WO2017026301A1 - 黄色ブドウ球菌不活化菌体とロイコシジンを混合したワクチン - Google Patents
黄色ブドウ球菌不活化菌体とロイコシジンを混合したワクチン Download PDFInfo
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/02—Bacterial antigens
- A61K39/085—Staphylococcus
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/305—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Micrococcaceae (F)
- C07K14/31—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Micrococcaceae (F) from Staphylococcus (G)
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
- A61K2039/52—Bacterial cells; Fungal cells; Protozoal cells
- A61K2039/521—Bacterial cells; Fungal cells; Protozoal cells inactivated (killed)
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/55—Medicinal preparations containing antigens or antibodies characterised by the host/recipient, e.g. newborn with maternal antibodies
- A61K2039/552—Veterinary vaccine
Definitions
- the present invention relates to a vaccine for protecting ruminants from diseases (mastitis) caused by Staphylococcus aureus, S. aureus.
- Staphylococcus aureus is one of the staphylococci that are resident bacteria (intestinal bacteria) of the skin and digestive tract (intestine) of humans and animals, and is known as one of the causative agents of purulent diseases and food poisoning in humans and animals. ing.
- the animal itself infected with food poisoning does not develop symptoms, there is a possibility that humans who have come into contact with the animal may be infected with food poisoning bacteria, or food poisoning bacteria may adhere to products produced by animals, It is known to significantly reduce the value of the animal itself and the value of products produced by the animal.
- Staphylococcus aureus is said to be strongly associated with bovine mastitis.
- Staphylococcus aureus is one of the major pathogens that cause infectious mastitis in cattle, and unlike environmental mastitis bacteria such as Escherichia coli and environmental streptococci, it is said that the curative effect of antibiotics is low. .
- At least 30 pathogenic factors as bacterial cell components of Staphylococcus aureus are known, and protein A, fibronectin binding protein, clamping factor, lipoteichoic acid and the like are known as molecules localized in cells.
- protein A, fibronectin binding protein, clamping factor, lipoteichoic acid and the like are known as molecules localized in cells.
- coagulase and staphylokinase are known as enzymes related to pathogenicity.
- enterotoxins that are closely related to food poisoning in humans, TSST-1 associated with sepsis, and leukocidin that exhibits toxic activity against leukocytes that are immune cells are known.
- the use of the supernatant component as a vaccine antigen has also been studied, but no studies have been made in consideration of the actual condition of mastitis-derived bacteria as an active ingredient contained in the supernatant, and as a result, no infection prevention effect has been achieved. . In addition, it is thought that the supernatant component alone cannot be expected to provide sufficient immunological protection. With respect to the fact that an effective vaccine against S. aureus bovine mastitis has not been developed so far, the above background is considered.
- a mastitis vaccine (Patent Document 1) or 1 containing Staphylococcus aureus as an immunogen and contained in a vaccine carrier composed of liposomes containing methylglutarylated polyglycidol.
- a method for treating or preventing S. aureus infection in a bird or mammal comprising administering to the bird or mammal a therapeutically effective amount of a pharmaceutical composition comprising one or more types of digestive enzymes ( Techniques such as Patent Document 2) are known, but in Japan, there are no vaccines that can be used against mastitis, including other pathogens, at sites such as ranches.
- the present inventors focused on leukocidin M / F, which is a staphylococcal toxin, and produced bovine mastitis on farms by producing antibodies against the toxin in the target animal. Has been found to be effective in preventing infections. Furthermore, the present inventors have found that in addition to the leukocidin M / F antigen, a bacterial antigen of Staphylococcus aureus is used in combination to prevent bovine mastitis infection more efficiently. The present invention has been completed by the present inventors based on the above findings.
- the gist of the present invention is as follows.
- An immunogenic composition comprising leukocidin M / F antigen and capable of imparting toxin neutralizing activity to ruminant animals to be administered;
- the above-mentioned leukocidin M / F antigen is a protein or peptide having at least part of an amino acid sequence constituting leukocidin M protein, or a protein or peptide having at least part of an amino acid sequence constituting leukocidin F protein [ 1) the immunogenic composition according to 1), [3]
- the aforementioned leukocidin M / F antigen is a protein or peptide having at least a part of an amino acid sequence constituting leukocidin M protein, and a protein or peptide having at least a part of the amino acid sequence constituting leukocidin F protein [ 1) the immunogenic composition according to 1), [4] The immunogenic composition according to any one of [1] to [3], wherein the leukocidin M /
- the immunogenic composition of the present invention in addition to the existing symptomatic treatment with antibiotic treatment and hygiene measures for infection prevention, in addition to the staphylococcus aureus such as active mastitis in ruminants including cattle It is possible to provide a preventive means for the disease from which it originates.
- FIG. 1 is a graph showing the results of an onset protection test using the S. aureus HK-3 strain inactivated cell antigen performed in Example 3.
- the vertical axis of the upper figure shows the number of somatic cells per mL of milk, and the horizontal axis shows the number of days (d).
- the vertical axis of the lower figure shows the number of bacteria per mL of milk, and the horizontal axis shows the number of days (d) (the same applies to the upper and lower figures of FIG. 3).
- FIG. 2 is a graph showing the results of an inflammatory effect reduction test on supernatant components using the leukocidin M / F concentrated inactivated antigen performed in Example 4.
- FIG. 3 is a graph showing the results of an infection protection test using leukocidin M / F concentrated inactivated antigen and inactivated whole cell antigen performed in Example 5-1.
- FIG. 4 is a graph showing the results of an infection protection test using leukocidin M / F concentrated inactivated antigen and inactivated whole cell antigen performed in Example 5-2.
- toxin neutralizing activity means the toxin of leukocidin M / F which is a bacterial toxin produced by Staphylococcus aureus when the immunogenic composition of the present invention is administered to ruminants.
- immunogenicity refers to the property of stimulating the immune system of ruminant animals, which are the administration target animals, to induce the production of antibodies against the aforementioned leukocidin M / F and S. aureus whole cells.
- An immunogenic composition refers to a composition that, when administered to a ruminant, causes an immune response in the ruminant.
- ruminants that are animals to be administered include cows, goats, sheep or deer. Of these, cattle are preferred because they can efficiently prevent diseases that are economically lossy for breeders, such as bovine mastitis.
