JP2019182751A - Vaccine formulation for neospora caninum infectious disease - Google Patents

Abstract

To provide a safe vaccine formulation that can shift a balance between Th1 type reaction and Th2 type reaction in a body of livestock such as bovine toward Th1 side, and a safe vaccine formulation that can induce effective immune response which is especially cellular immunity to Neospora caninum infection.SOLUTION: The present invention relates to a vaccine formulation for Neospora caninum infectious disease in which a variant or a derivative holding Neospora caninum cyclophilin or immunogenicity thereof is encapsulated, and which contains a liposome having oligosaccharide that can bond to a sugar chain recognition molecule on the surface of an antigen-presenting cell on the surface thereof.SELECTED DRAWING: Figure 1

Description

  The present invention relates to a vaccine preparation against Neospora protozoa infection. More specifically, the present invention relates to a vaccine preparation comprising cyclophilin, which is a protein of Neospora, encapsulated in a liposome having on its surface an oligosaccharide capable of binding to a sugar chain recognition molecule on the surface of an antigen-presenting cell.

  Neospora caninum is an intracellular parasitic protozoa with canine animals as final hosts and cattle, sheep, goats, deer, etc. as intermediate hosts, host epithelial cells, mononuclear cells in cerebrospinal fluid, Parasitize the nerve cells. Neospora protozoa are transmitted by horizontal infection by oocysts excreted in the feces of the final host or by vertical infection in intermediate hosts.

  Neospora parasites cause polymyositis and ascending paralysis in dogs, and frequently cause abnormal births in cattle, mainly miscarriages, stillbirth or calf neurological symptoms. In particular, when Neospora protozoa is infected during the mid-gestation when the mother is immunosuppressed, fetuses are vertically infected, and individuals who are congenitally infected with Neospora protozoa are born. Also, mother cows infected with Neospora protozoa are often unable to be milked. Thus, in the livestock industry, especially neospora infection of cattle is a serious problem that causes great economic loss.

  Administration of sulfa drugs, erythromycin, doxycycline, clindamycin, etc. is performed as treatment for Neospora protozoa infection. However, since it is not desirable to administer antibiotics to edible livestock, establishment of a method for preventing Neospora infection is an important issue.

  As an infection prevention method for Neospora protozoa, inactivated vaccines (Non-patent Document 1) and live vaccines containing inactivated Neospora protozoa and adjuvants have been studied. In addition to inactivated vaccines or live vaccines that use Neospora protozoa per se, vaccines that contain Neospora protozoan antigen proteins have also been developed. For example, vaccines containing recombinant proteins such as MIC1, MIC3, GRA2, GRA6, SAG1, and SRS2 have been proposed, and suppression of Neospora protozoa has been confirmed (Non-Patent Documents 2 and 3). However, since these recombinant vaccines use microorganisms or viruses as vectors for their production, the pathogenicity of the vectors can be a problem.

  Since Neospora protozoa are intracellular parasitic protozoa, induction of humoral immunity in Neospora protozoa infections is effective in blocking the spread of protozoa present outside the cell and inhibiting the function of pathogenic factors released from protozoa However, a direct effect on intracellular protozoa cannot be expected. On the other hand, since cellular immunity can destroy protozoa-infected cells, it is thought to contribute more directly to the elimination of intracellular protozoa.

  As an abiotic vaccine carrier capable of inducing cellular immunity, it is applied to liposomes with 2 to 11 sugar residues, particularly mannose oligosaccharides on the surface, that bind to sugar chain recognition molecules on the surface of antigen-presenting cells. A vaccine encapsulating an antigen has been reported (Patent Document 1). Liposomes having mannose oligosaccharides on the surface can be phagocytosed by antigen-presenting cells via mannose receptors, and cellular immunity can be induced by presenting antigens via MHC class I or II molecules.

  The present inventors have proposed a vaccine preparation for Neospora protozoa, in which Dense granule protein 7 (GRA7) or Apical membrane antigen 1 (AMA1), which is a protein derived from Neospora protozoa, is encapsulated in liposomes having the above-mentioned mannotrice on the surface. (Patent Document 2).

  Cellular immunity is induced by activation of macrophages and cytotoxic T cells by Th1 cytokines such as IFN-γ and IL-2 produced by Th1 cells, whereas humoral immunity is IL produced by Th2 cells. -4, Th2 cytokines such as IL-10 are induced by promoting differentiation of B cells into plasma cells and antibody production. In vivo, the immune response by Th1 cells (Th1-type reaction) and the immune response by Th2 cells (Th2-type reaction) are mutually controlled to maintain an equilibrium relationship. It is considered important to shift the state to the Th1 side.

JP-A-7-126185 International Publication No. 2010/32408 Pamphlet

Dubey et al., Clinical Microbiology Reviews, 2007, 20: 323-367 Ramamoorthy et al., Int. J. Parasitol., 2007, 37: 1531-1538 Nishikawa et al., Vaccine, 2001, 19: 1710-1716

  An object of the present invention is to provide a safe vaccine preparation that can shift the balance of Th1-type reaction and Th2-type reaction in livestock such as cattle to the Th1 side. A further object of the present invention is to provide a safe vaccine preparation that can induce an effective immune response against Neospora protozoa infection, particularly cellular immunity.

