Bovine mastitis vaccine

Abstract

To provide an excellent method for administering vaccines to cows with mastitis.SOLUTION: A vaccine composition for bovine mastitis is used, containing an antigen derived from Staphylococcus aureus and a bovine CCL28 component, to be administered subcutaneously or intramuscularly to the neck or rump of a cow. The composition may inhibit the growth of Staphylococcus aureus. An initial administration of the composition may significantly increase antibody titers.SELECTED DRAWING: Figure 6

Description

[Technical field]

[0001] The technical field of the invention relates to bovine mastitis vaccines. [Background technology]

[0002] Currently, Startvac (registered trademark) (Laboratorios Hipra SA), which contains Staphylococcus aureus and the like as antigens, is available as a bovine mastitis vaccine.

[0003] Meanwhile, the present inventors have produced a bovine mastitis vaccine for intranasal administration three times, which comprises killed Staphylococcus aureus bacteria coated with cationic cholesteryl pullulan (cCHP) (Non-Patent Document 1). [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Nagasawa et al., BMC Vet Res. 2019 Aug 9;15(1):286. Summary of the Invention [Problem to be solved by the invention]

[0005] The above-mentioned start-back requires three injections in total, one before and after the delivery of pregnant cows, which places a heavy burden on livestock producers and veterinarians.

[0006] The bovine mastitis vaccine coated with cCHP for three doses intranasally could not induce specific IgG antibodies in milk. In addition, it is known that the use of adjuvants that induce strong systemic immunity in nasal administration generally causes severe side effects such as facial nerve paralysis (Mutsch et al., N Engl J Med. 2004 Feb 26;350(9):896-903.), and in order to develop it as an animal vaccine, concerns about the residual cCHP must be eliminated from the perspective of food safety, and development has been suspended. Furthermore, nasal administration places a heavy burden on livestock producers and veterinarians, who must restrain the cows.

[0007] Meanwhile, the present inventors, through extensive research, have discovered an excellent method for administering a vaccine to cows suffering from mastitis, and in particular, this method shows good vaccine efficacy even in the first administration. [Means for solving the problem]

[0008] According to one aspect of the present invention, there is provided a vaccine composition for bovine mastitis, comprising an antigen derived from Staphylococcus aureus and a bovine CCL28 component, and administered subcutaneously or intramuscularly to the neck or rump of a bovine animal, and when administered to a bovine animal, the composition provides an excellent vaccine effect. [Brief description of the drawings]