- the present invention is an immunogenic composition
- an immunogenic composition comprising leukocidin M / F antigen and capable of imparting toxin neutralizing activity to ruminants which are animals to be administered.
- leucocidin M / F is a kind of bacterial toxin produced by Staphylococcus aureus, and refers to a dimer formed by binding leucocidin M protein (LukM) and leucocidin F protein (LukF).
- Leukocidin M protein is known to have a binding activity to neutrophils
- leukocidin F protein is known to have a neutrophil-damaging activity.
- the leukocidin M protein consists of an amino acid sequence of 308 amino acid residues (SEQ ID NO: 3), which is encoded by the 927 bp base sequence shown in SEQ ID NO: 1 in the gene sequence of S. aureus.
- the leukocidin F protein consists of an amino acid sequence of 322 amino acid residues (SEQ ID NO: 4), which is shown in SEQ ID NO: 2 via a 1 bp base downstream of the gene region encoding the leukocidin M protein. It is encoded by a 969 bp base sequence.
- the amino acid sequences and base sequences of the Leukocidin M protein and Leukocidin F protein can be confirmed with a known database. Known databases include DNA Data Bank of Japan, Medline and the like.
- leukocidin M / F antigen refers to an antigen capable of producing an antibody capable of binding to leukocidin M / F in a ruminant which is an animal to be administered.
- the leukocidin M / F antigen include a protein or peptide having at least a part of the amino acid sequence constituting the leukocidin M protein, or a protein or peptide having at least a part of the amino acid sequence constituting the leukocidin F protein.
- the protein or peptide having at least a portion refers to a protein or peptide having at least a portion of the amino acid sequence represented by SEQ ID NO: 3 or SEQ ID NO: 4, and the length of the amino acid sequence of these proteins or peptides.
- the protein having at least a part may be a protein in which a mutation is introduced into the amino acid sequence described in SEQ ID NO: 3 or SEQ ID NO: 4.
- the mutation-introduced protein is not particularly limited as long as it is capable of imparting toxin neutralizing activity to the ruminant animal to be administered.
- the mutation is introduced.
- the homology of the amino acid sequences of the proteins should be at least 70% or more, preferably 80% or more, more preferably 90% or more with respect to the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 4. Good.
- Examples of the amino acid residue mutation that can be introduced into the amino acid sequence shown in SEQ ID NO: 3 or 4 include substitution, deletion, insertion and / or addition of 1 to 2 amino acids.
- the amino acid to be substituted is desirably an amino acid having a chemical property close to that of the original amino acid in order to retain the three-dimensional structure of the original protein.
- conservative substitution include, for example, mutual substitution between nonpolar amino acids of Ala, Val, Leu, Ile, Pro, Met, Phe, Trp, Gly, Ser, Thr, Cys, Tyr, Asn, Gln. And the substitution between acidic amino acids Asp and Glu, and mutual substitution of basic amino acids with Lys, Arg, and His. Moreover, what is necessary is just to perform about mutations, such as the said deletion and insertion addition, using a well-known method.
- the protein used in the present invention does not need to include the entire amino acid sequence of leucocidin M / F as long as it can impart toxin neutralizing activity equivalent to leucocidin M / F existing in nature.
- the leukocidin M / F antigen is preferably a protein or peptide having at least a part of the amino acid sequence constituting the leukocidin M protein and a protein or peptide having at least a part of the amino acid sequence constituting the leukocidin F protein.
- the leukocidin M / F antigen can be prepared from the culture supernatant of S. aureus.
- Staphylococcus aureus can be cultured by the method described in the Examples below, and leucocidin M / F antigen can be obtained from the supernatant.
- the leucocidin M / F antigen may be synthesized.
- Examples of the synthesis method include a method using an apparatus such as an automatic protein synthesizer and a fully automatic peptide synthesizer.
- the leukocidin M / F antigen prepared from or synthesized from the culture supernatant of S. aureus can be imparted with a toxin neutralizing activity equivalent to that of leukocidin M / F existing in nature. If it is a thing, the length of an amino acid may be shortened or a specific amino acid may be modified by further applying an enzyme treatment or the like.
- the leucocidin M / F antigen may be inactivated.
- the leukocidin M / F antigen obtained from the culture supernatant has toxicity, and thus the safety can be enhanced by inactivation treatment.
- the inactivation treatment method include, but are not particularly limited to, a method in which the leucocidin M / F antigen is brought into contact with formalin or phenol, or subjected to heating treatment, ultraviolet irradiation, or the like.
- the immunogenic composition of the present invention contains a bacterial antigen of Staphylococcus aureus, thereby preventing bovine mastitis infection more efficiently.
- Examples of the cell antigen of S. aureus include inactivated whole cells of S. aureus or a part thereof.
- Examples of the inactivation treatment of Staphylococcus aureus include a method in which the cells of Staphylococcus aureus are brought into contact with formalin and phenol, or are subjected to heating treatment, ultraviolet irradiation, and the like, but are not particularly limited. Further, as a part of the inactivated whole cell body, the inactivated whole cell body is subjected to a treatment such as a decomposition by a physical treatment such as ultrasonic treatment or an enzyme treatment using a hydrolase such as lysostaphin. What you get.
- the S. aureus may be a commercially available strain or a strain stored in various research institutions.
- Staphylococcus aureus isolated from a site where the disease occurs using a known method may be used.
- Staphylococcus aureus isolated from ruminant milk is preferable.
- the immunogenic composition of the present invention includes, for example, a pharmacologically acceptable carrier or medium, specifically, sterilized water, physiological saline, vegetable oil, emulsifier, suspending agent, surfactant, stabilizer and the like. And an appropriate combination thereof, it is possible to produce antibodies against leukocidin M / F antigen and Staphylococcus aureus antigens and to enhance the protective effect against infection.
- a pharmacologically acceptable carrier or medium specifically, sterilized water, physiological saline, vegetable oil, emulsifier, suspending agent, surfactant, stabilizer and the like. And an appropriate combination thereof, it is possible to produce antibodies against leukocidin M / F antigen and Staphylococcus aureus antigens and to enhance the protective effect against infection.
- Various immunostimulants may be added to the immunogenic composition of the present invention.
- the immunogenic composition can be produced, for example, by mixing the various components such as leucocidin M / F antigen and, if necessary, a Staphylococcus aureus cell antigen.