  The present inventors encapsulate Neospora caninum cyclophilin (sometimes abbreviated as NcCyp) in liposomes having oligosaccharides that bind to sugar chain recognition molecules on the surface of antigen-presenting cells, and administer them to living bodies. As a result, it was found that Th1 cytokines were produced predominantly over Th2 cytokines, and cellular immunity against Neospora was preferentially induced, and the following inventions were completed.

(1) Neospora protozoa, including liposomes having oligosaccharides on the surface thereof, which are capable of binding to sugar chain recognition molecules on the surface of antigen-presenting cells, encapsulating cyclosporins derived from Neospora protozoa or immunogenic variants or derivatives thereof Vaccine preparation for infectious diseases.
(2) The variant is a protein that includes an amino acid sequence in which one or several amino acids are deleted, substituted, or added in the amino acid sequence constituting cyclophilin, and that can induce an immune response against Neospora protozoa (1) The vaccine preparation described in 1.
(3) A derivative is a protein in which one or several amino acid residues of cyclophilin or a variant thereof are chemically modified, a fusion protein of cyclophilin or a variant thereof with another polypeptide, and cyclophilin or a variant thereof is blood. The vaccine preparation according to (1), wherein the vaccine preparation is a protein selected from the group consisting of proteins modified with a polymer that enhances medium retention, and capable of inducing an immune response against Neospora protozoa.
(4) The vaccine preparation according to any one of (1) to (3), wherein cyclophilin is a protein consisting of the amino acid sequence represented by SEQ ID NO: 1.
(5) The vaccine preparation according to any one of (1) to (4), wherein the sugar chain recognition molecule on the surface of the antigen-presenting cell is a mannose receptor.
(6) The vaccine preparation according to any one of (1) to (5), wherein the oligosaccharide is composed of 2 to 11 sugar residues.
(7) The vaccine preparation according to any one of (1) to (6), wherein the oligosaccharide comprises 3 to 5 sugar residues.
(8) The vaccine preparation according to any one of (1) to (7), wherein the oligosaccharide contains two or more mannose sugar residues.
(9) The vaccine preparation according to any one of (1) to (8), further comprising a pharmaceutically acceptable carrier.
(10) The vaccine preparation according to (9), which is administered subcutaneously, intradermally, orally or nasally.

  The vaccine preparation provided by the present invention shifts the balance between Th1 type reaction and Th2 type reaction to the Th1 side, and can induce an immune response, particularly cellular immunity against Neospora protozoa infection. Has a high preventive effect.

It is a graph which shows the amount of alkaline phosphatase which RAW264.7 cell line (NF-kB / SEAP) which introduce | transduced the liposome (NcCyp-OML) which enclosed lipopolysaccharide (LPS), cyclophilin (NcCyp), or cyclophilin was introduce | transduced. Results are expressed as mean ± standard deviation. In the figure, * indicates that there is a significant difference (*: p <0.05, **: p <0.01, ***: p <0.001) with respect to the untreated group (mock). It is a graph which shows the IL-12 p40 amount which the mouse | mouth peritoneum origin macrophage which introduce | transduced LPS, NcCyp, or NcCyp-OML produces. Results are expressed as mean ± standard deviation. In the figure, different alphabets indicate significant differences between groups. It is a graph which shows the serum NcCyp specific IgG1 antibody (left figure) and IgG2a antibody (right figure) density | concentration when inoculating Neospora protozoa to the mouse | mouth which immunized NcCyp or NcCyp-OML. Results are expressed as mean ± standard deviation. In the figure, * indicates that there is a significant difference (p <0.05) from the pre-immunization (day 0) within the same group, and different alphabets indicate that there is a significant difference between samples with the same sampling date. . Proliferation of spleen cells (panel A) and IFN- in the culture supernatant when concanavalin A, Neospora protozoa lysate (NLA) or NcCyp is added to spleen cells collected from mice immunized with NcCyp or NcCyp-OML It is a graph which shows (gamma) production amount (panel B). Results are expressed as mean ± standard deviation. In the figure, # indicates that there is a significant difference (p <0.05) from the negative control (Medium) within the same inoculation group, and different alphabets indicate that there is a significant difference between the same antigen stimulations. From mice immunized with NcCyp or NcCyp-OML (panel A), mice immunized with NcGRA7, M3-NcGRA7 or M3-GST (panel B), mice immunized with NcAMA1, M3-NcAMA1 or M3-GST (panel C) It is a graph which shows the ratio of IFN-γ production amount and IL-4 production amount in the culture supernatant when the collected spleen cells are cultured under the corresponding antigen stimulation. It is a graph which shows the survival curve (panel A) of a mouse | mouth when a neospora protozoa is inoculated to the mouse | mouth immunized with NcCyp or NcCyp-OML, a clinical score (panel B), and the amount of protozoa in a brain tissue (panel C). Panel B results are expressed as mean ± standard error. In the figure, * indicates that there is a significant difference (p <0.05) from the PBS administration group. It is a graph which shows the survival curve of a C57BL / 6 mouse (panel A) and a TLR2 KO mouse (panel B) when a mouse | mouth immunized with NcCyp or NcCyp-OML is inoculated with Neospora protozoa. In the figure, * indicates that there is a significant difference (p <0.05) from the PBS administration group.

  The present invention includes a liposomal preparation having an oligosaccharide encapsulated on a surface of an antigen-presenting cell encapsulating a cyclophilin derived from a neospora protozoa or a mutant or derivative retaining its immunogenicity on the surface, The present invention relates to a vaccine preparation against Neospora protozoa infection.