[0009] [Figure 1] This shows the transition of IgA and IgG antibodies in milk from dairy cows that received both nasal and subcutaneous administration of nanogel. Each data point (black circle) in the figure indicates the antibody titer in milk collected from each nasal and subcutaneously administered dairy cow (milk collected from the udders of three dairy cows, n=3). An asterisk (*) indicates that a significant difference was observed between the average values ​​of each group 1 to 8 weeks after immunization compared to day 0 (P<0.05). [Diagram 2]This shows the transition of various cytokines and chemokines in milk from dairy cows that received both nasal and subcutaneous administration of nanogel. Each data point (black circle) in the figure indicates the concentration of various cytokines in milk extracted from each nasal and subcutaneously administered dairy cow (milk extracted from the udders of three dairy cows, n=3). An asterisk (*) indicates that a significant difference was observed between the average values ​​of each group 1 to 8 weeks after immunization compared to day 0 (P<0.05). [Diagram 3] Comparison of various cytokines and chemokines in mammary tissues of dairy cows administered nanogel nasally and subcutaneously. Black bars in the figure show the concentration of various cytokines in each mammary tissue from dairy cows administered nasally and subcutaneously (n=3). White bars show the concentration of various cytokines in each mammary tissue from non-immunized dairy cows (n=6 from 6 udders of 3 dairy cows). Asterisks (*) indicate significant differences (P<0.05) between the mean values ​​of each group of dairy cows administered nasally and subcutaneously compared to non-immunized dairy cows. [Figure 4] This shows a comparison of the proliferation ability of bovine mammary epithelial cell lines and fibroblast cell lines with the addition of CXCL12. The dotted black circle line in the figure shows the cell counts of bovine mammary epithelial cell lines and fibroblast cell lines without the addition of CXCL12. The solid black triangle line in the figure shows the cell counts of bovine mammary epithelial cell lines and fibroblast cell lines with the addition of CXCL12 (50 ng / mL of human-derived CXCL12). An asterisk (*) indicates that a significant difference was observed between the average values ​​of each group after 1 to 3 days of culture (P<0.05). [Diagram 5] The graph shows the transition of IgA and IgG antibodies in milk from dairy cows subcutaneously administered CCL28 and killed Staphylococcus aureus bacteria at the same time. The dotted black circle line in the graph shows the antibody titer in milk collected from dairy cows subcutaneously administered only killed Staphylococcus aureus bacteria (milk collected from the udders of three dairy cows, n=3). The solid black triangle line in the graph shows the antibody titer in milk collected from dairy cows subcutaneously administered killed Staphylococcus aureus bacteria + CCL28 (milk collected from the udders of three dairy cows, n=3). The asterisk (*) indicates that a significant difference was observed between the average values ​​of each group 1 to 3 weeks after immunization (P<0.05). [Figure 6] The figure shows the number of S. aureus in milk from dairy cows that were subcutaneously administered CCL28 and killed S. aureus bacteria at the same time. The black dotted circle line in the figure shows the number of S. aureus in milk collected from dairy cows that were subcutaneously administered only killed S. aureus bacteria (milk collected from the udders of three dairy cows, n=3). The black solid triangle line in the figure shows the number of S. aureus in milk collected from dairy cows that were subcutaneously administered killed S. aureus bacteria + CCL28 (milk collected from the udders of three dairy cows, n=3). The asterisk (*) indicates that a significant difference was observed between the average values ​​of each group 1 to 3 weeks after immunization (P<0.05). [Figure 7] The graph shows the progression of clinical symptoms in dairy cows subcutaneously administered CCL28 and killed Staphylococcus aureus at the same time. Clinical symptoms were scored from 0 to 3 based on Pinzon-Sanchez et al., J Dairy Sci. 2011 Jul;94(7):3397-410. and NAGASAWA et al., J Vet Med Sci. 2019 Jan;81(1):107-112. [Figure 8] This shows the progression of clinical symptoms in dairy cows given subcutaneous administration of CCL28 and killed Staphylococcus aureus simultaneously. The dotted black circle line in the figure shows the clinical symptom score in dairy cows given subcutaneous administration of killed Staphylococcus aureus alone (milk extracted from the udders of three dairy cows, n=3). The solid black triangle line in the figure shows the clinical symptom score in dairy cows given subcutaneous administration of killed Staphylococcus aureus + CCL28 (milk extracted from the udders of three dairy cows, n=3). An asterisk (*) indicates that a significant difference was observed between the average values ​​of each group 0, 1, 2, 3, and 7 days after immunization (P<0.05). [Figure 9] This shows the transition of IgA and IgG antibodies in milk from dairy cows that were given two subcutaneous doses of CCL28 and killed Staphylococcus aureus simultaneously before intramammary infection. The solid black circle line in the figure indicates the right hind quarter. The dotted black circle line in the figure indicates the left anterior quarter. The solid white circle line in the figure indicates the right anterior quarter. The dotted white circle line in the figure indicates the left hind quarter. [Figure 10]The figure shows the number of Staphylococcus aureus in milk from dairy cows that were given two subcutaneous doses of CCL28 and killed Staphylococcus aureus simultaneously before intramammary infection. The solid black circle line in the figure indicates the right hind quarter. The dotted black circle line in the figure indicates the left front quarter. [Figure 11] The progress of clinical symptoms in dairy cows that were subcutaneously administered CCL28 and killed Staphylococcus aureus twice before intramammary infection is shown. Clinical symptoms were scored from 0 to 3 based on Pinzon-Sanchez et al., J Dairy Sci. 2011 Jul;94(7):3397-410. and NAGASAWA et al., J Vet Med Sci. 2019 Jan;81(1):107-112. [Figure 12] This shows the progression of clinical symptoms in dairy cows that were subcutaneously administered CCL28 and killed Staphylococcus aureus twice before intramammary infection. The solid line in the figure shows the clinical symptom score in dairy cows that were subcutaneously administered killed Staphylococcus aureus + CCL28. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0010] Hereinafter, the embodiments of the present invention will be described in detail. Note that, in order to avoid repetition and complexity, the description of similar contents will be omitted as appropriate.

[0011] (1) Composition According to one embodiment of the present invention, a vaccine composition against bovine mastitis is provided. This composition may be a composition containing an antigen derived from Staphylococcus aureus and a bovine CCL28 component, and may be a composition administered subcutaneously or intramuscularly to the neck or rump of a bovine. In this case, administration of this composition to a bovine animal can provide an excellent vaccine effect.

[0012] In one embodiment of the present invention, excellent vaccine effects include, for example, induction of both specific IgA antibodies and IgG antibodies in milk, excellent safety, suppression of the number of Staphylococcus aureus in milk, improvement of clinical symptoms of mastitis, or the achievement of these effects with the first administration of the vaccine.

[0013] According to one embodiment of the present invention, there is provided a composition for preventing bovine mastitis, which comprises an antigen derived from Staphylococcus aureus and a bovine CCL28 component, and is administered subcutaneously or intramuscularly to the neck or rump of a bovine animal. When this composition is administered to a bovine animal, it provides an excellent effect of preventing mastitis.

[0014] According to one embodiment of the present invention, there is provided a composition for increasing IgA and IgG antibody titers, which comprises an antigen derived from Staphylococcus aureus and a bovine CCL28 component, and is administered subcutaneously or intramuscularly to the neck or rump of a cow. By administering this composition to a cow, it is possible to increase the IgA and IgG antibody titers in the cow's body.

[0015] According to one embodiment of the present invention, there is provided a composition for improving clinical symptoms in cows suffering from mastitis, the composition comprising an antigen derived from Staphylococcus aureus and a bovine CCL28 component, and being administered subcutaneously or intramuscularly to the neck or rump of a cow, and administration of the composition to a cow can improve the clinical symptoms.

[0016] According to one embodiment of the present invention, there is provided a composition for inhibiting the growth of Staphylococcus aureus, comprising an antigen derived from Staphylococcus aureus and a bovine CCL28 component, and administered subcutaneously or intramuscularly to the neck or rump of a cow, and administration of the composition to the cow can inhibit the growth of Staphylococcus aureus in the cow's body.

