Patent 6,908,620

The present invention relates to a vaccine for coccidiosis in chickens prepared from four attenuated Eimeria species: E. acervulina, E. maxima, E. mitis and E. tenella. The vaccine was similar to or superior to other anticoccidial drugs in stimulating protective immunity against coccidiosis.

Description of the Invention

FIELD OF THE INVENTION

The present invention relates to the preparation of immunogenic compositions and vaccines against diseases caused by coccidia. The present invention also provides for attenuated vaccines against coccidiosis.

BACKGROUND OF THE INVENTION

Coccidiosis is a disease caused by infection with one or more of the many species of coccidia which is a subdivision of the phylum Protozoa, intracellular protozoal parasites of the subphylum Apicomplexa and the genus Eimeria. The genus Eimeria contains the species of major economic importance in domestic birds, such as chickens, ducks, geese, guinea fowl, peafowl, pheasants, pigeons and turkeys. While coccidiosis occurs in practically all kinds of birds, the parasites are host specific and each species occurs in a single or in a limited group of related hosts. On the other hand, avian hosts are known to harbor more than one species of coccidia. Species of Eimeria that cause coccidiosis in chickens include E. acervulina, E. brunette, E. hagani, E. maxima, E. mitis, E. mivati, E. necatrix, E. praecox and E. tenella. E. acervulina is one of the most common species found in the litter of broiler houses. It has a great reproductive potential and is regarded as pathogenic because it produces a marked depression in gain of body weight, higher feed conversion and it produces gross lesions in the upper small intestine.

Among domesticated birds, chickens are the most susceptible to significant economic losses from coccidiosis, although losses can also occur within turkeys, geese, ducks, and guinea fowl. Coccidiosis has also produced serious losses in pheasants and quail raised in captivity. The effects of a coccidiosis infection can take the highly visible form of devastating flock mortality, but another undesirable effect is morbidity and/or weight loss which results from infection.

During the life cycle, the Eimeria parasite passes through a number of stages (see, e.g., U.S. Pat. No. 6,100,241 for an overview). The life cycle begins when the chicken ingests the infectious stage, known as the sporulated oocyst, during ground feeding or by inhalation of dust. The wall of the sporulated oocyst is ruptured by a combination of mechanical grinding action and chemical action in the gizzard and intestinal tract, resulting in the release of four sporocysts. The sporocysts pass into the duodenum where they are exposed to bile and digestive enzymes resulting in the release of two sporozites per sporocyst.

The sporozoites are mobile and search for suitable host epithelium cells in order to penetrate and reproduce in them. Following infection of an epithelium cell, the parasite enters the schizont phase of its life cycle, producing from 8 to 16 to >200 merozoites per schizont. Once released from the schizont, the merozoites are free to infect further epithelium cells. After two to five of these asexual reproduction cycles, the intracellular merozoites grow into sexual forms known as the female or macrogametocyte and the male or microgametocyte. Following fertilization of the macrogametocyte by the microgametes released from the microgametocyte, a zygote is formed which creates a cyst wall about itself. The newly formed oocyst is passed out of the infected chicken with the fecal droppings.

With the correct environmental conditions of temperature and humidity and sufficient oxygen in the air, the oocyst will sporulate into the infectious stage, ready to infect a new host and thereby spreading the disease. Thus, no intermediate host is required for transfer of the parasite from bird to bird.

The result of the Eimeria parasite infecting the digestive tract of a chicken may be a reduction in weight gain, increased feed conversion, cessation of egg production and, in some cases, death. The increase in intensive production of poultry has been accompanied by severe losses due to this parasite; indeed, coccidiosis has become an economically important parasitic disease.

In the past, several methods have been used in attempts to control coccidiosis. Prior to the advent of chemotherapeutic agents, improved sanitation using disinfectants, together with the mechanical removal of litter, was the main method employed; sufficient oocysts, however, usually remained to transmit the disease. The introduction of coccidiostatic agents in the feed or drinking water, in addition to good management, resulted in some success at disease control. Such agents have been found to suffer from a drop in effectiveness over the years, due partly to the development of drug resistant strains of coccidia. Furthermore, several chemotherapeutic agents have been found to leave residues in the meat, making it unsuitable for consumption.

U.S. Pat. Nos. 4,438,097; 4,639,372; 4,808,404; 5,055,292; 5,068,104; 5,387,414; 5,602,033; 5,614,195; 5,635,181; 5,637,487; 5,674,484; 5,677,438; 5,709,862; 5,780,289; 5,795,741; 5,814,320; 5,843,722; 5,846,527; 5,885,568; 5,932,225; 6,001,363 and 6,100,241 relate to coccidiosis vaccines, including live and recombinant vaccines. However, there are problems with existing coccidiosis vaccines, such as reduced efficacy, cross-infection with other parasites (e.g., Clostridium spp.) and poor bird performance. Thus, there exists a need for efficacious coccidiosis vaccines with reduced or non-existent cross-infection that do not adversely affect bird performance.

Citation or identification of any document in this application is not an admission that such document is available as prior art to the present invention.

SUMMARY OF THE INVENTION

DETAILED DESCRIPTION OF THE INVENTION

This invention is based, in part, on an attenuated coccidiosis vaccine that is efficacious in the face of virulent challenge, reduced cross-infection with Clostridium spp. and has better bird performance as defined by feed conversion rates when compared to other coccidiosis vaccines.

The invention relates to a mixture of sporulated oocysts from precocious strains of E. acervulina, E. maxima, E. mitis and E. tenella. Sporulated oocysts were isolated from a seed culture harvested from one or more chickens seeded with a culture of a precocious strain of E. acervulina, E. maxima, E. mitis or E. tenella, i.e., one or more chickens are seeded with either a precocious strain of E. acervulina, E. maxima, E. mitis or E. tenella resulting in four groups of chickens, each seeded with a different Eimeria strain.