- the immunogenic composition of the present invention can be suitably used as a vaccine against ruminants that are adversely affected by infection with Staphylococcus aureus.
- Adjuvants formulated when the immunogenic composition of the present invention is used as a vaccine include, for example, inorganic substances such as aluminum gel adjuvant, microorganisms or microorganism-derived substances (BCG, muramyl dipeptide, Bordetella pertussis, Hundred days Cough toxin, cholera toxin, etc.), surfactants (saponin, deoxycholic acid, etc.), emulsions of oily substances (mineral oil, vegetable oil, animal oil), etc. These can be used alone or in combination. can do.
- an adjuvant to be blended in the immunogenic composition of the present invention an oil adjuvant is preferable. More preferred is an oil adjuvant mainly composed of squalane.
- an anhydrous mannitol oleate-added squalane solution is preferable from the viewpoint of safety.
- the effect of this adjuvant is more remarkable than expected, and by blending this adjuvant, an excellent protective effect against Staphylococcus aureus and high safety can be obtained.
- the anhydrous mannitol oleate-added squalane liquid refers to a solution comprising anhydrous mannitol oleate and squalane.
- the immunogenic compositions of the present invention can also be used to prepare formulations for the treatment or prevention of diseases associated with S. aureus in ruminants.
- the disease associated with S. aureus is not particularly limited, but includes mastitis, suppurative disease, arthritis, conjunctivitis, dermatitis and the like.
- treatment refers to curing or ameliorating the symptom in a ruminant animal that has developed a disease due to infection with Staphylococcus aureus.
- prevention refers to inhibition of onset by prophylactic administration to ruminants in a state after infection with S. aureus but before the onset of the disease.
- an “immunologically effective amount” is a cellular immune response (T cell) or a humoral immune response (B cell or antibody) as measured by standard assays known to those skilled in the art. Or refers to the amount of antigen or immunogenic composition sufficient to induce both immune responses.
- the immunogenic composition of the present invention can be administered to animals by, for example, intraarterial injection, intravenous injection, subcutaneous injection, intranasal, transbronchial, intramuscular, or oral administration to those skilled in the art. It can be performed by a known method.
- the dosage varies depending on the body weight and age of the ruminant, the administration method, the purpose of use, etc., but those skilled in the art can appropriately select an appropriate dosage.
- Test strain Use a strain that satisfies the following conditions as a test strain for S. aureus.
- a strain in which the culture supernatant has a stronger damaging activity against bovine neutrophils than that of a strain without leucocidin M / F.
- [Method for preparing immunogen] Preparation of Whole Cell Antigen and Leukocidin M / F Antigen Staphylococcus aureus is obtained by transplanting the inoculum into a liquid medium such as Brain Heart Infusion broth (BHI medium) and shaking culture at 37 ° C. for 18 to 24 hours.
- BHI medium Brain Heart Infusion broth
- the culture method is not limited to this as long as the number of bacteria reaches 10 9 CFU / mL or more, and expression of CP antigen or leukocidin is recognized, and a known culture method can be used.
- the leukocidin toxin activity was measured according to the following procedure. 1) Polymorphonuclear (PMN) leukocytes and leukocidin PMN leukocytes were prepared from bovine peripheral blood by centrifugation using Ficoll (Pharmacia). As a positive control, BM1006 strain isolated from milk was cultured in a BHI medium at 37 ° C. for 20 hours, and the supernatant of a bacterial solution (leucosidine toxin activity: equivalent to 320 times) was used as a leucosidine reference solution.
- PMN Polymorphonuclear
- BM1006 strain isolated from milk was cultured in a BHI medium at 37 ° C. for 20 hours, and the supernatant of a bacterial solution (leucosidine toxin activity: equivalent to 320 times) was used as a leucosidine reference solution.
- PMN assay Measurement of leukocidin toxin activity (PMN assay) According to 1) above, PMN leukocytes were prepared from bovine peripheral blood and mixed with each specimen diluted stepwise in a flat bottom 96 well plate. The leucocidin toxin activity of the specimen was determined based on the maximum dilution factor at which 50% or more injury was observed per well. 3) Measurement of Leukocidin Neutralizing Antibody A sample diluted with RPMI-GH (RPMI1640 + 0.1% gelatin + 20 mM HEPES) and diluted 2-fold in a flat-bottom 96-well plate was diluted 50-fold with RPMI-GH. An equal amount of a reference solution was added and sensitized for 60 minutes at 37 ° C.
- RPMI-GH RPMI1640 + 0.1% gelatin + 20 mM HEPES
- the test substance in which the leucocidin antibody titer in the specimen is significantly higher than 4 times that of a control cow not administered with the test substance is referred to as “leucocidin toxin”. It is judged that neutralizing activity can be imparted.
- Adjuvant As a vaccine, an adjuvant for conferring immunity against active ingredients on milk is added. Although an oily adjuvant mainly composed of mineral oil or vegetable oil is basically used, an aluminum gel adjuvant or the like may be added thereto.
- Example 1 Selection of strains MLST analysis, leukocidin gene analysis For Staphylococcus aureus isolated and identified from cow's milk, DNA is extracted by a general method such as phenol chloroform extraction / ethanol precipitation method, boil method or commercially available kit method. It was used for.
- the MLST typing method is a molecular epidemiological analysis method based on the base sequence for determining and analyzing subspecies of bacterial isolates and other microorganisms. The MLST typing method (http://www.mlst.net/) This was carried out and analyzed in the same manner. Leukocidin gene possession survey was conducted according to the method described in Hata et al., J Clin Microbiol.
- the supernatant obtained by inoculating the candidate strain into a bacterial growth medium (Brain Heart Infusion Porcine broth) and shaking culture at 37 ° C. for 24 hours is a commercially available Eagle minimum essential medium ( Eg-MEM) was diluted stepwise, and equal amounts of granulocyte cells collected and purified from bovine peripheral blood were mixed and sensitized at 37 ° C. for 2 hours.
- the toxic activity of the candidate strain supernatant was evaluated with the leucocidin toxin activity as the maximum dilution factor showing a degenerative effect on granulocyte cells.