Neospora caninum cyclopilin (NcCyp)
NcCyp is a protein that has peptidyl-prolyl cis-trans isomerase (PPIase) activity and binding ability to cyclosporine, an immunosuppressant, and is a major component that promotes IFN-γ production in infected hosts Is known (Tuo et al., Infect Immun., 2005, 73 (8): 5093-5100.).

The amino acid sequence of NcCyp and the ORF sequence of the gene that encodes it are already known, and each of them is published in the public database GenBank as ACCESSION No. XP_003880037.1.
(Amino acid sequence), ACCESSION No. XM_003879988 (base sequence) is registered. The amino acid sequence of NcCyp that does not include a signal sequence is SEQ ID NO: 1, the base sequence of the DNA that encodes it is SEQ ID NO: 2, the amino acid sequence of the NcCyp that includes the signal sequence is SEQ ID NO: 3, and the base sequence of the DNA that encodes this Are shown in SEQ ID NO: 4, respectively.

  In the present invention, in addition to NcCyp, NcCyp mutants or derivatives that retain the immunogenicity of NcCyp, that is, can induce an immune response against Neospora protozoa are also used as proteins encapsulated in liposomes. Can do.

  Examples of variants include deletion, substitution or substitution of one or several, preferably 2-7, more preferably 2-5, most preferably 2-3 amino acid residues in the amino acid sequence of NcCyp. Mention may be made of proteins comprising the added amino acid sequence and retaining the immunogenicity of NcCyp.

  The substitution is preferably a so-called conservative substitution. Examples of such substitution include glycine (Gly) and proline (Pro), glycine and alanine (Ala) or valine (Val), leucine (Leu) and isoleucine (Ile), glutamic acid ( Amino acids such as Glu) and glutamine (Gln), aspartic acid (Asp) and asparagine (Asn), cysteine (Cys) and threonine (Thr), threonine and serine (Ser) or alanine, lysine (Lys) and arginine (Arg) Can be mentioned.

  Further, when calculating the identity with the NcCyp amino acid sequence, for example, using the initial condition parameters of BLAST, at least 70% or more, preferably 80% or more, more preferably 90% or more, more preferably 95% or more, Particularly preferred is a protein having an amino acid sequence having 97%, 98% or 99% identity and capable of inducing an immune response against Neospora protozoa as a variant of NcCyp. It is contained and can be used as a protein encapsulated in a liposome.

  In addition, a protein in which one or several amino acid residues of NcCyp or its variant are chemically modified, a fusion protein of NcCyp or its variant and another polypeptide, or NcCyp or its variant is converted to polyethylene glycol (PEG), etc. Proteins modified with macromolecules that increase the retention of blood in the blood and that can induce an immune response against Neospora protozoa can also be used as NcCyp derivatives as proteins encapsulated in liposomes it can.

Preparation of NcCyps
NcCyp may be isolated and purified from Neospora protozoa by known means. However, NcCyp is a set prepared by a recombinant protein production method using E. coli or other microorganisms, insect cells or animal cells as host cells. It is preferable to use a modified NcCyp. Construction of recombinant genes, introduction of expression vectors into host cells, expression of target proteins in host cells, and other genetic engineering techniques are based on the instructions in the experimental operation manual that explains various gene recombination operations in detail. Can be done.

  DNA encoding NcCyp, its mutants or derivatives (hereinafter collectively referred to as NcCyps) can be incorporated into an appropriate expression vector for recombinant production of NcCyps. The recombinant vector having DNA encoding NcCyps may be in any form such as circular or linear. In addition to the base sequence encoding NcCyps, such a recombinant vector may have other base sequences if necessary. Examples of other base sequences include enhancer sequences, promoter sequences, ribosome binding sequences, base sequences used for the purpose of copy number amplification, base sequences encoding peptides such as signal peptides and other polypeptides, and addition of poly A These include sequences, splicing sequences, and base sequences of genes that serve as selection markers.

  For gene recombination, a suitable synthetic DNA adapter is used to add a translation initiation codon or translation termination codon to DNA encoding NcCyps, or a new restriction enzyme cleavage sequence is newly generated or eliminated in the base sequence. It is also possible to do. These are within the scope of work normally performed by those skilled in the art, and those skilled in the art can arbitrarily and easily process DNA encoding NcCyps.

  Moreover, as an expression vector holding DNA encoding NcCyps, an appropriate vector according to the host to be used can be selected. In addition to plasmids, various viruses such as bacteriophage, baculovirus, retrovirus, vaccinia virus, etc. However, it is preferable to use a vector that can be used in a host expression system using microorganisms, particularly bacteria such as Escherichia.

  NcCyps can be expressed under the control of a promoter sequence unique to the NcCyp gene. Alternatively, another appropriate expression promoter can be linked upstream of the base sequence encoding NcCyps. Such an expression promoter may be appropriately selected depending on the host. For example, when the host is an Escherichia bacterium, preferably E. coli, the T7 promoter, lac promoter, trp promoter, λPL promoter, etc. In the case of bacteria, preferably B. subtillis, P43 promoter, vegI promoter, xylose-inducible promoter, tetracycline inducible promoter and the like can be mentioned. When the host is yeast, PHO5 promoter, GAP promoter, ADH promoter, etc. When the host is an animal cell, SV40-derived promoter, retrovirus promoter, cytomegalovirus (CMV) IE (immediate early) Examples include gene promoters, metallothionein promoters, heat shock promoters, SRα promoters, and the like.

