JPH10182483A - Coccidial antigen and coccidiosis vaccine - Google Patents

Abstract

(57) Abstract: An antigen useful as a coccidiosis vaccine, an antigen purified from oocysts of Eimeria tenella, a vaccine containing the antigen, and a method for inducing an immune response against coccidiosis using the vaccine. To provide. The induction of an immune response against coccidiosis in chickens, which can be extracted from oocysts of Eimeria tenella , has a molecular weight of 25 kDa on SDS-PAGE (under reduction) and an isoelectric point of 3.68. A method for inducing an immune response against chicken coccidiosis, which comprises administering to a chicken an antigen comprising a glycopeptide having the ability, a vaccine containing the antigen, and an immunologically effective amount of the vaccine.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to an antigen that can be used as a vaccine for coccidiosis in livestock, especially chickens, a vaccine containing the antigen, and a method for using the vaccine.

[0002]

2. Description of the Related Art In recent years, the poultry industry in Japan has been rapidly developed by mechanization and elimination. Already in 1994,
144 million laying hens and 104 broilers in Japan
100 million birds are bred, and the average number of birds bred per house is 16,000 for laying hens and 30,000 for broilers. Thus, the breeding environment of chickens is large and dense, and it can be said that there is always a risk of disease spread. Chicken coccidiosis has been known as one of the chicken diseases for a long time, but when the disease is affected, it extremely decreases the body weight gain effect and causes death, resulting in considerable economic loss. It is said that hundreds of millions of dollars of anticoccidial drugs are consumed annually worldwide, indicating the seriousness of the disease. In addition, there are also concerns about problems such as food contamination due to residues of these agents in chicken eggs and chicken meat and environmental pollution through feces.

[0003] parasite to develop chicken coccidiosis, Api Complexion box Gate, spore insect rope, coccidia subclass, true coccidiosis eyes, Eimeria suborder, a Eimeria species belonging to the eimeriidae (Eimeria). Each species has a parasitoid site and a complex life cycle. At present, nine Eimeria species have been identified, and the most commonly identified species in Japan are Eimeria acervulina (E. aceruvulin).
a), Eimeria tenella (E. tenella), Eimeria Neka trix (E.necatrix) and Eimeria maxima
(E. maxima) , and about half of chicken coccidiosis is caused by mixed infection (Oikawa
Hiro, Pathophysiology of chicken coccidiosis, Tsuchiyama Printing,
p.1-215, 1976). Coccidiosis includes acute coccidiosis and chronic coccidiosis. Acute symptoms include Eimeria tenella, Eimeria necatrix, and chronic symptoms include
Eimeria aserbrina and Eimeria maxima are known. Usually, chronic coccidiosis presents only symptoms, and the life cycle of the protozoan that causes it ends in a shorter time than the acute one.

[0004] Chickens infected with Eimeria die if their symptoms are severe, but chicks that have recovered are known to have antibody production about one week after infection and show resistance to subsequent infections. I have. In general, immunoreactivity is different for each species of Eimeria, and Eimeria maxima is highly immunogenic, relatively long compared to many species, and is known to maintain a high level of immune response (Long, Rose, Pa
rasitology 65 (3): 437-445,1972). In comparison, Eimeria tenella is a species whose immunity is slow to establish, and it is said that 2-3 immunizations are required to obtain immunity that can completely suppress oocyst production. .

At present, prevention and prevention of coccidiosis largely depend on drugs. Although drugs had not been used until 1936, a variety of drugs, including sulfa drugs, have been used since the discovery that adding at least 1.5% sulfur to feed reduced mortality from Eimeria tenella infection. Has been developed. However, despite administration by a rotation program, coccidiosis has not yet been eliminated due to the emergence of drug-resistant strains and strains with reduced drug sensitivity.

[0006] Under these circumstances, basic immunological research on coccidiosis has been carried out, and various findings have been obtained up to the present. When chicks become infected with coccidiosis, about one week after infection, humoral immunity
Specific antibodies of the IgG and IgM classes are produced in the serum.
It is known that the binding of these antibodies to protozoan surface antigens activates complement and causes lysis of sporozoites and merozoites (Witlock and Danforth,
Journal of Protozoology 29 (3): 3190-3196,1982). Opsonization is one of the effects of serum antibodies, but when sporozoites and merozoites sensitized with inactivated serum were inoculated into cultured peritoneal macrophages, the macrophages were compared with those sensitized with untreated serum. It has also been confirmed that the phagocytic activity of liposomes is clearly high. Although the antibody has such an action, the degree of infection protection in the chick body is very unclear. Coccidiosis is not always exposed to serum antibodies because it is caused by protozoa growing in cells of the intestinal mucosal tissue, and this is thought to be the cause of the reduced effects of serum antibodies .
Rose and colleagues believe that stimulation of sporozoites into immune chick intestinal tract tissue may increase vascular permeability in intestinal tract tissue, causing serum antibodies to leak into the intestinal tract and exert a protozoal killing effect ( Rose et al., Parasite Immunology 2 (3):
189-199 (1980)).

[0007] Next, regarding local immunity, coccidiosis does not develop unless it is transmitted by oral oocysts, so it is difficult to induce local immunity by intravenous or intramuscular inoculation. Davis et al., Eimeria
When the contents of chick cecum immunized with Tenella were sensitized to sporozoites and merozoites, the ability of chick kidney cells to inhibit the invasion of sporozoites and merozoites was observed (Davis et a
l., Immunology 36 (3): 471-477 (1979)). Subsequent studies have demonstrated that this ability to block entry is due to secreted IgA (SIgA). SIgA was not passed through the cecal mucosa, but was produced by plasma cells localized in epithelial tissue. However, these SIgAs had no effect on invading sporozoites, indicating that they had no strict neutralizing effect. From these results, it is generally considered that SIgA exerts its protective ability by preventing the invasion of sporozoites into intestinal mucosal tissues.