- Example 2 Specific immunogen preparation method Preparation of inactivated whole cell antigens Culture of cells: The S. aureus HK-3 strain obtained in Example 1 was inoculated on a manufacturing agar medium (Brain Heart Infusion Porcine + Bacto Agar), and cultured at 37 ° C. for 24 hours. The grown colonies were picked and transplanted into a production liquid medium (Brain Heart Infusion Porcine) and cultured with shaking at 37 ° C. for 18 to 24 hours. The number of bacteria reached 2 ⁇ 10 9 CFU / mL or more. This was used as the main culture broth.
- a manufacturing agar medium (Brain Heart Infusion Porcine + Bacto Agar)
- the grown colonies were picked and transplanted into a production liquid medium (Brain Heart Infusion Porcine) and cultured with shaking at 37 ° C. for 18 to 24 hours.
- the number of bacteria reached 2 ⁇ 10 9 CFU / mL or more. This was used as the main culture broth.
- inactivated whole cell antigen solution Formalin (formaldehyde) was added to the main culture broth at 0.4 v / v% and sensitized at 37 ° C. for 24 hours. After sensitization, the number of bacteria was appropriately adjusted with PBS so as to be 1 to 3 ⁇ 10 11 CFU / mL, and used for Example 3, Example 5-1, and Example 5-2 described later. 2. Preparation of Leukocidin M / F Concentrated Inactivated Antigen The supernatant was collected from the main culture by cooling centrifugation, and the precipitate was collected by the ammonium sulfate method, dissolved in PBS, and dialyzed to obtain leucocidin M / F antigen.
- the obtained leucocidin M / F antigen was prepared by preparing a recombinant protein based on SEQ ID NO: 3 and SEQ ID NO: 4, respectively, and performing Western blotting using immune serum prepared based on the recombinant protein. 3 and leucocidin F protein shown in SEQ ID NO: 4 were confirmed. And after adjusting so that it may become 25,600 U / mL or more as leukocidin toxin activity, 0.2% / v% of formalin (formaldehyde) was added, and what was sensitized for 24 hours at 37 degreeC below-mentioned Example 4, implementation It used for Example 5-1 and Example 5-2. The inactivated antigen after formalin sensitization was confirmed by measuring the leukocidin toxin activity and examining the disappearance of the toxin activity.
- Example 3 Onset protection test using inactivated cell antigen of HK-3 strain Among the immunogens prepared in Example 2, a vaccine prepared by using inactivated whole cells was used for milking cows (Holstein, 5 years old). ) was compared with control cattle (unvaccinated cattle) for protection against intramammary infection.
- an inactivated whole cell vaccine an inactivated bacteria-containing liquid adjusted to 4 ⁇ 10 10 CFU as the number of bacteria before inactivation was added to 2 mL injection amount per injection, and an adjuvant was added to this 4 Two intramuscular injections were made at weekly intervals.
- S. aureus HK-3 strain 500 CFU was inoculated into the breast two weeks after the second immunization.
- the somatic cells in the early stages of infection were higher in the breasts 1 and 2 of the test cows administered with the HK-3 strain inactivated bacterial antigen vaccine than in the breasts 1 and 2 of the control cow.
- the degree was limited, so the inactivated bacterial antigen vaccine alone could prevent the disease caused by Staphylococcus aureus in the cow's breast. It was determined that the preventive effect to suppress was insufficient.
- Example 4 Inflammatory effect reduction test on supernatant components using leukocidin M / F concentrated inactivated antigen Vaccine prepared using leukocidin M / F concentrated inactivated antigen among immunogens prepared in Example 2 was injected into milking cows (Holstein, age 3), and before and after that, a supernatant component containing leukocidin M / F was inoculated into the breast to compare the inflammation-reducing effect.
- leukocidin M / F concentrated inactivated antigen vaccine 5120 U of leukocidin M / F concentrated inactivated antigen was used as the leucocidin toxin activity before inactivation with respect to 2 mL injection amount per injection, and an adjuvant was added thereto. Two intramuscular injections were made at 4-week intervals.
- As a supernatant inoculation test 1280 U of concentrated supernatant of S. aureus HK-3 strain was inoculated into the breast before immunization and 2 weeks after the second immunization. After inoculating the concentrated supernatant, the number of milk somatic cells was evaluated as a mammary inflammation condition. The results are shown in FIG.
- the somatic cell counts (SCC, leukocytes and leukocytes) were obtained by inoculating concentrated supernatant in the breasts 1 and 2 of the test cow and the control cow (unvaccinated cow) before vaccine injection (before immunization).
- the number of cells in milk mainly composed of epithelial cells was intense inflammation exceeding 5 million / mL, whereas this was significantly increased after injection of leucocidin M / F concentrated inactivated antigen vaccine prepared from HK-3 strain. The reduction shows that breast inflammation is significantly reduced.
- S. aureus HK-3 strain was inoculated into the breast in the same manner as in the method described in Example 3 in this test cow.
- Example 5-1 Infection protection test by using together leukocidin M / F concentrated inactivated antigen and inactivated whole cell antigen Among the immunogens prepared in Example 2, inactivated whole cell body and leukocidin M After injecting a vaccine prepared using / F concentrated inactivated antigen into milking cows (Holstein, 4 years old), the protective effect against intramammary infection was compared with control cows (unvaccinated cows).
- inactivated whole cell vaccine As an inactivated whole cell vaccine, a liquid containing inactivated bacteria adjusted to 4 ⁇ 10 10 CFU as the number of bacteria before inactivation with respect to 2 mL of injection amount per injection, and 5120 U of leukocidin as the leukocidin toxin activity before inactivation M / F-concentrated inactivated antigen was added and adjuvant was added twice intramuscularly at 4-week intervals.
- the amount of ELISA antibody against whole cells and neutralizing antibody against leucocidin in the milk after the second injection of vaccine was higher in test cows after vaccine injection than in test cows before vaccine injection and unvaccinated control cows. It was confirmed that a remarkably high antibody was given to both the bacterial cells and leukocidin.
- Table 2 The antibody titer against bovine serum cells was measured by indirect ELISA using 1 to 3 ⁇ 10 7 CFU of inactivated whole cell antigen prepared by the same method as in Example 2 as an antigen.
- the breasts 1 and 2 of the test cows administered with the leukocidin M / F concentrated inactivated antigen and the inactivated whole cell antigen were compared with the breasts 1 and 2 of the control cow, The number of somatic cells at the early stage of infection was significantly reduced, and the number of bacteria excreted from milk was not confirmed. From the above results, it was found that the combination of the leukocidin M / F concentrated inactivated antigen and the HK-3 strain inactivated cell antigen combined with the yellow grape It can be seen that in addition to the effect of further reducing the degree of disease caused by cocci, an infection protection effect is also exhibited.