[0017] According to one embodiment of the present invention, there is provided a composition for treating bovine mastitis, which comprises an antigen derived from Staphylococcus aureus and a bovine CCL28 component, and is administered subcutaneously or intramuscularly to the neck or rump of a bovine animal, and when administered to a bovine animal, the composition provides an excellent effect of suppressing mastitis.

[0018] According to one embodiment of the present invention, there is provided a vaccine composition for bovine mastitis, comprising an antigen derived from Staphylococcus aureus and CCL28 or a variant thereof, or a nucleic acid encoding either of them, and administered subcutaneously or intramuscularly to the neck or rump of a bovine. When the composition is administered to a bovine, it provides an excellent vaccine effect.

[0019] According to one embodiment of the present invention, there is provided a composition for use in any of the following methods (2), comprising an antigen derived from Staphylococcus aureus and a bovine CCL28 component. When this composition is administered to a bovine, an excellent vaccine effect is obtained.

[0020] (2) Method According to one embodiment of the present invention, there is provided a method for preventing bovine mastitis, comprising subcutaneously or intramuscularly administering an antigen derived from Staphylococcus aureus and a bovine CCL28 component to the neck or rump of a bovine animal. This method provides an excellent effect in preventing mastitis. Hereinafter, both the antigen derived from Staphylococcus aureus and the bovine CCL28 component may be referred to as vaccine components.

[0021] According to one embodiment of the present invention, there is provided a method for increasing antibody titers of IgA and IgG, comprising subcutaneously or intramuscularly administering an antigen derived from Staphylococcus aureus and a bovine CCL28 component to the neck or rump of a bovine animal, which method can increase the antibody titers of IgA and IgG in the bovine body.

[0022] According to one embodiment of the present invention, there is provided a method for improving clinical symptoms in a cow suffering from mastitis, comprising subcutaneously or intramuscularly administering an antigen derived from Staphylococcus aureus and a bovine CCL28 component to the neck or rump of the cow, which can improve the clinical symptoms.

[0023] According to one embodiment of the present invention, there is provided a method for inhibiting the proliferation of Staphylococcus aureus, comprising subcutaneously or intramuscularly administering an antigen derived from Staphylococcus aureus and a bovine CCL28 component to the neck or buttocks of a cow. According to this method, the number of Staphylococcus aureus in the body of the cow can be suppressed.

[0024] According to one embodiment of the present invention, there is provided a method for treating bovine mastitis, comprising subcutaneously or intramuscularly administering an antigen derived from Staphylococcus aureus and a bovine CCL28 component to the neck or rump of a bovine animal, which method provides an excellent effect of suppressing mastitis.

[0025] According to one embodiment of the present invention, there is provided a method for preventing bovine mastitis, comprising subcutaneously or intramuscularly administering an antigen derived from Staphylococcus aureus and CCL28 or a variant thereof, or a nucleic acid encoding either of them, to the neck or rump of a bovine animal, which method provides an excellent vaccine effect.

[0026] According to one embodiment of the present invention, there is provided a method comprising subcutaneously or intramuscularly administering any one of the compositions described in (1) above to the neck or buttocks of a cow, which method provides excellent vaccine efficacy.

[0027] According to one embodiment of the present invention, there is provided a method for producing a vaccine composition, comprising mixing an antigen derived from Staphylococcus aureus and a bovine CCL28 component, and the composition obtained by this method can provide an excellent vaccine effect.

[0028] (3) Use According to one embodiment of the present invention, there is provided a use of an antigen derived from Staphylococcus aureus and a bovine CCL28 component for producing any of the compositions described in (1) above. The composition obtained by this use can provide an excellent vaccine effect.

[0029] An embodiment of each of the compositions, methods, and uses described above (each of the compositions (1), each of the methods (2), and each of the uses (3) above) will be described in more detail below. Each of the compositions, methods, and uses described above can be applied to the embodiments and explanations of each term described below.

[0030] In one embodiment of the present invention, the Staphylococcus aureus is a type of facultative anaerobic gram-positive cocci, and includes bacteria that can induce an inflammatory response by invading, colonizing, and growing in the mammary gland of a cow. Staphylococcus aureus is also written as Staphylococcus aureus (S. aureus). The Staphylococcus aureus may be a commercially available strain, a strain stored in various research institutions (e.g., BM1006 strain, SA003 strain, JE2 strain), a Staphylococcus aureus isolated from the milk of a cow suffering from mastitis, or a Staphylococcus aureus present in the body of a cow suffering from mastitis.

