 21 after exposure of control BALB / c mice (n = 6) and vaccine (. R? = G) »exposed with 1 x 10 * 3 (A» B) or 1 x lO "7 (OD) tachyzoites NC-1 and were given a score as described below. injury scores for individual animals. The line of points represents the average injury score for each group. The results demonstrate that animals immunized with a Neospora cell homogenate and stimulated with 1 x IO "7 tachyzoites NC-1 have mean lung injury scores (P <0 »01) and brain scores (P <0» 05> significantly lower than those obtained when compared with stimulated controls A. mean of control = 0 »5» average of the vaccinated = 0 »33. B. average of the control = 1.0» average of the vaccinated = 0.33 C. average of the control = 1.83 » average of the vaccinated = 0 »67 (P <0» 01). D. control mean = 1.83 »average of vaccinated Cough = 1.0 (P <0.05).

FIGURE 7. Survival curves after exposure of mice without athymic immune system. Mice without an immune system that receive only DPBS (no splenocytes = "no cells"); mice without an immune system receiving splenocytes from BALB / c mice injected with adjuvant ("adjuvant"); mice without an immune system receiving splenocytes from BALB / c mice injected with an NSA plus adjuvant preparation ("vaccine") (n = 6-7 mouse / group). The results demonstrate that the transfer of cells from vaccinated BALB / c mice to mice without an immune system results in an adoptive protective immunity "as shown by increased survival and by significant protection against a virulent exposure of NC -1.

DETAILED DESCRIPTION OF THE INVENTION Applicants have discovered that a homogenate prepared from Neospora cells is capable of inducing a protective response against neosporosis in mammals. The present invention thus provides a homogenate prepared from Neospora cells, this homogenate being capable of inducing a protective response against neosporosis in a mammal. The present invention further provides a vaccine for protecting a mammal against neosporosis that contains an immunologically effective amount of a homogenate prepared from Neospora cells, the homogenate being capable of inducing a protective response against neosporosis in a mammal and a vehicle veterinarily 5 acceptable. As used in the present »the term "neosporosis" refers to the infection of a mammal with ^^ cells of species or strain of Neospora »or any other symptom. "" disorder "event or pathology associated with the infection IO of the mammal with the cells of a species or strain of Neospora »or caused by it. The phrase "capable of inducing a protective response" is very frequently used herein to include the induction or exposure of any base response immunological in the animal in response to vaccination, which includes an antibody or a cell-mediated immune response or both, which serves to protect the vaccinated animal against neosporosis. The terms "protective response", "protection against", "protect", et., As used in The present invention refers not only to the prevention of neosporosis or absolute prevention of infection with a pathogen causing neosporosis, but also any detectable reduction in the degree or percentage of infection by such a pathogen, to any reduction detectable in the incidence of death or any detectable increase in survival time after * infection with a virulent strain of the pathogen »at any detectable reduction in the severity of the disease or in any symptom or disorder caused by the infection with the pathogen, including, for example, any detectable reduction in the percentage of formation or in the absolute number of lesions in one or more tissues in the vaccinated animal, or any detectable reduction in the incidence of abortions or transmission of the infection of a parental mammal to his offspring. -LO The phrase "immunologically effective amount" refers to that amount or dose of "homogenized" vaccine antigen or NSA preparation capable of inducing a protective response against neosporosis when administered to a member of a mammalian species after administration of a single dose "or after multiple administrations.

* - Preparation of the Neospora anisone The invention is based on e! discovery that a homogenate prepared from Neospora cells is capable of inducing a protective response against neosporosis in mammals. The cells used to produce the homogenate in the vaccine of the present invention can come from any strain of any species of the genus Neospora, these cells being able to be pathogenic or not, the homogenate capable of inducing a protective response against neosporosis in mammals. In a preferred embodiment the Neospora species is N. caninum, from which a homogenate can be usefully prepared is strain NC-1 which can be obtained from MARC 145 monkey kidney cells infected from the American Type Culture Collection »at 12301 Parklawn Drive» Rockville, MD 20852 »USA (ATCC r - of registry CRL-12231) and which includes strains derived from NC-1 by one or more transients ± r \ vi ro and / or -vn i vo. Strain NC-1 is also described in Dubey et al. 1988. J. A. Vet. Med. Assoc. 193: 1259: 1263 »whose publication is incorporated in -fcO the present as a reference. Meospora strains for use in accordance with the present invention can be isolated on the other hand from tissues or body fluids of infected animals using conventional isolation techniques, such as those described in the publications mentioned above. It is a non-limiting realization »the vaccine. present invention can be prepared using homogenates of cells from other Neospora species that are immunologically equivalent to N. caninum or used or homogenized from cells of other N. caninum strains that are immunologically equivalent to the N. caninum strain NC-1. . A Neospora species is "immunologically equivalent" to N. caninum when a homogenate prepared from cells of the immunologically equivalent species is able to induce in a mammal the production of antibodies that recognize one or more antigenic components present in a homogenate of N. cani um cells "as determined, for example, by Western blot analysis" and when the homogenate of cells of the immunologically equivalent species is able to induce a protective response against neosporosis in mammals. Likewise, a N. caninum strain is "immunologically equivalent" to the strain NC-1 of N. caninum when a homogenate prepared from the cells of the immunologically equivalent strain is able to induce in a mammal the production of antibodies that recognize one or more components present in a cell homogenate of N. can? m strain NC-1 (see FIG. 1). and when the homogenate prepared from cells of the immunologically equivalent strain is capable of inducing a protective response against neosporosis in mammals. The Neurospora cells for use in accordance with the present invention can be used directly and without subsequent modification. On the other hand, said cells can be modified, for example, by genetic manipulation to add »increase» to suppress or reduce the expression of one or more routes or metabolic products »or antigenic properties, such as, for example, a particular surface antigen or a virulence factor »or likewise» modify said routes, products or properties. Said routes »products or properties» whose expression can be added, increased or modified in a useful way in the cells are preferably those that serve to trigger or stimulate the induction of a protective response against neosporosis in a mammal vaccinated with the corresponding homogenate. For example, Neospora cells can be genetically modified to add or detectably increase the expression of one or more antigenic components that are useful in triggering or stimulating the induction of a protective response. In a non-limiting embodiment, the Neospora cells are genetically modified to add or increase detectably the "" "expression of one or more immunodominant antigens" as 1-0 those visualized by SDA-PAGE separation and Western blot analysis of the NSA preparation as described below "and including those identified antigens having molecular weights selected from the group consisting approximately of 17-19 , 28-30 »33» 37, 46 »56 kD. On the other hand or additionally »the cells can be modified to suppress or reduce in a detectable way the expression of one or more antigenic components normally associated with unmodified Neospora cells or a homogenate prepared from them. In a non-limiting embodiment, the Neospora cells are genetically modified to suppress or reduce in a detectable manner the expression of one or more antigenic components, such as those that can be visualized by SDS-PAGE separation and Western transference analysis of the NSA preparation as described below, and including those identified antigenic components having molecular weights selected from the group consisting between about 17-19, 28-30, 33, 37, 46, 48 and 56 kD. In this way »vaccines can be produced in such a way that they are 5" marked "or" - "marked", which allows to distinguish the animals that have been vaccinated from those that have been naturally infected with the pathogen. ---- The procedures by which protozoal cells »like those of Neospora» can be modified They are generally known in the art and include the introduction of random mutations "eg, by exposure to chemical mutagenic agents or radiation" followed by the selection of a desired mutant phenotype. On the other hand »or additionally» the Neospora cells can be Modify by directed genetic modification »such as that performed by procedures such as» for example »homologous recombination» as described for example by Cruz and Bervely »1990, Nature 348: 171-173; Cruz et al. »1991» Proc. Nati Acad. Sci. USA 83: 7170-7174; Donald and Ross »1994» Mol. Biochem.