[0008] On the other hand, in Toxoplasma which is closely related to Eimeria, it is considered that cellular immunity is mainly involved. However, in Eimeria, since the parasitic site is limited to intestinal mucosal epithelial cells, the involvement of cellular immunity is not so large. Have been considered not. However, a number of reports on in vitro cell-mediated immunity have been made in recent years. For example, reports on macrophage activation by interferon (Lilleh
oj, Avian Desease 37 (3): 731-740 (1993)), and in cell-mediated immunity, activated macrophages increase phagocytic activity by the opsonic action of IgG antibodies bound to protozoa,
It can be assumed that the protozoa are dissolved or destroyed.

[0009] In vivo, it is considered that the immune mechanism is complicated and the immune response is established.
Based on these findings, live vaccines have been developed, and virulent and attenuated strains have been used. In virulent strains, a method of conferring immunity while adjusting the number of oocysts (Nakai et
l., Avian Desease 36 (4): 1034-1036 (1992)), and administering a drug-sensitive strain as a vaccine strain to give immunity,
A method for controlling coccidium with a drug is known. Attenuated strains are known to have a less virulent passage in chicken eggs and those that have been passaged with a pre-patent period (Shirley
and Millard, Avian Pathology 15: 629-638 (1986)). However, it has also been pointed out that these live vaccines have a risk of spreading infectious oocysts in the field and a risk of emergence of antigenic mutant field strains.

[0010]

As a solution to the problems of the live vaccine, a recombinant vaccine has attracted attention. This is a vaccine using an antigen produced by a gene recombination technique, and a search is made for an insect antigen having a protective ability for infection. For example,
Crane et al., Infection and Immunity 59
(4): 1271-1277 (1991)) isolates an antigen having a very high ability to protect against infection from insects, but there is no report that it definitely suppresses coccidiosis. In addition, Tokuyo Sho 61-
JP-A-502586 discloses a DNA obtained from a cDNA or a genomic library of Eimeria tenella sporosoid.
Although it has been reported about NA and its expressed protein (about 290 amino acid residues), what is being produced is a fusion protein of β-galactosidase and globose sporeworm antigen. It just indicates that it has fallen.

On the other hand, various reports have been made on attempts to use a protein purified from Eimeria tenella sporosoid as a vaccine. For example, JP
In JP-18724, 13 peptides were separated by SDS-PAGE from a suspension extract of Eimeria tenellas sporosoid or sporosoid-forming oocysts,
JP-A-61-69798 discloses a purified protein of Eimeria tenella sporosoid having a molecular weight of 25 kD.
a (17 kDa and 8 kDa are linked by a disulfide bond)
JP-A-2-264731 discloses the purification of five major glycolipid complex proteins from Eimeria tenella sporosoid (molecular weight 32 kDa, 29 kDa,
6 kDa, 25 kDa, 18 kDa). Further, JP-A-2-35085 discloses that a recombinant protein immunogen using a cDNA of Eimeria tenella sporosoid or DNA obtained from a genomic library and a purified protein of Eimeria tenella sporosoid are used as vaccines. It has been reported about. However, in these reports, although the severity of cecal lesions is evaluated as a score, it is difficult to prevent infection by the vaccine in any case, and the effect on the lesions is considered to be insufficient. Therefore, it can be said that the search for an antigen having the ability to protect against infection is a future research topic.

[0012] Accordingly, a first object of the present invention is to provide an antigen purified from Eimeria tenella oocysts, which is useful as a coccidiosis vaccine. A second object of the present invention is to provide a vaccine containing the antigen. Still another object of the present invention is to provide a method for inducing an immune response against coccidiosis using the vaccine.

[0013]

Means for Solving the Problems The present inventors have developed a monoclonal antibody (KC-1 antibody) that recognizes the surface antigen of Eimeria tenella sporozoites,
In addition, when the sporozoites are reacted with the host cell extract, the reaction between the KC-1 antibody and the sporozoites disappears, and the antigen recognized by the KC-1 antibody (KC-1
Focusing on the suggestion that the (antigen) is a host cell adhesion molecule, as a result of intensive studies, the extract derived from oocysts of Eimeria tenella was purified by monoclonal KC-1 antibody binding affinity chromatography. Successfully purified an antigen useful as a vaccine. The present invention has been completed based on this fact.

That is, the gist of the present invention is as follows: (1) SDS-P which can be extracted from oocysts of Eimeria tenella
An antigen consisting of a glycopeptide having an ability to induce an immune response to coccidiosis in chicken, which has a molecular weight of 25 kDa under AGE (under reduction) and an isoelectric point of 3.68, and (2) recognizes a sporozoite surface antigen The antigen according to (1), which is purified by affinity chromatography to which a monoclonal antibody is bound, (3) the monoclonal antibody, wherein the monoclonal antibody is a monoclonal KC-1 antibody produced by a FERM P-15954 hybridoma And (4) a vaccine comprising the antigen according to any of (1) to (3); and (5) administering to the chicken an immunologically effective amount of the vaccine according to (4). A method of inducing an immune response against coccidiosis in chickens.