[0031] In one embodiment of the present invention, the antigen derived from Staphylococcus aureus includes an antigen having a property of inducing the production of antibodies against Staphylococcus aureus when administered to cattle (so-called immunogenicity). The antigen derived from Staphylococcus aureus may be, for example, inactivated Staphylococcus aureus or a part thereof. The antigen derived from Staphylococcus aureus may be, for example, an immunogenic protein of Staphylococcus aureus, or a nucleic acid encoding the protein. The preparation of inactivated Staphylococcus aureus can be appropriately performed using a known method such as formalin treatment, phenol treatment, heat treatment, or ultraviolet irradiation treatment. The part of inactivated Staphylococcus aureus has immunogenicity, and may be, for example, a bacterial fragment obtained by subjecting Staphylococcus aureus to a physical treatment such as ultrasonic treatment, or a bacterial decomposition product obtained by subjecting Staphylococcus aureus to an enzymatic treatment using a hydrolase (lysostaphin, etc.). The immunogenic protein of Staphylococcus aureus may be isolated and purified from Staphylococcus aureus, or may be artificially synthesized (for example, chemically synthesized). The protein may be an immunogenic membrane surface protein or secretory protein. The method for producing or using a vaccine protein may be, for example, the method or theory described in Camussone et al., Res Vet Sci. 2022 Jul;145:177-187., Zeng et al., Vaccines (Basel). 2020 Mar; 8(1): 134., or Landrum et al., Hum Vaccin Immunother. 2017 Apr; 13(4): 791-801. The nucleic acid may be, for example, a plasmid. The plasmid may be, for example, an expression vector. The expression vector may include a DNA region encoding the above protein, and the DNA region may be operably linked to an appropriate transcriptional regulatory region (e.g., a promoter). The nucleic acid may be a DNA strand or an mRNA strand.Methods for producing or using nucleic acids encoding vaccines or adjuvants can utilize, for example, the methods and theories described in Yin et al., Vet Immunol Immunopathol. 2009 Dec 15; 132(2-4): 270-4., Lee et al., Acta Biomater. 2018 Oct 15; 80: 31-47., or Kwon et al., Arch Pharm Res. 2022; 45(4): 245-262.

[0032] In one embodiment of the present invention, the cattle include, for example, animals of the subfamily Bovinae or genus Bos. The cattle may include, for example, animals of the scientific name Bos taurus. The cattle may include, for example, Holstein, Guernsey, Jersey, Ayrshire, or Brown Swiss. The cattle may be dairy cattle. The cattle may be cattle before they develop mastitis, or before they are diagnosed as having mastitis, or healthy cattle. The cattle may be pregnant or non-pregnant. The cattle may be, for example, cattle in the growing stage, lactation stage, or dry stage. The lactation stage may be, for example, up to 300 days after the day of parturition. When the vaccine composition is administered to a lactating cattle, it may be administered to a cattle that is, for example, 11 to 300 days after parturition.

[0033] In one embodiment of the present invention, the mastitis includes inflammation caused by, for example, Staphylococcus aureus infection of the mammary gland. Clinical symptoms of mastitis may include, for example, abnormalities in milk (e.g., lumps), local abnormalities in the mammary gland (e.g., swelling), or systemic abnormalities (e.g., loss of appetite or fever). Mastitis may be, for example, subclinical mastitis or clinical mastitis.

[0034] In one embodiment of the present invention, the vaccine includes, for example, a pharmaceutical for promoting antibody production against pathogens in the body and acquiring immunity against infectious diseases. The vaccine includes, for example, an inactivated vaccine or a live vaccine. The inactivated vaccine may include inactivated Staphylococcus aureus or a part thereof, an immunogenic Staphylococcus aureus protein, or a nucleic acid encoding the protein. The vaccine composition includes a composition used as a vaccine. The vaccine effect includes an effect caused by administration of the vaccine.

[0035] In one embodiment of the present invention, the prevention includes, for example, inhibiting the onset of mastitis or inhibiting the progression of mastitis (for example inhibiting the aggravation).

[0036] In one embodiment of the invention, the composition or vaccine components are preferably administered to the bovine neck or rump. Also, the composition or vaccine components are preferably administered to the bovine subcutaneously or intramuscularly, which provides a particularly good vaccine effect.

[0037] In one embodiment of the present invention, the composition and the method may be, for example, a composition or a method that significantly increases the antibody titer. The composition and the method may be, for example, a composition or a method that significantly increases the antibody titer by a single administration. The antibody titer may be an antibody titer of IgA and IgG.

[0038] In one embodiment of the present invention, the number of times the composition or vaccine components are administered to cattle may be, for example, one or more times. The number of times may be, for example, 1, 2, 3, or 4 times. The composition of one embodiment of the present invention is excellent in terms of preventive effect and management because it can show good vaccine effect even with the first administration. In addition, although the effect is seen with the first administration, good vaccine effect is also shown with two administrations. In one embodiment of the present invention, the number of times of administration may include the case where only the number of administrations is the same.

[0039] In one embodiment of the present invention, the bovine CCL28 component includes a molecule derived from bovine CCL28 or a nucleic acid encoding the molecule. CCL28 is a protein known as a type of chemokine. The bovine CCL28 component may be obtained by extraction or purification from bovines, a protein expression system, chemical synthesis, or by purchasing from a manufacturer. The bovine CCL28 component has, for example, CCL28 activity, or generates CCL28 activity in vivo when administered to bovines. The CCL28 activity may be, for example, chemokine activity or binding activity to CCR10. The chemokine activity includes, for example, cell migration activity. The cell migration activity includes, for example, cell migration activity of CCR10-expressing cells or IgA antibody-producing cells, or homing-inducing activity of CCR10-expressing cells or IgA antibody-producing cells to the mammary gland. Migration includes, for example, migration into the breast. Bovine CCL28 includes, for example, a protein with UniProt primary accession number A6YT15. The amino acid sequence of bovine CCL28 includes, for example, the amino acid sequence shown in SEQ ID NO: 1 (SEQ ID NO: 1, MGMQQTGLALLALAACVAFRPSEAILPIASSCCTEVSHHISRRLLERVTTCRIQRADGDCDLAAVILHVKRRRVCVSPHNHVIKQWMNEQAAKKEAKGNICHKKRHHGRRNSKGAHRERQETHGHKTPY). Full-length bovine CCL28 has a signal peptide at the N-terminus. This signal peptide is not considered to be an essential part when it is intended to be administered from outside the body to inside the body. Furthermore, CCL28 without a signal peptide is considered to have the same chemokine activity as full-length CCL28. The amino acid sequence of full-length CCL28 excluding the putative signal peptide is, for example, the amino acid sequence shown in SEQ ID NO: 2 (SEQ ID NO: 2, SEAILPIASSCCTEVSHHISRRLLERVTTCRIQRADGDCDLAAVILHVKRRRVCVSPHNHVIKQWMNEQAAKKEAKGNICHKKRHHGRRNSKGAHRERQETHGHKTPY). When vaccinating cattle, a nucleic acid encoding bovine CCL28 can also be used as a bovine CCL28 component.In one embodiment of the present invention, the bovine CCL28 component may be, for example, a protein selected from the group consisting of a protein comprising the amino acid sequence shown in SEQ ID NO: 1 or 2, and a protein comprising an amino acid sequence having 90% or more homology to the amino acid sequence shown in SEQ ID NO: 1 or 2, or a biologically active fragment thereof, or a nucleic acid encoding the protein or biologically active fragment thereof. In one embodiment of the present invention, the biologically active fragment is a molecule derived from bovine CCL28 and comprises a polypeptide having CCL28 activity.