Parasitol. 63: 243-253; Titus et al. »1995» Proc. Nati Sci. USA 92: 10267-10271, whose publications are incorporated herein by reference. Such genetic modification is within the possibilities of the technique and can be performed using recombination techniques generally known "such as those described for example in Maniatis" and others. »1989» Molecular Cloning. A Laboratory Manual »Cold Spring Harbor Laboratory Press» Cold Spring Harbor, N.Y.; Ausubel, et al., 1989 »Current Protocols in Molecular Biology, Greene Publishing Associates & Wiley Interscience, N.Y .; and Sambrook, et al., 1989 Molecular Cloning. A Laboratory Manual, 2"Ed. Cold Spring Harbor Laboratory Press» Cold Spring Harbor »NY» said publications being incorporated herein by reference, Once obtained, the Neospora cells for use in the present invention can be cultured iv. by infection of any recipient cell line »preferentially a mammalian cell line» with tachyzoites from Tas species or from 1 to Neospora strain according to the techniques described in 1. The cellular lines of mammals in which the cells can be cultured. Neospora tachyzoites are, for example, human foreskin fibroblasts (Lindsay et al., 1993, Am. J. Vet. Res. 54: 103-106), bovine cardiopulmonary aortic endothelial cells (Marsh et al., 1995, supra), bovine monocytes (Lindsay and Dubey, 1989, supra), and monkey kidney cells, etc. For example, N. caninum tachyzoites can be cultured in monolayers of human foreskin fibroblasts Hs68 (ATCC registration number CRL 1635) (Líndsay et al. 1993, above). Similarly, bradyzoites can be cultured and manipulated. Mammalian cell cultures can be grown and cell cultures that have been infected with Neospora can be maintained in any of the many culture media described in the art. For example, stationary monolayer cultures of cardiopulmonary aortic endothelial cells infected with N. caninum tachyzoites can be grown in Dulbecco's Minimum Essential Medium (DMEM: Gibco Laboratories, NY) supplemented with heat inactivated fetal bovine serum (SBF) or equine adult (SE) at 10% (v / v), 2 mM L-glutamine, 50 U / ml penicillin and 50 μg / ml streptomycin (Conrad et al. 1993, above). The monolayers of human foreskin fibroblasts Hs68 can be maintained in RPMI 1640 containing 2% SBF (v / v) »1.0 M sodium pyruvate» 1 x 10"* U / ml penic l na, 1 x 10" * μg / ml of streptomycin, 2-ercaptoethanol 5 x 10-2 and O »3 mg / ml of L-glutamine (maintenance medium). The monolayer cultures of human foreskin fibroblasts Hs6B infected with Neospora can be maintained in identical media, but in which the SBF has been increased to 10% (v / v) (growth medium). Monolayer cultures of mammalian cells infected with Neospora are typically maintained under standard conditions for tissue culture, such as »37 ° C and CO» 5%. The tachyzoites are typically transferred to uninfected monolayer cultures when 70-90% of the mammalian cells in the culture have been infected, which can be determined microscopically using conventional techniques. The tachyzoites can be harvested from the cultures of infected mammalian cells by lysing the host cells using any conventional technique and collecting the tachyzoites, for example, by filtration or by centrifugation. The cells that can be used to produce the cell homogenate of the invention are preferably tachyzoites, but can also be bradyzoites or oocysts or some combination thereof. Also, the cells used in the present invention can be either viable cells or cells that have previously been inactivated, for example by treatment with inactive chemical agents such as formaldehyde or glutaraldehyde, among others, or by treatment with radiation. , or by exposure or a pH or extreme temperature "or some combinations thereof. The production of the homogenate of the invention is not limited to any particular homogenization or disintegration process. Neospora cells can be homogenized or disrupted using any technique known in the art "including, but not limited to freezing / thawing" osmotic shock »crushing» sonication »use of a polytron» mixer or tissue homogenizer or some combination of the same. As used herein, the term "homogenized" refers to a preparation prepared by homogenization or disintegration of the complete Neospora cells. The homogenate of the present invention can contain all the components produced by the homogenization or disintegration of the complete Neospora cells, which implies a preparation of "whole cells". On the other hand, the homogenate of the present invention may consist of a fraction of the total content of the homogenized or disrupted Neospora cells, the fraction being prepared from the preparation of the whole cells using one or more fractionation steps, isolation or purification. known in the art »which include for example» centrifugation »filtration» dialysis »preparative gel electrophoresis» affinity chromatography »ion exchange chromatography, gel permeation chromatography, ammonium sulfate precipitation, or some combination thereof so that the fraction resulting from the preparation of whole cells retains the ability to induce a protective response against neosporosis in mammals. A fraction of this type can be an enriched membrane fraction or a fraction enriched in soluble asthmatic topical components. Such fractions are prepared and easily tested using only the usual preparation and determination methods. Preparation and use of vaccines The present invention provides a vaccine against neosporosis which contains a immunologically effective amount of a homogenate prepared from Neospora cells., this homogenate being capable of inducing a protective response against neosporosis in a mammal and a veterinarily acceptable vehicle. The present invention further provides a method for the preparation of a vaccine that protects a mammal against neosporosis "comprising the homogenization of Neospora cells to produce a homogenate capable of inducing a protective response against neosporosis in a mammal and the combination of an immunologically effective amount of the homogenate with a veterinarily acceptable carrier in a form suitable for administration to the mammal. The vaccine may simply contain a cell homogenate prepared in a culture fluid taken directly from a Neospora cell culture, which is administered directly to the mammal, or instead may contain a cellular homogenate combined with a veterinarily acceptable carrier selected from those known in the art »so that it is appropriate for the route of administration. For example, the vaccine of the present invention can be formulated following the accepted convention of combining the homogenate or a fraction thereof with buffers, carriers, stabilizers, diluents, preservatives and / or conventional solubilizers. The vaccine can also be formulated to facilitate sustained release. The diluents can be water, saline, dextrose, ethanol, glycerol and the like. The additives to obtain isotonicity can be sodium chloride, dextrose, mannitol, sorbitol and lactose, among others. The stabilizers can be albumin, among others. Other vehicles and additives for suitable vaccines are known or will be apparent to those skilled in the art. See for example »Remington's Pharmaceutical Science» 18th Ed. »1990» Mack publishing »which are included here for reference. The vaccine of the present invention may further contain one or more "munomodulatory components" such as, for example, an adjuvant or cytokine. Non-limiting examples of adjuvants are the RIBI adjuvant system (Ribi Inc. »Hamilton, MT), alum, mineral gels, such as aluminum hydroxide gel,» oil-in-water emulsions, water-in-oil emulsions »such as, for example, complete and incomplete adjuvants of Freund »block copolymers (CytRx» Atlanta »GA), QS.21 (Cambridge Biotech Inc., Cambridge MA) and SAF-M (Chiron, Eryville CA) »adjuvant AMPHIGEN®» saponin »Qu l A or other fraction of saponin» monophosphorus 1 lipid A and adjuvant 1 ipido-amine Avridine.

