Patent Application
20140286978
The present invention relates to a cytotoxic T lymphocyte (CTL) inducer, and a method for preventing or treating a viral disease or a cancer utilizing the same.
Simian virus 40 (SV40) is one of simian polyomaviruses belonging to Papovaviridae, and is a tumor virus which is small and has no envelope.
An outer shell of the SV40 virus is composed of a major capsid protein called VP1. When a VP1 gene is highly expressed in an insect cell or the like, VP1 is synthesized in a large amount, and is self-organized in a nucleus of a cell to form many virus-like particles (VLPs). It has been proposed by the present inventors that a drug or the like is encapsulated into this VLP, and the resultant is utilized as drug delivery (WO 2006/004173).
It has been confirmed by the present inventors that a normal VLP becomes not to be formed in many cases when an exogenous peptide is inserted into VP1, but normal particles are formed when an exogenous peptide is inserted into a DE loop or an HI loop in VP1. It is possible to insert an exogenous peptide into a DE loop or an HI loop utilizing this nature to change cell directionality of a VLP (Patent Document 1).
Preparation of a vaccine utilizing this VLP has previously been conducted. For example, Patent Document 2 describes a CTL vaccine containing a VLP with a CTL epitope peptide bound thereto.
Patent Document 1: WO 2006/088229
Patent Document 2: JP 2009-197001 A
In order to destroy a virus-infected cell or the like, a virus-derived peptide is utilized as a vaccine. However, since a peptide is poor in retention and is easily degraded, it is usually administered with an adjuvant. However, many of adjuvants have some side effects, and there is a problem with administration to a human.
The present invention was made under the aforementioned technical background, and an object thereof is to provide a means which allows a peptide to exhibit a sufficient effect as a vaccine even in the absence of an adjuvant.
In order to solve the aforementioned problems, the present inventors intensively continued study and, as a result, found out that a VLP composed of altered VP1 in which a T cell epitope peptide is inserted into a DE loop or an HI loop exhibits the strong CTL inductivity.
A VLP composed of altered VP1 in which an exogenous peptide is inserted into a DE loop or an HI loop is disclosed in Patent Document 1. However, in Patent Document 1, an exogenous peptide is inserted exclusively for changing cell directionality of a VLP, and an idea of inducing a CTL using an exogenous peptide is not disclosed in Patent Document 1. In addition, in claim 10 or the like of Patent Document 1, use of an antigenic epitope as an exogenous peptide is described, but this antigenic epitope is used to enable a VLP to bind to an antibody. That is, the “epitope” described in Patent Document 1 is an epitope which is recognized by an antibody, but is not an epitope which is recognized by a T cell (T cell epitope).
A CTL vaccine containing a VLP with a CTL epitope peptide bound thereto is disclosed in Patent Document 2 (claim 182, etc.). However, Patent Document 2 does not disclose insertion of a CTL epitope peptide into a DE loop and/or an HI loop of VP1 of simian virus 40. The CTL inducer of the present invention has a sufficient CTL inducing effect even without an immunostimulator, but a vaccine disclosed in Cited Reference 2 contains an immunostimulator such as a non-methylated CpG-containing oligonucleotide (claim 1), and this point is also different from the CTL inducer of the present invention.
The present invention was completed based on the aforementioned findings.
That is, the present invention provides the following (1) to (10).
(1) A CTL inducer containing a virus-like particle composed of VP1 of simian virus 40, wherein a T cell epitope peptide is inserted into a DE loop and/or an HI loop of the VP1.
(2) The CTL inducer according to the item (1), wherein the T cell epitope peptide is a peptide derived from a virus protein.
(3) The CTL inducer according to the item (1), wherein the T cell epitope peptide is a peptide derived from HA, NA, M1, M2, NP, NS1, NS2, PA, PB1, PB2 or PB1-F2 of an influenza virus.
(4) The CTL inducer according to the item (1), wherein the T cell epitope peptide is a peptide derived from a protein specific to a cancer cell.
(5) The CTL inducer according to the item (1), wherein the T cell epitope peptide is a peptide derived from Melan-A/MART-1, gp100, MAGE-A3, MAGE-A10, CEA, HER2/new, NY-E50-1, WT-1 or hTERT.
(6) The CTL inducer according to the item (1), wherein the T cell epitope peptide is a peptide consisting of an amino acid sequence described in any of SEQ ID Nos.: 1 to 39.
(7) A method for preventing or treating a viral disease of an animal other than a human, including administering, to an animal other than a human, a virus-like particle composed of VP1 of simian virus 40 in which a T cell epitope peptide derived from a virus protein is inserted into a DE loop and/or an HI loop of the VP1.
(8) A method for preventing or treating a cancer of an animal other than a human, including administering, to an animal other than a human, a virus-like particle composed of VP1 of simian virus 40 in which a T cell epitope peptide derived from a protein specific to a cancer cell is inserted into a DE loop and/or an HI loop of the VP1.
(9) A method for preventing or treating a viral disease of a human, including administering, to a human, a virus-like particle composed of VP1 of simian virus 40 in which a T cell epitope peptide derived from a virus protein is inserted into a DE loop and/or an HI loop of the VP1.
(10) A method for preventing or treating a cancer of a human, including administering, to a human, a virus-like particle composed of VP1 of simian virus 40 in which a T cell epitope peptide derived from a protein specific to a cancer cell is inserted into a DE loop and/or an HI loop of the VP1.
The VLP contained in the CTL inducer of the present invention has the strong CTL inducing effect even without an adjuvant. Therefore, the CTL inducer of the present invention can prevent or treat a viral disease or a cancer without generating a side effect.
The present invention will be explained in detail below.
The CTL inducer of the present invention contains a virus-like particle composed of VP1 of simian virus 40, and is characterized in that a T cell epitope peptide is inserted into a DE loop and/or an HI loop of the VP1.
The T cell epitope peptide used in the present invention is not particularly limited, as far as it can induce a CTL. As the T cell epitope peptide, a peptide derived from a virus protein and a peptide derived from a protein specific to a cancer cell are known and, in the present invention, all of these peptides can be used. Herein, the “protein specific to a cancer cell” refers to a protein which is not expressed in a normal cell and is expressed only in a cancer cell, or a protein, expression of which is increased in a cancer cell as compared with that in a normal cell.