[0015]

BEST MODE FOR CARRYING OUT THE INVENTION The physicochemical characteristics of the purified antigen (KC-1 antigen) extracted from the oocysts of Eimeria tenella of the present invention are as follows. 1. Molecular weight: 25 kDa (SDS-PAGE (under reduction)
Does not separate into subunits by SDS-PAGE under reducing conditions.) 2. Isoelectric point: 3.68. 3. Glycoprotein: It is shown to contain sugar by the periodate method. 4. Eimeria at each stage: present in sporozoites of Eimeria tenella, but not in unsporulated oocysts, second generation merozoites, macrogammates and microgamates.

The antigen of the present invention derived from the oocysts of Eimeria tenella is purified as follows. i) Purification of oocyst-derived proteins. Eimeria tenella NIAH strain derived from a livestock hygiene laboratory passaged by the present inventors was orally inoculated with 1.0 x 10 ⁴ sporulated oocysts on a 7-day-old broiler or 10-day-old layer, and 6 to 8 days after inoculation. Collect eye stool. After removing contaminants from the collected feces oocysts, spores are formed. Fine contaminants are removed from the oocysts after spore formation by a saturated salt suspension method, and the oocysts are suspended in a 2.5% potassium dichromate solution. Use the one stored at 4 ° C.

The oocyst is centrifugally washed three times with distilled water to remove potassium dichromate. A bleaching agent approximately 5 times the volume of the oocyst after centrifugation is added, treated at 0 ° C. for 10 minutes, and centrifugally washed three times with distilled water to obtain a purified oocyst. The suspension is centrifugally washed three times with a 0.1 M Tris buffer (pH 6.8) containing 1 mM of PMSF and 200 Kallikrein units / ml of aprotinin, and resuspended in the same buffer. Add 1/3 volume of glass beads of oocyst suspension, sonicate 10 times under ice-cooling, and add 10 times to the above buffer.
Add an equal volume of extraction buffer containing mM EDTA and 3% NP-40 to the oocyst crushed solution, and leave at 4 ° C overnight. After the extract is centrifuged and the protein concentration of the supernatant is determined, it is stored at -80 ° C until use.

Ii) Purification of the KC-1 antigen. A. Purification by anion exchange chromatography Using DE-52 (manufactured by Whatman) as an anion exchange carrier, the above-described oocyst-derived protein is suspended in a double volume of Tris buffer, applied to a column, and adsorbed. . After the adsorption, the plate is washed with about three times the volume of Tris buffer until the protein is completely eluted. 1M NaCl
Perform elution using. The antigen activity was measured using an ELISA method. The solution is collected into five (1) to (5), desalted, and lyophilized.

B. Purification by affinity chromatography (1) Preparation of KC-1 antibody The KC-1 antibody which is the monoclonal antibody of the present invention is prepared as follows. The purified oocyst was treated with a Teflon homogenizer under ice-cooling for 40 minutes to destroy the oocyst wall, washed, and then 0.3% (w / v) trypsin-0.1 M phosphoric acid supplemented with 10% (v / v) chicken bile. In a buffer solution (pH 8.0), the mixture was treated at 41 ° C. for 60 minutes.
After centrifugal washing using 1M phosphate buffer (pH 7.0),
The released sporozoites were obtained by the method of Schmats et al. (J. Protozoo
l., 31: 181-183 (1984)). 2.5x10 ^Five purified sporozoites were suspended in PBS and transferred to BALB / C mice.
Inoculate the tail vein or intraperitoneally three times at weekly intervals. One week later, the same amount of sporozoite is injected into the tail vein for final immunization. The spleen was removed from the mouse 3 days after the final immunization, and RP
A spleen cell suspension is prepared using MI1640 medium. Myeloma cells include P3X63-Ag8.653 strain (ATCC CRL 15
80) is used. The splenocytes and myeloma cells were mixed at a ratio of 10: 1, and 50% w / v polyethylene glycol 4000 was mixed.
(Roth), and RPMI1640 medium is added to perform cell fusion. The fused cells are selected using HAT medium to obtain a hybridoma. Hybridomas producing antibodies against the oocyst antigen of Eimeria tenella are screened by ELISA, and hybridomas that respond to sporozoites are established by IFA. The hybridoma is
Mouse-Mouse hybridoma KC-1 is displayed, and FERM P-15954 (address: 1-3-1 Higashi, Tsukuba-shi, Ibaraki, Japan (postal code 305)) at the Institute of Biotechnology and Industrial Technology, Ministry of International Trade and Industry of Japan. Deposit date; November 2, 1996
0). Hybridoma (FER
MP-15595) previously administered with pristane
/ C mice are inoculated intraperitoneally and 10-14 days later, ascites containing high concentration of monoclonal KC-1 antibody is collected. The recovered ascites is purified by Affigel Protein A (manufactured by Bio-rad) in immunoblot or ELISA, and the antibody is purified by the following method in the other cases. First, using a desalting column (manufactured by Bio-rad), the ascites is exchanged with an Affigelhydrazine coupling buffer (pH 5.5) (manufactured by Bio-rad). Next, the buffer-exchanged ascites was passed through DEAE Affigel Blue (manufactured by Bio-rad) at a flow rate of 1.0 ml / min while monitoring the absorbance at 280 nm. Collect and use as one antibody. (2) Pretreatment of KC-1 antibody Sodium periodate was added to the purified KC-1 antibody as an Ig.
G Antibody is oxidized by adding antibody: sodium periodate = 10: 1 (volume ratio), inverting and mixing at room temperature for 1 hour while shielding from light. Affigel hydrazine coupling buffer (pH 5.5) diluted 10-fold (Bio-rad
Desalting column (Bio-Rad) equilibrated with
The KC-1 antibody was immediately desalted using
Determine the concentration of G antibody using a protein assay kit. (3) Antibody coupling Affigel hydrazine coupling buffer (pH 5.5
Hydrazine gel (Bio)
-Rad) in the coupling buffer. The pre-treated KC-1 antibody according to the above (2), which has been oxidized and desalted, is added to the gel so as to obtain a 1.0 mg / ml gel, and the mixture is mixed by inversion at room temperature for 24 hours. Do. After the reaction, Econo column (Bio-Rad)
Transfer the gel to the column, wash the column with an equal volume of PBS + 0.5 M NaCl, collect the eluate, and calculate the coupling efficiency. Use 10 volumes of PBS + 0.5 M NaCl (0.02%
Wash with NaN ₃ ) and use as an affinity column. (4) Adsorption of sample Before use, the affinity column is optimized.
The column at 4 ° C. is returned to room temperature, the buffer selected for antigen elution is added in an amount twice the column volume, and the column is washed with a 10-fold volume of sample apply buffer (PBS). The three fractions (Nos. 1 to 3) having high activity in A
A sample was prepared by adding PBS. After the sample is adsorbed on the column, the column is washed with 5 to 10 volumes of PBS, and the column is washed with 2 volumes of PBS + 0.5M NaCl to remove non-specific binding. After washing the column with 5 volumes of PBS, an antigen elution buffer is applied. (5) Elution of antigen 5M Gu-HCl (pH 2.5) is used as an antigen elution buffer, and the eluted fraction is neutralized with twice the volume of a phosphate buffer (pH 8.0). The column is regenerated by neutralizing with 2 volumes of phosphate buffer (pH 8.0) and washing with 10 volumes of PBS (adding 0.02% NaN ₃ ). (6) Activity measurement The antigen activity of each fraction is measured by an ELISA method using a KC-1 antibody. The fraction whose activity has been confirmed is desalted and lyophilized to obtain the purified antigen of the present invention. The purified antigen is
Store at -80 ° C until use.