Specific non-limiting examples of oil-in-water emulsions useful for the vaccine of the invention are SEAM62 and SEAM1 / 2, the components of which are presented below. Other immunomodulatory agents that can be included in the vaccine are, for example, »one or more interleukins» interferons or other known cytokines. The vaccine may be kept in solution or in the form of a Tiofil ized form that can be reconstituted with a sterile diluent solution before administration. The vaccine of the present invention can optionally be formulated for sustained release of the antigen. Examples of such sustained-release formulations are homogenized in combination with combinations of biocompatible polymers such as, for example, poly (lactic acid), poly (lactic acid-co-glycolic acid), hypromellose, hyaluronic acid, the collagen and the like. The structure, selection and use of degradable polymers in pharmaceutical pharmaceutical vehicles has been discussed in various publications, such as A. Domb and others, 1992, Polymers for Advanced Technologies 3: 279-292, which is incorporated into the present as a reference. An additional guide for the selection and use of polymers in pharmaceutical formulations can be found in the text by M. Chasin and R. Langer (ed.), 1990 »" Biodegradable Polymers as Drug Delivery Systems "in: Drugs and the Pharmaceutical Sciences . Vol. 45 »M. Dekker, NY» which is incorporated herein by reference. On the other hand »or additionally» the homogenate can be microencapsulated to improve the administration and efficiency. Methods for microencapsulation of antigens are well known in the art "and include the techniques described for example" in U.S. Patent 3,137,631, U.S. Patent 3,959,457, U.S. Patent 4,205,060, U.S. Patent 4,606,940. »U.S. Patent 4,744,933; U.S. Patent 5,132,117; and International Publication WO 95 / 2B227, all of which are incorporated herein by reference. Liposomes can also be used to provide sustained release of! homogenized from the invention. Details on how to prepare and use the formulations in the form of liposomes can be found among others "in US patent 4,016,100» the patent ^ US 4,452,747 »US Patent 4,921,706» W U.S. Patent 4,927,637 to U.S. Pat.

