Patent Application
20210187002
The present invention relates to an antimalarial drug, a malaria treatment method, a screening method for a candidate substance for malaria treatment, a malaria severity marker, a method for testing a risk of severe malaria, and a test reagent.
Vaccines are being developed to prevent malaria infection and to inhibit the symptoms from becoming severe when infected. However, currently, no clinically effective malaria vaccine has been developed.
Accordingly, it is an object of the present invention to provide a new antimalarial drug.
In order to achieve the above object, the present invention provides an antimalarial drug including: a binding inhibitor that inhibits binding between a RIFIN protein and a leukocyte immunoglobulin-like receptor subfamily B member 1 (LILRB1) protein; an inducer of the binding inhibitor; or an expression inhibitor of RIFIN or LILRB1.
The present invention also provides a malaria treatment method (hereinafter, also referred to as the “treatment method”), including: administering the antimalarial drug according to the present invention to a patient.
The present invention also provides a method for screening a candidate substance for malaria treatment (hereinafter, also referred to as the “screening method”), including: selecting, as a candidate substance for malaria treatment, a binding inhibitor that inhibits binding between a RIFIN protein and a leukocyte immunoglobulin-like receptor subfamily B member 1 (LILRB1) protein, an inducer of the binding inhibitor; or an expression inhibitor of RIFIN or LILRB1 from a test substance.
The present invention also provides a malaria severity marker (hereinafter, also referred to as the “marker”), wherein the marker is RIFIN.
The present invention also provides a method for testing a risk of severe malaria (hereinafter, also referred to as the “test method”), including: measuring an expression of RIFIN in a biological sample of a subject.
The present invention also provides a test reagent for use in the test method according to the present invention, including: a reagent for measuring an expression of RIFIN.
The present invention can provide a new antimalarial drug.
In the present invention, the meaning of “treatment” includes avoidance (prevention), inhibition (arrest), or suppression of the progression of symptoms; avoidance (prevention), inhibition (arrest), or suppression from becoming severe; improvement or amelioration of symptoms; or improvement of prognosis. The “treatment” may be any of these meanings.
In the present invention, the meaning of “becoming severe” includes cerebral malaria and severe anemia, and may mean either or both. The cerebral malaria is defined, for example, as follows: Blantyre coma score<3. The severe anaemia is defined, for example, as follows: blood haemoglobin<5 g/dl. The Blantyre coma score is, for example, the sum of the following endpoints (a), (b), and (c). The method for measuring the blood haemoglobin level is, for example, a cyanmethaemoglobin method.
(Endpoints)
The present invention will be described below with reference to examples. The present invention, however, is not limited to the following description. In addition, regarding the descriptions of the respective inventions, reference can be made to each other unless otherwise stated.
As described above, the antimalarial drug of the present invention includes: a binding inhibitor that inhibits binding between a RIFIN protein and a leukocyte immunoglobulin-like receptor subfamily B member 1 (LILRB1) protein; an inducer of the binding inhibitor; or an expression inhibitor of RIFIN or LILRB1. The antimalarial drug of the present invention is characterized in that it includes a binding inhibitor that inhibits binding between a RIFIN protein and a LILRB1 protein, an inducer of the binding inhibitor, or an expression inhibitor of RIFIN or LILRB1, and other configurations and conditions are not particularly limited. According to the antimalarial drug of the present invention, malaria can be treated. As will be described below, the antimalarial drug of the present invention can avoid (prevent), for example, malaria from becoming severe. Thus, the antimalarial drug of the present invention can be referred to as, for example, a drug for avoiding (preventing) malaria from becoming severe.
As a result of intensive studies, the inventors of the present invention have found that RIFIN expressed in infected erythrocytes infected with Plasmodium falciparum binds to LILRB1 expressed in immune system cells such as B cells and NK cells and suppresses the function of the immune system cells. The inventors of the present invention have also found that binding of LILRB1 protein, i.e., expression of LILRB1-binding RIFIN protein is higher in erythrocytes of severe malaria patients than in erythrocytes of mild malaria patients (non-severe malaria patients). Furthermore, the inventors of the present invention have found that, causing a binding inhibitor that inhibits the binding between a RIFIN protein and a LILRB1 protein to coexist, generation of a signal through a LILRB1 protein by a RIFIN protein can be inhibited. From these findings, the inventors of the present invention have found that Plasmodium falciparum evades the immune system through the binding between a RIFIN protein and a LILRB1 protein and causes malaria to become severe, and have established the present invention. According to the antimalarial drug of the present invention, for example, the binding between a RIFIN protein and a LILRB1 protein can be directly or indirectly inhibited, so that the generation of a signal through a LILRB1 protein by a RIFIN protein can be inhibited. Therefore, according to the antimalarial drug of the present invention, for example, the suppression of the function of the immune system cells by the binding between a RIFIN protein and a LILRB1 protein can be prevented or released. Therefore, the antimalarial drug of the present invention can treat malaria, such as by preventing malaria from becoming severe, for example.