[0040] In one embodiment of the invention, the bovine CCL28 component may be administered as an adjuvant. Adjuvants include substances used to enhance the effectiveness (e.g., immunogenicity) of a vaccine, for example when administered together with an antigen.

[0041] In one embodiment of the present invention, the content of the bovine CCL28 component in the composition or the amount administered per dose may be 0.1 to 10 mg. From the viewpoint of exerting a more excellent vaccine effect, this amount is preferably 0.1 to 5 mg, more preferably 0.4 to 3 mg, even more preferably 0.6 to 2 mg, and most preferably 0.8 to 1.2 mg. This amount may be, for example, 0.1, 0.4, 0.6, 0.8, 0.9, 1, 1.1, 1.2, 1.4, 1.6, 1.8, 2, 3, 4, 5, 8, or 10 mg, or may be any one of these values ​​or more, or may be within a range between any two of these values. The dosage includes the dosage per cow. The dosage includes the dosage per day.

[0042] In one embodiment of the present invention, when the bovine CCL28 component is in the form of a nucleic acid, the nucleic acid may be, for example, a plasmid. The plasmid may be, for example, an expression vector. The expression vector may include a DNA region encoding the above protein, and the DNA region may be operably linked to an appropriate transcriptional regulatory region (e.g., a promoter). The nucleic acid may be a DNA strand or an mRNA strand. The method for producing and using the nucleic acid encoding the vaccine or adjuvant may be, for example, the method or theory described in Yin et al., Vet Immunol Immunopathol. 2009 Dec 15; 132(2-4): 270-4., Lee et al., Acta Biomater. 2018 Oct 15; 80: 31-47., or Kwon et al., Arch Pharm Res. 2022; 45(4): 245-262.

[0043] In one embodiment of the present invention, the content of the antigen derived from Staphylococcus aureus in the composition or the amount of the antigen administered per dose is 1×10 8 ~5×10 11 From the viewpoint of exerting a more excellent vaccine effect, this amount is preferably 1×10 8 ~5×10 11 CFU is preferred, 1 x 10 9 ~3×10 11 CFU is more preferred, 1 x 10 10 ~3×10 11 CFU is more preferred, 8 x 10 10 ~1.2×10 11 CFU is most preferred. This amount is, for example, 1×10 8 , 1×10 9 , 5×10 9 , 1×10 10 , 5×10 10 , 8×10 10 , 9×10 10 , 1×10 11 , 1.1×10 11 , 1.2×10 11 , 1.5×10 11 , 3×10 11 , or 5 × 10 11The vaccine composition containing an antigen derived from Staphylococcus aureus may be a whole cell vaccine composition. The content in the composition or the amount per dose is 1×10 8 ~5×10 11 The amount of the inactivated Staphylococcus aureus may be sufficient to induce an immune response induced by the inactivated CFU. The CFU can be measured, for example, by the following procedure: (1) The number of live bacteria before inactivation is counted by shaking culture at 37°C in 40 mL of BHI medium, diluting 400-fold, and then measuring the number of live bacteria at OD600nm (0.45 = 6×10 8 CFU / mL) (theoretical value: 6×10 8 x 40 mL x 400 = 9.6 x 10 12 CFU / 40 mL = 2.4 x 10 11 (2) The bacterial solution was diluted to 100% (CFU / mL) at a concentration 2 to 4 times higher than the theoretical value and at a concentration 10 -9 , 10- 10 (3) Prepare a dilution series of the above, inoculate 100 μL onto a medium containing BHI agarose, and count the following day. (4) Use the concentration that gives the closest count to the theoretical value, and adjust to each CFU number. (5) Inactivate the virus.

[0044] In one embodiment of the present invention, the dose of the composition or the total dose of the vaccine components may be 1 to 10 mL per dose. This amount is preferably 1 to 9 mL, more preferably 1 to 8 mL, even more preferably 1 to 4 mL, and most preferably 1 to 2 mL. This amount may be, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mL, or may be any one of these values ​​or more, or may be within a range between any two of these values.