No. 4,944,948"U.S. Patent 5,005,050 and U.S. Patent 5,009,965" all of which are incorporated herein by reference. The present invention further provides a method for the protection of a mammal against neosporosis "comprising administering to the mammal a vaccine containing an immunologically effective amount of a homogenate prepared from Neospora cells" the homogenate being capable of inducing a protective response against neosporosis in a mammal and a vehicle veterinarily acceptable. The vaccine is preferably administered parenterally, for example, or subcutaneously or by intramuscular injection. However »the vaccine can also be administered by intraperitoneal or intravenous injection or by other pathways »including for example, oral, intranasal» rectal »vaginal» intraocular route or by combining several of these routes. The person skilled in the art will know how to formulate the composition of the vaccine according to the chosen route. Effective dosing can be determined by conventional means »starting with a low dose of homogenate and then increasing the dosage while controlling the effects. Many factors must be taken into account when determining an optimal dose per animal.Fundamental among these »the species» the size of the animal »w the age of! animal., the general condition of the animal, the presence of other drugs in the animal, the virulence of a particular species or strain of Neospora against which the animal is to be vaccinated and the like. The actual dose is preferably chosen once the results of other animal studies have been considered. Vaccine regimens can also be to select based on the factors described above. The vaccine of the invention can be administered at any time during the life of a particular animal depending on several factors, such as the time at which neosporosis occurs in other animals. etc. The vaccine can be To administer to animals at weaning age or earlier or to more mature animals, for example, as a pre-crossover vaccine as a protection against neosporosis to avoid a congenital disease related to Neospora or an abortion. Effective protection may require only one primary vaccination or one or more booster vaccines may be required for total protection. A method to detect if adequate immunological protection has been acquired is to determine the seroconversion and the titres of antibodies in the animal after 1 vaccination. The timing of the vaccination and the number of booster doses "if applicable, will preferably be determined by a veterinarian based on the analysis of all relevant factors" some of which have been described previously. The amount of homogenized in the vaccine preferably ranges from about IO to about -LO l.OOO μg protein / ml and more preferably from about 100 to approximately 500 μg protein / ml. An appropriate dosage size ranges from about 0.5 ml to about 10. O ml and more preferably from about 1.0 ml to approximately 5.0 m! . Generally, in the case of a dose, the amount of cellular homogenate administered to an animal is preferably from about 10 to about 10000 μg of protein and more preferably from about 100 to about 500 μg of 0 proinin. The vaccine of the present invention is useful for protecting mammals against infection or disease caused by Neospora. As used herein, the term "mammal" refers to any mammal species that can be protected against neosporosis using the vaccine of the invention "and which includes dogs, cows, goats, sheep and horses" among others. The vaccine is useful to protect both pregnant and non-pregnant mammals. The present invention further provides a combination vaccine for the protection of a mammal against neosporosis and »optionally» one or more pathological diseases or disorders which may affect the mammal »this combination vaccine comprising an immunologically effective amount of a first composition that contains a homogenate prepared from Neospora cells »said homogenate being capable of inducing a protective response against neosporosis in a mammal» an immunologically effective amount of a second composition capable of inducing a protective response against a disease or pathological disorder which may affect to the mammal "and a veterinarily acceptable vehicle such as those described above. The second composition of the combination vaccine is selected on the basis of its ability to induce a protective response against neosporosis or another disease or pathological disorder that affects the members of mammalian species "as is known in the art. Any immunogenic composition known to be useful in a vaccine composition in the particular mammalian species can be used in the second composition of the combination vaccine. Such immunogenic compositions include but are not limited to those which provide protection against pathogens selected from the group consisting of bovine herpesvirus (infectious rhinotraquei tis bovine)., bovine respiratory syncytial virus, bovine viral diarrhea virus, nfluenza virus type I, II or III »Leptospira spp.» Campylobacter spp., Staphylococcus aureus »Streptococcus agalactiae, MycopTasma spp. , Klebsiel la spp., Sal onel la spp., Rotavirus, coronavirus, rabies. Pasteurella haemolytica »Pasteurella m? Ltocida» Clostridia spp. »Tetanus toxoid» E. col i. Cryptosporidium spp., T Eimer a spp., And other eukaryotic parasites among others. The combination vaccine of the present invention may further include one or more additional immunomodulatory components "including for example" an adjuvant or cytokine and which may optionally be formulated for a sustained release or kept in lyophilized form or both "as has been previously described. The present invention further provides a device for the vaccination of a mammal against neosporosis which contains a first container having a vaccine containing an immunologically effective amount of a homogenate prepared from Neospora cells, this homogenate being capable of inducing a protective response against neosporosis in a mammal and a second container having a veterinarily acceptable vehicle or diluent suitable for mixing with the contents of the first container. The vaccine may be stored in lyophilized form or reconstituted by addition of the vehicle or diluent, Anti-Neospora Antibodies The production of polyclonal and monoclonal antibodies that bind to one or more antigenic components specific to Neospora is within the scope of the invention. Said antibodies can be specific for any of the antigenic components associated with the Neospora cells or for a homogenate PREPARED from them. In a non-limiting embodiment, antibodies can be obtained against one or more antigenic components visualized in the Western blot of Figure 1, which include those antigenic components identified with molecular weights selected from the group consisting of about 17-19 »28- 30 »33» 37 »46, 48 and 56 kD. said antibodies may be useful as reagents for the differential diagnosis of neosporosis, for the detection of Neospora-specific antigens in histological sections or in cells »samples of tissues or fluids from an animal, as for example» in ELISA or Western blotting or to quantitate the amount of antigen in a vaccine preparation Antibodies can be obtained against any of the antigenic components present in a Neospora 5 cell homogenate such as those of the NSA preparation described below. Various host animals can be used, including but not limited to cows, horses, rabbits, goats, sheep and mice, according to known immunological techniques to produce antibodies against one or more antigenic components specific to Neospora. Various adjuvants, such as those described above, can be used to increase the immune response to stimulate antibody production. Potential antibodies can be obtained from immunized animals that have been tested for -EO determine the specificity against the specific antigenic components of Neospora using standard techniques. On the other hand »monoclonal antibodies to a specific antigenic component of Neospora can be prepared using any technique that provides for the production of antibody by continuous cell lines in culture. These include »but are not limited to the technique of I hybridoma, originally described by Kohler and Milstein (Nature, 1975, 256: 495-497); the human B-cell hybridoma technique (Kosbor et al., 1983, In unology Today 4:72; Cote et al., 1983, Proc. Nati Acad. Sci. USA 80: 2026-2030) and the EBV hybridoma technique (Cole et al., 1985, Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96). On the other hand »the techniques described for the production of single chain antibodies (see for example» the US Pat. No. 4,946,778) can be adapted to produce single chain antibodies specific for Neospora antigen. These publications are incorporated herein by reference. Antibody fragments containing specific binding sites with a specific Neospora antigenic behavior are also included within the scope of the present invention and can be obtained by known techniques. Such fragments include but are not limited to the F (abt) .- »fragments, which can be obtained by reducing the disulfide bridges of the F (ab ').?» Fragments. On the other hand »Fab expression banks (Huse et al. 1989» Science 246: 1275-1281) can be constructed to allow rapid identification of Fab fragments having the desired specificity for a Neospora-specific antigen. The antibodies and antibody fragments of the present invention may further contain a detectable marker "as a fluorescent signal" radioactive label or enzyme "as is known in the art" to facilitate the detection of an antibody specifically bound in any of the assays diagnoses mentioned above. Techniques for production and use of monoclonal antibodies and antibody fragments are well known in the art and are described more extensively "among others" in Harlow and Lane, 1988, Antibodies: a Laboratory Manual, Cold Spring Harbor Laboratory and in J.W. Goldin »1986, Monoclonal Antibodies: Principles and Practice, Academic Press, London, which are incorporated herein by reference.

The following examples are offered to further illustrate, but are not limited to, the compositions and methods of the invention.

EXAMPLE 1 Preparation of the vaccine against Neospora Maintenance of Neospora cultures Tachyzoites from N. caninum strain NC-1 were maintained in MARC-145 monkey kidney cell monolayers (USDA »ARS, Clay Center, NE) in tissue culture flasks at 37 ° C and CO ,,, 5% in Opti-MEM ™ medium (Gibco BRL) containing 1% FBS (v / v). 100 U / ml of penicillin »100 μg / ml streptomycin and 2 mM glutamycin. The tachyzoites were harvested from the infected cell cultures when approximately 60-90% of the MARC-145 host cells had been lysed »as determined by microscopic examination by observing the cytopathic effects in the monolayers. The remaining infected cells containing intracellular tachyzoites were removed using a cell scraper and combined with e! culture medium containing free extracellular tachyzoites. The preparation (infected cells plus free tachyzoites) was centrifuged (1.876 x g »10 min, 4 ° C) and the pellet was resuspended in 5 ml of Hank's balanced salt solution (HBSS) (Gibco BRL). The suspension was passed five times through * a 22-gauge needle, centrifuged as before, resuspended in 5 ml of HBBS and passed five times through a 28 gauge needle. The material was centrifuged as before and the sediment was resuspended in 5 ml of HBBS, followed by passage through a sterile 5 μM filter to remove cellular debris from the host. The material was centrifuged as before, the parasitic sediment containing the free tachyzoites was resuspended in HBBS and the total number of viable tachyzoites was determined using an O acymeter and trypan blue.