In the present invention, RIFIN is derived from Plasmodium falciparum, for example. The RIFIN derived from Plasmodium falciparum can be referred to, for example, from information registered in an existing database. Specifically, examples of the protein of RIFIN of Plasmodium falciparum include the following amino acid sequences. The character strings shown after “Genbank:” in parentheses are the accession numbers in the Genbank of the respective RIFIN proteins (hereinafter, the same applies). Examples of mRNA of RIFIN of Plasmodium falciparum include base sequences registered with the NCBI accession numbers shown in Table 1 below.
Plasmodium falciparum RIFIN protein 1
Plasmodium falciparum RIFIN protein 2
Plasmodium falciparum RIFIN protein 3
Plasmodium falciparum RIFIN protein 4
Plasmodium falciparum RIFIN protein 5
Plasmodium falciparum RIFIN protein 6
Plasmodium falciparum RIFIN protein 7
Plasmodium falciparum RIFIN protein 8
Plasmodium falciparum RIFIN protein 9
Plasmodium falciparum RIFIN protein 10
Plasmodium falciparum RIFIN protein 11
Plasmodium falciparum RIFIN protein 12
Plasmodium falciparum RIFIN protein 13
Plasmodium falciparum RIFIN protein 14
Plasmodium falciparum RIFIN protein 15
Plasmodium falciparum RIFIN protein 16
Plasmodium falciparum RIFIN protein 17
Plasmodium falciparum RIFIN protein 18
Plasmodium falciparum RIFIN protein 19
Plasmodium falciparum RIFIN protein 20
Plasmodium falciparum RIFIN protein 21
Plasmodium falciparum RIFIN protein 22
Plasmodium falciparum RIFIN protein 23
Plasmodium falciparum RIFIN protein 24
Plasmodium falciparum RIFIN protein 25
Plasmodium falciparum RIFIN protein 26
Plasmodium falciparum RIFIN protein 27
Plasmodium falciparum RIFIN protein 28
Plasmodium falciparum RIFIN protein 29
Plasmodium falciparum RIFIN protein 30
Plasmodium falciparum RIFIN protein 31
Plasmodium falciparum RIFIN protein 32
Plasmodium falciparum RIFIN protein 33
The RIFIN preferably include, for example, a LILRB1-binding RIFIN. Examples of the LILRB1-binding RIFIN include the RIFINs described below. Preferable examples of the RIFIN include PF3D7_1254800 (SEQ ID NO: 4), PF3D7_0223100 (SEQ ID NO: 3),
PF3D7_1100400 (SEQ ID NO: 6), PF3D7_0632700 (SEQ ID NO: 9), PF3D7_0700200 (SEQ ID NO: 5), and PF3D7_0100200 (SEQ ID NO: 1) because of their stronger binding to LILRB1. The character strings shown after “Genbank:” in parentheses are the accession numbers in the Genbank of the respective RIFIN proteins.
In the present invention, the origin of leukocyte immunoglobulin-like receptor subfamily B member 1 (LILRB1) is, for example, human. The human-derived LILRB1 can be referred to, for example, from information registered in an existing database. Specific examples of the human-derived LILRB1 include the following base sequence (SEQ ID NO: 34) registered with NCBI accession NO. NM_001081637.2 as an mRNA and the following amino acid sequence (SEQ ID NO: 35) registered with NCBI accession NO. NP_001075106.2 as a protein. It is to be noted that, in the base sequence of LILRB1 mRNA and the amino acid sequence of LILRB1 protein described below, the underlined region is a region corresponding to the extracellular region of LILRB1 used for the preparation of LILRB1-Fc in Examples to be described below.