C. Identification of Antigen by SDS-PAGE, Western Blotting and Isoelectric Focusing SD of the Purified Antigen
S-PAGE (under reduction) and Western blotting are performed by conventional methods to confirm that the purified antigen of the present invention is recognized by the KC-1 antibody, and to identify the molecular weight. 63 kDa,
Bands were detected at 25 kDa and 12 kDa, of which only the 25 kDa protein had antigenicity. Further, by isoelectric focusing, the pI of the 25 kDa protein is 3.68.

Eimeria has very strict site specificity not only for the host but also for the invasion of a different host (Augustine, Avian Desease 34 (1): 196-202 (199)
0)). This suggests that a very specific recognition mechanism exists between the host cell and the sporozoite, and that the action mediates entry into the host. In recent years, electron microscopy has confirmed that insect-derived polypeptides are present on the host cell surface immediately after invasion. This is presumed to be due to the initiation of invasion after the binding action between the sporozoite surface molecule and the host cell surface molecule, and the loss of the sporozoite surface molecule during the invasion. Until now, sporozoites with various substances have been used to identify their surface molecules.
In vitro penetration inhibition tests have been performed. As a result, host cells can be converted to enzymes or cationic substances (Augustine, Journal of Pa
rasitology 66 (3): 498-505 (1980), Augustine and Danf
orth, Journal of Immunology 14 (9): 3190-3196 (1984))
Or lectin (Augustine, Poultry Science 64 (1
2): 2296-2299 (1985)), it is clear that invasion is inhibited.

It has been confirmed that the monoclonal antibody KC-1 produced by the present inventors significantly inhibits the invasion of Eimeria tenella sporozoites by 60% or more, and recognizes a sporozoite molecule involved in cell invasion. it is conceivable that. Cryptosporidiu belonging to the Eimeriaceae family
In sp. m parvum , sporozoite surface glycoproteins that govern growth in vivo have been identified (Tilley et al., Inf.
Section and Immunity59 (3): 1002-1007 (1991)), Trypano
In soma cruzi and Plasmodium falciparum , it is known that host cells are recognized through sialic acid (Schenkman, Cell 65 (7): 1117-1126 (1991), Perkins,
Journal of Immunology 141 (9): 3190-3196 (1988)).

The vaccine of the present invention contains an effective amount of the purified antigen.
The following adjuvants may be included. Suitable adjuvants for vaccination include Freund
d's) complete or incomplete adjuvants; inorganic gels such as aluminum hydroxide, alumina hydrate, calcium chloride dihydrate, alum; saponin, alginate gel,
Surfactants such as phosphatidylcholine and lecithin; β
Polysaccharides such as glucan and chitosan; synthetic polymers such as polyoxypropylene / polyoxyethylene and acrylic acid / methacrylic acid copolymers; natural immunostimulating substances such as muramyl dipeptide, lipopolysaccharide, cytokines; levamisole, tuftsin and the like , But are not limited thereto. The adjuvant is usually administered with the antigen, but may be given before or after the antigen administration. Antigens can also be administered incorporated into liposomes, oligomannose-coated liposomes or other microcarriers. Furthermore, it is also possible to use a mixture of antigens from other Eimeria species, thereby inducing protective immunity against a plurality of coccidial infections.

The vaccine of the present invention can be administered by a usual oral or transdermal administration method, preferably by intraperitoneal, intramuscular or subcutaneous injection, or by nasal or eye drops. It is also possible to administer from such as. Furthermore, it can be administered orally or by respiration by drinking water, spraying, or atomizing. The dose of the vaccine is usually 0.001 to 5 mg / kg as the amount of antigen.
As for body weight, the dose may be increased or the number of administrations may be increased depending on the degree of prevention required.