- Example 5-2 Infection protection test using a combination of leukocidin M / F concentrated inactivated antigen and inactivated whole cell antigen After two immunizations in the same manner as in Example 5-1, 2 Week test and control cows were inoculated intramammary with 100 CFU of S. aureus HK-3 strain as a viable challenge test. After inoculation with live bacteria, the number of milk somatic cells was evaluated as a malignant inflammatory condition, and the number of bacteria excreted from milk was evaluated over time as the degree of infection. The results are shown in FIG. From the results shown in the upper and lower diagrams of FIG.
- the breasts of the test cows administered with the leukocidin M / F concentrated inactivated antigen and the inactivated whole cell antigen were obtained from the somatic cell count and milk as compared with the breast of the control cow. There was a significant reduction in both the number of bacteria excreted. From the above results, it was found that the combination of the leukocidin M / F concentrated inactivated antigen and the HK-3 strain inactivated cell antigen combined with the yellow grape It can be seen that in addition to the effect of further reducing the degree of disease caused by cocci, an infection protection effect is also exhibited.
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Abstract
Description
また、本発明は、前記免疫原性組成物を反芻動物に投与して、黄色ブドウ球菌に対する免疫応答を誘発する方法を提供することを目的とする。
さらに、本発明者らは、前記ロイコシジンM/F抗原に加えて、黄色ブドウ球菌の菌体抗原を併用することで、より効率よく牛乳房炎の感染の予防することを見出した。
本発明は、上記の知見に基づいて本発明者らが完成させたものである。
〔1〕ロイコシジンM/F抗原を含有してなり、かつ、投与対象動物である反芻動物に毒素中和活性を付与することが可能な免疫原性組成物、
〔2〕ロイコシジンM/F抗原が、ロイコシジンMタンパク質を構成するアミノ酸配列の少なくとも一部を有するタンパク質もしくはペプチド、またはロイコシジンFタンパク質を構成するアミノ酸配列の少なくとも一部を有するタンパク質もしくはペプチドである前記〔1〕に記載の免疫原性組成物、
〔3〕ロイコシジンM/F抗原が、ロイコシジンMタンパク質を構成するアミノ酸配列の少なくとも一部を有するタンパク質もしくはペプチド、およびロイコシジンFタンパク質を構成するアミノ酸配列の少なくとも一部を有するタンパク質もしくはペプチドである前記〔1〕に記載の免疫原性組成物、
〔4〕前記ロイコシジンM/F抗原が、黄色ブドウ球菌(Staphylococcus aureus)の培養上清から得られたものである前記〔1〕~〔3〕のいずれかに記載の免疫原性組成物、
〔5〕前記ロイコシジンM/F抗原が、合成されたものである前記〔1〕~〔3〕のいずれかに記載の免疫原性組成物、
〔6〕前記ロイコシジンM/F抗原が、不活化処理されたものである前記〔1〕~〔5〕のいずれかに記載の免疫原性組成物、
〔7〕さらに黄色ブドウ球菌の菌体抗原を含有する前記〔1〕~〔6〕のいずれかに記載の免疫原性組成物、
〔8〕前記黄色ブドウ球菌の菌体抗原が、黄色ブドウ球菌の不活化全菌体またはその一部である前記〔7〕に記載の免疫原性組成物、
〔9〕前記黄色ブドウ球菌が、反芻動物の乳汁より分離された黄色ブドウ球菌である前記〔7〕または〔8〕に記載の免疫原性組成物、
〔10〕前記反芻動物がウシ、ヤギ、ヒツジまたはシカである前記〔1〕~〔9〕のいずれかに記載の免疫原性組成物、
〔11〕ワクチンとして使用するための、前記〔1〕~〔10〕のいずれかに記載の免疫原性組成物、
〔12〕反芻動物における黄色ブドウ球菌に関連する疾患の治療または予防用の製剤を調製するための、前記〔1〕~〔10〕のいずれかに記載の免疫原性組成物、
〔13〕免疫学的に効果的な量の前記〔1〕~〔12〕のいずれか1項に記載の免疫原性組成物を投与対象動物である反芻動物に投与することを含む、黄色ブドウ球菌(Staphylococcus aureus)に対する免疫応答を誘発する方法、
〔14〕免疫応答が、反芻動物における黄色ブドウ球菌に関連する疾患もしくは症状を予防するかもしくは低減させる、前記〔13〕に記載の方法、
に関する。
毒素中和活性を反芻動物に付与することが可能であることについては、具体的には、後述の実施例に記載の手順に基づいて、確認することができる。
前記ロイコシジンMタンパク質は、308アミノ酸残基のアミノ酸配列(配列番号3)からなり、これは黄色ブドウ球菌が有する遺伝子配列において配列番号1に示される927bpの塩基配列でコードされる。