[0045] In one embodiment of the present invention, the composition may further include a component other than the vaccine component, and the method may include a step of administering a component other than the vaccine component. The other component may include, for example, an adjuvant. The other component may be a sustained-release agent. From the viewpoint of exerting a better vaccine effect, the sustained-release agent is preferably a polyacrylate (e.g., sodium polyacrylate). The other component may be, for example, aluminum hydroxide. In one embodiment of the present invention, the composition may not further include an adjuvant. The content of the other component may be 0.1, 0.15, 0.2, 0.25, 0.3, 0.4, 0.5, 1, 2, 5, or 10 w / v% of the composition, or may be equal to or greater than any of these values, or may be within a range between any two of these values. This value may be, for example, 0.1 to 10, 0.1 to 3, 0.1 to 0.25, 0.1 to 0.2, 0.15 to 0.4, 0.15 to 0.3, 0.15 to 0.25, or 0.15 to 0.2 w / v %.

[0046] In one embodiment of the invention, the composition may further comprise an antigen derived from E. coli, and the method may comprise the step of administering the antigen derived from E. coli. The antigen derived from E. coli may be, for example, inactivated E. coli or a part thereof, an immunogenic E. coli protein, or a nucleic acid encoding the protein.

[0047] In one embodiment of the invention, the composition may further comprise a pharma- ceutically acceptable carrier (excluding nanogels), and the method may comprise the step of administering a pharma- ceutically acceptable carrier (excluding nanogels).

[0048] In one embodiment of the invention, the initial administration of the composition or vaccine components to a cow may be carried out, for example, in a lactating cow.

[0049] In one embodiment of the invention, a booster dose may be administered after the composition or vaccine components are administered. The composition or vaccine components may be used for the booster dose. The booster or additional dose may be administered 7 days or more after the previous dose (e.g., the first dose). This may be, for example, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 120, 150, or 180 days, or any number of days after, or within a range of any two of, the number of days. This number of days may be, for example, 7 to 180, 11 to 180, 12 to 100, 12 to 60, 12 to 30, 13 to 60, 13 to 50, 13 to 30, 13 to 20, 14 to 60, 14 to 50, 14 to 30, or 14 to 20 days. The number of booster administrations may be, for example, 1, 2, or 3 times.

[0050] In one embodiment of the present invention, the homology (%) may be calculated by, for example, calculating the ratio of the number of amino acids that are homologous between a plurality of amino acid sequences according to a method known in the art. Before calculating the ratio, the sequences to be compared are aligned, and gaps are introduced into a part of the sequence if necessary to maximize the ratio of homologous / identical amino acids. Alignment methods, ratio calculation methods, and computer programs related thereto are conventionally well known in the art. In this specification, homology includes identity, and the homology ratio can be replaced with the identity ratio. The homology is calculated, for example, using global or local alignment. The former may be calculated using the Needleman-Wunsch algorithm (Needleman et al., J Mol Biol. 1970 Mar;48(3):443-53.), or may be calculated using EMBOSS Needle (Rice et al., EMBOSS User's Guide: Practical Bioinformatics, 25 July 2011.). Default settings for EMBOSS Needle may include, for example, MATRIX: BLOSUM62, Gap Open Penalty: 10, Gap Extend Penalty: 0.5, Output formats: pair, End Gap Penalty: false, End Gap Open Penalty: 10, End Gap Extend Penalty: 0.5. The latter may be determined by the BLAST algorithm (Altschul et al., J Mol Biol. 1990 Oct 5;215(3):403-10.) or calculated using blastp (The BLAST Sequence Analysis Tool. The NCBI Handbook, 2nd edition. March 15, 2013.).The default settings of blastp may include, for example, MATRIX: BLOSUM62, Gap Open Penalty: 11, Extension: 1, Compositional adjustments: Conditional compositional score matrix adjustment. Homology may be expressed as (number of homologous amino acids / number of amino acids in the amino acid sequence to be compared)×100. Homology is preferably calculated using global alignment. Unless otherwise specified, the homology of the test subject being equal to or greater than a particular value includes a value equal to or greater than a particular value calculated by at least one of the above algorithms. Any of the above algorithms may be used with default settings.

[0051] In one embodiment of the present invention, the above-mentioned 90% or more may be, for example, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, or may be any of these values ​​or more, or may be within a range between any two of these values. From the viewpoint of further maintaining functional equivalence, this percentage is preferably 95% or more, more preferably 97% or more, even more preferably 98% or more, and particularly preferably 99% or more.

[0052] In one embodiment of the present invention, when the bovine CCL28 component is in the form of a protein, the protein may have an amino acid sequence having one to several amino acid deletions, substitutions, insertions or additions relative to the amino acid sequence shown in SEQ ID NO: 1 or 2. The number may be, for example, 15, 10, 8, 6, 5, 4, 3, 2, or 1, or may be any one of these values ​​or less, or may be within a range between any two of these values. From the viewpoint of further maintaining functional equivalence, this number is preferably 15 or less, more preferably 10 or less, even more preferably 5 or less, and particularly preferably 3 or less.

[0053] In one embodiment of the present invention, conservative amino acid substitutions (see, e.g., French et al., J Mol Evol (1983) 19, 171-175) are preferred when substitutions are made to the original sequence. In one embodiment of the present invention, conservative amino acid substitutions include replacing an amino acid residue with an amino acid residue having a side chain of similar properties. Amino acid residues are classified into several families according to their side chains, such as basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan). Conservative amino acid substitutions are preferably between amino acid residues within the same family.