Examples of the peptide derived from a virus protein include peptides derived from proteins such as HA, NA, M1, M2, NP, NS1, NS2, PA, PB1, PB2 and PB1-F2 of an influenza virus. Among them, peptides derived from proteins such as M 1, NP, NS1, PA, PB 1 and PB2 of an influenza virus are preferable. Examples of specific peptides include GILGFVFTL (SEQ ID No.: 1), ASCMGLIY (SEQ ID No.: 2), SIIPSGPLK (SEQ ID No.: 3), and ILGFVFTLTV (SEQ ID No.: 4), which are peptides derived from M1 of an influenza virus, CTELKLSDY (SEQ ID No.: 5), LPFEKSTVM (SEQ ID No.: 6), ILRGSVAHK (SEQ ID No.: 7), ELRSRYWAI (SEQ ID No.: 8), and SRYWAIRTR (SEQ ID No.: 9), which are peptides derived from NP, NMLSTVLGV (SEQ ID No.: 10), RIFLAMITYI (SEQ ID No.: 11), and VSDGGPNLY (SEQ ID No.: 12), which are peptides derived from PB1, VLTGNLQTL (SEQ ID No.: 13), which is a peptide derived from PB2, and IILKANFSV (SEQ ID No.: 14), which is a peptide derived from NS1.
Examples of a peptide derived from a protein of a virus other than an influenza virus include peptides derived from Gag, Pol, Env, Tat, Nef and Rev of HIV, peptides derived from E1, E2, Core, NS2, NS3, NS4 and NS5 of a hepatitis C virus (HCV), and peptides derived from E6 and E7 of a human papilloma virus (HPV).
Examples of the peptide derived from a protein specific to a cancer cell include peptides derived from proteins such as Melan-A/MART-1, gp100, MAGE-A3, MAGE-A10, CEA, HER2/new, NY-E50-1, WT-1, and hTERT. Among them, peptides derived from proteins such as HER2/new, WT-1, and MAGE-A3 are preferable. Examples of specific peptides include ALCRWGLLL (SEQ ID No.: 15), QLFEDNYAL (SEQ ID No.: 16), KIFGSLAFL (SEQ ID No.: 17), ILHNGAYSL (SEQ ID No.: 18), IISAVVGIL (SEQ ID No.: 19), VVLGVVFGI (SEQ ID No.: 20), RLLQETELV (SEQ ID No.: 21), VMAGVGSPYV (SEQ ID No.: 22), CLTSTVQLV (SEQ ID No.: 23), QLMPYGCLL (SEQ ID No.: 24), YLEDVRLV (SEQ ID No.: 25), VLVKSPNHV (SEQ ID No.: 26), YMIMVKCWMI (SEQ ID No.: 27), ELVSEFSRM (SEQ ID No.: 28), VLRENTSPK (SEQ ID No.: 29), RWGLLLALL (SEQ ID No.: 30), TYLPTNASL (SEQ ID No.: 31), and PYVSRLLGI (SEQ ID No.: 32), which are peptides derived from HER2/new, and RMFPNAPYL (SEQ ID No.: 33), SLGEQQYSV (SEQ ID No.: 34), CMTWNQMNL (SEQ ID No.: 35), YMFPNAPYL (SEQ ID No.: 36), CMTWNQMNL (SEQ ID No.: 37), CYTWNQMNL (SEQ ID No.: 38), and RWPSCQKKF (SEQ ID No.: 39), which are peptides derived from WT-1.
The length of the T cell epitope peptide to be inserted into a loop is not limited as far as a CTL can be induced, but is usually 3 to 27 amino acids, preferably 8 to 11 amino acids, more preferably 9 amino acids.
The DE loop is located at the 127th to 146th positions counted from an N end of VP1, and the HI loop is located at the 268th to 277th positions (WO 2006/088229). The T cell epitope peptide may be inserted into any of these loops. As for the DE loop, it is preferable that the T cell epitope peptide is inserted so as to replace an amino acid sequence of the 137th to 138th positions with the epitope peptide. As for the HI loop, it is preferable that the T cell epitope peptide is inserted so as to replace an amino acid sequence of the 273rd to 274th positions with the epitope peptide. Into both ends of the T cell epitope peptide to be inserted into the loop, usually, 1 to several spacer amino acids, for example, glycine, alanine, and serine are inserted (WO 2006/088229). The number of spacer amino acids is usually 1 to 9, preferably 1 to 6, further preferably 3 to 6.
The T cell epitope peptide may be inserted into both of the DE loop and the HI loop, or may be inserted into only one of them. When it is inserted into both of them, the same T cell epitope peptide may be inserted, or different T cell epitope peptides may be inserted. Further, two or more T cell epitope peptides may be inserted into the loop in series.
Since a method for preparing the virus-like particle composed of VP1 in which an exogenous peptide is inserted into a loop is described in WO 2006/088229, a virus-like particle composed of VP1 in which the T cell epitope peptide is inserted into a loop can be prepared according to this method. For example, an objective virus-like particle can be prepared by preparing an altered VP1 gene in which a nucleotide sequence encoding the T cell epitope peptide is inserted, and highly expressing this altered VP1 gene in an insect cell or the like using a baculovirus vector or the like.
The CTL inducer of the present invention is formulated into a form of a general pharmaceutical composition in accordance with the nature of a substance as an active ingredient, and direct delivery of the composition can be generally attained by parenteral injection (e.g. subcutaneous injection, intraperitoneal injection, intravenous injection, intramuscular injection, or injection into an interstitial space of a tissue). Examples of other administration methods include mucous membrane administration (e.g. oral, transnasal, or lung), administration via eyes, transdermal administration and suppositories.
That is, when administered parenterally, the CTL inducer can be administered in a dosage form such as injectables, pernasal preparations, local preparations (transdermal preparations, etc.) and rectal preparations. When administered orally, the CTL inducer can be administered in a dosage form which is usually used in the art. Examples of the injectables include sterile solutions or suspensions and emulsions, specifically, water, a water-propylene glycol solution, a buffering solution, and 0.4% physiological saline. Further, when the CTL inducer is formulated into a liquid preparation, it can be freeze-preserved, or can be preserved after removing water by lyophilization or the like. Lyophilized preparations are used by adding distilled water for injection or the like and redissolved before use. Examples of the local preparations include creams, ointments, lotions, and transdermal preparations. Examples of the oral preparations or the rectal preparations include capsules, tablets, pills, powders, drops, suppositories, and solutions.