CTCTGGGCTGAACCAGGCTCTGTGATCACCCAGGGGAGTCCTGTGACCCTC
AAAACAGCACCCTGGATTACACGGATCCCACAGGAGCTTGTGAAGAAGGGC
CAGTTCCCCATCCCATCCATCACCTGGGAACACACAGGGCGGTATCGCTGT
TACTATGGTAGCGACACTGCAGGCCGCTCAGAGAGCAGTGACCCCCTGGAG
CTGGTGGTGACAGGAGCCTACATCAAACCCACCCTCTCAGCCCAGCCCAGC
CCCGTGGTGAACTCAGGAGGGAATGTAACCCTCCAGTGTGACTCACAGGTG
GCATTTGATGGCTTCATTCTGTGTAAGGAAGGAGAAGATGAACACCCACAA
TGCCTGAACTCCCAGCCCCATGCCCGTGGGTCGTCCCGCGCCATCTTCTCC
GTGGGCCCCGTGAGCCCGAGTCGCAGGTGGTGGTACAGGTGCTATGCTTAT
GACTCGAACTCTCCCTATGAGTGGTCTCTACCCAGTGATCTCCTGGAGCTC
CTGGTCCTAGGTGTTTCTAAGAAGCCATCACTCTCAGTGCAGCCAGGTCCT
ATCGTGGCCCCTGAGGAGACCCTGACTCTGCAGTGTGGCTCTGATGCTGGC
TACAACAGATTTGTTCTGTATAAGGACGGGGAACGTGACTTCCTTCAGCTC
GCTGGCGCACAGCCCCAGGCTGGGCTCTCCCAGGCCAACTTCACCCTGGGC
CCTGTGAGCCGCTCCTACGGGGGCCAGTACAGATGCTACGGTGCACACAAC
CTCTCCTCCGAGTGGTCGGCCCCCAGCGACCCCCTGGACATCCTGATCGCA
GGACAGTTCTATGACAGAGTCTCCCTCTCGGTGCAGCCGGGCCCCACGGTG
GCCTCAGGAGAGAACGTGACCCTGCTGTGTCAGTCACAGGGATGGATGCAA
ACTTTCCTTCTGACCAAGGAGGGGGCAGCTGATGACCCATGGCGTCTAAGA
TCAACGTACCAATCTCAAAAATACCAGGCTGAATTCCCCATGGGTCCTGTG
ACCTCAGCCCATGCGGGGACCTACAGGTGCTACGGCTCACAGAGCTCCAAA
CCCTACCTGCTGACTCACCCCAGTGACCCCCTGGAGCTCGTGGTCTCAGGA
CCGTCTGGGGGCCCCAGCTCCCCGACAACAGGCCCCACCTCCACATCTGCA
GGCCCTGAGGACCAGCCCCTCACCCCCACCGGGTCGGATCCCCAGAGTGGT
CTGGGAAGGCACCTGGGGGTTGTGATCGGCATCTTGGTGGCCGTCATCCTA
QGGQETQEYRLYREKKTAPWITRIPQELVKKGQFPIPSITWEHTGRYRC
YYGSDTAGRSESSDPLELVVTGAYIKPTLSAQPSPVVNSGGNVTLQCDS
QVAFDGFILCKEGEDEHPQCLNSQPHARGSSRAIFSVGPVSPSRRWWYR
CYAYDSNSPYEWSLPSDLLELLVLGVSKKPSLSVQPGPIVAPEETLTLQ
CGSDAGYNRFVLYKDGERDFLQLAGAQPQAGLSQANFTLGPVSRSYGGQ
YRCYGAHNLSSEWSAPSDPLDILIAGQFYDRVSLSVQPGPTVASGENVT
LLCQSQGWMQTFLLTKEGAADDPWRLRSTYQSQKYQAEFPMGPVTSAHA
GTYRCYGSQSSKPYLLTHPSDPLELVVSGPSGGPSSPTTGPTSTSAGPE
DQPLTPTGSDPQSGLGRHLGVVIGILVAVILLLLLLLLLFLILRHRRQG
In the present invention, the binding inhibitor is only required to inhibit the binding between a RIFIN protein and a LILRB1 protein. The inhibition may be, for example, direct inhibition or indirect inhibition. The inhibition means, for example, that the formation amount of the complex of a RIFIN protein and a LILRB1 protein when the complex is formed in the presence of the binding inhibitor is (e.g., significantly) reduced compared to the formation amount of the complex when the complex is formed in the absence of the binding inhibitor, for example. The formation of the complex of a RIFIN protein and a LILRB1 protein can be measured, for example, with reference to from Examples described below, by bringing a labeled RIFIN protein or LILRB1 protein into contact with a subject expressing a LILRB1 protein or a RIFIN protein, and detecting the label in the subject after the contact. The measurement can be performed using, for example, a flow cytometer or the like.