また、前記ロイコシジンFタンパク質は、322アミノ酸残基のアミノ酸配列(配列番号4)からなり、これは前記ロイコシジンMタンパク質をコードする遺伝子領域の下流側に1bpの塩基を介して配列番号2に示される969bpの塩基配列でコードされる。
なお、前記ロイコシジンMタンパク質およびロイコシジンFタンパク質のアミノ酸配列や塩基配列は、公知のデータベースで確認することができる。公知のデータベースとしては、DNA Data Bank of Japan, Medlineなどが挙げられる。
ロイコシジンM/F抗原としては、ロイコシジンMタンパク質を構成するアミノ酸配列の少なくとも一部を有するタンパク質もしくはペプチド、またはロイコシジンFタンパク質を構成するアミノ酸配列の少なくとも一部を有するタンパク質もしくはペプチドが挙げられる。
また、前記少なくとも一部を有するタンパク質は、配列番号3または配列番号4に記載のアミノ酸配列に変異を導入されたものでもよい。この場合、変異を導入されたタンパク質としては、投与対象動物である反芻動物に毒素中和活性を付与することが可能なものであれば変異の程度は特に限定はなく、例えば、変異を導入されたタンパク質のアミノ酸配列の相同性は、配列番号3または配列番号4のアミノ酸配列に対して、少なくとも70%以上、好ましくは80%以上、より好ましくは90%以上の相同性を有していればよい。配列番号3または配列番号4に記載のアミノ酸配列に導入しうるアミノ酸残基の変異としては、1~2個のアミノ酸が置換、欠失、挿入及び/又は付加することが挙げられる。例えば、置換するアミノ酸は、元の蛋白質の立体構造を保持するために、元のアミノ酸に近い化学的性質のアミノ酸であることが望ましい。このような保存的置換の具体例として、例えば、Ala、Val、Leu、Ile、Pro、Met、Phe、Trpの非極性アミノ酸間の相互置換、Gly、Ser、Thr、Cys、Tyr、Asn、Glnの非荷電性アミノ酸間の置換、酸性アミノ酸であるAspとGluとの置換、及び塩基性アミノ酸のLys、Arg、Hisによる相互置換を挙げることができる。また、前記欠失、挿入付加などの変異についても、公知の手法を用いて行えばよい。
例えば、後述の実施例に記載の方法によって、黄色ブドウ球菌を培養し、その上清からロイコシジンM/F抗原を得ることができる。
前記合成の方法としては、例えば、タンパク質自動合成装置、全自動ペプチド合成装置などの装置を用いる方法などが挙げられる。
例えば、前記培養上清から得られるロイコシジンM/F抗原は、毒性を有するため、不活化処理することで安全性を高めることができる。
不活化処理の方法としては、前記ロイコシジンM/F抗原をホルマリンやフェノールと接触させたり、加温処理、紫外線照射などに供したりする方法が挙げられるが、特に限定はない。
黄色ブドウ球菌の不活化処理としては、黄色ブドウ球菌の菌体をホルマリン、フェノールと接触させたり、加温処理、紫外線照射などに供したりする方法などが挙げられるが、特に限定はない。
また、前記不活化全菌体の一部としては、前記不活化全菌体を超音波処理等の物理的処理またはリゾスタフィン等の加水分解酵素を用いた酵素処理などで分解等の処理を施して得られるものをいう。
無水マンニトールオレイン酸エステル加スクワラン液とは、無水マンニトールオレイン酸エステルおよびスクワランからなる溶液をいう。
前記黄色ブドウ球菌に関連する疾患としては、特に限定はないが、乳房炎、化膿性疾患、関節炎、結膜炎、皮膚炎などが挙げられる。
また、予防とは、黄色ブドウ球菌に感染した後で疾患を発症する前の状態に有る反芻動物への予防的な投与によって、発症を阻害することをいう。
黄色ブドウ球菌の試験用菌株の選定として以下の条件を満たすものを使用する。
1.野外で黄色ブドウ球菌が原因と考えられる乳房炎を発症した牛の乳汁より分離された菌株。黄色ブドウ球菌としての同定方法は一般的な同定方法に準ずる。200CFU程度を牛の乳房に接種することで急性乳房炎を発症する。
2.遺伝子プロファイルによりCP5型・MLST型別-CC97型で、かつ、ロイコシジンM/Fを保有する菌株。この他にフィブロネクチン結合蛋白、クランピングファクターといった既知の病原因子の遺伝子保有も含まれる。
3.培養上清が、ロイコシジンM/F非保有株のそれに比べ牛の好中球に対して強い傷害活性を有する菌株。
1.全菌体抗原及びロイコシジンM/F抗原の調製
黄色ブドウ球菌の培養は種菌をBrain Heart Infusion broth(BHI培地)といった液体培地に移植し、37℃で18~24時間振とう培養等により得る。培養方法は、菌数が109CFU/mL以上に達すること、CP抗原やロイコシジンの発現が認められるものであればこれに限定されるものではなく、公知の培養方法が利用できる。
全菌体抗原は上記培養菌液を遠心処理して集菌した後、ホルマリン(ホルムアルデヒド)を加えて37℃で24時間感作することで不活化する。ホルマリン濃度は抗原性を損なわず不活化が達成される範囲でよい。
培養菌液より冷却遠心分離により上清を採取し、硫酸アンモニウム法により沈渣を回収しPBSに溶解後、透析を行う。硫酸アンモニウムの濃度はロイコシジンM/F分画を沈殿可能な範囲でよい。また、他のポリエチレングリコール法等を用いてもよい。得られた濃縮ロイコシジンM/Fに対してホルマリン(ホルムアルデヒド)を加えて37℃で24時間感作することで不活化する。
抗原量として、一回注射量あたり不活化菌体を不活化前の菌数として4×1010CFU/dose、これに加え不活化濃縮上清を不活化前のロイコシジン毒素活性として5120U/doseになるように添加する。
1)多形核(PMN)白血球及びロイコシジン
PMN白血球は、牛末梢血よりフィコール(Pharmacia社)を用いた遠心分離法により調製した。陽性対照として、牛乳汁から分離したBM1006株をBHI培地で37℃、20時間培養した菌液の上清(ロイコシジン毒素活性:320倍相当)をロイコシジン参照液として用いた。
2)ロイコシジン毒素活性の測定(PMNアッセイ)
前記1)に従い、牛末梢血よりPMN白血球を調製し、平底96ウェルプレート内で階段希釈した各検体と混合した。1ウェルあたり50%以上の傷害が見られた最大希釈倍率をもとに検体のロイコシジン毒素活性を求めた。
3)ロイコシジン中和抗体の測定