The aforementioned dosage forms are formulated into preparations together with pharmaceutically acceptable excipients and additives by a procedure which is usually performed in the art. Examples of the pharmaceutically acceptable excipients and additives include carriers, binders, perfumes, buffers, thickeners, colorants, stabilizers, emulsifiers, dispersants, suspending agents, antiseptics, pH adjusting agents, tonicity adjusting agents, and wetting-out agents. Further, examples of the pharmaceutically acceptable carriers include magnesium carbonate, lactose, pectin, starch, and methylcellulose.
Since the CTL inducer of the present invention has a sufficient CTL inducing effect even without an adjuvant, it does not have to contain an adjuvant, but may contain an adjuvant. Examples of the adjuvant to be used include aluminum hydroxide gel, complete freund's adjuvant, incomplete freund's adjuvant, bordetella pertussis adjuvant, poly(I,C), and CpG-DNA.
The dose of the T cell epitope peptide in the preparation and number of doses of the preparation are different according to the kind of the T cell epitope peptide, the symptom, age and weight of the administration subject, and the dosage form, but the dose is usually 0.01 μg to 1 mg, preferably 0.1 μg to 500 μg, more preferably 1.0 μg to 100 μg, and it is preferable that such a dose is administered once in a few days to a few months. For example, in initial immunization (i.e. therapeutic or preventive administration), as for an adult patient, 1.0 μg to 500 μg of a peptide is administered, followed by boosting administration of 1.0 μg to 100 μg of a peptide in accordance with a boosting therapy over a few weeks to a few months, depending on the response and state of a patient by measurement of the specific CTL activity in the blood of the patient.
The CTL inducer of the present invention can be used as a vaccine for preventing or treating a viral disease, or a vaccine for preventing or treating various cancers.
The CTL inducer of the present invention can be administered to a human or an animal other than a human. Examples of the animal other than a human include livestock (e.g. cow, horse, pig, and chicken), pet animals (e.g. dog and cat), and experimental animals (mouse and rat).
The CTL inducer of the present invention can also be utilized in the following preventive or treatment methods.
(A) A method for preventing or treating a viral disease of a human, including administering, to a human, a virus-like particle composed of VP1 of simian virus 40 in which a T cell epitope peptide derived from a virus protein is inserted into a DE loop and/or an HI loop of the VP1.
(B) A method for preventing or treating a viral disease of an animal other than a human, including administering, to an animal other than a human, a virus-like particle composed of VP1 of simian virus 40 in which a T cell epitope peptide derived from a virus protein is inserted into a DE loop and/or an HI loop of the VP1.
(C) A method for preventing or treating a cancer of a human, including administering, to a human, a virus-like particle composed of VP1 of simian virus 40 in which a T cell epitope peptide derived from a protein specific to a cancer cell is inserted into a DE loop and/or an HI loop of the VP1.
(D) A method for preventing or treating a cancer of an animal other than a human, including administering, to an animal other than a human, a virus-like particle composed of VP1 of simian virus 40 in which a T cell epitope peptide derived from a protein specific to a cancer cell is inserted into a DE loop and/or an HI loop of the VP1.
The present invention will be explained in more detail below by way of examples.
A CTL epitope sequence (GILGFVFTL) (this epitope sequence is an epitope sequence for HLA-A*0201 of human MHC class I) of an influenza virus particle internal protein M1 was inserted into SV40 VP1. Specifically, a DE loop region (137-138 amino acid region, classically, 4th Ala of a VP1 gene as amino acid No. 1) or an HI loop region (273-274 amino acid region) of VP1 was replaced with GILGFVFTL to prepare a M1 CTL epitope-inserted SV40 VP1 insertion mutant (M1-DE-VP1, M1-H1-VP1). The mutant was prepared by the Overhang PCR method by employing pFastBac 1-SV40 wild type VP1 encoding SV40 VP1 as a template. The following primers were used.
Employing 10 ng of a template, each 50 pmol of primers were added to prepare 30 μL of a mixed solution having the concentrations of 2.5 units of a KOP polymerase, 0.2 mM dNTPs, 1 mM MgCl2, 6 mM (NH4)2SO4, 10 mM KCl, 120 mM Tris-HCl (pH 8.0), 0.1% Triton X-100, and 0.001% BSA.
In PCR, in both of 1st round and 2nd round, after incubation at 98° C. for 60 seconds, the following cycle was repeated 25 times (98° C. 15 seconds, 59° C. 15 seconds, 74° C. 30 seconds) and, finally, incubation was performed at 74° C. for 1 minute and 30 seconds, and the procedure was migrated to 4° C.
PCR fragments prepared by the Overhang PCR were cut with restriction enzymes KpnI and SalI, and inserted between KpnI and SalI sites of a pFastBac 1 plasmid vector to obtain M1-DE-VP1 and M1-H1-VP1 plasmids.
M1-DE-VP1 and M1-H1-VP1 plasmids were introduced into Escherichia coli DH10bac (Invitrogen) harboring a baculovirus genome to transform the Escherichia coli, and thereby a recombinant baculovirus genome with M1-DE-VP1 and M1-H1-VP1 incorporated therein was prepared. The recombinant baculovirus genome was transfected into a Sf-9 cell and, after 3 days, the supernatant was recovered to obtain a solution containing a recombinant baculovirus. The recombinant baculovirus titer was elevated by infecting a part of this solution with a Sf-9 cell again, to obtain a stock solution of the recombinant baculovirus.