The binding inhibitor may be a binding substance that binds to the RIFIN protein, a binding substance that binds to the LILRB1 protein, or the like. When the binding inhibitor is a binding substance that binds to the RIFIN protein, the binding inhibitor is preferably a binding substance that binds to a variable region of the RIFIN protein. The binding substance that binds to the variable region of the RIFIN protein binds to a part or all of the variable region of the RIFIN protein, for example. The variable and conserved regions of the RIFIN protein mean the amino acid regions described below, for example.
(Variable and Conserved Regions of RIFIN Protein)
The type of the binding inhibitor is not particularly limited, and examples thereof include low molecular weight compounds, peptides, proteins, and nucleic acids. As a specific example, when the binding inhibitor is a peptide or a protein, the binding inhibitor may be, for example, an antibody or an antigen binding fragment thereof. The antibody may be, for example, a monoclonal antibody or a polyclonal antibody (hereinafter, the same applies). When the binding inhibitor is a peptide or a protein, the binding inhibitor may be, for example, a protein or a peptide that binds to a RIFIN protein, a protein or a peptide that binds to a LILRB1 protein, or the like. The protein or peptide that binds to a RIFIN protein may be, for example, an antibody that binds to a RIFIN protein or an antigen-binding fragment thereof, a solubilized product of a LILRB1 protein or a RIFIN protein-binding fragment thereof, or the like. The solubilized product of a LILRB1 protein may be, for example, a fusion protein of the Fc-region (fragment crystallizable region) of the antibody and a LILRB1 protein or a RIFIN protein-binding fragment thereof. The protein or peptide that binds to the LILRB1 protein may be, for example, an antibody that binds to a LILRB1 protein or an antigen-binding fragment thereof, a decoy peptide of a RIFIN protein, or the like. The decoy peptide of a RIFIN protein is, for example, a peptide that inhibits the binding of a RIFIN protein to a LILRB1 protein and that does not activate a LILRB1 protein. The activation of a LILRB1 protein means, for example, that the function of the LILRB1 protein is exerted. The peptide may be, for example, a cyclic peptide or a special cyclic peptide (hereinafter, the same applies). When the binding inhibitor is a nucleic acid, the binding inhibitor may be, for example, a binding nucleic acid molecule that binds to a RIFIN protein, a binding nucleic acid molecule that binds to a LILRB1 protein, or the like. The binding nucleic acid molecule may be, for example, DNA, RNA, or an aptamer consisting of DNA and RNA. For example, one type of the binding inhibitors may be used alone, or two or more types of them may be used in combination.
The inducer of the binding inhibitor is, when administered to a patient (hereinafter, also referred to as an “administration subject”), a substance that induces the binding inhibitor in the administration subject, for example. The binding inhibitor is preferably an antibody. Examples of the type of the inducer include a peptide, a protein, and a nucleic acid. The inducer is preferably used in combination with the adjuvant to be described below, for example, so that the binding inhibitor can be efficiently induced. The inducer may be, for example, the RIFIN protein or a part of the RIFIN protein, the LILRB1 protein or a part of the LILRB1 protein, or a polynucleotide encoding at least one of them. The part of the RIFIN protein preferably includes a variable region of a RIFIN protein, for example, so that a binding inhibitor that effectively inhibits the binding between a RIFIN protein and a LILRB1 protein can be induced. In this case, the inducer includes, for example, a part or all of the variable region of a RIFIN protein. When the inducer is the polynucleotide, the polynucleotide is functionally linked to, for example, a vector. The vector may be, for example, a known vector such as an adenovirus vector. The polynucleotide may be, for example, a DNA consisting of a deoxyribonucleotide, an RNA consisting of a ribonucleotide, or a polynucleotide consisting of DNA and RNA. The inducer may induce the expression of the binding inhibitor in the administration subject, for example. In this case, examples of the inducer include a polynucleotide encoding a synthetase of a low molecular weight compound, a peptide, or a protein; a polynucleotide containing a nucleic acid serving as a template of the nucleic acid serving as the binding inhibitor; and a vector containing the same. For example, one type of the inducers may be used alone or two or more types of them may be used in combination.