Advantageously, the Eimeria strain is a precocious strain. Precocious strains are derived from field species are not pathogenic when administered at the right dose. In an advantageous embodiment, the Eimeria strain is a precocious strain of the respective microorganism as described in Avian Pathology, 17: 305-314, 1988 entitled "Eimeria of American Chickens: Characteristics of Six Attenuated Strains Produced by Selection for Precocious Development, P. L. Long and Joyce K. Johnson, the disclosure of which is incorporated by reference in its entirety. Microorganisms can be attenuated by their selection for precocious development as described as described in Avian Pathology, 17: 305-314, 1988 entitled "Eimeria of American Chickens: Characteristics of Six Attenuated Strains Produced by Selection for Precocious Development, P. L. Long and Joyce K. Johnson, the disclosure of which is incorporated by reference in its entirety. Briefly, the microorganisms are attenuated by selecting for an earlier pre-patent period, i.e., when the oocysts first show up in the feces. Such methods are well known to one of skill in the art and constitute routine experimentation.

Stock cultures of the Eimeria strains for the seed cultures include, but are not limited to, the following. The parent of the Eimeria acervulina, obtained from T. K. Jeffers at Hess and Clark Laboratories in 1969, was thought to have been isolated by Dr. M. M. Farr at USDA, Beltsville, Md., which was derived from a single oocyst. The Eimeria maxima culture was derived from an interbred mixture of 10 purified isolates obtained from Georgia, Delaware, Maryland, Virginia and Texas. The parent of the Eimeria mitis culture was isolated from Gainesville, Ga. in July 1978 and was purified by single oocyst isolation. The parent of the Eimeria tenella culture was obtained from a culture maintained at Pennsylvania State University by Dr. Patten since the early 1960's, and was acquired by the University of Georgia in 1982. Other precocious Eimeria strains include LS100 precocious E. Acervulina isolate 809-13, and LS precocious E. mitis, obtained from Merck Research Laboratories, which were obtained from Dr. Peter Long. Alternatively, attenuated precocious Eimeria lines that have been deposited as sporocysts at the European Collection of Animal Cell Cultures ("ECACC") as patent deposits (see, e.g., U.S. Pat. No. 5,055,292, the disclosure of which is incorporated by reference in its entirety) are useful stock cultures to generate the Eimeria seed cultures described herein. Specifically, deposits of E. acervulina (deposit no. ECACC 86072203), E. maxima (deposit nos. ECACC 86112011 and 86112012), E. mitis (deposit no. ECACC 86072206) and E. tenella (deposit no. ECACC 86072201) as described in U.S. Pat. No. 5,055,292 are useful stock cultures for the seed cultures of the present invention.

Advantageously, the microorganisms are attenuated by their selection for precocious development as described above. In another advantageous embodiment, the culture is pathogen-free. The stock cultures described above are advantageously maintained in the liquid or vapor phase of liquid nitrogen. Such methods are known to one of skill in the art.

In an advantageous embodiment, the chickens are two to eight weeks old. Sporulated oocysts are passed successively, without limitations to the passage, in chickens until the number of oocysts are sufficient to be used as seed for production. Advantageously, the cultures should not be held for longer than 12 months in order to maintain viability/infectivity.

In an advantageous embodiment, dedicated facilities are maintained for each Eimeria species. Advantageously, a sufficient volume of sporulated oocysts (seed) is mixed with feed or alternatively, is administered orally to provide each chicken with a minimum dose. In an advantageous embodiment, about 5000 to about 15,000 oocytes are seeded per chicken to generate the seed culture.

The sporulated oocysts from the seed culture are isolated from the bird feces, advantageously by centrifugation. In an advantageous embodiment, the harvest is as follows. Droppings are homogenized at an approximate ratio of 10% (w/v) in water. Large particles are removed by passing homogenate through screens. Solids are separated by either centrifugation, screening or by holding at 5±3° C. up to 24 hours. If solids are separated by holding at 5±3° C., they are further concentrated by centrifugation. The supernatant is discarded, and the solids are resuspended in a saturated NaCl (80% w/v) solution in water. The resulting solution is centrifuged. The oocysts are collected (removed) from the top of the liquid, and resuspended in water. Optionally, the remaining liquid is diluted to 20-40% NaCl with water and centrifuged. The pellet is then resuspended in a saturated NaCl solution and re-centrifuged, until no additional oocysts are recovered. The oocysts are washed no more than twice. The oocysts are washed free of salt by repeated, resuspension centrifugation cycles-followed by resuspension in a 0.5% solution of sodium hypochlorite for 10 to 15 minutes. The oocysts are then washed free of the sodium hypochlorite solution by repeated (3×) centrifugation and resuspension steps. The final resuspension is made in a 2.5% aqueous solution of potassium dichromate (K₂C_rO₇). The oocysts were then transferred to sporulation vessels. Sporulation is facilitated by sparging the suspensions with air for a period not to exceed 72 hours at 27±3° C. Following sporulation the oocysts are held at 5±3° C. until the final product is produced.