With a recombinant baculovirus with M1-DE-VP1 and M1-H1-VP1 incorporated therein, 3×107 Sf-9 cells were infected at M.O.I. (multiplicity of infection)=0.05 to 0.2. Three days after infection, the Sf-9 cells were recovered and then washed with PBS (−), the cells were resuspended in 200 μl of a 50% Opti-prep™ solution (20 mM Tris-HCl (pH 7.9), 50% Opti-prep™), and ultrasonic-crushed to obtain a lysate solution (
A part of the prepared lysate and pellet solution was electrophoresed by SDS-PAGE, and stained by CBB staining (
To the lysate solution (100 μl) and the pellet solution (100 μl) containing M1-DE-VLP and M1-HI-VLP was added 5 μg of a CpG-olygodeoxyncleotide5002 (TCCATGACGTTCTTGATGTT: SEQ ID No.: 52) adjuvant as an adjuvant, and this was immunized into an 8-week old transgenic mouse by a footpad, heel joint, intravenous, intraperitoneal, intramuscular or intranasal route. As the transgenic mouse, a transgenic mouse, a base of which is C57BL/6, and which expresses human β2m further fused with a chimera of HLA-A*0201 and H-2 Db was used. In this mouse, since mouse β2m and H-2 Db are knocked out, it is thought that mouse-derived MHC class I is not exposed to the cell surface.
One week or two weeks after immunization, the spleen and lung were isolated from the mouse, and placed on a φ6 cm dish containing 5 ml of an RPMI-1640 medium, respectively. Using a pincette, the spleen was kneaded well in the medium, a solution containing lymphocytes which had been eluted into the medium was transferred to a 15 ml tube, the φ6 cm dish was washed again with 5 ml of the RPMI-1640 medium, and the supernatant was added to the 15 ml tube to a total amount of 10 ml Immediately, while a tissue section accumulated on the bottom of the 15 ml tube was left, the supernatant was transferred again to a fresh 15 ml tube and centrifuged at room temperature and 1,200 rpm for 5 minutes to transfer lymphocytes to pellets. After the supernatant was removed and the pellets were loosened, in order to remove erythrocytes, 250 μl of a NH4Cl-Tris solution was added and the materials were mixed, thereafter, 10 ml of an RPMI-1640 medium was added quickly, and the mixture was centrifuged at room temperature and 1,2000 rpm for 5 minutes to transfer lymphocytes to pellets. After the supernatant was removed and the pellets were loosened, 10 ml of an RPMI 1640 medium was added again, the mixture was transferred to a fresh 15 ml tube with a pipette so as not to absorb denatured erythrocytes, and this was centrifuged again at room temperature and 1,200 rpm for 5 minutes. After the supernatant was removed, pellets were loosened, suspended in 10 ml of an RPMI-1640 medium again, and the suspension was centrifuged at room temperature and 1,200 rpm for 5 minutes. The supernatant was removed, and the remainder was finally suspended in 2 ml of a 10% FCS-mixed RPMI-1640 medium. In order to count lymphocytes, 10 μA of the suspended solution was added to 490 μl of a 2% acetic acid solution, the cell number was counted with a Burker-Turk hemacytometer, and the cells were diluted with a 10% FCS-mixed RPMI-1640 medium or concentrated so that the cell number became 2×107 cells/ml.
The isolated lung was transferred to a φ6 cm dish with no medium mixed therein, minced well with scissors, suspended in an RPMI-1640 medium with 10 ml of 50 units/ml Collagenase Type 1 and 10% FCS mixed therein, and incubated with a 5% CO2 incubator at 37° C. for 1 hour. After incubation, the suspension was passed through 100 μm of a cell strainer made of Nylon (BD Falcon) to recover the supernatant in a 50 ml tube, a tissue fragment remaining in the strainer was ground with a cell scraper, thereafter, the ground tissue fragment was further passed through 10 ml of a 10% FCS-mixed RPMI-1640 medium, and a total amount of 20 ml of a solution was recovered in a 50 ml tube. The recovered solution was centrifuged at room temperature and 1,200 rpm for 5 minutes. After the supernatant was removed and pellets were loosened, 10 ml of an RPMI-1640 medium was added, and the suspension was transferred to a 15 ml tube. The 15 ml tube was centrifuged at room temperature and 1,200 rpm for 5 minutes, the supernatant was removed and pellets were loosened, thereafter, 10 ml of an RPMI-1640 medium was further added, and the mixture was centrifuged at room temperature and 1,200 rpm for 5 minutes, thereby, cells were washed. After the supernatant was removed, pellets were loosened, and finally, 10 ml of a 10% FCS-mixed RPMI-1640 medium was added, the mixture was centrifuged at room temperature and 1,200 rpm for 5 minutes, the supernatant was removed and pellets were loosened and, thereafter, 250 μl of a 10% FCS-mixed RPMI-1640 medium was added.
In order to examine that a CTL which is reacted with a CTL epitope sequence (GILGFVFTL) of M1 to be induced is present in lymphocytes which were recovered from the spleen and lung by the aforementioned method, ICS analysis was performed. BD GolgiPlug™ (BD) which had been diluted 25-fold with a 10% FCS-mixed RPMI-1640 medium was added to a 96-well round bottom plate in an amount of 5 μA per well, and 100 μA of a 20 μM peptide (GILGFVFTL, Operon) of a CTL epitope of M1, which had been diluted with a 10% FCS-mixed RPMI-1640 medium, was further added thereto. As a negative control, 100 μl of a 10% RCS-mixed RPMI-1640 medium not containing a peptide was added.
To this well were added 100 μl of lymphocytes prepared from the spleen and lymphocytes prepared from the lung, which had been prepared as described above, and the mixture was incubated with a 5% CO2 incubator at 37° C. for 5 hours.
After incubation, the mixture was spun down at 4° C. and 1,400 rpm to remove the supernatant and the cells were loosened with a vortex mixer, a FACS buffer (2% FCS, 0.1% sodium azide, 1×PBS (−)) was added in an amount of 200 μl per well, the mixture was spun down again at 4° C. and 1,400 rpm to remove the supernatant, the cells were loosened, and thereafter, 100 μl of Mouse BD Fc Block™ (BD Pharmingen) which had been diluted with a FACS buffer to 5 μg/ml was added, and the mixture was incubated at 4° C. for 10 minutes.
After incubation, the mixture was spun down at 4° C. and 1,400 rpm to remove the supernatant, and the cells were loosened, a FACS buffer was added in an amount of 200 μl per well, the mixture was spun down again at 4° C. and 1,400 rpm to remove the supernatant, and the washing operation with a FACS buffer was performed again. To the loosened cells was added FICT Rat Anti-Mouse CD8a Clone: 53-6.7 (BD Pharmingen) which had been diluted with a FACS buffer to 10 μg/ml, in an amount of 50 μl per well, and this was incubated in a dark place at 4° C. for 30 minutes.