The expression inhibitor is not particularly limited, and examples thereof include a substance that inhibits an expression of mRNA of a RIFIN gene or a LILRB1 gene; a substance that cleaves expressed mRNA, and a substance that inhibits translation of a protein from the expressed mRNA. Specific examples of the expression inhibitor include a RNA-interfering agent such as siRNA, an antisense, and a ribozyme. The expression inhibitor may be, for example, a gene editing system capable of editing the base sequence of at least one of a RIFIN gene of Plasmodium falciparum and a LILRB1 gene of the patient. The genetic editing system may be, for example, a known gene editing system such as ZFN, TALEN, CRISPR-Cas9, or the like or a known mRNA editing system such as CRISPR-Cas13, or the like. For example, one type of the expression inhibitors may be used alone or two or more types of them may be used in combination.
The antimalarial drug of the present invention is only required to include any one or more of the binding inhibitor, the inducer, and the expression inhibitor, and may include two or all of them.
When the type of the antimalarial drug of the present invention is a peptide, a protein, or a nucleic acid, the peptide, the protein, and the nucleic acid may be modified and/or varied with one or more amino acids or one or more nucleotides, for example.
The modification is not particularly limited, and may be, for example, modification of the N-terminal, the C-terminal, or the side chain of a peptide or a protein, modification of a base, a sugar, a phosphate group, or a sugar phosphate backbone of a nucleic acid, or the like.
When the antimalarial drug of the present invention is a peptide or a protein, the variant of the peptide or the protein may be, for example, a peptide or a protein consisting of an amino acid sequence in which one to several amino acids are deleted, substituted, inserted and/or added in the amino acid sequence of the peptide or the protein, and/or a peptide or a protein consisting of an amino acid sequence having 80% or more identity with respect to the amino acid sequence of the peptide or protein. When the peptide or protein is a RIFIN protein or a part thereof, the variant binds to a LILRB1 protein, for example. Also, when the peptide or the protein is a LILRB1 protein or a part thereof, the variant binds to a RIFIN protein, for example. When the peptide or the protein is an antibody that binds to a RIFIN protein or an antigen-binding fragment thereof, the variant binds to a LILRB1 protein, for example. When the peptide or the protein is an antibody that binds to a LILRB1 protein or an antigen-binding fragment thereof, the variant binds to a RIFIN protein, for example. The foregoing numerical range of “one to several” include, for example, 1 to 76, 1 to 56, 1 to 37, 1 to 18, 1 to 15, 1 to 11, 1 to 7, 1 to 3, or 1 or 2. In the present invention, for example, the numerical range regarding the number of amino acids or the like discloses all the positive integers falling within that range. That is, for example, the numerical range of “1 to 5” discloses all of “1, 2, 3, 4, and 5” (the same applies hereinafter). The “identity” is, for example, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more. The “identity” can be calculated by default parameters using analysis software such as BLAST, FASTA, or the like (hereinafter, the same applies).
When the antimalarial drug of the present invention is a nucleic acid, the variant of the nucleic acid may be, for example, a polynucleotide consisting of a base sequence in which one to several bases are deleted, substituted, inserted and/or added in the base sequence of the nucleic acid, and/or a polynucleotide consisting of a base sequence having 80% or more identity with respect to the base sequence of the nucleic acid. When the nucleic acid is a nucleic acid encoding a RIFIN protein or a part thereof, the protein or the peptide encoded by the variant binds to a LILRB1 protein, for example. Also, when the nucleic acid is a nucleic acid encoding a LILRB1 protein or a part thereof, the protein or the peptide encoded by the variant binds to a RIFIN protein, for example. When the nucleic acid is a nucleic acid that binds to a RIFIN protein, the variant binds to a LILRB1 protein, for example. When the nucleic acid is a nucleic acid that binds to a LILRB1 protein, the variant binds to a RIFIN protein, for example. The foregoing numerical range of “one to several” include, for example, 1 to 228, 1 to 168, 1 to 111, 1 to 54, 1 to 45, 1 to 33, 1 to 21, 1 to 9, 1 to 6, 1 to 3, or 1 or 2. The “identity” is, for example, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more.
The antimalarial drug of the present invention may contain other components, such as, for example, a pharmaceutically acceptable carrier. In this case, the antimalarial drug of the present invention can also be referred to as a “pharmaceutical composition”, for example. The other components are not particularly limited, and examples thereof include preservatives, antioxidants, chelating agents, stabilizing agents, emulsifying agents, dispersing agents, suspending agents, and thickening agents. Examples of the preservative include thimerosal and 2-phenoxyethanol. Examples of the chelating agent include an ethylenediaminetetraacetic acid and a glycol ether diaminetetraacetic acid.