  Examples of host cells include Escherichia, Bacillus, Corynebacterium, Brevibacterium, Serratia, Pseudomonas, Pseudomonas, Arthrobacter (Arthrobacter) genus, Erwinia genus, Methylobacterium genus and Rhodobacter genus bacteria, Streptomyces genus, Zymomonas genus and Saccharomyces genus fungus Can be mentioned. Insect cells such as silkworms, HEK293 cells, MEF cells, Vero cells, Hela cells, CHO cells, WI38 cells, BHK cells, COS-7 cells, MDCK cells, C127 cells, HKG cells, and human kidney cell lines Is also available.

  Examples of transformation methods for introducing an expression vector into host cells include electroporation, alkali metal, calcium phosphate precipitation, DEAE dextran, and microinjection.

  NcCyps can be obtained by culturing a transformed cell into which an expression vector has been introduced, expressing the protein in the cell, collecting the target protein from the cell or medium, and purifying it. Culture of the transformed cells may be performed according to conventional methods according to the properties of the host cell such as carbon assimilation and auxotrophy, the selection marker contained in the introduced recombinant gene, promoter and the like.

  NcCyps can be purified by appropriately selecting an appropriate method from methods usually used for protein purification. That is, salting-out method, ultrafiltration method, isoelectric point precipitation method, gel filtration method, electrophoresis method, various affinity chromatography such as ion exchange chromatography, hydrophobic chromatography and antibody chromatography, chromatofocusing method, adsorption An appropriate method may be appropriately selected from commonly used methods such as chromatography and reverse phase chromatography, and purification may be performed in an appropriate order using an HPLC system or the like if necessary.

  Further, when NcCyps is expressed as a fusion protein with another functional protein or peptide, it is preferable to employ a purification method characteristic of the functional protein or peptide. Such functional proteins or peptides and the corresponding purification methods include histidine tags consisting of about 6 to 10 consecutive histidine residues, nickel immobilized affinity chromatography, glutathione S-transferase (GST) and glutathione immobilized affinity. Examples include chromatography. NcCyps can be recovered by cleaving the purified fusion protein with an appropriate protease (thrombin, trypsin, etc.).

  Furthermore, a cell-free synthesis method using a recombinant DNA molecule is one of genetic engineering production methods.

  NcCyps can also be produced using a commercially available suitable peptide synthesizer, for example, by organic chemical synthesis methods such as the Fmoc method (fluorenylmethyloxycarbonyl method) and the tBoc method (t-butyloxycarbonyl method). Is produced by introducing the above-described nucleic acid, particularly DNA incorporated into an expression vector, into a suitable expression system using a suitable host cell selected from prokaryotes or eukaryotes by genetic recombination technology. Is preferred.

Liposome and its preparation The liposome used in the present invention is a liposome having on its surface an oligosaccharide capable of binding to a sugar chain recognition molecule on the surface of an antigen-presenting cell. Such liposomes are described in JP-A-7-126185, US5759572, WO2010 / 32408, US2011 / 0250265, and US8821882, together with their constitution and production method. These documents are incorporated herein by reference in their entirety.

  “Sugar chain recognition molecule on antigen-presenting cell surface” refers to Fc receptor, complement receptor, scavenger receptor, mannose receptor, lipopolysaccharide (LPS) receptor present on the surface of antigen-presenting cells such as macrophages and dendritic cells, Sugar chains that are directly or indirectly involved in phagocytosis of bacteria, such as CD11b / CD18 (CR3) and Toll-like receptors, uptake of glycoproteins in foreign substances and antigen presentation to the cell surface It refers to all molecules with binding lectin-like properties. A suitable example is the mannose receptor.

  The oligosaccharide on the surface of the liposome is not particularly limited as long as it can bind to the sugar chain recognition molecule on the surface of the antigen-presenting cell. Constituent sugar residues include D-mannose (D-Man), L-fucose (L-Fuc), D-acetylglucosamine (D-GlcNAc), D-glucose (D-Glc), D-galactose (D- Gal), D-acetylgalactosamine (D-GalNAc), D-rhamnose (D-Rha), etc., but oligosaccharides containing D-Man, such as oligosaccharides consisting only of D-Man or D-Man And oligosaccharides consisting of D-GlcNAc are preferred, and oligosaccharides consisting only of D-Man are particularly preferred. Oligosaccharides consisting only of D-Man include mannobiose (Man2), mannotriose (Man3), mannotetraose (Man4), mannopentaose (Man5), mannohexaose (Man6), mannohepta Aus (Man7) can be mentioned.

  The binding mode of each sugar residue constituting the oligosaccharide is not particularly limited, and examples include α1 → 2 bond, α1 → 3 bond, α1 → 4 bond, α1 → 6 bond, β1 → 4 bond, and the like. They may be bonded in a straight chain or branched.

  The number of sugar residues constituting the oligosaccharide is preferably 2 to 11, particularly 3 to 11, and particularly preferably about 3 to 5.

  The lipid constituting the liposome in the present invention may be a normal lipid known to constitute a liposome. For example, fatty acids having a saturated or unsaturated acyl group, phospholipids, sterols, etc. Can be mentioned. Further, it may contain phospholipids derived from natural products such as egg yolk lecithin and soybean lecithin. These can be used alone or in combination of two or more.