After incubation, after an operation of washing with 200 μl of a FACS buffer was performed two times, to the loosened cells was added 100 μl of BD Cytofix/Cytoperm™ (BD Biosciences) per well, and this was incubated in a dark place at 4° C. for 20 minutes. After incubation, after the same operation as the washing operation which had been performed with a FACS buffer was performed with 200 μl of 1×BD Perm/Wash™ (BD Biosciences) two times, to the loosened cells was added 50 μl of PE anti-mouse IFN-γ Clone: XMG1.2 (BioLegend) which had been diluted with 1×BD Perm/Wash™ to 10 μg/ml, and the mixture was incubated in a dark place at 4° C. for 30 minutes.
After incubation, after a washing operation with 200 μl of 1×BD Perm/Wash™ was performed two times, to the loosened cells was added 100 μl of a FACS fixation buffer (1% formaldehyde, 1×FACS buffer) per well, and this was incubated in a dark place at 4° C. overnight.
After incubation, after 400 μl of a FACS buffer was added to a 5 ml polystyrene tube, and a sample fixed with 100 μl of a FACS fixation buffer was added thereto, dot plot analysis was performed with FACScan (BD). For the analysis, the software of Cell Quest (BD) was used. The result of two-dimensional analysis of ICS is shown in
As shown in
As shown in
In order to examine that a CTL having the cytotoxic activity, which is reacted with a CTL epitope sequence (GILGFVFTL) of M1 to be induced, is present in lymphocytes which were recovered from the spleen by the method of Example 5, CD107-ICS analysis was performed. To a 96-well round bottom plate were added BD GolgiPlug™ (BD) which had been diluted 25-fold with a 10% FCS-mixed RPMI-1640 medium in an amount of 5 μl per well, BD Golgi Stop™ (BD) which had been diluted 25-fold with a 10% FCS-mixed RPMI-1640 medium in an amount of 5 μl per well, and 0.8 μg of FITC Rat Anti-Mouse CD107a Clone: 1D4B (BD Pharmingen), and 100 μl of a 20 μM peptide (GILGFVFTL, Operon) of a CTL epitope of M1 which had been diluted with a 10% FCS-mixed RPMI-1640 medium was further added thereto. As a negative control, 100 μl of a 10% RCS-mixed RPMI-1640 medium not containing a peptide was added.
After 100 μl of lymphocytes prepared from the spleen, which had been prepared in Example 5, were added to this well, the mixture was incubated with a 5% CO2 incubator in a dark place at 37° C. for 6 hours.
After incubation, the mixture was spun down at 4° C. and 1,400 rpm to remove the supernatant and the cells were loosened with a vortex mixer, a FACS buffer (2% FCS, 0.1% sodium azide, 1×PBS (−)) was added in an amount of 200 μA per well, the mixture was spun down again at 4° C. and 1,400 rpm to remove the supernatant, the cells were loosened, and thereafter, 100 μl of Mouse BD Fc Block™ (BD Pharmingen) which had been diluted with a FACS buffer to 5 μg/ml was added, and the mixture was incubated at 4° C. for 10 minutes.
After incubation, the mixture was spun down at 4° C. and 1,400 rpm to remove the supernatant, and the cells were loosened, a FACS buffer was added in an amount of 200 μl per well, the mixture was spun down again at 4° C. and 1,400 rpm to remove the supernatant, and the washing operation with a FACS buffer was performed again. To the loosened cells was added PE/Cy5 Rat Anti-Mouse CD8a Clone: 53-6.7 (BD Pharmingen) which had been diluted with a FACS buffer to 10 μg/ml, in an amount of 50 μl per well, and this was incubated in a dark place at 4° C. for 30 minutes.
After incubation, after an operation of washing with 200 μl of a FACS buffer was performed two times, to the loosened cells was added 100 μl of BD Cytofix/Cytoperm™ (BD Biosciences) per well, and this was incubated in a dark place at 4° C. for 20 minutes. After incubation, after the same operation as the washing operation which had been performed with a FACS buffer was performed with 200 μl of 1×BD Perm/Wash™ (BD Biosciences) two times, to the loosened cells was added 50 μl of PE anti-mouse IFN-γ Clone: XMG1.2 (BioLegend) which had been diluted with 1×BD Perm/Wash™ to 10 μg/ml, and the mixture was incubated in a dark place at 4° C. for 30 minutes.
After incubation, after a washing operation with 200 μl of 1×BD Perm/Wash™ was performed two times, to the loosened cells was added 100 μl of a FACS fixation buffer (1% formaldehyde, 1×FACS buffer) per well, and this was incubated in a dark place at 4° C. overnight.
After incubation, after 400 μl of a FACS buffer was added to a 5 ml polystyrene tube, and a sample fixed with 100 μl of a FACS fixation buffer was added thereto, dot plot analysis was performed with FACScan (BD). For the analysis, the software of Cell Quest (BD) was used. The result of two-dimensional analysis of CD107-ICS is shown in
As shown in
In order to show that the CD8 positive, CD107 positive, intracellular IFN-γ positive cell shown in Example 7 damages a cell, 51Chrome release analysis was performed. Lymphocytes of the spleen were prepared from a non-immunized mouse by the same method as that of Example 5. To 2.4×108 cells of spleen lymphocytes of a non-immunized mouse in 2 ml of a 10% FCS-mixed RPMI-1640 medium was added 4 μl of a 10 mM peptide (GILGFVFTL, Operon) of a CTL epitope of M1, and the mixture was incubated with a 5% CO2 incubator at 37° C. for 2 hours in a 15 ml tube. After incubation, 20 Gy (gray) X-ray was radiated. After the radiation, the mixture was centrifuged at room temperature and 1,2000 rpm for 5 minutes to transfer lymphocytes to pellets. After the supernatant was removed and pellets were loosened, 2 ml of a 10% FCS-mixed RPMI-1640 medium was added, and in order to count lymphocytes, 10 μl of a lymphocyte solution was added to 490 μl of a 2% acetic acid solution, the cell number was calculated with a Burker-Turk hemacytometer, and this was diluted with a 10% FCS-mixed RPMI-1640 medium to 5×106 cells/ml.