In another embodiment, oocysts to be used in accord with the present vaccination method can be prepared by any of several methods known to those skilled in the a rt. Such methods include those described in J. F. Ryley at al., Parasitology 73:311-326, 1976, P. L. Long et al., Folia Veterinaria Latina VI#3, 201-217, 1976, and U.S. Pat. No. 6,627,205, the disclosures of which are incorporated by reference in their entireties. According to one method, commercial broiler chickens, approximately 2 weeks old, are infected with the Eimeria species of interest by oral gavage of an appropriate dose of sporulated oocysts. Well known procedures for collection and purification of oocysts from infected birds are then followed. For most species of Eimeria , feces are collected from infected birds 5-7 days post-infection, blended and filtered to remove debris, then centrifuged at a speed sufficient to pellet the remaining fecal material. The pellet is resuspended in a saturated salt solution, in which the oocysts float and most of the contaminating debris can be removed by centrifugation. The oocyst suspension is then diluted to lower the salt concentration. The oocysts are washed repeatedly to remove the salt and resuspended in potassium dichromate solution (2.5% w/v). The oocyst suspension is incubated at 29 C with shaking (e.g., 140 rpm) for approximately 72 hours to induce sporulation of the oocysts. Alternatively, the oocysts can be treated with sodium hypochlorite and then sporulated. The number of sporulated oocysts/ml is determined by direct count using a hemocytometer or McMaster slide, and the culture is stored under refrigeration until needed.

To prepare sporocysts, the potassium dichromate is removed from the oocyst suspension described above by repeated washing of the oocysts, which involves collection of oocysts by centrifugation and resuspending in deionized or distilled water. When the dichromate has been removed as judged by the lack of yellowish-orange coloration, the oocyst suspension is mixed with an equal volume of sodium hypochlorite (bleach) and incubated at room temperature for 15 minutes. The bleach is then removed by repeated washings, and the oocysts are resuspended in physiological saline or deionized water. Oocysts can be broken to release sporocysts using a variety of known techniques. For example, oocysts can be broken to release sporocysts by mixing the oocysts with glass beads of 1-4 mm diameter and shaking by hand, vortex mixer, or shaking incubator, or using a hand-held homogenizer. Unbroken oocysts and oocysts walls can be separated from the released sporocysts by differential centrifugation in 50% PERCOLL, a colloidal suspension of polyvinyl pyrrolidone coated silica particles (sold by Pharmacia Biotech) or 1 M sucrose as described in Dulski et al., Avian Diseases, 32: 235-239, 1988. The sporocysts can be used in the present vaccination method either mixed with or separated from the unbroken oocysts and oocysts walls. Advantageously, the dose of sporocysts is separated from the oocysts and oocysts walls.

In an advantageous embodiment, the specifications for an acceptable harvest of the seed culture are as follows. First, the ratio of sporulated oocysts to total oocysts was determined. Only harvests meeting or exceeding >40% sporulation are considered acceptable. Second, the size, shape and appearance of each oocyst harvest must be characteristic of the species intended to be produced. For example, parameters to be considered in characterizing the Eimeria species include, but are not limited to, DNA-based technologies, DNA buoyant density, enzyme variation, host and site specificity, immunological specificity, pathogenicity, pre-patent period and sporulation time (see, e.g., Long & Joyner, J Protozool. 1984 November; 31(4): 535-41 and Shirley, Acta Vet Hung. 1997; 45(3): 331-47, the disclosures of which are incorporated by reference).

The isolated sporulated oocysts from the seed cultures described herein are combined to formulate a mixture of sporulated oocysts from precocious strains of E. acervulina, E. maxima, E. mitis and E. tenella. Generally, the mixture is about 10 to about 1000 oocysts of E. acervulina, about 10 to about 100 oocysts of E. maxima, about 10 to about 1000 oocysts of E. mitis and about 10 to about 1000 oocysts of E. tenella. Advantageously, the range of sporulated oocysts in the mixture is about 125 to about 500 oocysts of E. acervulina, about 25 to about 100 oocysts of E. maxima, about 125 to about 500 oocysts of E. mitis and about 25 to about 500 oocysts of E. tenella. In one embodiment, a low dose is about 125 oocysts of E. acervulina, about 25 oocysts of E. maxima, about 125 oocysts of E. mitis and about 25 oocysts of E. tenella. In another embodiment, a medium dose is about 250 oocysts of E. acervulina, about 50 oocysts of E. maxima, about 250 oocysts of E. mitis and about 50 oocysts of E. tenella. In yet another embodiment, a high dose is about 500 oocysts of E. acervulina, about 100 oocysts of E. maxima, about 500 oocysts of E. mitis and about 100 oocysts of E. tenella.

The invention also relates to specific ratios of sporulated oocysts isolated from precocious strains of E. acervulina, E. maxima, E. mitis and E. tenella, wherein the ratio of E. acervulina:E. maxima:E. mitis:E. tenella is about 10:1 to 2:10:2 to 10 (i.e., for every 10 sporocysts of E. acervulina, there are about 1 to 2 sporocysts of E. maxima, about 10 sporocysts of E. mitis and about 2 to 10 sporocysts of E. tenella). Advantageously, the ratio of E. acervulina:E. maxima:E. mitis:E. tenella is about 5:1:5:1 (i.e., 10:2:10:2).

Advantageously, the mixture is about 500 oocysts of E. acervulina, about 50 to about 100 oocysts of E. maxima, about 500 oocysts of E. mitis and about 100 to about 500 oocysts of E. tenella. In a more advantageous embodiment, the mixture is about 500 oocysts of E. acervulina, about 100 oocysts of E. maxima, about 500 oocysts of E. mitis and about 100 oocysts of E. tenella.

Advantageously, the oocysts are suspended in a preservative consisting of a 0.01M phosphate buffered saline solution containing gentamicin. In another embodiment, the oocysts are suspended in any one of a variety of preservatives or organic acids such as, but not limited to, acetic acid, citric acid, potassium dichromate or propionic acid. For example, but not by limitation, sufficient sterile, 0.01M phosphate buffered saline containing not more than 30 mcg/ml gentamicin, is used to yield 2 ml per bottle for a 2,000 dose presentation, 5 ml per bottle for a 5,000 dose presentation and 10 ml per bottle for a 10,000 dose presentation. Advantageously, the oocysts are stored in sterile, borosilicate glass vials. For example, but not by limitation, the oocysts are aseptically filled into vaccine vials with a semi-automatic or automatic dispenser, stoppers are mechanically or manually inserted and aluminum seals are placed and crimped.