The vaccine of the present invention is useful for preventing protozoan infection caused by coccidial infections such as Eimeria tenella. That is, by administering an immunologically effective amount of the vaccine of the present invention to chickens, an immune response against coccidiosis in chickens can be induced. In order to evaluate the effect of the vaccine of the present invention, after inoculation with the vaccine of the present invention, the number of oocysts was adjusted in advance so that the lesion index became +3, which is severe infection, and the mice were challenged with infection. amount,
The cecal length and lesion index are evaluated. The evaluation method is described in Example 4 described later.

[0026]

EXAMPLES The present invention will be described in more detail with reference to the following Examples, which should not be construed as limiting the present invention.

Materials (1) Protozoa to be Tested The Eimeria tenella NIAH strain from the Livestock Hygiene Laboratory passaged by the present inventors was used in all experiments. Passage was performed on a 7-day-old broiler or 10-day-old layer with Eimeria tenella sporulated oocysts 1.0X
10 ^four were orally inoculated, was recovered 6-8 days of feces. The oocysts in the collected feces were removed after removal of impurities.
Spores were formed by culturing at 7 to 29 ° C. for 3 days. The oocysts after spore formation were freed of fine impurities by a saturated salt suspension method, suspended in a 2.5% potassium dichromate solution, and stored at 4 ° C. Oocysts were counted on a Bürkerturk hemocytometer.

(2) Test antibody Mouse monoclonal prepared by the present inventors using purified spirozoites of Eimeria tenella as an immunizing antigen
The KC-1 antibody (see Example 2 below) was used. The antibody isotype was IgG3κ and cross-reacted with the interior of primary immature schizonts, primary merozoites, primary mature schizonts and sporulated oocysts after fecal release in chicken cecal sections infected with Eimeria tenella (Table 1). It has been confirmed that there is no cross-reactivity with the air-dried sporozoites of Eimeria aserbrina, Eimeria Brunetti, Eimeria maxima, Eimeria Mitis, Eimeria Necatrix, and Eimeria Plecox (Table 2). In addition, it has been confirmed that the entry of Eimeria tenella sporozoites into PCK cells is inhibited, and this antibody is considered to recognize sporozoite molecules involved in Eimeria tenella cell invasion. Since the KC-1 antibody was in the state of ascites, the antibody was purified using Affigel Protein A (manufactured by Bio-rad) in the immunoblot and ELISA methods, and the other methods were used in the following method. First, using a desalting column (manufactured by Bio-rad), ascites was purified from Affigelhydrazine coupling buffer (pH 5.5) (Bi-
o-rad). Next, the buffer exchanged ascites was monitored in DEAE Affigel Blue (manufactured by Bio-rad) at an absorbance of 280 nm, and a flow rate of 1.0 m
What passed through at 1 / min and eluted in the void volume was collected and used as a purified antibody.

[0029]

[Table 1]

[0030]

[Table 2]

Embodiment 1 Preparation of oocyst antigen The preserved oocyst described in the above (1) was centrifugally washed (3,000 rpm, 10 min) with distilled water three times to remove potassium dichromate. Bleach (Purex, Oyralux) about 5 times the amount of sediment and disinfect (10 minutes under ice cooling)
Min) to obtain a purified oocyst, which was centrifugally washed (3,000 rpm, 10 min) three times with distilled water. The cells were centrifugally washed three times with 0.1 M Tris buffer (pH 6.8) containing 1 mM of PMSF and 200 Kallikrein units / ml of aprotinin, and resuspended in 0.5 ml of the same buffer (3 × 10 ⁷ oocysts / 0.5 ml). 1 / of the oocyst suspension
The oocysts were crushed by adding 3 volumes of glass beads (0.3 mm) and performing the treatment 10 times under ice cooling using an ultrasonic device (25 W, 30 seconds; Ohtake works, Tokyo). 10mM EDT
A and the buffer containing 3% NP-40 as an extraction buffer,
An equal amount was added to the oocyst crushed liquid, and the mixture was allowed to stand at 4 ° C. overnight.
The extract is centrifuged (15000 rpm, 10 minutes), and the protein concentration of the supernatant is determined using the Bio-Rad Protein Assay Kit (Bio-Rad
(Richmond, CA)
Stored at ° C.

Embodiment 2 FIG. Separation and purification of KC-1 antigen Purification by anion exchange chromatography DE-52 (manufactured by Whatman) was used as an anion exchange carrier, and the column size was 3 × 11 cm. The buffer is
Elution was performed using a 20 mM Tris buffer (pH 8.0) and a final concentration of 1 M NaCl using a gradient mixer (Pharmacia). First, the oocyst antigen obtained in Example 1 was suspended in twice the volume of Tris buffer, applied to a column, and adsorbed. After the adsorption, the column was washed with about three times the column volume of Tris buffer until the protein was completely eluted. Elution was started using 2 column volumes of 1 M NaCl. The antigen activity was measured by ELISA. The fractions in which the activity was confirmed were collected into five Nos. 1 to 5, desalted, and lyophilized. The fraction with particularly high activity (No. 3) was subjected to SDS-PAGE and Western blotting. The polypeptide was confirmed by the method described in B (9).