RPMI-GH(RPMI1640+0.1%ゼラチン+20mM HEPES)を希釈液とし、平底96ウェルプレート内で2倍階段希釈した検体に対し、RPMI-GHで50倍に希釈したロイコシジン参照液を等量加え、37℃で60分間感作した。ゼラチンコーティングをした平底96ウェルプレートに中和感作液を80μL添加した後、4×106cell/mLに調整したPMN白血球含有液20μLを添加してプレートミキサーで攪拌後、37℃で感作をおこなった。1ウェルあたり50%以上の傷害が見られたものをロイコシジン毒素活性陽性とし、ロイコシジン毒素活性を抑制した最大希釈倍率をもとに検体のロイコシジン抗体価を求めた。本発明では、被験物を牛に投与した場合に、被験物を投与していない対照牛と比べて、前記検体でのロイコシジン抗体価を4倍以上に有意に高くできた被験物を「ロイコシジン毒素中和活性を付与することが可能」と判断する。
ワクチンとしては、乳汁に対して有効成分に対する免疫を付与させるためのアジュバントを添加する。鉱物油あるいは植物油を主体とした油性アジュバントを基本とするが、これにアルミニウムゲルアジュバント等を加えてもよい。
1.MLST解析、ロイコシジン遺伝子解析
牛乳汁より分離、同定した黄色ブドウ球菌について、フェノールクロロホルム抽出・エタノール沈殿法、ボイル法や市販キットを用いた方法などの一般的手法によりDNAを抽出し、MLST型別法に供した。MLST型別法はバクテリア分離体や他の微生物の亜種決定及び解析を行なう塩基配列に基づいた分子疫学的解析手法であり、MLST型別法(http://www.mlst.net/)に準じて実施、解析した。ロイコシジン遺伝子保有調査はHataら、J Clin Microbiol. 2010.48:2130-2139 2003; 10(2):272-277に記載の方法に準じて実施した。
2.培養上清の好中球傷害活性評価
候補菌株を細菌増殖用培地(Brain Heart Infusion Porcine broth)に接種し、37℃で24時間振とう培養して得た上清を市販のイーグル最小必須培地(Eg-MEM)で階段希釈し、これに牛末梢血より採取、精製した顆粒球細胞を等量混合して37℃で2時間感作させた。顆粒球細胞に対して変性効果を示す最大希釈倍率をロイコシジン毒素活性として候補株上清の毒素活性を評価した。
なお、上清中では、ロイコシジンEとロイコシジンDとの複合体(LukE-LukD)、ヘモリジン(溶血素)(hlg)、ロイコシジンMおよび/またはロイコシジンF(LukM(/F))の有無を公知の手法に基づいて調べ、表中では、検出したものを「+」で示す。
得られた結果を表1に示す。
表1に示す結果より、MLST型別においてCC97型かつ、ロイコシジンM/F遺伝子を保有し、毒素活性に優れたS. aureus HK-3株を選抜し、以下の試験に供した。
1.不活化全菌体抗原の調製
菌体の培養:
実施例1で得られたS. aureus HK-3株を製造用寒天培地(Brain Heart Infusion Porcine + Bacto Agar)に接種し、37℃で24時間静置培養した。発育した集落を釣菌して製造用液体培地(Brain Heart Infusion Porcine)に移植し、37℃で18~24時間振とう培養したもので、菌数が2×109CFU/mL以上に達したものを本培養菌液とした。
不活化全菌体抗原液の調製:
本培養菌液にホルマリン(ホルムアルデヒド)を0.4v/v%加え、37℃で24時間感作した。感作後、菌数を1~3×1011CFU/mLとなるようにPBSで適宜調整し、後述の実施例3、実施例5-1及び実施例5-2に供した。
2.ロイコシジンM/F濃縮不活化抗原の調製
本培養菌液から冷却遠心分離により上清を採取し、硫酸アンモニウム法により沈渣を回収しPBSに溶解後、透析を行い、ロイコシジンM/F抗原を得た。なお、得られたロイコシジンM/F抗原は、配列番号3及び配列番号4を基にそれぞれ組換え蛋白を作製し、それを元に作製した免疫血清を用いてウエスタンブロッティングを行うことで、配列番号3に示されるロイコシジンMタンパク質と、配列番号4に示されるロイコシジンFタンパク質とを含むことを確認した。そして、ロイコシジン毒素活性として25,600U/mL以上となるように調整した後、ホルマリン(ホルムアルデヒド)を0.2v/v%加え、37℃で24時間感作したものを後述の実施例4、実施例5-1及び実施例5-2に供した。
なお、ホルマリン感作後に不活化抗原となっていることは、前記ロイコシジン毒素活性を測定して、毒素活性が消失していることを調べて確認した。
実施例2で調製した免疫原のうち、不活化全菌体を用いて調製したワクチンを搾乳牛(ホルスタイン、5齢)に注射した後、乳房内感染に対する防御効果を対照牛(ワクチン未接種牛)と比較した。
不活化全菌体ワクチンとして、一回あたりの注射量2mLに対して不活化前の菌数として4×1010CFUに調整した不活化菌含有液を加え、これにアジュバントを加えたものを4週間隔で2回筋肉内注射した。生菌攻撃試験として、2回の免疫後2週間後にS. aureus HK-3株 500CFUを乳房内に接種した。生菌接種後、乳房炎症状として乳汁体細胞数、および感染の程度として乳汁からの菌排泄数をそれぞれ経日的に評価した。結果を図1に示す。
図1の上図および下図に示す結果より、HK-3株不活化菌体抗原ワクチンを投与した試験牛の乳房1、2では、対照牛の乳房1、2に比べ、感染初期での体細胞数上昇の早期回復と乳汁からの菌排泄数の軽減効果が見られたが程度は限定的であったことから、不活化菌体抗原ワクチン単独では牛の乳房における黄色ブドウ球菌による疾患の発病を抑える予防効果は不十分であると判定した。
実施例2で調製した免疫原のうち、ロイコシジンM/F濃縮不活化抗原を用いて調製したワクチンを搾乳牛(ホルスタイン、3齢)に注射し、その前後でロイコシジンM/Fを含む上清成分を乳房内に接種して炎症軽減効果を比較した。ロイコシジンM/F濃縮不活化抗原ワクチンとして、一回あたりの注射量2mLに対して不活化前のロイコシジン毒素活性として5120UのロイコシジンM/F濃縮不活化抗原を用い、これにアジュバントを加えたものを4週間隔で2回筋肉内注射した。上清接種試験として、免疫前及び2回の免疫後2週間後にS. aureus HK-3株の濃縮上清1280Uを乳房内に接種した。前記濃縮上清接種後、乳房炎症状として乳汁体細胞数を評価した。結果を図2に示す。
図2の上図および下図に示す結果から、ワクチン注射前(免疫前)の試験牛及び対照牛(ワクチン未接種牛)の乳房1、2では濃縮上清接種により体細胞数(SCC、白血球と上皮細胞を主体とする乳汁中の細胞数)500万/mLを超える強い炎症が惹起されたのに対し、HK-3株より調製したロイコシジンM/F濃縮不活化抗原ワクチン注射後ではこれが大幅に軽減されたことから、乳房の炎症が有意に弱められていることがわかる。