In another embodiment, oocysts are suspended in sterile distilled water containing a suspending agent, for example a polysaccharide suspending agent such as a gum, e.g. xanthan gum or gum acacia, a cellulose derivative, e.g. carboxymethyl cellulose, hydroxypropyl methyl cellulose or microcrystalline cellulose, carageenan, sodium alginate, pectin or starch; a polypeptide suspending agent such as gelatin; a synthetic polymer suspending agent such as polyacrylic acid; or a silicate suspending agent such as magnesium aluminium silicate (see, e.g., U.S. Pat. No. 5,055,292, the disclosure of which is incorporated by reference in its entirety).

The present invention also provides for verifying the sporulated oocysts are characteristic of the precocious strain of E. acervulina, E. maxima, E. mitis or E. tenella. Advantageously, all oocysts are attenuated, in that they are precocious. In another advantageous embodiment, the size, shape and appearance of each oocyst harvest must be characteristic of the species intended to be produced. In yet another advantageous embodiment, the mixture of sporulated oocysts is tested for purity, extraneous pathogens, and/or deaths or severe lesions of the test animals, e.g., chickens. The characteristics of the various Eimeria species are fully set out by Long P. L. and Reid W. M. (1982: A Guide for the Diagnosis of Coccidiosis in Chickens; University of Georgia Research Report 404) and Joyner L. P. (1978: Identification and Diagnosis, Avian Coccidiosis, Poultry Science Symposium No. 13, British Poultry Science Ltd), the disclosures of which are incorporated by reference in their entireties.

The invention also relates to testing the efficacy of the mixture of sporulated oocysts from precocious strains of E. acervulina, E. maxima, E. mitis and E. tenella. Advantageously, the testing relates to administering a challenge dose of about 100,000 to about 500,000 oocysts of E. acervulina and about 10,000 to about 100,000 oocysts of E. maxima, about 100,000 to about 500,000 oocysts of E. mitis, or about 10,000 to about 100,000 oocysts of E. tenella to the animal. In a more advantageous embodiment, the challenge dose is about 200,000 oocysts of E. acervulina and about 20,000 to about 50,000 oocysts of E. maxima, about 200,000 oocysts of E. mitis, or about 20,000 to about 50,000 oocysts of E. tenella.

The invention further provides for determining bird performance as defined by feed conversion rates as a result of administering the the mixture of sporulated oocysts from precocious strains of E. acervulina, E. maxima, E. mitis and E. tenella to the birds. Feed conversion efficiency is defined as the as pounds of feed to produce a pound of meat. A common result is about 1.90 or 2.00. One point in feed conversion in common lingo is 0.01, which equals about 0.5% (half of a percent). If the metric system is used, the feed conversion is Kg of feed per Kg of meat, and is still proportional to the above.

The present invention relates to immunizing a chicken, advantageously a broiler chicken. However, methods of making the vaccine described herein can be extrapolated to other animals infected by Eimeria , in particular avians such as, but not limited to, a chicken, duck, goose, guinea fowl, peafowl, pheasant, pigeon, quail or turkey, or in a less advantageous embodiment, a rabbit.

The invention encompasses an immunogenic or vaccine composition comprising a mixture of sporulated oocysts isolated from precocious strains of E. acervulina, E. maxima, E. mitis and E. tenella. The isolated sporulated oocysts are combined to formulate a composition of sporulated oocysts from precocious strains of E. acervulina, E. maxima, E. mitis and E. tenella. Generally, the mixture is about 10 to about 1000 oocysts of E. acervulina, about 10 to about 100 oocysts of E. maxima, about 10 to about 1000 oocysts of E. mitis and about 10 to about 1000 oocysts of E. tenella. Advantageously, the range of sporulated oocysts in the composition is about 125 to about 500 oocysts of E. acervulina, about 25 to about 100 oocysts of E. maxima, about 125 to about 500 oocysts of E. mitis and about 25 to about 250 oocysts of E. tenella. In one embodiment, a low dose is about 125 oocysts of E. acervulina, about 25 oocysts of E. maxima, about 125 oocysts of E. mitis and about 25 oocysts of E. tenella. In another embodiment, a medium dose is about 250 oocysts of E. acervulina, about 50 oocysts of E. maxima, about 250 oocysts of E. mitis and about 50 oocysts of E. tenella. In yet another embodiment, a high dose is about 500 oocysts of E. acervulina, about 100 oocysts of E. maxima, about 500 oocysts of E. mitis and about 100 oocysts of E. tenella.

Advantageously, the composition is about 500 oocysts of E. acervulina, about 50 to about 100 oocysts of E. maxima, about 500 oocysts of E. mitis and about 100 to about 500 oocysts of E. tenella. In a more advantageous embodiment, the composition is about 500 oocysts of E. acervulina, about 100 oocysts of E. maxima, about 500 oocysts of E. mitis and about 100 oocysts of E. tenella.

The composition also relates to specific ratios of sporulated oocysts isolated from precocious strains of E. acervulina, E. maxima, E. mitis and E. tenella, wherein the ratio of E. acervulina:E. maxima:E. mitis:E. tenella is about 10:1 to 2:10:2 to 10 (i.e., 10 sporocysts of E. acervulina to about 1 to 2 sporocysts of E. maxima to about 10 sporocysts of E. mitis to about 2 to 10 sporocysts of E. tenella). Advantageously, the ratio of E. acervulina:E. maxima:E. mitis:E. tenella is about 5:1:5:1 (i.e., 10:2:10:2).