Simultaneously, about 50 μg of M1-DE-VLP was immunized by intramuscular injection, and about 50 μg of M1-HI-VLP was immunized by intraperitoneal injection and, one week after immunization, the spleen was isolated from a mouse, and placed on a φ6 cm dish containing 5 ml of an RPMI-1640 medium. Using a pincette, the spleen was kneaded well in the medium, a solution containing lymphocytes which had been eluted into the medium was transferred to a 15 ml tube, the φ6 cm dish was washed again with 5 ml of the RPMI-1640 medium, and the supernatant was added to the 15 ml tube to a total amount of 10 ml. Since the blood was not removed from the immunized lymphocytes, immediately, while a tissue section accumulated on the bottom of the 15 ml tube was left, the supernatant was transferred again to a fresh 15 ml tube and centrifuged at room temperature and 1,200 rpm for 5 minutes to transfer lymphocytes to pellets. After the supernatant was removed and pellets were loosened, 2 ml of a 10% FCS-mixed RPMI-1640 medium was added, and in order to count lymphocytes, 10 μl of a lymphocyte solution was added to 490 μl of a 2% acetic acid solution, the cell number was calculated with a Burker-Turk hemacytometer, and this was diluted with a 10% FCS-mixed RPMI-1640 medium to 5×106 cells/ml.
Into a 48-well plate were mixed 500 μl of 5×106 cells/ml of lymphocytes which had been incubated with a peptide of a CTL epitope of M1 and irradiated with an X-ray, and which had not been immunized, and 500 μl of 5×106 cells/ml of immunized lymphocytes, per well. Twenty four wells per sample were prepared. After mixing, the mixture was incubated with a 5% CO2 incubator at 37° C. for 7 days.
After incubation for 7 days, lymphocytes in 24 wells were collected in one 50 ml tube, and centrifuged at room temperature and 1,200 rpm for 5 minutes to transfer lymphocytes to pellets. After the supernatant was removed and pellets were loosened, 2 ml of a 10% FCS-mixed RPMI-1640 medium was added, and in order to count lymphocytes, 20 μl of a lymphocyte solution was added to 20 μl of a 0.4% trypan blue solution (Gibco), the cell number was calculated with a Burker-Turk hemacytometer, and the cells were diluted with a 10% FCS-mixed RPMI-1640 medium or concentrated, so that the cell number became 7.5×106 cells/ml. The prepared lymphocytes were used as effector cells.
Simultaneously, each 1 ml of 1×106 cells/ml of RMA-HHD cultured cells were dispensed into two 15 ml tubes, 5 μl of a 10 mM peptide (GILGFVFTL, Operon) of a CTL epitope of M1 was added to one of the tubes, and this was incubated with a 5% CO2 incubator at 37° C. for 2 hours. No peptide was added to the other tube. After incubation, the tube was centrifuged at room temperature and 1,000 rpm for 5 minutes to transfer cells to pellets. After the supernatant was removed and pellets were loosened, 100 Ki (microsievert) of a Na251CrO4 solution wad added, and this was incubated with a 5% CO2 incubator at 37° C. for 30 minutes. After incubation, the tube was centrifuged at room temperature and 1,000 rpm for 5 minutes to transfer cells to pellets. After the supernatant was removed and pellets were loosened, 1 ml of a 10% FCS-mixed RPMI-1640 medium was added for a washing operation, this was centrifuged at room temperature and 1,200 rpm for 5 minutes to transfer lymphocytes to pellets, and the supernatant was removed. This washing operation was repeated five times. Finally, after the supernatant was removed, 1 ml of a 10% FCS-mixed RPMI-1640 medium was added. Into a fresh 15 ml tube were mixed 500 μl of this cell solution and 9.5 ml of a 10% FCS-mixed RPMI 1640 medium to dilute the cells 20-fold. This cell solution was used as target cells.
Into a 96-well plate was dispensed 100 μl of target cells (5×103 cells), 100 μl of the effector cells (7.5×105 cells) were added thereto so that the ratio of effector cell:target cell became 150:1, 20 μl of the effector cells (1.5×105 cells) and 80 μl of a 10% FCS-mixed RPMI-1640 medium were added thereto so that the ratio of effector cell:target cell became 30:1, and 10 μl of the effector cells (7.5×104 cells) and 90 μl of a 10% FCS-mixed RPMI-1640 medium were added thereto so that the ratio of effector cell:target cell became 15:1. In addition, 100 μl of a 5% Triton-X 100 solution as a positive control, and 100 μl of a 10% FCS-mixed RPMI-1640 medium as a negative control were added. After mixing, the mixture was incubated with a 5% CO2 incubator at 37° C. for 4 hours.
After incubation, the supernatant was recovered using Supernatant collection system (Molecular Devices), a gamma ray of released 51Cr was counted and analyzed with AUTO WELL GAMMA SYSTEM (ARC-380CL, Aloka) and ALOKA RIA Program (ARCAS ver. 3.11, Aloka). The result of 51Chrome release analysis is shown in
As shown in
In order to analyze that the CTL which selectively damages a cell presenting a peptide (GILGFVFTL) of a CTL epitope of M1, shown in Example 8, selectively damages a cell actually presenting a peptide (GILGFVFTL) of a CTL epitope of M1 in an animal body, In vivo CTR analysis was performed. About 50 μg of M1-DE-VLP was immunized by intramuscular injection, and about 50 μg of M1-HI-VLP was immunized by intraperitoneal injection.