[0054] In one embodiment of the present invention, the protein includes a protein that can be used as an adjuvant, such as a chemokine. The protein may be fused to, for example, a peptide (e.g., a tag).

[0055] In one embodiment of the present invention, the CCL28 mutant may be, for example, a protein having an amino acid sequence that is 90% or more homologous to the amino acid sequence shown in SEQ ID NO: 1 or 2, or a protein having an amino acid sequence in which one to several amino acids have been deleted, substituted, inserted, or added relative to the amino acid sequence shown in SEQ ID NO: 1 or 2.

[0056] In one embodiment of the invention, the composition or vaccine components may be administered prior to the onset of mastitis or prior to being diagnosed with mastitis.

[0057] In one embodiment of the invention, the vaccine composition includes a composition for use in a vaccine application, for example, a composition for preventing or controlling bovine mastitis.

[0058] In one embodiment of the present invention, the composition may be prepared by any method known in the technical field of pharmaceuticals, for example, by mixing an active ingredient with one or more pharma- ceutically acceptable carriers. The carrier may be, for example, a liquid (e.g., saline or buffer solution). The content of the carrier may be, for example, a pharma-ceutically effective amount. The pharma-ceutically effective amount may be, for example, an amount sufficient for pharma-ceutically stabilization or delivery of the active ingredient. The composition may be a pharmaceutical composition. The composition may include a stabilizer, a buffer, or a pH adjuster. The dosage, administration interval, administration method, and administration route may be appropriately selected depending on the symptoms of the subject, etc. The composition preferably includes a prophylactically effective amount of the active ingredient. In one embodiment of the present invention, the prophylactically effective amount includes an amount necessary for clinically observed suppression of symptoms in the subject. In one embodiment of the present invention, pharma-ceutically acceptable includes a state suitable for use within the scope of sound medical judgment and commensurate with a reasonable benefit / risk ratio.

[0059] In one embodiment of the invention, the vaccine components may be administered simultaneously or separately or as a composition containing them.

[0060] In one embodiment of the present invention, the amino acid is a general term for an organic compound having an amino group and a carboxyl group. When the antibody according to the embodiment of the present invention contains a "specific amino acid sequence", any of the amino acids in the amino acid sequence may be chemically modified. Any of the amino acids in the amino acid sequence may form a salt or a solvate. Any of the amino acids in the amino acid sequence may be L-type or D-type. Even in such cases, the antibody according to the embodiment of the present invention can be said to contain the above-mentioned "specific amino acid sequence". Examples of chemical modifications that amino acids contained in proteins undergo in vivo include N-terminal modifications (e.g., acetylation, myristoylation, etc.), C-terminal modifications (e.g., amidation, glycosylphosphatidylinositol addition, etc.), and side chain modifications (e.g., phosphorylation, sugar chain addition, etc.).

[0061] In one embodiment of the present invention, "significantly" may mean, for example, a state where a statistically significant difference is evaluated using a Student's t-test (one-tailed or two-tailed) and p<0.05 or p<0.01, or a state where a substantial difference is generated.

[0062] All publications cited herein are incorporated by reference in their entirety. In this specification, "or" is used when "at least one or more" of the items listed in the text can be employed. The same applies to "or." When it is stated herein that "within a range of two values," the range includes the two values ​​themselves. In this specification, "A to B" includes A and B as well as values ​​between A and B.

[0063] Although the embodiments of the present invention have been described above, these are merely examples of forms that may be included in the present invention, and the present invention is not limited to these, and various configurations other than those described above may be adopted. Furthermore, the present invention may be adopted in combination with each of the configurations or features described in the above embodiments, or independently. EXAMPLES

[0064] The present invention will be further explained below with reference to examples, but is not limited to these.

[0065] Example 1 Previously, vaccination by nasal administration of nanogel was able to induce IgA antibodies in milk, but not IgG antibodies (Nagasawa et al., BMC Vet Res. 2019 Aug 9;15(1):286.). Therefore, we administered nanogel intranasally to dairy cows (Holstein cows) three times, and then administered sodium polyacrylate (SP) subcutaneously once two weeks later. As a result, we were able to induce both specific IgA and IgG antibodies (Figure 1).

[0066] However, vaccination by a combination of intranasal and subcutaneous administration is more effective than the commercially available vaccine (Startback (Laboratorios Hipra SA)), but has the disadvantage of requiring more frequent administrations.

[0067] To search for potential adjuvants, we analyzed the expression of six cytokines or chemokines (CXCL6, CXCL12, CCL2, CCL28, IL-8 (CXCL8), and IFN-γ) in milk and mammary tissues of cows immunized with intranasal administration of nanogel and subcutaneous administration of SP. As a result, it was revealed that the expression of CCL28 was increased in milk (Figure 2), and that the expressions of CCL28 and CXCL12 were increased in mammary tissues (Figure 3).

[0068] When human-derived CXCL12 was added to a mammary epithelial cell line, cell proliferation was suppressed. In addition, when CXCL12 was added to a fibroblast cell line, cell proliferation was promoted, suggesting that CXCL12 may act as an exacerbating factor for mastitis by inducing fibroblast proliferation (Figure 4) (mammary epithelial cells are the cells that produce milk, and when mastitis occurs, mammary epithelial cells are replaced by fibroblasts, reducing milk production). Therefore, CCL28 remained as a candidate adjuvant.