The term of "immunogenic composition" covers herein any composition able, once it has been administered to an animal, e.g., avian, to elicit a protective immune response against the parasite or antigen or immunogen or epitope. The term of "vaccine" covers herein any composition able, once it has been administered to the animal, e.g., avian, to induce a protective immune response against the virus, or to efficaciously protect the animal against the parasitic invention.

Immunogenic compositions or vaccines according to the invention can also include the pathogen or immunogen, antigen or epitope of the pathogen and at least one immunogen, antigen or epitope of another pathogen, parasite or virus, i.e., the coccidiosis vaccine is combined with another avian vaccine. Such an immunogen, antigen or epitope may e.g. be of bacterial, or parasitic or viral origin or an inactivated or attenuated form of the pathogen, parasite or virus. The invention also comprehends kits to prepare these combination compositions, as well as methods for making these combination compositions and the use of the components of these combination compositions to prepare the combination compositions. Accordingly, the invention involves a kit for preparing the combination immunogenic or vaccine compositions of the invention; for instance, such a kit that comprises (a) an organism, pathogen or virus or antigen or epitope thereof (advantageously a pathogen as mentioned herein) and (b) an organism, pathogen or virus or immunogen, antigen or epitope thereof (advantageously a virus or immunogen, antigen or epitope thereof, but other pathogens as herein mentioned are also contemplated) that is different than (a), in separate containers, optionally in the same package, and optionally with instructions for admixture and/or administration.

Immunogenic compositions and/or vaccines according to the invention can include Eimeria culture or preparation (e.g., inactivated or attenuated Eimeria , or an immunogen or antigen or epitope thereof), and at least one immunogen, antigen or epitope of another avian pathogen (including without limitation the pathogen in inactivated or attenuated form). For avian multivalent immunogenic compositions and multivalent vaccines, the additional avian pathogen(s), as to which additional avian antigen(s) or immunogen(s) or epitope(s) thereof are included in and/or expressed by the multivalent immunogenic compositions and multivalent vaccines, are viruses, diseases, or pathogens of the Marek's disease virus (MDV) (e.g., serotypes 1 and 2, advantageously 1), Newcastle disease virus (NDV), paramyxoviruses other than Newcastle disease (PMV2 to PMV7), infectious bronchitis virus (IBV), infectious anaemia virus or chicken anemia virus (CAV), infectious laryngotracheitis virus (ILTV), infectious bursal disease virus (IBDV), encephalomyelitis virus or avian encephalomyelitis virus (AEV or avian leukosis virus ALV), virus of hemorragic enteritis of turkeys (HEV), pneumovirosis virus (TRTV), fowl plague virus (avian influenza), chicken hydropericarditis virus, avian reoviruses, coccidiosis, egg drop syndrome (EDS76), fowl pox, inclusion body hepatitis (adenovirus), lymphoproliferative disease of turkeys, reticuloendotheliosis in chickens, reticuloendotheliosis in turkeys, rotavirus enteritis, and turkey rhinotracheitis, Clostridium spp., Escherichia coli, Mycoplasma gallinarum, Mycoplasma gallisepticum, Haemophilus avium, Pasteurella gallinarum, Pasteurella multocida gallicida, and mixtures thereof. Advantageously, for MDV the immunogen is advantageously gB and/or gD, e.g., gB and gD, for NDV the immunogen is advantageously HN and/or F, e.g., HN and F; for IBDV the immunogen advantageously is VP2; for IBV the immunogen is advantageously S (more advantageously S1) and/or M and/or N, e.g., S (or S1) and M and/or N; for CAV the immunogen is advantageously VP1 and/or VP2; for ILTV the immunogen is advantageously gB and/or gD; for AEV the immunogen advantageously is env and/or gag/pro, e.g., env and gag/pro or gag/pro; for HEV the immunogen is advantageously the 100K protein and/or hexon; for TRTV the immunogen is advantageously F and/or G, and for fowl plague the immunogen is advantageously HA and/or N and/or NP, e.g., HA and N and/or NP. Thus, the invention also involves methods for making these compositions, as well as kits therefore.

An immunogenic composition or vaccine according to the invention that also comprises such an additional immunogenic component (additional immunogen, antigen or epitope) has the advantage that it induces an immune response or protection against several infections or maladies or causative agents thereof at the same time. This additional immunogenic component can be an attenuated or inactivated micro-organism, a recombinant construct or sub-units (e.g. proteins, glycoproteins, polypeptides, or epitopes). Epitope determination procedures, such as, generating overlapping peptide libraries (Hemmer et al., Immunology Today, 1998, 19 (4), 163-168), Pepscan (Geysen H. M. et al., Proc. Nat. Acad. Sci. USA, 1984, 81 (13), 3998-4002; Geysen H. M. et al., Proc. Nat. Acad. Sci. USA, 1985, 82 (1), 178-182; Van der Zee R. et al., Eur. J. Immunol., 1989, 19 (1), 43-47; Geysen H. M., Southeast Asian J. Trop. Med. Public Health, 1990, 21 (4), 523-533; Multipin Peptide Synthesis Kits de Chiron) and algorithms (De Groot A. et al., Nature Biotechnology, 1999, 17, 533-561), can be used in the practice of the invention, to determine epitopes of immunogens, antigens, polypeptides, glycoproteins and the like, without undue experimentation. From that information, one can construct nucleic acid molecules encoding such an epitope, and from that knowledge and knowledge in the art, one can construct vectors or constructs, e.g., recombinant viruses or vectors or plasmids that express immunogens, epitopes or antigens; all without undue experimentation.