When the antimalarial drug of the present invention includes the inducer, the antimalarial drug of the present invention preferably includes an adjuvant as described above. In this case, the antimalarial drug of the present invention can also be referred to, for example, as a “vaccine” or a “vaccine composition”. The adjuvant is not particularly limited, and examples thereof include known adjuvants such as aluminum hydroxide, aluminum phosphate, aluminum chloride, lipopolysaccharide (LPS), Poly (I:C) (Polyinosinic-polycytidylic acid), complete Freund's adjuvant, incomplete Freund's adjuvant, Toll like receptor stimulators including CpG oligonucleotides, and the like, and saponins.
The antimalarial drug of the present invention can directly or indirectly inhibit the binding between a RIFIN protein and a LILRB1 protein, for example, by administrating on to an administration subject. As a specific example, the binding inhibitor inhibits, by binding to a RIFIN protein or a LILRB1 protein, binding of a LILRB1 protein to a RIFIN protein or a RIFIN protein to a LILRB1 protein, for example. The inducer induces the binding inhibitor, for example, in the administration subject, thereby inhibiting binding of a LILRB1 protein to a RIFIN protein or binding of a RIFIN protein to a LILRB1 protein. In addition, the expression inhibitor inhibits, by inhibiting expression of a RIFIN protein or a LILRB1 protein in the administration subject, binding of a LILRB1 protein to a RIFIN protein or binding of a RIFIN protein to a LILRB1 protein, for example.
For example, as described above, the antimalarial drug of the present invention can directly or indirectly inhibit the binding between a RIFIN protein and a LILRB1 protein, so that the generation of a signal through a LILRB1 protein by a RIFIN protein can be inhibited or the signal can be suppressed. Therefore, according to the antimalarial drug of the present invention, for example, the suppression of the function of the immune system cells by the binding between a RIFIN protein and a LILRB1 protein can be prevented or released. Thus, the antimalarial drug of the present invention may contain, for example, a signal inhibitor that inhibits a signal through a LILRB1 protein in addition to or in place of the binding inhibitor, the inducer, and/or the expression inhibitor. Examples of the signal inhibitor include an antagonist of LILRB1 protein, an active inhibitor of a signal transduction protein of LILRB1 protein, the binding inhibitor, the inducer, and the expression inhibitor. The type of the signal inhibitor is not particularly limited, and examples thereof include low molecular weight compounds, peptides, proteins, and nucleic acids. The inhibition of the signal means, for example, that the amount of a signal through a LILRB1 protein when the complex of a ligand (e.g., RIFIN protein) of a LILRB1 protein and a LILRB1 protein is formed in the presence of the signal inhibitor is (e.g., significantly) reduced compared to the amount of a signal through a LILRB1 protein in the absence of the signal inhibitor, for example. The amount of the signal can be measured, for example, with reference to the Examples described below, using reporter cells expressing reporter genes when a signal through a LILRB1 protein is generated. The measurement can be performed using, for example, a flow cytometer or the like.
In the present invention, examples of the administration subject (patient) include humans and non-human animals excluding humans. Examples of the non-human animals include mice, rats, dogs, monkeys, rabbits, sheep, horses, guinea pigs, and cats.
The dose of the antimalarial drug of the present invention is not particularly limited, and can be set as appropriate depending on the type of the antimalarial drug, the type, symptom, and age of the administration subject, and the administration method, for example. As a specific example, when the antimalarial drug of the present invention is a peptide and the antimalarial drug is administered to a human, the dose of the peptide per administration is 0.8 to 30 mg or 10 to 30 mg, for example. The number of administrations of the peptide is not particularly limited, and is 1 to 3 times, for example.
When the antimalarial drug of the present invention is a protein and the antimalarial drug is administered to a human, the dose of the protein per administration is 1 to 100 mg or 10 to 1000 mg, for example. The number of administrations of the protein is not particularly limited, and is 1 to 5 times, for example.
The administration form (dosage form) of the antimalarial drug of the present invention is not particularly limited, and examples thereof include solutions, suspensions, emulsions, injections, sprays, and powders.