One week after immunization, the spleen was isolated from three non-immunized mice, and placed on a φ6 cm dish containing 5 ml of an RPMI-1640 medium. Using a pincette, the spleen was kneaded well in the medium, a solution containing lymphocytes which had been eluted into the medium was transferred to a 15 ml tube, the φ6 cm dish was washed again with 5 ml of the RPMI-1640 medium, and the supernatant was added to the 15 ml tube to a total amount of 10 ml. Since blood was not removed from non-immunized lymphocytes, immediately, while a tissue section accumulated on a bottom of the 15 ml tube was left, the supernatant was transferred again to a fresh 15 ml tube and centrifuged at room temperature and 1,200 rpm for 5 minutes to transfer lymphocytes to pellets. After the supernatant was removed and pellets were loosened, 2 ml of a 10% FCS-mixed RPMI-1640 medium was added. Into two 15 ml tubes were dispensed each 1 ml of prepared non-immunized lymphocytes, 1 μl of a 10 mM peptide (GILGFVFTL, Operon) of a CTL epitope of M1 was added to one of the tubes, and this was incubated with a 5% CO2 incubator at 37° C. for 1 hour. After incubation, the mixture was centrifuged at room temperature and 1,2000 rpm for 5 minutes to transfer lymphocytes to pellets. After the supernatant was removed and pellets were loosened, 10 ml of an RPMI-1640 medium was added again, the mixture was centrifuged at room temperature and 1,2000 rpm to transfer lymphocytes to pellets. After the supernatant was removed and pellets were loosened, 20 ml of a 0.1% BSA-mixed PBS (−) solution was added, 10 μl of a 5 mM carboxyfluorescein diacetate succinimidyl ester (CFSE) (Molecular Probes) was added to a lymphocyte solution which had been incubated with a peptide (GILGFVFTL) of a CTL epitope, 1 μl of 5 mM CFSE was added to a lymphocyte solution which had not been incubated with a peptide, the materials were mixed well and, thereafter, the mixture was shaking-cultured with a water bath at 37° C. for 10 minutes. After shaking, the mixture was centrifuged at room temperature and 1,200 rpm for 5 minutes to transfer lymphocytes to pellets. After the supernatant was removed and pellets were loosened, 10 ml of a 10% FCS-mixed RPMI-1640 medium was added, and the mixture was centrifuged at room temperature and 1,200 rpm for 5 minutes to transfer lymphocytes to pellets. After the supernatant was removed and pellets were loosened, 2 ml of a 10% FCS-mixed RPMI-1640 medium was added, and in order to count lymphocytes, 10 μl of a lymphocyte solution was added to 490 μl of a 2% acetic acid solution, the cell number was calculated with a Burker-Turk hemacytometer, and the cells were diluted with a 10% FCS-mixed RPMI-1640 medium or concentrated, so that the cell number became 5×107 cells/ml. The same amount of 5×107 cells/ml of lymphocytes which had not been incubated with a peptide labeled with CFSE and 5×107 cells/ml of lymphocytes which had been incubated therewith were mixed to obtain a CFSE-labeled lymphocyte solution.
Each of a non-immunized mouse, and a mouse after one week from immunization with about 50 μg of M1-DE-VLP by intramuscular injection and about 50 μg of M1-HI-VLP by intraperitoneal injection was intravenously injected with 200 μl of the above-prepared CFSE-labeled lymphocyte solution, and left for one day.
After one day had passed, the spleen was isolated from the mouse, and placed on a φ6 cm dish containing a 5 ml of an RPMI-1640 medium. Using a pincette, the spleen was kneaded well in the medium, a solution containing lymphocytes which had been eluted into the medium was transferred to a 15 ml tube, the φ6 cm dish was washed again with 5 ml of the RPMI-1640 medium, and the supernatant was added to the 15 ml tube to a total amount of 10 ml. Since blood was not removed from lymphocytes, immediately, while a tissue section accumulated on the bottom of the 15 ml tube was left, the supernatant was transferred again to a fresh 15 ml tube and centrifuged at room temperature and 1,200 rpm for 5 minutes to transfer lymphocytes to pellets. After the supernatant was removed and pellets were loosened, 1 ml of a FACS fixation buffer (1% formaldehyde, 1×FACS buffer) was added. To a 5 ml polystyrene tube was added 500 μl of a FACS buffer, 200 μl of a lymphocyte solution which had been fixed with the FACS fixation buffer was added thereto, and histogram analysis was performed with FACScan (BD). For the analysis, the software of Cell Quest (BD) was used. The result of In vivo CTL analysis is shown in
As shown in
According to the same method as that of Example 5, lymphocytes, from which erythrocytes had been removed, were purified from the spleen of a C57BL/6 mouse, and diluted with a 10% FCS-mixed RPMI-1640 medium to 1×107 cells/ml. To a 96-well plate was added a) 5 μl of a 50% Opti-prep™ solution with no substance added thereto (20 mM Tris-HCl (pH 7.9), 50% Opti-prep™), b) 5 μl of a M1-DE-VLP solution prepared in Example 3, or c) 5 μl of a M1-HI-VLP solution, 50 μl of the above-prepared lymphocytes were added thereto, and a 10% FCS-mixed RPMI-1640 medium was added thereto so that the total amount per well became 2004 After mixing by pipetting, this plate was incubated with a 5% CO2 incubator at 37° C. for 6 hours or 24 hours.
After incubation, after the mixture was spun down at 4° C. and 1,400 rpm to remove the supernatant and the cells were loosened with a vortex mixer, 200 μl of a FACS buffer (2% FCS, 0.1% sodium azide, 1×PBS (−)) was added per well, the mixture was spun down again at 4° C. and 1,400 rpm to remove the supernatant, and the cells were loosened, thereafter, 100 μl of FITC American Hamster Anti-Mouse CD80 Clone: 16-10A1 (BD Pharmingen), FITC Rat Anti-Mouse CD86 Clone: GL-1 (BD Pharmingen), or FITC American Hamster Anti-Mouse CD40 Clone: HM40-3 (BD Pharmingen), which had been diluted with a FACS buffer to 10 μg/ml, was added, and this was incubated in a dark place at 4° C. for 30 minutes.
After incubation, after an operation of washing with 200 μl of a FACS buffer was performed two times, to loosened cells was added 100 μl of a FACS fixation buffer (1% formaldehyde, 1×FACS buffer) per well, and the mixture was incubated in a dark place at 4° C. overnight.
After incubation, 400 μl of a FACS buffer was added to a 5 ml polystyrene tube, a sample which had been fixed with 100 μl of a FACS fixation buffer was added thereto, and histogram analysis was performed with FACScan (BD). For the analysis, the software of Cell Quest (BD) was used. The result of histogram is shown in
As shown in
In order to analyze the influence of a CTL which selectively damages a cell presenting a peptide (GILGFVFTL) of a CTL epitope of M1 on influenza virus infection, a virus challenge experiment was performed. About 50 μg of M1-DE-VLP was immunized into a HLA-A2 transgenic mouse (HHD mouse) by intramuscular injection, and about 50 μg of M1-HI-VLP was immunized into the mouse by intraperitoneal injection.