Preparation of the homogenate (Neospora antigen) The viable tachyzoites prepared as described above were adjusted to a cell density 5 of 2 x 10s / ml in Dulbecco's phosphate buffer (DBPS) saline. For each ml of tachyzoite suspension, 5 μl of a protease inhibitor stock solution A and B were added. The protease A inhibitor stock solution contains 1 ml of EDTA solution at 5 ml of Hz0) and 4 ml of H- jO. The stock solution 0 of protease inhibitor B contains 1 ml of NEM (N-ethyl maleimide) solution (prepared by adding 312 mg of NEM (Sigma Che ica! Co.) to 2.5 ml of ethanol), 1 ml of pepstatin solution ( prepared by adding 3.43 mg of pepstatin (Sigma) to 5 ml of ethanol) »3 ml of PMSF solution (5-phenyl fluoride Imeti-Isulfonyl) (prepared by adding 291 mg of PMSF (Sigma) to 5 ml of ethanol) and 1 ml of TPCK (N-tosyl-L-phenylalanine chloromethyl ketone) (prepared by adding 176 mg of TPCK (Sigma) to 5 ml of ethanol). The tachyzoite preparation (-20 ° C) was frozen and thawed (room temperature) three times and then shaken (Branson So if? Er 250, Branson Inc.) with a constant yield (4 minutes / cycle) during three cycles on ice. AND! The resulting homogenate was called the Neospora antigen (NSA) preparation. The protein concentration of the NSA preparation was determined using a commercial O (Pierce BCA) assay. They were prepared in the NSA preparation and stored at -20 ° C or -70 ° C until further use for the vaccine and for in vitro tests (eg, Western blotting, cell proliferation). The NSA preparation did not contain any viable tachyzoite, as could be determined by the lack of in vitro growth in MARC-145 cells and by the inability to kill mice with the nu / nu and immunodeficiency gene.

Formulation of the vaccine The vaccine tested herein contains the NSA preparation prepared as described above and a veterinarily acceptable adjuvant. One of the two adjuvants, SEAM62 or SEAMl / 2, was used as adjuvant. SEAM62 is an oil-in-water emulsion containing 5% squalene (v / v) 5 (Sigma), detergent SPANR 85 AL 1% (v / v) (ICI Surfactants), detergent TWEENR 80 0.7% (v / v) (ICI Sur actants), ethanol a! * 2.5% (v / v), 200 μg / ml Quil A, 100 μg / ml cholesterol, lecithin a! 0.5% (v / v) and thimerosal at 0.01% (Sigma). SEAM1 / 2 is an oil-in-water emulsion containing 5% squalene (v / v). SPANR detergent at 1% (v / v), detergent TWEEN * SO at 0.7% (v / v), ethanol at 2.5% (v / v), 100 μg / ml Quil A, 50 μg / ml cholesterol and thimerosal at 0.01%. The vaccines were prepared by adding equal volumes of the NSA preparation and the adjuvant (SEAM64 or SEAM1 / 2), then mixing gently and stored at A ° C for the primary and recall mediations. The protein concentration in the final vaccine in both experiments was 250 μg of NSA protein / ml. Control vaccines contained adjuvant alone (SEAM62 in Example 2 presented later, SEAM1 / 2 in Example 3 presented below). EXAMPLE 2 IMMUNIZATION AND EXPOSURE OF IMMUNOCOMPETENT MICE The purpose of this two-part study was to demonstrate the ability of a homogenate of Neospora cells, in this case of tachyzoites of the NC-1 strain of N. caninum to induce a protective response in an immunocompetent mouse. In this first part of the study, 8-week-old female BALB / c mice (n = 10 / group) were immunized in the -5 day O and again on day 21 or with the adjuvant SEAM62 only (control) or with the NSA preparation plus the SEAM62 adjuvant (vaccine). Fifteen days after the last immunization, individual serum samples were collected at random from 3 mice per group and stored at -20 ° C for analysis of parasite-specific antibodies by Western blot (Figure 1). The post-immunization Western blot analysis was performed as follows. The NSA preparation was fractionated alongside the molecular weight markers (Novex »San Diego» CA) under standard non-reducing conditions by preparative gel electrophoresis (SDS-PAGE) using previously molded 12% sodium dodec sulfate 1 -pol gels. iacri lick (Novex). After electrophoresis, the separated proteins were transferred to a PVDF membrane (Millipore »Bedford» MA) which was then rinsed in washing buffer (phosphate buffer saline (pH 7.5) / 0.5% Tween 20 detergent. ) »Was air dried and individual membrane strips were cut (approximately 8 μg of NSA / strip protein). The strips were incubated overnight at room temperature in blocking buffer (wash buffer containing 5% skim milk). After two brief washings the strips were incubated for 1 h at room temperature with the primary antiserum samples (dilution 1: 200 in wash buffer) obtained at 15 days after the last immunization from 3 individual mice of the control group (adjuvant) (figure 1, columns 1-3) or from 3 vaccinated mice (figure 1 »columns 4-6). After rinsing twice with wash buffer, the strips were incubated with goat anti-mouse IgG conjugated with alkaline phosphatase (Kirkegaard &Perry) (1: 10,000 dilution in wash buffer) for 1 h at room temperature, rinsed twice with wash buffer and n immunoreactivity proteins were detected using the chromogenic substrate BCIP / NBT (5-bromo-4-c! gold-3-i dol i 1-phosphate / tetrazole nitro blue) (Kirkegard & amp; Perry). As can be seen in the Western transference of Figure 1, the serum antibodies collected from the three mice IO immunized with the most adjuvant NSA preparation (columns 4-6) reacted with the proteins of the NSA preparation having molecular weights of about 17-19 »28-30» 33 »37» 46 »48 and 56 KD. The immunoreactive profiles of the serum samples of the three mice immunized with the NSA preparation plus adjuvant were essentially identical with each other and was due to the administration of the NSA preparation »since it was not W • detected NSA-specific antibodies in control cough mice that were only given adjuvant (columns 1-3). 20 In the second part of the study, 4-week-old female BALB / c mice (n = 16 / group) were immunized in e! day O and on day 14 or with the NSA preparation plus the adjuvant SEAM1 / 2 (vaccine) or with the SEAM1 / 2 adjuvant alone (control). Seven days after the last immunization » randomly selected 4 donor mice from each group. Serum was collected from each mouse »was combined and stored at 20 ° C 3B until e! moment of its analysis by i muno luorescence. The splenocytes of the donor groups were then prepared using the standard procedures. In summary, the assembled spleens of each group were disaggregated using mesh sieves for tissues to obtain a single cell suspension and the erythrocytes were lysed in 0.83% HCl at Tris. After counting the viable cells »aliquots were used of each of the two preparations of combined splenocytes (vaccine »control) for the antigen-specific lymphocyte proliferation and cytokine proliferation assays» as described below. The rest of the combined splenocytes were used for an adoptive transfer to nude mice deficient in T cells (see example 3 below). The rest of the mice were sub-divided into 4 groups (n = 6 / group) for subsequent exposure. Day 22 post-immunization, to the groups (n = 6 / group) of BALB / ca mice that had been administered the NSA preparation plus the adjuvant SEAM1 / 2 or the adjuvant SEAM1 / 2 were only stimulated subcutaneously with 1 x 10 3 or 1 x 10"7 NC-1 tachyzoites Three weeks after challenge "individual serum samples were collected from all the mice" the mice were euthanized and their tissues were individually harvested (spleen, lung and brain) for further processing and perform the tests, as described below.