The administration method of the antimalarial drug of the present invention is not particularly limited, and may be, for example, intravenous injection, intramuscular injection, subcutaneous administration, intradermal administration, transdermal administration, rectal administration, intraperitoneal administration, local administration, transnasal administration, or sublingual administration.
The method for producing the antimalarial drug of the present invention is not particularly limited, and, for example, a known method can be adopted depending on the type of the antimalarial drug. When the antimalarial drug of the present invention includes a low-molecular compound, the low-molecular compound can be produced by a known method, for example, depending on its structure.
When the antimalarial drug of the present invention includes a peptide, examples of the method for producing the peptide include a chemical synthesis method, a method for producing the peptide by degradation of a protein containing the peptide, and a synthesis method using recombinant DNA technology. In the chemical synthesis method, the peptide can be produced by a known organic synthesis method using, for example, a protecting group such as a benzyloxycarbonyl group (Cbz), a tert-butoxycarbonyl group (Boc), a fluorenylmethoxycarbonyl group (Fmoc), or the like. When the peptide is produced by the degradation of the protein containing the peptide, the peptide can be produced, for example, by degrading the protein containing the peptide with a known proteolytic enzyme such as a protease, a peptidase, or the like. The degradation condition of the protein containing the peptide can be set as appropriate depending on, for example, the type of the protein containing the peptide, the substrate specificity of the proteolytic enzyme, and the like. When the peptide is produced using the recombinant DNA technology, for example, an expression vector containing a polynucleotide encoding the peptide, is created, then, an expression system of the peptide is produced, and the expressed peptide is isolated, thereby producing the peptide. The expression system can be produced, for example, by introducing the expression vector into a host. Examples of the host include known hosts such as animal cells, plant cells, insect cells, and bacteria. When the peptide is produced using the recombinant DNA technology, for example, a polynucleotide encoding the peptide and a known cell-free translation system may be used. The peptide can be produced by isolating the peptide translated from the polynucleotide by the cell-free translation system.
When the antimalarial drug of the present invention includes a protein, examples of the method for producing the protein include a chemical synthesis method and a synthesis method using recombinant DNA technology. Regarding the method for producing the protein, for example, reference can be made to the description as to the method for producing the peptide by replacing “peptide” with “protein”. When the protein contains an antibody, the method for producing the antibody may be, for example, the same as the method for producing the protein, a method for immunizing the animal with an antigen of the antibody and then collecting a serum, or a method for culturing cells such as hybridomas that produce the antibody.
When the antimalarial drug of the present invention includes a nucleic acid, the method for producing the nucleic acid is not particularly limited, and examples thereof include a chemical synthesis method such as a phosphoramidite method and a synthesis method using recombinant DNA technology. When the nucleic acid contains an aptamer, the aptamer can be obtained by the SELEX method using the RIFIN protein or the LILRB1 protein, for example.
<Malaria Treatment Method>
The malaria treatment method of the present invention is characterized in that the method includes administering the antimalarial drug of the present invention to a patient, as described above. The treatment method of the present invention is characterized in that the method includes administering the antimalarial drug of the present invention to a patient, and the other steps and conditions are not particularly limited. According to the treatment method of the present invention, it is possible to treat malaria such as preventing malaria from becoming severe. Regarding the treatment method of the present invention, for example, reference can be made to the description as to the antimalarial drug of the present invention.
The treatment method of the present invention may be performed, for example, on a patient who is determined to be at risk of malaria becoming severe in the test method of the present invention to be described below. In this case, the treatment method of the present invention may be carried out, for example, in combination with the test method of the present invention. Regarding the specific steps of testing the risk of severe malaria, reference can be made to the description as to the test method of the present invention. The patient may be a patient not infected with malaria, a patient infected with malaria, or a patient with unknown malaria status.
<Screening Method for Candidate Substance for Malaria Treatment>
As described above, the method for screening a candidate substance for malaria treatment of the present invention includes: selecting, as a candidate substance for malaria treatment, a binding inhibitor that inhibits binding between a RIFIN protein and a leukocyte immunoglobulin-like receptor subfamily B member 1 (LILRB1) protein, an inducer of the binding inhibitor; or an expression inhibitor of RIFIN or LILRB1 from a test substance. The screening method of the present invention is characterized in that the candidate substance for malaria treatment is a binding inhibitor that inhibits binding between a RIFIN protein and a LILRB1 protein, an inducer of the binding inhibitor; or an expression inhibitor of RIFIN or LILRB1, and other steps and condition are not particularly limited. Regarding the screening method of the present invention, reference can be made to the description as to the antimalarial drug and treatment method of the present invention, for example.