  Fatty acid residues in fatty acids and phospholipids can be arbitrarily selected. Preferred fatty acids or fatty acid residues include saturated or unsaturated fatty acids having 4 to 30 carbon atoms, more preferably saturated fatty acids having 12 to 24 carbon atoms such as lauric acid, myristic acid, palmitic acid, stearic acid, oleic acid, Mention may be made of unsaturated fatty acids having 14 to 22 carbon atoms and 1 to 6 unsaturated bonds, such as linoleic acid, linolenic acid and arachidonic acid. These can be used singly or in a mixture, and in a lipid having two or more fatty acid residues, these groups may be the same or different.

  Suitable examples of phospholipids include phosphatidylethanolamines such as phosphatidylethanolamine (PA), dipalmitoylphosphatidylethanolamine (DPPE), distearoylphosphatidylethanolamine (DSPE); phosphatidylserine (PS), dipalmitoylphosphatidylserine (DPPS), phosphatidyl serines such as distearoyl phosphatidyl serine (DSPS); phosphatidyl cholines such as phosphatidyl choline (PC), dipalmitoyl phosphatidyl choline (DPPC); distearoyl phosphatidyl choline (DSPC); phosphatidic acid (PA), dipalmitoyl phosphatidic acid (DPPA), phosphatidic acids such as distearoyl phosphatidic acid (DSPA), and the like.

  Examples of sterols include cholesterol (Chol), 3β- [N- (dimethylaminoethane) carbamoyl] cholesterol (DC-Chol), N- (trimethylammonioethyl) carbamoylcholesterol (TC-Chol), and the like. .

The oligosaccharide can be introduced to the surface of the liposome by using an artificial glycolipid prepared by binding the oligosaccharide and the lipid as a constituent lipid of the liposome. Artificial glycolipids react with an aldehyde group possessed by an oligosaccharide with a phospholipid having an amino group to form a Schiff base, which is then reduced, preferably chemically reduced, for example NaBH ₃ CN, according to a conventional method. Can be prepared by binding oligosaccharides and lipids (for example, Tsujio Mizuochi, Carbohydrate Engineering, pages 224-232, 1992). The oligosaccharide on the surface of the liposome can be confirmed by the occurrence of an aggregation reaction of the liposome when a sugar chain recognition molecule present on the surface of the antigen-presenting cell or a part thereof is added. The amount of oligosaccharide introduced into the liposome surface may be 0.5 μg to 500 μg with respect to 1 mg of liposome-constituting lipid.

  The amount of NcCyps encapsulated in the liposome is not particularly limited and can be appropriately adjusted according to the administration route, but it is generally desirable to be 0.1 μg to 500 μg per 1 mg of lipid used in the liposome.

  Encapsulation of NcCyps in liposomes can be performed by dissolving NcCyps in an aqueous solvent used when hydrating a lipid membrane. For example, after dissolving the lipid and artificial glycolipid constituting the lipid membrane of the liposome in an organic solvent such as chloroform, an aqueous solvent containing NcCyps is added to the dried product obtained by distilling off the organic solvent, and then subjected to ultrasonic treatment or By emulsifying with vortex or the like, a liposome encapsulating NcCyps can be prepared. In addition, it can be prepared by an ethanol injection method, an ether injection method, a reverse phase evaporation method, a surfactant method, a freezing / thawing method, or the like. In addition to NcCyps, drugs, nucleic acids, peptides, proteins, or complexes thereof useful for preventing or treating Neospora protozoa can be encapsulated together.

  As long as the liposome in the present invention is a closed vesicle having a lipid membrane composed of a lipid bilayer, the number of lipid membranes is not particularly limited, and even if it is a multilamellar vesicle, it is also a single sheet. It may be a membrane liposome (uni lamella vesicle). The average particle size of the liposome is not particularly limited. For example, the average particle size can be adjusted by repeating filtration with a filter having a desired pore size.

Vaccine preparation The vaccine preparation of the present invention can be used in the form of a dispersion in which the above-mentioned liposomes are dispersed in an appropriate aqueous solvent such as physiological saline, phosphate buffer, citrate buffer, and acetate buffer. Dispersions include sugars, polyhydric alcohols, water-soluble polymers, nonionic surfactants, antioxidants, pH regulators, hydration promoters, proteins such as albumin or casein, proteins such as bovine serum or skim milk Pharmaceutically acceptable carriers and additives such as contained substances may be added as appropriate. The vaccine preparation of the present invention can also be used in the form of a dry preparation obtained by lyophilizing the above dispersion.

  The vaccine preparation of the present invention may contain an adjuvant or the like as desired. Examples of adjuvants include Freund's complete adjuvant, Freund's incomplete adjuvant, saponin, aluminum hydroxide, aluminum phosphate, aluminum oxide, N-acetylmuramyl-L-threonyl-D-isoglutamine (thr-MDP), N- Acetylnormuryl-L-alanyl-D-isoglutamine (CGP11637, nor-MDP), N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2- (1'-2'-dipalmitoyl- sn-glycero-3-hydroxyphosphoryloxy) -ethylamine (CGP19835A, MTP-PE) and the like can be mentioned, but are not limited thereto.

  The administration method of the vaccine preparation of the present invention is not particularly limited, but administration by subcutaneous injection, intravenous injection or intramuscular injection is preferable.

  The dose is usually in the range of 0.01 μg to 100,000 μg of antigen per dose, and the target animal, for example, the species of mammals such as cattle, sheep, goats, deer, dogs, their physical condition, age, etc. Is adjusted as appropriate.