The pharmaceutically or veterinarily acceptable carriers or vehicles or excipients are well known to the one skilled in the art. For example, a pharmaceutically or veterinarily acceptable carrier or vehicle or excipient can be a 0.9% NaCl (e.g., saline) solution or a phosphate buffer. The pharmaceutically or veterinarily acceptable carrier or vehicle or excipients may be any compound or combination of compounds facilitating the administration of the vector (or protein expressed from an inventive vector in vitro); advantageously, the carrier, vehicle or excipient may facilitate transfection and/or improve preservation of the vector (or protein). Doses and dose volumes are herein discussed in the general description of immunization and vaccination methods, and can also be determined by the skilled artisan from this disclosure read in conjunction with the knowledge in the art, without any undue experimentation.

The immunogenic compositions and vaccines according to the invention may comprise or consist essentially of one or more adjuvants. Suitable adjuvants for use in the practice of the present invention are (1) polymers of acrylic or methacrylic acid, maleic anhydride and alkenyl derivative polymers, (2) immunostimulating sequences (ISS), such as oligodeoxyribonucleotide sequences having one ore more non-methylated CpG units (Klinman et al., Proc. Natl. Acad. Sci., USA, 1996, 93, 2879-2883; WO98/16247), (3) an oil in water emulsion, such as the SPT emulsion described on p 147 of "Vaccine Design, The Subunit and Adjuvant Approach" published by M. Powell, M. Newman, Plenum Press 1995, and the emulsion MF59 described on p 183 of the same work, (4) cation lipids containing a quaternary ammonium salt, (5) cytokines, (6) aluminum hydroxide or aluminum phosphate or (7) other adjuvants discussed in any document cited and incorporated by reference into the instant application, or (8) any combinations or mixtures thereof.

The oil in water emulsion (3), which is especially appropriate for viral vectors, can be based on: light liquid paraffin oil (European pharmacopoeia type), isoprenoid oil such as squalane, squalene, oil resulting from the oligomerization of alkenes, e.g. isobutene or decene, esters of acids or alcohols having a straight-chain alkyl group, such as vegetable oils, ethyl oleate, propylene glycol, di(caprylate/caprate), glycerol tri(caprylate/caprate) and propylene glycol dioleate, or esters of branched, fatty alcohols or acids, especially isostearic acid esters. The oil is used in combination with emulsifiers to form an emulsion. The emulsifiers may be nonionic surfactants, such as: esters of on the one hand sorbitan, mannide (e.g. anhydromannitol oleate), glycerol, polyglycerol or propylene glycol and on the other hand oleic, isostearic, ricinoleic or hydroxystearic acids, said esters being optionally ethoxylated, or polyoxypropylene-polyoxyethylene copolymer blocks, such as Pluronic, e.g., L121.

Among the type (1) adjuvant polymers, preference is given to polymers of crosslinked acrylic or methacrylic acid, especially crosslinked by polyalkenyl ethers of sugars or polyalcohols. These compounds are known under the name carbomer (Pharmeuropa, vol. 8, no. 2, June 1996). One skilled in the art can also refer to U.S. Pat. No. 2,909,462, which provides such acrylic polymers crosslinked by a polyhydroxyl compound having at least three hydroxyl groups, preferably no more than eight such groups, the hydrogen atoms of at least three hydroxyl groups being replaced by unsaturated, aliphatic radicals having at least two carbon atoms. The preferred radicals are those containing 2 to 4 carbon atoms, e.g. vinyls, allyls and other ethylenically unsaturated groups. The unsaturated radicals can also contain other substituents, such as methyl. Products sold under the name Carbopol (BF Goodrich, Ohio, USA) are especially suitable. They are crosslinked by allyl saccharose or by allyl pentaerythritol. Among them, reference is made to Carbopol 974P, 934P and 971P.

As to the maleic anhydride-alkenyl derivative copolymers, preference is given to EMA (Monsanto), which are straight-chain or crosslinked ethylene-maleic anhydride copolymers and they are, for example, crosslinked by divinyl ether. Reference is also made to J. Fields et al., Nature 186: 778-780, Jun. 4, 1960.

These polymers are soluble in water or physiological salt solution (20 g/l NaCl) and the pH can be adjusted to 7.3 to 7.4, e.g., by soda (NaOH), to provide the adjuvant solution in which the expression vector(s) can be incorporated. The polymer concentration in the final vaccine composition can range between 0.01 and 1.5% w/v, advantageously 0.05 to 1% w/v and preferably 0.1 to 0.4% w/v.

The cationic lipids (4) containing a quaternary ammonium salt which are advantageously but not exclusively suitable for plasmids. Among these cationic lipids, preference is given to DMRIE (N-(2-hydroxyethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-1-propane ammonium; WO96/34109), preferably associated with a neutral lipid, preferably DOPE (dioleoyl-phosphatidyl-ethanol amine; Behr J. P., 1994, Bioconjugate Chemistry, 5, 382-389), to form DMRIE-DOPE.

Advantageously, the mixture with the adjuvant is formed extemporaneously and preferably contemporaneously with administration of the preparation or shortly before administration of the preparation; for instance, shortly before or prior to administration, the plasmid-adjuvant mixture is formed, advantageously so as to give enough time prior to administration for the mixture to form a complex, e.g. between about 10 and about 60 minutes prior to administration, such as approximately 30 minutes prior to administration.

When DOPE is present, the DMRIE:DOPE molar ratio is preferably about 95: about 5 to about 5:about 95, more advantageously about 1: about 1, e.g., 1:1.

The DMRIE or DMRIE-DOPE adjuvant:plasmid weight ratio can be between about 50: about 1 and about 1: about 10, such as about 10: about 1 and about 1:about 5, and preferably about 1: about 1 and about 1:about 2, e.g., 1:1 and 1:2.