• The assays for the titration of immunofluorescent antibodies (IFA) before and after exposure were carried out in the following manner. Viable NC-1 tachyzoites (5 x 10"') were added to each of the wells of a 96 well flat bottom plate The wells were air dried and the plates were stored at -20 ° C until use. Serum test samples collected on day 21 post-immunization (day O exposure) and day 21 after challenge were assayed for IFA titers.Since with an initial serum dilution of 1:50, serial double dilutions were added. to the wells and incubated for 30 min at room temperature After two washes in carbonate buffer for rinsing, the wells were incubated with anti-mouse IgG conjugated with fluorescein isothiocyanate (Fab) s + IGM (Southern Biotechnology, Birmingham., AL.) The plates were washed and 50 μl of 50% glycerol diluted in rinse buffer was added to each well The plates were stored at 4 ° C until they were observed in a fluorescence microscope equipped with a filter for e. mission at 510 nm The titres of the antibodies are based on the highest dilution of the immune serum which produces a detectable fluorescence. Based on the results of the IFA titration assays, the vaccine of the invention is able to induce a humoral immunity response that results in the production of antibodies reactive towards the complete tachyzoites (Figure 2). The mean IFA titre of 4 combined randomized serum samples from mice immunized with vaccine was > 25,000 compared to the average title of < 50 obtained by using the combined serum of the control mice. The geometric mean titres after the explosion were significantly higher in the vaccinated animals than in the controls (P <0, OOl) and higher than the titres before exposure, which indicates that the vaccine induces a memory of immune response in the animals vaccinated to which the booster dose was administered after the subsequent exposure with the parasite. The ability of the NSA preparation to induce the cellular immune response (T cells) was determined as follows. Seven days after the last immunization, the mice were euthanized once they had been injected with adjuvant only (control) and the mice that had been injected with the more adjuvanted NSA preparation (vaccine) and were removed. the spleens. The combined spleens (4 per group) were disaggregated using mesh sieves for tissues to obtain a single-cell suspension, followed by lysis of the erythrocytes in 0.83% HCl Cl buffered with Tris. After counting the viable cells »the cells were suspended in complete RPMI 1640 medium containing 10% heat-inactivated SBF» penicillin (100 U / ml) »streptomycin (10 μg / ml), 5 x 10_β M of β - ercaptoethanol, 2 nM L-glutamine, 5 μg / ml insulin, 1 μg / ml transferrin and 10 μg / ml selenium. For proliferation assays, the cells were placed in 96-well flat bottom plates at the rate of 5 x 10H cells per well. The cells were incubated with complete medium with or without 5-fold serial dilutions of the NSA preparation, starting with 10 μg of NSA / ml protein in quadrupled wells in a final volume of 200 μl. The plates were incubated at 37 ° C in CO-2 7% for 72 hours. Proliferation of T ucfoctes was determined by giving a pulse to the splenocyte cultures of 0.33 μCi of C3H-3 thymidine for another 18 to 24 hours. The cells were harvested on filters using a MACHIII cell harvester (TomTech, Orange, CT.) And the radioactivity incorporation was determined with a scintillation counter (Wallac »Turku, Finland). The results are 'expressed in figure 3 as \ cpm (mean cpm with NSA less cpm medium with medium only). Splenocytes from vaccinated mice, but not of the control mice, prolified vitro after exposure with the NSA preparation. These results of the in vitro cell proliferation assay demonstrate that the vaccine of the present invention can induce cellular immunity responses (T cells). For the cytokine assays, the cells were placed in 96-well flat bottom plates at the rate of 5 x 10 cells per well. The cells were incubated with complete medium with or without the NSA preparation (final concentration of 1 μg NSA protein / ml) in quadrupled wells in a final volume of 200 μl. The plates were incubated at 37 ° C in C0-. 7% and supernatants devoid of cells were collected at 24-hour intervals for 4 days and stored at -20 ° C until the time of their assay. The presence of specific cytokines in the collected samples was determined by a two-site immunosorbent assay (ELISA) using a commercial panel of unconjugated antibodies and cytokine-specific conjugates, recombinant cytokine patterns before and protocols suggested by the manufacturer (PharMingen » San Diego »CA.), The results are expressed in pg / ml» from which the background activity of the cytokine was subtracted in the wells of cells incubated without NSA. The production before exposure of splenic antigen-specific cytokines from the donor is shown in Fig. 4. "demonstrating that the vaccine of the present invention can induce both types of cellular immunity responses" of type 1 (IFN-, IL-2 ) and type 2 (IL-6 »IL-10), after immunization. Induction of IFN - ¡>; it is a remarkable entity »since recently it has been shown that this cytokine plays a protective role against murine neosporosis (Khan et al.» 1997 »Experimental Parasitology, 35: 24-34), likewise» IFN-t > it seems that it is necessary for the protection of the host against the related parasite Apicomplexa, T. gondi i (Suzuki et al. 1988, Science 240: 516-518). Induction of IFN- by the vaccine of the present invention may also be involved in the ability of the vaccine to protect munocompetent mice »as well as in its ability to induce memory T cells capable of secreting IFN- after transference The ability of the killed vaccine to induce IL-6 and IL-10 may also be important in the ability of the vaccine to adopt such cells in aty ictic and immunodeficient mice as described in the following example 3. to protect against neosporosis »since both cytokines have been shown to play an important role in protecting the host against? _ lt) gond i (Suzuki and others» 19 7, In. Immun. 65: 2339-2345; t Neyer and others »1997» In ect. Immun. »65: 1675-1682). The ability of the vaccine of the present invention to induce cellular immunity responses (T cells) was also established by examining day 21 after the shows the antigen-specific splenic proliferation as described below for day 21 post-immunization of the mice. As can be seen in figure 5, the responses of the T lymphocytes to the NSA preparation after exposure with 1 x 10 = or 1 x 10"7 tachyzoites NC-1 were significantly higher (P <0.01, P <0.05) in the vaccinated animals than in the controls. The ability of the vaccine of the invention to protect animals against Neospora-induced encephalitis was determined by measuring the number of lesions in the sections of lung and brain tissue after infection of the vaccinated mice and control with 2 different exposure doses. On # 21 day after exposure, pulmonary and cerebral tissues (6 / group) were individually collected and fixed in 10% formalin with neutral buffer, sectioned and stained with hematoxylin and eosin, using histological techniques serious. The stained lung and brain sections were coded and blindly scored without having prior knowledge of the treatment. Scores were given to the lung and brain lesions using the following system: (O) within normal limits, (1) light, (2) mild, (3) moderate, (4) severe, and (5) marked. The scores of the brain and lung lesions after exposure of the BALB / c mice are presented in Figure 6 (ad) »demonstrating that the animals immunized with the vaccine of the present invention have significantly less lung lesions (P < 0.01) and cerebral (P <0.05) compared to controls after a parasitic exposure of 1 x 10"7 tachyzoites, as well as" average scores of brain and lung lesions are numerically higher in control mice compared with mice vaccinated after a parasitic exposure of 1x10 s tachyzoites NC- The difference in statistically significant differences in lesion scores between vaccinated mice and control mice with an exposure dose of 1 x 1045 can attributed to a mouse isolated from the vaccine group that had a score of 2 for both the lung and the brain.