  The vaccine formulations of the present invention may be given on a single dose schedule or preferably on a combined dose schedule. In a combined dosing schedule, 1 to 10 individual doses are given at the start of the inoculation, followed by time intervals required to maintain and / or enhance the immune response, for example 1 to 4 as the second dose. Another dose may be given after a month. If necessary, subsequent administrations can be given after several months. The dosage regimen will also be determined, at least in part, by the needs of the individual and will depend on the judgment of the veterinarian.

  The vaccine preparation of the present invention may be used for the purpose of preventing infection with Neospora protozoa, but can also be used as a therapeutic vaccine that is administered to subjects infected with Neospora protozoa to eliminate Neospora protozoa.

  Accordingly, the present invention also provides a method for treating and / or preventing Neospora protozoal infection comprising administering an effective amount of the above vaccine formulation to an animal in need thereof. The present invention further provides a method of eliciting a specific immune response against Neospora protozoa in an animal comprising administering an effective amount of the above vaccine formulation to the animal in need thereof.

EXAMPLES Hereinafter, although an Example demonstrates this invention concretely, this invention is not limited by these Examples.

Example 1 Preparation of Liposomes (1) PCR was performed using primer DNA designed to amplify cyclophilin gene (GenBank ACCESSION No. XM_003879988) using Neospora caninum genomic DNA as a template. The increase by-product was incorporated into the BamH I and EcoR I sites of the expression vector pGEX-4T1, and a vector expressing a fusion protein of glutathione S-transferase (GST) and NcCyp consisting of the amino acid sequence shown in SEQ ID NO: 1 was constructed. Escherichia coli DH5α was transformed with this vector, and the fusion protein was expressed in the cells. The fusion protein is recovered from the bacterial cells, purified and endotoxin removed using Glutathione Sepharose 4B (Amersham Pharmacia Biotech) and Detoxi-Gel ™ Endotoxin Removing Gel (Pierce), and GST is cleaved and removed to produce NcCyp as a recombinant protein. / Phosphate buffer was obtained.

(2) Mannotriose (Man3) 2.5 to 5 mg having a structure of Manα1 → 6 (Manα1 → 3) Man was dissolved in 600 μl of distilled water to prepare an oligosaccharide solution. Dipalmitoylphosphatidylethanolamine (DPPE) was dissolved in a chloroform / methanol (1: 1 volume ratio) mixture at a concentration of 5 mg / ml to prepare a DPPE solution. Further, NaBH ₃ CN was dissolved in methanol at a concentration of 10 mg / ml to prepare a NaBH ₃ CN solution. 9.4 ml of the DPPE solution and 1 ml of the NaBH ₃ CN solution were added to 600 μl of the oligosaccharide solution, mixed with stirring, and reacted at 60 ° C. for 16 hours. Mannotriose dipalmitoyl phosphatidylethanolamine (M3-DPPE) was prepared by passing the reaction mixture through a silica gel column and a C18 reverse phase column.

(3) Chol, DPPC, and M3-DPPE of (2) were mixed and dissolved at 10: 10: 1 (molar ratio) in 2 mL of chloroform in a 10 mL flask, and then the chloroform was distilled off to prepare a lipid film. . To this lipid film, 1.4 ml of NcCyp / phosphate buffer prepared in (1) (NcCyp content 2.0 mg) was added, and Vortexed in a 40 ° C. water bath to prepare liposomes. After passing through a 1 μm filter 5 times while applying pressure at 0.2 to 1 MPa, the solution was centrifuged 3 times with PBS (-), and liposomes encapsulated with NcCyp (NcCyp-OML, average particle size 1.0 μm) were obtained. Obtained.

Example 2 Evaluation of immunity induction ability of NcCyp-OML (in vitro)
(1) RAW264.7 cells
Murine macrophage-like RAW264.7 cell line that produces secreted alkaline phosphatase (NF-kB / SEAP) by NF-κB activation in 2 × 10 ⁶ cells / ml in Dulbecco's modified Eagle medium containing 10% fetal bovine serum It was suspended at a concentration and dispensed into a 96-well plate at 0.1 ml / well. Next, lipopolysaccharide (LPS) (final concentrations 50 μg / ml, 100 μg / ml), NcCyp (final concentrations 10 nM, 100 nM), NcCyp-OML (final concentrations 10 nM, 100 nM) were added to each well, and 37 ° C., 5% CO 2 Incubated under ₂ for 24 hours. The amount of secreted alkaline phosphatase in the culture supernatant after the incubation was measured using Secreted Alkaline Phosphatase Reporter Assay Kit (Novas Biologicals). Statistical analysis of measured values was performed by multiple comparison test of one-way ANOVA and Tukey Kramer.

  The results are shown in FIG. The amount of alkaline phosphatase was significantly higher in the NcCyp-OML group than in the untreated group (mock), and activation of NF-κB was confirmed by the addition of NcCyp-OML.

(2) Mouse peritoneal macrophages
A mouse abdominal cavity-derived macrophage obtained from a C57BL / 6 mouse (male, 8 weeks old) according to a conventional method was suspended in Dulbecco's modified Eagle medium containing 10% fetal bovine serum at a concentration of 3 × 10 ⁶ cells / ml, and 0.1 ml / Wells were dispensed into 96 well plates. Next, add lipopolysaccharide (LPS) (final concentration 10 ng / ml), NcCyp (final concentration 10 nM, 100 nM), NcCyp-OML (final concentration 10 nM, 100 nM) to each well, at 37 ° C, 5% CO ₂ Incubated for 20 hours. The IL-12 p40 concentration in the culture supernatant after incubation was measured using an IL-12 p40 Mouse ELISA Kit (Thermo Fisher Scientific). Statistical analysis was performed by one-way ANOVA and Tukey Kramer's multiple comparison test.