B. Purification by affinity chromatography (1) Preparation of monoclonal KC-1 antibody The purified oocyst was treated with a Teflon homogenizer under ice-cooling for 40 minutes to destroy the oocyst wall, and after washing, 10%
(V / v) 4% in 0.3% (w / v) trypsin-0.1 M phosphate buffer (pH 8.0) supplemented with chicken bile.
Treated at 1 ° C for 60 minutes. 0.1 M phosphate buffer (pH
7.0) and centrifugally washed using the sporozoites released.
Purification was carried out by the method of Schmats et al. (J. Protozool., 31: 181-183 (1984)). 2.5x10 ⁵ purified sporozoites in PBS
And inoculated into the tail vein or intraperitoneally three times at two-week intervals in BALB / C mice. One week later, the same amount of sporozoite was injected into the tail vein for final immunization. The spleen was removed from the mouse three days after the final immunization, and a spleen cell suspension was prepared using RPMI1640 medium. P3X63-Ag8 for myeloma cells.
Strain 653 (ATCC CRL 1580) was used. The splenocytes and myeloma cells were mixed at a ratio of 10: 1, and 50% w /
v RPMI containing polyethylene glycol 4000 (from Roth)
1640 medium was added to perform cell fusion. The fused cells were selected using HAT medium to obtain hybridomas. Hybridomas producing antibodies against the oocyst antigen of Eimeria tenella were screened by ELISA and
Hybridoma K reacting to sporozoites by method A
C-1 (FERM P-15954) was established. 1x
10 ⁶ were inoculated intraperitoneally BALB / C mice previously administered with pristane to the hybridomas were collected ascites containing high concentrations of monoclonal KC-1 antibody after 10-14 days.
The recovered ascites is purified by the method described above, and purified KC-
One antibody was obtained. (2) Pretreatment of KC-1 antibody The purified antibody was oxidized and buffer exchanged. That is,
Antibody buffer exchange was performed on a desalting column (Bio-Rad) equilibrated with 10-fold diluted Affigelhydrazine coupling buffer (pH 5.5) (Bio-Rad). Sodium periodate (NaIO ₄ ) was dissolved in distilled water to a concentration of 20.8 mg / ml. NaIO ₄ was added so that IgG: NaIO ₄ = 10: 1 (volume ratio), and the mixture was inverted and mixed at room temperature for 1 hour under light shielding. Immediately, IgG was desalted with a coupling buffer, and the concentration of the collected IgG was measured using a protein assay kit. (3) Antibody coupling 3 ml of Affigelhydrazine gel (manufactured by Bio-Rad) washed 3 times or more with the coupling buffer was suspended in 5 ml of the coupling buffer. The oxidized and desalted purified antibody was added to a 1.0 mg / ml gel, and the mixture was mixed by inversion at room temperature for 24 hours. After the reaction, transfer the gel to an Econo column, and
The column was washed with S + 0.5M NaCl. Collect the eluate,
The coupling efficiency was calculated. Use 10 times the volume of PBS +
Wash with 0.5M NaCl (add 0.02% NaN ₃ ), 4 ℃ until use
Saved in. (4) Optimization of column Before use, the affinity column was optimized. The column at 4 ° C. was returned to room temperature, and the buffer selected for antigen elution was added in an amount twice the column volume. Further, the column was washed with a 10-fold amount of sample application buffer (PBS). (5) Sample adsorption Two fractions of the three fractions (Nos.
A sample was prepared by adding PBS. Sample adsorption was performed at room temperature for 5 hours or at 4 ° C. overnight. Wash the column with 5 to 10 volumes of PBS and remove 2 volumes of PBS + 0.5 M NaCl to remove non-specific binding.
The column was washed with. After washing the column with a 5-fold amount of PBS, an antigen elution buffer described later was applied. (6) Elution of antigen 5 M Gu-HCl (pH 2.5) was used as an antigen elution buffer, and the eluted fraction was neutralized with twice the volume of a phosphate buffer (pH 8.0). The column was regenerated by neutralizing with 2 volumes of phosphate buffer (pH 8.0) and washing with 10 volumes of PBS (adding 0.02% NaN ₃ ). (7) Activity measurement The activity was measured using an ELISA method, and the fraction for which the activity was confirmed was desalted, lyophilized, and used until used.
Stored at 80 ° C. FIG. 1 shows the results of affinity chromatography. (8) SDS-PAGE SDS-PAGE and Western blotting of the active fraction were performed. SDS-PAGE was performed using an electrophoresis apparatus (Cima).
emmli method (Laemmli, UK Nature 227: 680-685 (197
0)). CBB staining or Western blotting was performed using a 12.5% polyacrylamide gel (FIG. 2). (9) Western blotting Western blotting is a semi-dry transfer device (Bi
o-Rad), using Towbin et al. (Towbin, H. et al.,
Proc. Natl. Acad. Sci. USA. 76: 4350-4354 (1979)). Detection of the membrane-transferred antigen was performed by an enzyme-antibody ABC method using ABC (avidin-biotin complex) reagent (manufactured by Vector).