EXAMPLE 3 Exposure of mice and immunode icts after the adoptive transfer of splenocytes The objective of this study was to determine whether splenic lymphocytes from vaccinated immunocompetent mice could be used for an adoptive transfer of ^. protection of nude mice immunodeficient in T cells (nu / nu mice or "naked" mice, Charles River Labs) »such and ID as demonstrated by a higher survival after a virulent NC-1 exposure. Each of the nude mice (n = 7 / group) received an intravenous injection of 1? 10"7 splenocytes (in 0.1 ml of DPBS) obtained from vaccinated BALB / c mice or control (adjuvant) seven days after the last immunization. The nu / u control mice received only DPBS? R • (0.1 ml). On day 22 (24 hours after the transference) all the nude mice were stimulated subcutaneously with x 10 s tachyzoites NC-1. Naked mice stimulated were controlled to determine the existence of clinical signs of neosporosis and death from day 14 after exposure. Survival curves after exposure of nude nude mice are presented in Figure 7.

Nude mice that had received DBPS alone (non-splenocytes = "non-cells") were highly susceptible to exposure with the NC-1 tachyzoites. On day 21 after challenge "none of the mice in this group had survived. The nude mice that had received splenocytes from the BALB / c mice injected with them or the NSA preparation plus adjuvant or adjuvant alone, lived longer. Eighty percent of the nude mice that had received splenocytes from the BALB / c ^^ mice injected with adjuvant only succumbed w finally to infection and only 1 mouse from this group ID was alive at the end of the riment (day 48 after the exhibition). In contrast, 100% of the nude mice that had received splenocytes from the BALB / c mice injected with the most adjuvant NSA preparation survived parasitic sure throughout the entire rimental period. These results demonstrate that the splenocytes from the vaccinated donors are capable of conferring adoptive protective immunity against neosporosis, providing further evidence of the efficacy of the vaccine of the present invention. All patents, patent applications and publications cited above are incorporated herein by reference in their entirety. The present invention is not limited in scope by the specific embodiments described, which are intended to be only lustrations of the individual aspects of the invention. Unionally equivalent compositions and procedures are within the scope of the invention. Thus, the various modifications of the invention, in addition to those presented and described herein, will be apparent to those skilled in the art from the foregoing description. It is intended that such modi cations be within the scope of the accompanying indications.

Claims (26)

# NOVELTY OF THE INVENTION CLAIMS

1. - A homogenate prepared from Neospora cells »which is able to induce a protective response against neosporosis in a mammal.

2. The homogenate of claim 1 wherein the Neospora species from which the homogenized XO is prepared is N. caninum.

3. The homogenate of the rei indication 1 »which is able to induce the production of antibodies that recognize one or more antigenic components present a homogenate of cells of the strain NC-1 of N. caninum. 15 4.- The homogenate of the rei indication 3, in which the Neospora species from which the homogenate is prepared is N. camnum 5.- The homogenate of the reagent 4, in the that the strain of N. caninum from which it is prepared in homogenate is 1 to NC-1 6.- The homogenate of reiviication 1 which is prepared from tachyzoites 7.- A vaccine for protect a mammal against neosporosis »which comprises a quantity munologically 25 effective of a homogenate prepared from cells of Neospora »being this homogenate capable of inducing a protective response against neosporosis in a mammal and a veterinarily acceptable vehicle. 8. The rei-indication vaccine 7, in which the Neospora species from which the homogenate is prepared is N. caninum. 9. The vaccine of claim 7 which is able to induce the production of antibodies that recognize one or more antigenic components present in a homogenate of cells of the strain NC-1 of N. caninum. 10. The vaccine of 1 a rei indication 9, in which the Neospora species from which it is prepared e. homogenate of 1 to vaccine is N. caninum. 11. The vaccine of reiication 10, in which the strain of N. caninum from which the homogenate of the vaccine is prepared is NC-1. 12. The vaccine of claim 7, wherein the homogenate is prepared from tachyzoites. 13. The vaccine of claim 7, which additionally contains one or more immunomodulatory components. 1

4. The vaccine of claim 13 »in which the component munomodu! Additional designer is an adjuvant. 1

5. The vaccine of re-indication 14, wherein the adjuvant is selected from the group consisting of the RIBI adjuvant system (Ribi Inc.), alum »aluminum hydroxide gel» oil-in-water emulsions »an emulsion from oil to water »a water-in-oil emulsion» # block copolymers »QS-21» SAF-M, the adjuvant AMPHIGENR < saponin Quil A »monophosphor 1 lipid A and adjuvant 1 ipido-arninic Avridina. 1

6. The vaccine of claim 15, wherein the adjuvant is an oil-in-water emulsion selected from the group consisting of SEAM62 and SEAM1 / 2. 1