The cytokine or cytokines (5) can be in protein form in the immunogenic or vaccine composition, or can be co-expressed in the host with the immunogen or immunogens or epitope(s) thereof. Preference is given to the co-expression of the cytokine or cytokines, either by the same vector as that expressing the immunogen or immunogens or epitope(s) thereof, or by a separate vector therefor.

The invention comprehends preparing such combination compositions; for instance by admixing the active components, advantageously together and with an adjuvant, carrier, cytokine, and/or diluent.

Cytokines that may be used in the present invention include, but are not limited to, granulocyte colony stimulating factor (G-CSF), granulocyte/macrophage colony stimulating factor (GM-CSF), interferon α (IFN α), interferon β (IFN β), interferon γ, (IFN γ), interleukin-1α (IL-1α), interleukin-1 β (IL-1 β), interleukin-2 (IL-2), interleukin-3 (IL-3), interleukin-4 (IL-4), interleukin-5 (IL-5), interleukin-6 (IL-6), interleukin-7 (IL-7), interleukin-8 (IL-8), interleukin-9 (IL-9), interleukin-10 (IL-10), interleukin-11 (IL-11), interleukin-12 (IL-12), tumor necrosis factor α (TNF α), tumor necrosis factor β (TNF β), and transforming growth factor β (TGF β). It is understood that cytokines can be co-administered and/or sequentially administered with the immunogenic or vaccine composition of the present invention. Thus, for instance, the vaccine of the instant invention can also contain an exogenous nucleic acid molecule that expresses in vivo a suitable cytokine, e.g., a cytokine matched to this host to be vaccinated or in which an immunological response is to be elicited (for instance, an avian cytokine for preparations to be administered to birds).

The invention also provides eliciting an immune response or inducing an immunological or protective response comprising administering an effective amount of the immunogenic or vaccine composition comprising a mixture of sporulated oocysts isolated from precocious strains of E. acervulina, E. maxima, E. mitis and E. tenella to elicit or induce the response in an animal. Advantageously, the animal is an avian such as, but not limited to, a chicken, duck, goose, guinea fowl, peafowl, pheasant, pigeon, quail or turkey. In the most advantageous embodiment, the avian is a chicken, advantageously a broiler chicken. The method of eliciting an immune response or inducing an immunological response can also include administering an adjuvant, a cytokine or both.

The effective amount to elicit an immune response or induce an immunological or protective response is about 10 to about 1000 oocysts of E. acervulina, about 10 to about 100 oocysts of E. maxima, about 10 to about 1000 oocysts of E. mitis and about 10 to about 1000 oocysts of E. tenella. Advantageously, the effective amount to elicit an immune response or induce an immunological or protective response is about 500 oocysts of E. acervulina, about 50 to about 100 oocysts of E. maxima, about 500 oocysts of E. mitis and about 100 to about 500 oocysts of E. tenella. More advantageously, the effective amount is about 500 oocysts of E. acervulina, about 100 oocysts of E. maxima, about 500 oocysts of E. mitis and about 100 oocysts of E. tenella. In another embodiment, the effective amount is sufficient to resist a challenge dose of about 100,000 to about 500,000 oocysts of E. acervulina and about 10,000 to about 100,000 oocysts of E. maxima, about 100,000 to about 500,000 oocysts of E. mitis, or about 10,000 to about 100,000 oocysts of E. tenella to the animal. More advantageously, the challenge dose is about 200,000 oocysts of E. acervulina and about 20,000 to about 50,000 oocysts of E. maxima, about 200,000 oocysts of E. mitis, or about 20,000 to about 50,000 oocysts of E. tenella.

The effective amount to elicit an immune response or induce an immunological or protective response also relates to specific ratios of sporulated oocysts isolated from precocious strains of E. acervulina, E. maxima, E. mitis and E. tenella, wherein the ratio of E. acervulina:E. maxima:E. mitis:E. tenella is about 10:1 to 2:10:2 to 10 (i.e., for every 10 sporocysts of E. acervulina, there are about 1 to 2 sporocysts of E. maxima, about 10 sporocysts of E. mitis and about 2 to 10 sporocysts of E. tenella). Advantageously, the ratio of E. acervulina:E. maxima:E. mitis:E. tenella is about 5:1:5:1 (i.e., 10:2:10:2).

Another aspect of the present invention is a method of immunization or a method of vaccination using the immunogenic compositions or the vaccine compositions according to the invention, respectively.

The method includes at least one administration to an animal of an efficient amount of the immunogenic composition or vaccine according to the invention. The animal may be male or female. This administration may be notably done by intramuscular (IM), intradermal (ID) or subcutaneous (SC) injection or via intranasal or oral administration, wherein oral administration includes but is not limited to administration on feed or in drinking water, gels, or sprays. The immunogenic composition or the vaccine according to the invention can be administered by a syringe or a needleless apparatus (like for example Pigjet or Biojector (Bioject, Oreg., USA)). In an advantageous embodiment, the administration is oral.

The compositions according to the invention may also be administered to other mammals, e.g. mice or laboratory animal, for instance to generate polyclonal antibodies, or to prepare hybridomas for monoclonal antibodies.