次いで、引き続き、本試験牛に実施例3で記載した方法と同様にS. aureus HK-3株を乳房内に接種した。その結果、M/F濃縮不活化抗原ワクチン注射牛の乳房1、2において乳汁からの菌排泄について軽減効果は見られなかった。
以上の結果から、ロイコシジンM/F濃縮不活化抗原には、S. aureus HK-3株による炎症惹起に対する抑制効果が認められたことから、試験牛に毒素中和活性を付与できたことがわかる。ただし、黄色ブドウ球菌の排泄量が有意に低減せず、感染防御効果は認められなかった。したがって、前記ロイコシジンM/F濃縮不活化抗原には、黄色ブドウ球菌による疾患の程度を軽減する効果があることがわかる。
実施例2で調製した免疫原のうち、不活化全菌体及びロイコシジンM/F濃縮不活化抗原を用いて調製したワクチンを搾乳牛(ホルスタイン、4齢)に注射した後、乳房内感染に対する防御効果を対照牛(ワクチン未接種牛)と比較した。
不活化全菌体ワクチンとして、一回あたりの注射量2mLに対して不活化前の菌数として4×1010CFUに調整した不活化菌含有液及び不活化前のロイコシジン毒素活性として5120UのロイコシジンM/F濃縮不活化抗原を加え、これにアジュバントを加えたものを4週間隔で2回筋肉内注射した。ワクチン2回注射後の乳汁における全菌体に対するELISA抗体、ロイコシジンに対する中和抗体の量は、ワクチン注射後の試験牛では、ワクチン注射前の試験牛およびワクチン未接種の対照牛と比べて、全菌体及びロイコシジン双方に対して顕著に高い抗体付与がなされていることが確認された。
これらの結果を表2に示す。
なお、実施例2と同様の方法で作製した1~3×107CFUの不活化全菌体抗原を抗原として間接ELISA法で牛血清の菌体に対する抗体価を測定した。
図3の上図、下図に示す結果から、ロイコシジンM/F濃縮不活化抗原と不活化全菌体抗原とを投与した試験牛の乳房1、2では、対照牛の乳房1、2に比べ、感染初期での体細胞数を有意に低減し、しかも乳汁からの菌排泄数が確認されなかった。
以上の結果から、ロイコシジンM/F濃縮不活化抗原とHK-3株不活化菌体抗原とを併用することで、ロイコシジンM/F濃縮不活化抗原を単独で使用した場合と比べて、黄色ブドウ球菌による疾患の程度をより軽減する効果に加えて、感染防御効果も発揮されることがわかる。
実施例5-1と同様の方法で2回の免疫を行った後2週目の試験牛および対照牛に、生菌攻撃試験として、S. aureus HK-3株100CFUを乳房内に接種した。生菌接種後、乳房炎症状として乳汁体細胞数、および感染の程度として乳汁からの菌排泄数をそれぞれ経時的に評価した。結果を図4に示す。
図4の上図、下図に示す結果から、ロイコシジンM/F濃縮不活化抗原と不活化全菌体抗原とを投与した試験牛の乳房では、対照牛の乳房に比べ、体細胞数及び乳汁からの菌排泄数双方において有意な低減が認められた。
以上の結果から、ロイコシジンM/F濃縮不活化抗原とHK-3株不活化菌体抗原とを併用することで、ロイコシジンM/F濃縮不活化抗原を単独で使用した場合と比べて、黄色ブドウ球菌による疾患の程度をより軽減する効果に加えて、感染防御効果も発揮されることがわかる。
Claims (14)
- ロイコシジンM/F抗原を含有してなり、かつ、投与対象動物である反芻動物に毒素中和活性を付与することが可能な免疫原性組成物。
- ロイコシジンM/F抗原が、ロイコシジンMタンパク質を構成するアミノ酸配列の少なくとも一部を有するタンパク質もしくはペプチド、またはロイコシジンFタンパク質を構成するアミノ酸配列の少なくとも一部を有するタンパク質もしくはペプチドである請求項1に記載の免疫原性組成物。
- ロイコシジンM/F抗原が、ロイコシジンMタンパク質を構成するアミノ酸配列の少なくとも一部を有するタンパク質もしくはペプチド、およびロイコシジンFタンパク質を構成するアミノ酸配列の少なくとも一部を有するタンパク質もしくはペプチドである請求項1に記載の免疫原性組成物。
- 前記ロイコシジンM/F抗原が、黄色ブドウ球菌(Staphylococcus aureus)の培養上清から得られたものである請求項1~3のいずれか1項に記載の免疫原性組成物。
- 前記ロイコシジンM/F抗原が、合成されたものである請求項1~3のいずれか1項に記載の免疫原性組成物。
- 前記ロイコシジンM/F抗原が、不活化処理されたものである請求項1~5のいずれか1項に記載の免疫原性組成物。
- さらに黄色ブドウ球菌の菌体抗原を含有する請求項1~6のいずれか1項に記載の免疫原性組成物。
- 前記黄色ブドウ球菌の菌体抗原が、黄色ブドウ球菌の不活化全菌体またはその一部である請求項7に記載の免疫原性組成物。
- 前記黄色ブドウ球菌が、反芻動物の乳汁より分離された黄色ブドウ球菌である請求項7または8に記載の免疫原性組成物。
- 前記反芻動物がウシ、ヤギ、ヒツジまたはシカである請求項1~9のいずれか1項に記載の免疫原性組成物。
- ワクチンとして使用するための、請求項1~10のいずれか1項に記載の免疫原性組成物。
- 反芻動物における黄色ブドウ球菌に関連する疾患の治療または予防用の製剤を調製するための、請求項1~10のいずれか1項に記載の免疫原性組成物。
- 免疫学的に効果的な量の請求項1~12のいずれか1項に記載の免疫原性組成物を投与対象動物である反芻動物に投与することを含む、黄色ブドウ球菌(Staphylococcus aureus)に対する免疫応答を誘発する方法。
- 免疫応答が、反芻動物における黄色ブドウ球菌に関連する疾患もしくは症状を予防するかもしくは低減させる、請求項13に記載の方法。
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ALONZO, F. ET AL.: "The Bicomponent Pore-Forming Leucocidins of Staphylococcus aureus", MICROBIOLOGY AND MOLECULAR BIOLOGY REVIEWS, vol. 78, no. 2, June 2014 (2014-06-01), pages 199 - 230, XP002750719, ISSN: 1092-2172 * |
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