One week after immunization, influenza viruses (A/H1N1/PR8 and A/H3N2/Aichi) were diluted with PBS (−) so that the tissue culture influenza virus infectious dose (TCID50) became 100 TCID50/20 μl, and an immunized mouse was anesthetized and nasally infected with 20 μl of this diluted solution.
Four days after infection, the mouse lung was taken out, 1 ml of PBS (−) was added, the mouse lung was ground with a tissue homogenizer (glass homogenizer, warty, 10 ml, 812-770-02, Ikemoto Scientific Technology Co., Ltd.) until the tissue became invisible, and the solution was transferred to a 15 ml tube. The tube was centrifuged with a centrifuge at 4° C. and 2000 rpm for 5 minutes, and the supernatant was transferred to a 2 ml tube equipped with a screw cap. In order to calculate the amount of influenza virus from hemagglutination by adding erythrocytes of chicken, 100 μl of the supernatant from grinding of the mouse lung was added to a lane A of a round bottom 96-well plate, 90 μl of a 5% FCS-mixed DMEM medium wad added to lanes B to G and, as a negative control, 100 μl of a 5% FCS-mixed DMEM medium was added to a lane H. A repeating operation of transferring 10 μl of the liquid from the lane A to the lane B, suspending it by pipetting and, thereafter, transferring 10 μl of the liquid from the lane B to the lane C, and suspending it was performed up to the lane G, to prepare a 10-fold dilution series of the supernatant of lung infected with an influenza virus. In order to calculate the average, five series were prepared per one sample. After each 100 μl of 2.5×105 cells/ml of MDCK cells were added to all of the series, the mixture was incubated with a 5% CO2 incubator at 35° C. for 24 hours. After incubation, after all of the culturing supernatant was removed with a pipette and 200 μl of a 0.0002% trypsin-mixed (2 μl/ml trypsin) DMEM medium was added, the mixture was further incubated with a 5% CO2 incubator at 35° C. for 3 days. After incubation, 50 μl of 0.8% chicken blood (chicken stored blood, 0109-1, Nippon Bio-Test Laboratories Inc.) was added, and this was allowed to stand at 4° C. for 1 hour. From the distribution of hemagglutination, the tissue culture influenza virus infectious dose (TCID50) was calculated.
As shown in
Further, from the influenza virus (A/H1N1/PR8) infection, the weight of mouse was weighed every day for 2 weeks, and the change in weight was recorded regarding the weight before infection as 100%. As shown in
According to the same methods as those of Examples 1 to 3, a VLP in which a CTL epitope sequence (RMFPNAPYL) for HLA-A2 of WT1 known as a cancer antigen is inserted in place of a CTL epitope sequence (GILGFVFTL) of M1 was prepared (WT1WT-DE-VLP, WT1WT-HI-VLP) (FIG. 9-a). According to the same methods as those of Examples 4 to 6, a HLA-A2 transgenic mouse was immunized to prepare lymphocytes, and ICS analysis on a CTL epitope sequence of WT1 was performed (FIG. 9-b). Further, according to the same method as that of Example 8, 51Chrome release analysis on a CTL epitope sequence of WT1 was performed.
It was revealed that, as shown in
According to the same methods as those of Examples 3 to 6, ICS analysis of M1-DE-VLP (
On the other hand, an IFA emulsion in which a peptide (GILGFVFTL) of a CTL epitope of M1 is suspended was prepared by diluting 50 μl of a GILGFVFTL peptide (Operon), which had been dissolved in a DMSO solvent having the concentration of 20 μg/μl, with 450 μl of PBS (−), and kneading the diluted peptide with 500 μl of an incomplete freund's adjuvant (IFA) (Sigma) by the Two-syringe method (GP syringe connector, NIPRO) (Inject 2 ml, B. BRAUN) Immunization was performed by injecting 100 μl of the prepared emulsion into the inguinal area subcutaneously (s.c.). A mouse was subjected to first immunization (
As shown in
According to the same method as that of Example 5, lymphocytes from which erythrocytes had been removed were purified from the spleen of a HLA-A2 transgenic mouse, and diluted with a 10% FCS-mixed RPMI-1640 medium to 1×107 cells/ml. To a 96-well plate was added a) 5 μl of a 50% Opti-prep™ solution with no substance added thereto (20 mM Tris-HCl (pH 7.9), 50% Opti-prep™), b) 5 μl of a highly purified wild type SV40 VP1-VLP (500 ng/μl), c) 5 μl of a M1-DE-VLP solution prepared in Example 3, or d) 5 IA of a M1-HI-VLP solution, 50 μl of the above-prepared lymphocytes were added thereto, and a 10% FCS-mixed RPMI-1640 medium was added thereto so that the total amount per well became 200 μl. Alternatively, 150 μl of LPS (Sigma) which had been diluted with a 10% FCS-mixed RPMI-1640 medium to 100 ng/μl was added to 50 μl of the above-purified lymphocytes. After mixing by pipetting, this plate was incubated with a 5% CO2 incubator at 37° C. for 6 hours or 24 hours. After incubation, the mixture was spun down at 4° C. and 1,400 rpm, and the supernatant was recovered in an Eppendorf tube, the remainder was spun down at 4° C. and 15,000 rpm, and the supernatant was recovered into a fresh Eppendorf tube. According to the protocol of the kit, 50 μl of the recovered culturing supernatant was analyzed by an enzyme-linked immunosorbent assay (ELISA) (Mouse Total IL-12 ELISA Kit, Thermo SCIENTIFIC) of IL-12, and the concentration of IL-12 in the culturing supernatant was calculated (
As shown in
Since the present invention is useful as a preventive or a therapeutic for a viral disease and a cancer, it can be utilized in an industrial field such as pharmacy.
The present specification includes the contents described in the specification and/or the drawings of Japanese patent applications (Patent application Nos. 2011-107874 and 2011-191208) which are the basis of priority claim of the present application. Further, all publications, patents and patent applications cited in the present specification are incorporated herein by reference, as they are.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2011-107874 | May 2011 | JP | national |
| 2011-191208 | Sep 2011 | JP | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/JP2012/060910 | 4/24/2012 | WO | 00 | 6/6/2014 |