Embodiment 3 FIG. Analysis of purified antigen (KC-1 antigen) SDS-PAGE under reducing conditions, molecular weight 63 kDa,
Bands of 25 kDa and 12 kDa were detected (FIG. 2). Only 25 kDa of these three bands reacted with the KC-1 antibody after Western blotting (FIG. 2). 2 by SDS-PAGE under reducing conditions
Since an antibody positive band of 5 kDa or less was not detected, it was considered that the 25 kDa protein did not take a subunit structure. Oocysts were suspended in Tris buffer containing protease inhibitors and sonicated. To this was added an equal volume of Tris buffer containing NP-40, and the mixture was allowed to stand at 4 ° C. overnight, and then the soluble oocyst antigen contained in the centrifuged supernatant was analyzed by Fast System (Pharmacia). After performing isoelectric focusing, Western blotting was performed, and immunostaining was performed using the monoclonal antibody KC-1, and a band reacting with the antibody was detected. The result showed that the isoelectric point of the 25 kDa protein was 3.68.
For the above soluble oocyst antigen,
-PAGE was performed and Western blotting was performed on the PVDF membrane.
The blotted PVDF membrane was treated with 0.5% sodium periodate at 4 ° C. for 2 hours, and then immunostained with the monoclonal antibody KC-1. No positive reaction was observed. This indicates that the sugar is involved in the epitope recognized by the monoclonal antibody KC-1.
It means that the antigen contains a sugar. Similar results were obtained using the purified antigen.

The chickens were orally infected with Eimeria tenella, and the cecum was periodically sampled to prepare tissue sections containing protozoa at each developmental stage. When these were immunostained using the monoclonal antibody KC-1, oocysts, sporozoites, primary schizonts, and primary merozoites in the process of sporulation showed a positive reaction, but non-spore-forming oocysts, secondary schizonts, Second generation merozoites, macrogammates and microgamates were negative. These facts indicate that the KC-1 antigen is contained in oocysts, sporozoites, primary schizonts and primary merozoites during sporulation, but not in other developmental stages. Eimeria Acerbrina, Eimeria Brunetti, Eimeria Maxima, Eimeria Mitis,
When sporozoites were prepared from the oocysts of Eimeria Necatrix and Eimeria Plecox by artificial shelling, smeared and dried on a slide glass, and immunostained using the monoclonal antibody KC-1, positive reactions were observed. But only Eimeria tenella sporozoites reacted with the KC-1 antibody.

Embodiment 4 FIG. Use of vaccine containing KC-1 antigen In vivo infection experiments were performed using KC-1 antigen as a component vaccine. We challenged KC-1 immunized chickens and normal chickens and observed differences in blood antibody induction. In addition, the number of immunizations was increased, and the degree of lesions against the challenge infection was actually evaluated. As a test animal, broiler,
Eimeria tenella NIAH strain was used as a test protozoan.

A. Measurement of Antibody Titer Due to Coccidial Infection (1) Experimental System The weight of broilers was measured at the age of 2 days, and the broilers were divided into groups of two birds. One group was a non-immune control group and two groups were an immunized group. Immunization was performed at 2 days and 9 days of age, and both groups were weakly challenged at 16 days of age (3.0 × 10 ² / bird). (2) Antigen purification The purification was performed according to the method described in Example 2. (3) Preparation of immunizing antigen The initial immunization used Freund's complete adjuvant. The protein was dissolved in PBS to a concentration of 10 μg per bird. The mixture was mixed so that adjuvant: protein = 10: 8, and the mixture was stirred with a syringe until a gentle emulsion was formed. The booster was performed in the same manner as the primary immunization except that Freund's incomplete adjuvant was used. (4) Blood collection Blood was collected from the wing vein of 2, 5, 8, 11, 14, 17, 20, 23, and 26 day old chickens. (5) Measurement of antibody titer ELISA was performed using a high binding type 96-well flat bottom EIA plate (Costa
r (Cambridge, MA), Voller et al.
r, A. et al., Bull.World Health Organ. 53: 55-65 (19
76)). 50 mM in each well of the plate
Antigen diluted to 5 μg / ml with carbonate buffer (pH 9.6)
The mixture was dispensed at 4 ° C. overnight. After removing the antigen, add 200 μl of PBS (-) supplemented with 1% BSA, and add
Time blocked. After removing the blocking solution, 0.1%
The antiserum diluted 200-fold with PBS containing BSA was added in an amount of 100 μl each, and reacted at room temperature for 1 hour. After removal of antiserum, 5
Washed once and diluted to 1 μg / ml with PBST containing 0.1% BSA to give a peroxidase (HRP) -labeled rabbit anti-chicken IgG antibody (H + L
(Strand specific; Cappel, West chester, PA) was added in an amount of 100 μl, and the mixture was reacted at room temperature for 1 hour. After washing 5 times with PBST,
The substrate solution was added in an amount of 100 μl each, and reacted at room temperature for 30 minutes while shielding the light, and the absorbance at 405 nm was measured using a microplate reader.

FIG. 3 shows the results of changes in the antibody titer. Non-immunized group (group 1), immunized group (group 2) at 2 days of age before antigen inoculation
Both showed high antibody titers. Thereafter, in the non-immunized group, the antibody titer increased at the age of 5 days, but continued to decrease until the age of 23 days. At 26 days, 10 days after challenge, antibody titers are increasing. In the immunized group, the same reaction as that in the non-immunized group was observed until the age of 9 days, but after the second immunization, the antibody titer level was slightly higher than that in the non-immunized group. Very high levels of antibody titer were observed 10 days after challenge.