  The results are shown in FIG. Addition of NcCyp-OML induced IL-12p40 production from macrophages.

  From the above, it was confirmed that NcCyp-OML activates NFκB more strongly than NcCyp and induces the production of inflammatory cytokines from macrophages.

Example 3 Evaluation of the ability of NcCyp-OML to induce NcCyp-specific antibody production (in vivo)
BALB / c mice (female, 8 weeks old) were immunized subcutaneously with NcCyp, NcCyp-OML or PBS 3 times at intervals of 2 weeks (25 pmol protein per dose), and Neospora protozoa (2 weeks after the final immunization) 1 × 10 ⁶ ) was inoculated intraperitoneally. Serum was collected 2 days before each immunization day, and NcCyp-specific IgG1 and IgG2a antibodies were detected by ELISA. The ELISA method was performed using NcCyp purified recombinant protein as an antigen, primary antibody as a 100-fold diluted mouse serum, secondary antibody as a 4,000-fold diluted HRP-labeled anti-mouse IgG and IgG2a antibody, and ABTS as a substrate. Six mice were used in one test group in one experiment, and similar results were obtained in two experiments. Statistical analysis of measured values was performed by two-way ANOVA and Tukey Kramer's multiple comparison test.

  A representative one-time experiment result is shown in FIG. NcCyp-OML immunization confirmed the production of NcCyp-specific IgG1 antibodies. In addition, on the 49th day after the first immunization after Neospora protozoa infection, the production of NcCyp-specific IgG2a antibody was confirmed in the NcCyp-OML immunization group and the NcCyp immunization group compared with the PBS administration group, and NcCyp-OML immunization was confirmed. More antibody production was induced.

  From the above, immunization with NcCyp-OML induces the production of both Th2 type IgG1 antibody and Th1 type IgG2a antibody, which are specific antibodies to NcCyp, and the efficiency of specific antibody induction by encapsulating NcCyp in OML Has been shown to improve.

Example 4 Evaluation of NcCyp-OML's ability to induce NcCyp-specific cytokine production (mouse-derived spleen cells)
(1) Spleen cell proliferation and IFN-γ production
BALB / c mice (female, 8 weeks old) were immunized subcutaneously with NcCyp, NcCyp-OML, and PBS three times at intervals of 2 weeks (25 pmol protein per dose). Two weeks after the final immunization, the spleen was removed and homogenized to prepare a spleen cell suspension (5 × 10 ⁶ cells / ml RPMI1640 medium). Concanavalin A (ConA) (final concentration 0.5 μg / ml, 5 μg / ml), Neospora protozoa lysate (NLA) (final concentrations 10 μg / ml, 50 μg / ml), NcCyp (final concentration 10 μg / ml, Incubated for 48 hours at 37 ° C., 5% CO _{2 with} or without 50 μg / ml) or as a negative control (Medium). Spleen cell proliferation using Cell Counting Kit-8 reagent (CCK-8, Dojindo Laboratories, Kumamoto, Japan) and IFN-γ concentration in culture supernatant using IFN-γ Mouse ELISA Kit (Thermo Fisher Scientific) Measured. Four mice were used in one test group. Statistical analysis of measured values was performed by two-way ANOVA and Tukey Kramer's multiple comparison test.

  The results are shown in FIG. NcCyp-OML immunization group-derived spleen cells showed increased proliferation under NcCyp stimulation (FIG. 4A) and increased IFN-γ production (FIG. 4B).

(2) Comparison with other Neospora protozoan-derived antigen-encapsulated liposomes In the same manner as in Example 1, liposomes encapsulating Neospora dense granule protein 7 (NcGRA7), which is a GST fusion recombinant protein, instead of NcCyp (M3-NcGRA7) ), A liposome (M3-NcAMA1) encapsulating Neospora Apical membrane antigen 1 (NcAMA1), which is a GST fusion recombinant protein, or a liposome encapsulating GST (M3-GST) was prepared. BALB / c mice (female, 6 weeks old) were subcutaneously immunized 3 times at intervals of 2 weeks (25 pmol protein per time) with NcGRA7, M3-NcGRA7, NcAMA1, M3-NcAMA1, and PBS. Two weeks after the final immunization, the spleen was removed and homogenized to prepare a spleen cell suspension (5 × 10 ⁶ cells / ml RPMI1640 medium). Concanavalin A (ConA) (final concentration 2 μg / ml), Neospora protozoa lysate (NLA) (final concentration 50 μg / ml), NcGRA7 (final concentration 10 μg / ml), NcAMA1 (final concentration 10 μg / ml) Or without adding anything as a negative control (Medium), after culturing at 37 ° C. under 5% CO ₂ for 48 hours, the IFN-γ concentration in the culture supernatant was measured in the same manner as in (1). . The concentration of IL-4 in the culture supernatant was measured using IL-4 Mouse ELISA Kit (Thermo Fisher Scientific).

  For comparison between the immunized antigens, the measured values of the above IFN-γ and IL-4 concentrations and the measured values of IFN-γ and IL-4 measured in (1) were measured under CoA stimulation. Internal correction was performed by dividing by the corresponding measured value, and the value of IFN-γ / IL-4 representing Th1 response directivity was calculated.