7. The rei-indication vaccine 13"in which the additional immunomodulatory component is a cytokine. 1

8. Vaccination vaccine 7, in which the lt) Neospora cells from which the homogenate is prepared, have been modified to suppress the expression of one or more antigenic components normally associated with the cells of Meospora or a homogenate prepared from them. 1

9. A method for the preparation of a vaccine that protects a mammal against neosporosis, which * comprises the homogenization of the Neospora cells to form a homogenate capable of inducing a protective response against neosporosis in a mammal and the 0 combination of an immunologically effective amount of the homogenate with a veterinarily acceptable carrier. 20.- The procedure of the rection 19 »in which the Neospora species from which the homogenate is prepared is N. caninum. 21. The method of claim 19, wherein the vaccine is capable of inducing the production of antibodies that recognize one or more antigenic components present in a homogenate of cells of the NC-1 strain of N. caninum. 22. The method of claim 21, in which the Neospora species from which the homogenate of the vaccine is prepared is N. caninum. 23. The method of claim 22, in e! that the strain of N. caninum from which the homogenate of the vaccine is prepared is NC-1. 24.- The procedure of the rei indication 19, in the ID that the cells that are homogenized are tachyzoites. 25.- The procedure of rei indication 24, in e! that the tachyzoites are homogenized by ionization / thawing and sonication. 26.- E! rei-indication procedure 19, which 15 comprises, in addition, the addition of one or more immunomodulatory components to the vaccine. 27, The method of claim 26, wherein the additional immunomodulatory component is an adjuvant. 28. The method of claim 26, wherein the additional immunomodulatory component is a cytokine. 29.- The use of a homogenate prepared from Neospora cells, in combination with a veterinarily acceptable vehicle, to prepare a vaccine to protect a mammal against neosporosis, said homogenate 5 is able to induce a protective response against neosporosis in said mammal. # 30.- The use of claim 29, in which the species of Neospora from which the homogenate is prepared is N. caninum. 31. The use of claim 29, wherein the vaccine is capable of inducing the production of antibodies that recognize one or more antigenic components present in a homogenate of cells of the NC-1 strain of N. caninum. 32. The use of claim 31, wherein the Neospora species, from which the * 0 homogenate from 1 to vaccine is prepared "is N. caninum. 33. The use of claim 32, in which the N. caninum strain »from which the homogenate of the vaccine is prepared, is NC-1. 34.- The use of claim 29 »in which the homogenate of the vaccine is prepared from tachyzoites. 35.- The use of claim 29 »wherein the vaccine additionally contains one or more immunomodulatory components. 36.- The use of reivi cation 35, in which the 0 additional immunomodulatory component is an adjuvant. 37. The use of claim 35, wherein the additional immunomodulatory component is a cytokine. 38.- The use of re ind cation 29, in which the vaccine is administered to a mammal of a selected species 5 among the group consisting of dogs »cows» goats, sheep and cabal! os. # 39.- A combination vaccine for the protection of a mammal against neosporosis and »optionally, one or more diseases or pathological conditions that may affect the mammal» whose vaccine combination contains an immunologically effective amount of a first composition containing a homogenate prepared from Neospora cells »which is capable of inducing a protective response against neosporosis in a mammal» an immunologically effective amount of a second composition capable of inducing a protective response against a disease or pathological disorder which may affect to a mammal »and an acceptable veterinary vehicle. 40.- The combination vaccine of the reification 39 »in which the Neospora species from which it is prepared e! homogenized from the first composition is N. cani num. 41. The combination vaccine of claim 39 which is able to induce the production of antibodies that recognize one or more antigenic components present in a homogenate of cells of the NC-1 strain of N. caninum. 42.- The combination vaccine of claim 41 »wherein the Neospora species from which the homogenate of the first composition is prepared is N. cani nu. 5 43.- The combination re-indication vaccine 42 »in which the N. caninum strain from which it is prepared in homogenized from the first composition is NC-1. 44.- The combination vaccine of claim 39 »in which the homogenate of the first composition is prepared from tachyzoites. 45.- The combination vaccine of the claim 39 »which additionally comprises one or more components in unmodules. 46.- The combination vaccine of claim 45 »wherein the additional immunomodulatory component is an IX) adjuvant. 47.- The combination vaccine of claim 45 »wherein the additional immunomodulatory component is a cytokine. 48.- The combination vaccine of the claim 39"in which the second composition is capable of inducing a protective response in the mammal against a pathogen selected from the group consisting of e. bovine herpes virus »bovine respiratory syncytial virus» bovine viral diarrhea virus »parainf 1 type I, II or III virus, 20 Leptospira spp. , Ca pylobacter spp. , Staphy1 ococcus aureus, Streptococcus agal acti ae, Mycopl asthma spp. , Klebsiel 1 a spp. , Sal monel la spp., Rotavirus, coronavirus »rabies, Pasteurel 1 a haemolyt ca, Pasteurel 1 a muí ocida, Cl ostridia spp., Toxoid Tetanus »E. cabbage, Cryptosporidi um spp. - E me a spp. , Y 25 Neospora spp. 49.- A team for the vaccination of a mammal against neosporosis, which contains a first container with an immunologically effective amount of a homogenate prepared from Neospora cells, this homogenate being capable of inducing a protective response against neosporosis in a mammal and a second container having a vehicle or dye veterinar amente acceptable. 50.- The kit of claim 49, wherein the Meospora species from which the homogenate is prepared is N. caninum. The equipment of claim 49, wherein the homogenate is capable of inducing the production of antibodies that recognize one or more antigenic components present in a homogenate of cells of the NC-1 strain of N. caninum. . 52. The equipment of claim 51, wherein the Neospora species from which the homogenate is prepared is N. caninum. 53. The equipment of claim 52, wherein the strain of N. caninum from which the homogenate is prepared is 1 to NC-1. 54. The equipment of claim 49, wherein the homogenate is prepared from tachyzoites. 55.- The equipment of claim 49, in which the homogenate is in lyophilized form. 56.- A specific antibody of an antigenic component present in a homogenate of Neospora cells. 57.- The reagent antibody 56, which is specific for an anti-co gene component present in a homogenate prepared from N. cani um cells. 58.- The rei-indication antibody 57, which is specific for an antigenic component present in a homogenate prepared from cells of the NC-1 strain of N. caninum. 59. The antibody of claim 58, wherein the antigenic component has a selected molecular weight F between the group consisting of 17-19, 28-30, 33, 37, 46 »48 and ld 56 kD. The antibody of claim 56, which additionally contains a detectable marker.