[0069] Bovine CCL28 was produced using a recombinant protein cloning and expression system using Escherichia coli. The amino acid sequence of CCL28 is shown in SEQ ID NO: 1. The produced bovine CCL28 was diluted to 1 mg and 1 × 10 11 A prototype vaccine was prepared in a total volume of 8 mL by mixing CFU of formalin-inactivated mastitis-derived Staphylococcus aureus (BM1006 strain. For the origin of the strain, see Hata et al., J Vet Med Sci. 2010 May;72(5):647-52. Formalin inactivation was performed according to the procedure in Kiku et al., J Vet Med Sci. 2016 Oct 1;78(9):1505-1510.) and 0.2 w / v% SP with respect to the total volume into physiological saline. In addition, a control formulation with the same composition except that it did not contain CCL28 was prepared. The prototype vaccine and control formulation were each administered once subcutaneously to the neck of dairy cows (Holstein cows, lactating period). Two weeks later, an intramammary infection experiment was conducted (the infection method followed the procedure of Nagasawa et al., Anim Sci J. 2018 Jan;89(1):259-266.). As a result, it was confirmed that administration of the prototype vaccine induced IgA (mucosal immunity) and IgG (systemic immunity) in the milk (Figure 5). Furthermore, a single subcutaneous administration of the prototype vaccine suppressed the proliferation of Staphylococcus aureus in the milk (Figure 6) and reduced clinical symptoms (Figures 7 and 8). On the other hand, these effects were not observed when the control formulation was administered.

[0070] As described above, administration of inactivated S. aureus and CCL28 has been shown to have an excellent vaccine effect against bovine mastitis. Specifically, this administration method was shown to induce both specific IgA and IgG antibodies in milk, to be highly safe, to suppress the number of S. aureus in milk, and to improve clinical symptoms. In particular, it was surprising that these effects were observed with a single administration of the vaccine.

[0071] Bovine mastitis, when caused by Escherichia coli or streptococcus, can present with acute or peracute symptoms, and symptoms can lead to astasia or poor prognosis within half a day to a day. In addition, Staphylococcus aureus mastitis can also present with acute or peracute symptoms from chronic infection, so a fast-acting vaccine that is effective on the first dose is very beneficial. Bovine mastitis is also a disease that can be considered an occupational disease for dairy cows, so when using a mastitis vaccine, it is assumed that it will be administered to all cows under management. Therefore, a vaccine that is effective with a single dose will lead to labor savings in managing dairy cow herds.

[0072] Example 2 The bovine CCL28 prepared was used at 1 mg and 1 × 10 11 CFU of formalin-inactivated mastitis-derived Staphylococcus aureus (BM1006 strain) and 0.2 w / v% SP were mixed with physiological saline to prepare a prototype vaccine in a total volume of 8 mL. The prototype vaccine was administered once subcutaneously to the neck of one milking cow (Holstein cow, lactating). Two weeks later, the above prototype vaccine was administered subcutaneously to the neck of the same cow. Two weeks after the second subcutaneous administration, an intramammary infection experiment (infection method was according to the procedure of Nagasawa et al., Anim Sci J. 2018 Jan;89(1):259-266.) was performed. As a result, it was confirmed that administration of the prototype vaccine induced IgA (mucosal immunity) and IgG (systemic immunity) in milk to the same extent as a single subcutaneous administration (Figure 9), suppressed the growth of Staphylococcus aureus in milk (Figure 10), and reduced clinical symptoms (Figures 11 and 12).

[0073] The present invention has been described above based on the embodiments. However, these embodiments are merely illustrative, and it will be understood by those skilled in the art that various modifications are possible and that such modifications are also within the scope of the present invention.

Claims

1. A vaccine composition against bovine mastitis, comprising an antigen derived from Staphylococcus aureus and a bovine CCL28 component, the composition being administered subcutaneously or intramuscularly into the neck or rump of a cow.

2. The composition of claim 1, which inhibits the growth of Staphylococcus aureus.

3. The composition of claim 1, which significantly increases antibody titers upon initial administration.

4. The composition of claim 3, wherein the antibodies are IgA and IgG.

5. The composition of claim 1, which ameliorates clinical symptoms of mastitis.

6. The composition of claim 1, wherein the first administration improves clinical symptoms of mastitis.

7. The composition of claim 1, wherein the bovine CCL28 component is administered as an adjuvant.

8. The composition according to claim 1, wherein the content of the bovine CCL28 component is 0.1 to 10 mg.

9. The content of the antigen derived from Staphylococcus aureus is 1×10 8 CFU to 5 × 10 11 The composition of claim 1, which is a CFU.

10. The composition of claim 1, wherein the dosage of the composition is 1 to 10 mL.

11. The composition of claim 1, wherein the bovine CCL28 component is a protein having chemokine activity or a biologically active fragment thereof having chemokine activity selected from the group consisting of (i) a protein comprising the amino acid sequence shown in SEQ ID NO: 1 or 2, and (ii) a protein comprising an amino acid sequence having 90% or more homology to the amino acid sequence shown in SEQ ID NO: 1 or 2, or a nucleic acid encoding the protein or the biologically active fragment thereof.

12. A method for preventing mastitis in cattle, comprising administering to the cattle a composition according to any one of claims 1 to 11.

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