  The results are shown in FIG. Compared to immunization with M3-NcGRA7 (FIG. 5B) or M3-NcAMA1 (FIG. 5C), the spleen cells of mice immunized with NcCyp-OML show higher IFN-γ / IL-4 values were shown (FIG. 5A).

  From the above, it was shown that NcCyp-OML has a higher ability to shift the balance of Th1-type response and Th2-type response to the Th1 side compared to existing OML vaccines.

Example 5 Evaluation of the efficacy of NcCyp-OML in an infected mouse model
BALB / c mice (female, 8 weeks old) were immunized subcutaneously with NcCyp, NcCyp-OML or PBS 3 times at intervals of 2 weeks (25 pmol protein per dose), and Neospora protozoa 2 weeks after the final immunization (1 × 10 ⁶ ) was inoculated intraperitoneally. Mice were evaluated for clinical scores from -2 to 32 days after Neospora infection. The clinical score was the sum of physical symptoms, behavior, neurological symptoms, dehydration / decreased appetite, and other five items (Reichel and Ellis, Int J Parasitol 2009, 39: 1173-1187). In addition, brain tissue of surviving mice was collected 32 days after infection, and the amount of protozoa contained per 50 ng of brain tissue DNA was quantified (Abe et al., Parasit Vectors. 2015, 6; 8: 5). Six mice in one test group were used in one experiment, and the results of a total of two experiments were shown (12 mice in one test group). Protozoa in brain tissue of surviving mice 32 days after infection using Log-rank (Mantel-Cox) test for survival rate of mice 32 days after infection and multiple comparison test of ANOVA and Bonferroni with two-way clinical scores The quantity was statistically analyzed by one-way ANOVA and Tukey Kramer's multiple comparison test.

  The survival curve of mice 32 days after infection is shown in FIG. 6A. In the NcCyp immunized group and the NcCyp-OML immunized group, a significantly higher survival rate was confirmed than in the PBS-administered group.

  Moreover, the clinical score of a mouse | mouth is shown to FIG. 6B. In the NcCyp immunization group and the NcCyp-OML immunization group, the clinical score was significantly lower than that of the PBS administration group, and the effect of suppressing clinical symptoms by NcCyp or NcCyp-OML immunization was confirmed.

  Furthermore, FIG. 6C shows the amount of protozoa in the brain tissue of surviving mice 32 days after infection. In the NcCyp-OML immunized group, the amount of protozoa was significantly lower than that in the PBS-administered group.

  From the above, it was shown that NcCyp-OML is effective against Neospora protozoa infection.

Example 6 Evaluation of the efficacy of NcCyp-OML in TLR2 KO mice
C57BL / 6 mice and TLR2 KO mice deficient in Toll-like receptor 2 gene (male, 8 weeks old) were immunized subcutaneously 3 times at intervals of 2 weeks (25 pmol each time) with NcCyp, NcCyp-OML or PBS. Protein), and Neospora protozoa (1 × 10 ⁶ ) were inoculated intraperitoneally two weeks after the final immunization. Eight mice in one test group were used in one experiment. The survival rate of mice 70 days after infection was statistically analyzed by Log-rank (Mantel-Cox) test.

  The survival curve of C57BL / 6 mice 70 days after infection is shown in FIG. 7A, and the survival curve of TLR2 KO mice is shown in FIG. 7B. In the case of C57BL / 6 mice, a significantly higher survival rate was confirmed in the NcCyp-OML immunized group than in the PBS-administered group. On the other hand, in the case of TLR2 KO mice, no significant difference was observed between the NcCyp-OML immunized group and the PBS administration group.

  From the above, it was shown that TLR2 is required for induction of protective immunity by NcCyp-OML.

Claims (10)

  Neospora protozoa infection comprising a liposome having a oligosaccharide capable of binding to a sugar chain recognition molecule on the surface of an antigen-presenting cell encapsulated with a cyclophilin derived from Neospora protozoa or a variant or derivative retaining its immunogenicity Vaccine formulation.   The mutant according to claim 1, wherein the mutant comprises an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence constituting cyclophilin, and is a protein capable of inducing an immune response against Neospora protozoa. Vaccine formulation.   Derivatives are proteins in which one or several amino acid residues of cyclophilin or a variant thereof are chemically modified, a fusion protein of cyclophilin or a variant thereof with another polypeptide, and cyclophilin or a variant thereof is in the blood The vaccine preparation according to claim 1, wherein the vaccine preparation is a protein selected from the group consisting of proteins modified with macromolecules that enhances the ability to induce an immune response against Neospora protozoa.   The vaccine formulation as described in any one of Claims 1-3 whose cyclophilin is protein which consists of an amino acid sequence shown by sequence number 1.   The vaccine preparation according to any one of claims 1 to 4, wherein the sugar chain recognition molecule on the surface of the antigen-presenting cell is a mannose receptor.   The vaccine preparation according to any one of claims 1 to 5, wherein the oligosaccharide comprises 2 to 11 sugar residues.   The vaccine preparation according to any one of claims 1 to 6, wherein the oligosaccharide comprises 3 to 5 sugar residues.   The vaccine preparation according to any one of claims 1 to 7, wherein the oligosaccharide comprises two or more mannose sugar residues.   The vaccine preparation according to any one of claims 1 to 8, further comprising a pharmaceutically acceptable carrier. The vaccine preparation according to claim 9, which is administered subcutaneously, intradermally, orally or nasally.

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