The present invention provides for the immunization of animals, advantageously avians. Methods for administering coccidiosis vaccines are described in U.S. Pat. Nos. 4,438,097; 4,639,372; 4,808,404; 5,055,292; 5,068,104; 5,387,414; 5,602,033; 5,614,195; 5,635,181; 5,637,487; 5,674,484; 5,677,438; 5,709,862; 5,780,289; 5,795,741; 5,814,320; 5,843,722; 5,846,527; 5,885,568; 5,932,225; 6,001,363 and 6,100,241, the disclosures of which are incorporated by reference in their entireties. The method comprises administering to the animal an effective immunizing dose of the vaccine of the present invention. The vaccine may be administered by any of the methods well known to those skilled in the art, for example, by intramuscular, subcutaneous, intraperitoneal, intravenous, intranasally, orally, intradermal, intrabursal (just above the chickens vent), in ovo, or ocularly. Methods of administration are known to those skilled in the art. For example, U.S. Pat. Nos. 5,693,622;. 5,589,466; 5,580,859; and 5,566,064 are hereby incorporated by reference in their entirety. Birds may also be administered vaccines in a spray cabinet. Birds may also be administered the vaccine in ovo, as described in U.S. Pat. Nos. 4,458,630 and 6,627,205, the disclosures of which are incorporated by reference.

Advantageously, birds are administered vaccines in a spray cabinet, i.e., a cabinet in which the birds are placed and exposed to a vapor containing vaccine, or by course spray. In another advantageous embodiment, the immunogenic or vaccine composition is administered orally. Alternatively, the immunogenic or vaccine composition can be administered in the drinking water or the feed.

The invention encompasses an immunogenic or vaccine composition comprising a mixture of sporulated oocysts from precocious strains of E. acervulina, E. maxima, E. mitis and E. tenella. The sporulated oocysts from precocious strains of E. acervulina, E. maxima, E. mitis and E. tenella are isolated from the seed cultures described herein. Generally, the dose range of sporulated oocysts in the composition is about 10 to about 1000 oocysts of E. acervulina, about 10 to about 100 oocysts of E. maxima, about 10 to about 1000 oocysts of E. mitis and about 10 to about 1000 oocysts of E. tenella. Advantageously, the dose range of sporulated oocysts in the composition is about 125 to about 500 oocysts of E. acervulina, about 25 to about 100 oocysts of E. maxima, about 125 to about 500 oocysts of E. mitis and about 25 to about 500 oocysts of E. tenella. In one embodiment, a low dose is about 125 oocysts of E. acervulina, about 25 oocysts of E. maxima, about 125 oocysts of E. mitis and about 25 oocysts of E. tenella. In another embodiment, a medium dose is about 250 oocysts of E. acervulina, about 50 oocysts of E. maxima, about 250 oocysts of E. mitis and about 50 oocysts of E. tenella. In yet another embodiment, a high dose is about 500 oocysts of E. acervulina, about 100 oocysts of E. maxima, about 500 oocysts of E. mitis and about 100 oocysts of E. tenella.

Advantageously, the dose of the immunogenic or vaccine composition is about 500 oocysts of E. acervulina, about 50 to about 100 oocysts of E. maxima, about 500 oocysts of E. mitis and about 100 to about 500 oocysts of E. tenella. In a more advantageous embodiment, the dose is about 500 oocysts of E. acervulina, about 100 oocysts of E. maxima, about 500 oocysts of E. mitis and about 100 oocysts of E. tenella.

The invention also relates to specific ratios of sporulated oocysts isolated from precocious strains of E. acervulina, E. maxima, E. mitis and E. tenella, wherein the ratio of E. acervulina:E. maxima:E. mitis:E. tenella is about 10:1 to 2:10:2 to 10 (i.e., for every 10 sporocysts of E. acervulina, there are about 1 to 2 sporocysts of E. maxima, about 10 sporocysts of E. mitis and about 2 to 10 sporocysts of E. tenella). Advantageously, the ratio of E. acervulina:E. maxima:E. mitis:E. tenella is about 5:1:5:1 (i.e., 10:2:10:2).

Advantageously, the oocysts are suspended in a preservative consisting of a 0.01M phosphate buffered saline solution containing gentamicin. In another embodiment, the oocysts are suspended in any one of a variety of preservatives or organic acids such as, but not limited to, acetic acid, citric acid, potassium dichromate or propionic acid. For example, but not by limitation, sufficient sterile, 0.01M phosphate buffered saline containing not more than 30 mcg/ml gentamicin, is used to yield 2 ml per bottle for a 2,000 dose presentation, 5 ml per bottle for a 5,000 dose presentation and 10 ml per bottle for a 10,000 dose presentation. Advantageously, the oocysts are stored in sterile, borosilicate glass vials. For example, but not by limitation, the oocysts are aseptically filled into vaccine vials with a semi-automatic or automatic dispenser, stoppers are mechanically or manually inserted and aluminum seals are placed and crimped.

The vaccine is marketed as a multiple dose containing, 2000 dose vials, 5000 dose vials, 010,000 dose vials or 20,000 dose vials. The expiration date of the product shall not exceed 13 months from the date of the potency test initiation.

Animals, advantageously avians, can be vaccinated at any suitable age, and are usually about one to three days old before first vaccination. Advantageously, the animals are vaccinated once. Optionally, when two doses of vaccine are used, the first is normally given when the animals are 3 days to a week old and subsequently after a further 1-10 weeks dependent upon the type of animal being vaccinated.

The use, dosage and route of administration for each animal species in an advantageous embodiment is as follows. The immunogenic or vaccine composition of the present invention was used for the vaccination of healthy chickens one day of age or older, as an aid in the prevention of disease due to coccidiosis. Advantageously, the dosage was one dose per chicken by coarse water spray of 20 ml per 100 chickens.

Claim 1 of 13 Claims

1. An immunogenic or vaccine composition for protection against E. acervulina, E. maxima, E. mitis and E. tenella consisting of a mixture of sporulated oocysts isolated from precocious strains of E. acervulina, E. maxima, E. mitis and E. tenella, wherein the mixture is about 500 oocysts of E. acervulina, about 50 to about 100 oocysts of E. maxima, about 500 oocysts of E. mitis and about 100 to about 250 oocysts of E. tenella combined.

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