B. In Vivo Administration of Vaccine Containing KC-1 Antigen (1) Experimental System At the age of 2 days, broilers were weighed and divided into three groups per group. One group was a control group (no challenge infection), two groups were a non-immune group, three groups were an oocyst-extracted antigen-immunized group, and four groups were a purified antigen (KC-1 antigen) -immunized group. Immunization on days 2, 9
Performed at 16 days of age, groups 2-4 were challenged at 23 days of age (2 × 10
³ birds / wing). The number of oocysts was previously set so that the lesion index was +3, which is severe infection (2.0 × 10 ³ /
wing). Samples were collected 7 days after challenge (30 days old), and the lesion index, cecal length, and weight gain (23 days to 30 days old) were evaluated. (2) Antigen purification The oocyst-extracted antigen used for the three groups was prepared in the same manner as in Example 1. KC-1 antigen, which was a purified antigen of four groups, was prepared in the same manner as in Example 2. (3) Preparation of immunizing antigen The amount of protein of the immunizing antigen was 10 per bird in each of the three groups and the four groups.
μg and carried out in the same manner as in A. above. (4) Blood collection Blood was collected from the subwing vein on days 2, 5, 8, 11, 14, 17, 20, and 23 for antibody titer measurement. (5) Measurement of antibody titer It was performed in the same manner as in A. (FIG. 4) (6) Evaluation of Cecal Length and Weight Gain The cecal length was measured by extending on wet paper (FIG. 5). The lesion index was determined by Johnson & Reid (EXP. PARASITOL., 28, 30-36
(1970)) was slightly changed to make a judgment (Table 3). After determination of cecal length, body weight gain (FIG. 6) and lesion index, t-test was performed.

[0040]

[Table 3]

FIG. 4 shows the results of changes in the antibody titer. In the non-immune and non-challenged infected group (control group: 1 group), the antibody titer increased at 5 days of age, but the antibody titer continued to decrease until 14 days of age, and thereafter maintained almost the same level until 26 days of age. . In the non-immune and challenge infection group (group 2), the antibody titer slightly increased at 5 days of age, and then continued to decrease, and at 23 days of age, it was almost the same as that of the 1 group. 3 days after challenge (26 days old)
Then, there was no change in the antibody titer. In the oocyst-extracted antigen-immunized and challenged infection group (group 3), although the antibody titer slightly decreased from the age of 5 days, it continued to maintain a high level up to 23 days of age as compared to the other groups. Already 3 days after challenge infection, a high increase in antibody titer was observed. In the group immunized with KC-1 antigen and challenged with infection (group 4), the antibody titer gradually decreased after immunization at 2 days of age, but the antibody titer increased after the second immunization, and then maintained at a high level. did. After 23 days of challenge, 3
A very high increase in antibody titer was observed on day.

The results of the lesion index are shown in Table 4. The control group 1 had a lesion index of 0, showed no cecal atrophy or enlarged cecal wall, and the cecum was normal. In the two groups subjected to the challenge infection, blood and cecal core were contained in the cecum, the terminal cecum was clearly deformed, and atrophy was extremely severe. There were almost no normal parts on the whole, and the lesion index was 3. In the three groups subjected to oocyst extraction antigen immunization, the contents of the cecum contained a small amount of blood, there was almost no deformation of the outer shape, and slight punctate whitening and punctate spots were observed. The lesion index was 2. In the 4 groups immunized with KC-1 antigen, cecal atrophy was 2
The reduction was considerably reduced as compared with the group, and reduction of the lesion was observed. Bleeding spots were also reduced as compared to the three groups, and no core was observed in the cecal contents. Also, no deformation of the cecal end was observed. The lesion index is 1.0 to 2.0
was. There was a significant difference (p <0.05) between the 4 groups immunized with the purified antigen and the 2 groups not immunized, indicating that the immunized group reduced lesions.

[0043]

[Table 4]

FIG. 5 shows the results of the cecal length. Atrophy was the most severe in the two groups that had the challenge infection. Although there was no significant difference among the 3 groups and the 4 groups, the KC-1 antigen-immunized group (4 groups) performed better. In addition, a significant difference (p <
0.05), and an immune effect by the KC-1 antigen was observed.

FIG. 6 shows the results of the weight gain. Although no statistically significant difference was observed between the groups, the oocyst-extracted antigen-immunized group (Group 3) showed almost the same low weight gain as the challenge infection group (Group 2), but the purified antigen The immunized group (group 4) showed a high weight gain almost the same as the non-infected group (group 1). From this, it is considered that the immunization of the purified antigen can suppress the influence of coccidial infection on chickens to a low level.

[0046]

Industrial Applicability According to the present invention, an antigen purified from oocysts of Eimeria tenella and a vaccine containing the antigen, which are useful as a coccidiosis vaccine, are provided. Also provided is a method of using the vaccine to induce an immune response against coccidiosis.

[Brief description of the drawings]

FIG. 1 is a graph showing the results of the purification of KC-1 antigen by affinity chromatography.

FIG. 2 is a photograph of electrophoresis showing the results of SDS-PAGE and Western blotting of Example 2B.

FIG. 3 is a graph showing changes in antibody titer after immunization in Example 4A.

FIG. 4 is a graph showing changes in antibody titer after immunization in Example 4B.

FIG. 5 is a graph showing differences in cecal length.

FIG. 6 is a graph showing a difference in body weight gain.

Claims (5)

[Claims] [Claim 1] Eimeria tenella (Eimeria tenella)
An antigen consisting of a glycopeptide capable of inducing an immune response to coccidiosis in chickens, having a molecular weight of 25 kDa on SDS-PAGE (under reduction) and an isoelectric point of 3.68, which can be extracted from oocysts. 2. The antigen according to claim 1, which is purified by affinity chromatography to which a monoclonal antibody recognizing a sporozoite surface antigen is bound. 3. The method of claim 1, wherein the monoclonal antibody is FERMP.
The antigen according to claim 2, which is a monoclonal KC-1 antibody produced by the hybridoma of No. -15954. 4. A vaccine comprising the antigen according to claim 1. 5. A method for inducing an immune response against chicken coccidiosis, which comprises administering an immunologically effective amount of the vaccine according to claim 4 to chickens.