Examples of the type of the binding substance include a low molecular weight compound, a peptide, a protein, and a nucleic acid. The RIFIN is preferably a LILRB1-binding RIFIN so that it can effectively treat malaria, for example.
The screening method of the present invention includes: detecting binding between the RIFIN protein and the LILRB1 protein in the presence of the RIFIN protein, the LILRB1 protein, and the test substance; and selecting, as the candidate substance for malaria treatment, the test substance that inhibits binding between the RIFIN protein and the LILRB1 protein, for example. In the detecting, the method of detecting the binding between a RIFIN protein and a LILRB1 protein is not particularly limited, and, for example, a known method of detecting the binding between proteins can be adopted, and reference can be made to the Examples to be described below.
The screening method of the present invention includes: administering the test substance to a living organism (administration subject); collecting a biological sample from the living organism; detecting binding between the RIFIN protein and the LILRB1 protein in the presence of the RIFIN protein, theLILRB1 protein, and the biological sample; and selecting, as the candidate substance for malaria treatment, the test substance that inhibits binding between the RIFIN protein and the LILRB1 protein, for example. In the administering, regarding the administration condition of the test substance, for example, reference can be made to the description as to the antimalarial drug of the present invention. The administering and the collecting are optional steps and may not be included. In such a case, in the detecting, a biological sample collected from a living organism to which the test substance has been administered is used as the biological sample, for example. The biological sample is not particularly limited, and examples thereof include blood, plasma, and serum.
The screening method of the present invention includes: bringing the test substance into contact with the RIFIN protein or the LILRB1 protein; detecting binding between the RIFIN protein or the LILRB1 protein and the test substance; and selecting, as the candidate substance for malaria treatment, the test substance binding to the RIFIN protein or the LILRB1 protein, for example.
The screening method of the present invention includes: causing the test substance to be coexist in an expression system of the RIFIN or the LILRB1 to express the RIFIN or the LILRB1; detecting an expression of the RIFIN or the LILRB1 in the expression system; and selecting, as the candidate substance for malaria treatment, the test substance with which the expression level of the RIFIN or the LILRB1 is lower than that of a control expression system in which the test substance is not present, for example. In detecting, the expression of the detection target may be the expression of the RIFIN protein or the LILRB1 protein, or the transcription of an mRNA of a RIFIN gene or a LILRB1 gene, for example. The methods for detecting the expression of the protein and the expression of the mRNA are not particularly limited, and known methods can be adopted, for example.
The screening method of the present invention may select, in addition to or in place of the binding inhibitor, the inducer, and/or the expression inhibitor, a signal inhibitor that inhibits a signal through a LILRB1 protein as a candidate substance for malaria treatment, for example. Examples of the signal inhibitor include an antagonist of LILRB1 protein, an active inhibitor of the signal transduction molecule such as a signal transduction protein of LILRB1 protein and the like, the binding inhibitor, the inducer, and the expression inhibitor. When the screening method of the present invention selects a signal inhibitor as the candidate substance for malaria treatment, the screening method of the present invention includes: detecting a signal through a LILRB1 protein in the presence of a ligand of the LILRB1 protein such as the RIFIN protein, the LILRB1 protein, and the test substance; and selecting the test substance that inhibits a signal through the LILRB1 protein as the candidate substance for malaria treatment, for example. In the detecting, the method for detecting a signal through the LILRB1 protein is not particularly limited, and, for example, a known detection method of signal transduction molecule can be adopted, and, for example, reporter cells in the Examples to be described below can be used.
<Malaria Severity Marker>
The malaria severity marker of the present invention is characterized in that the marker is RIFIN, as described above. The marker of the present invention is characterized in that RIFIN is used as the marker, and the other configurations and conditions are not particularly limited. The marker of the present invention can test the risk of severe malaria in the subject by measuring the expression of RIFIN in the biological sample of the subject, for example. Regarding the marker of the present invention, for example, reference can be made to the description as to the antimalarial drug, treatment method, and screening method of the present invention. The malaria is, for example, malaria caused by infection of the Plasmodium falciparum. The RIFIN is preferably a LILRB1-binding RIFIN because it is more correlated with the severe malaria, for example.
<Test Method>
The method for testing the risk of severe malaria of the present invention is characterized in that it includes measuring an expression of RIFIN in a biological sample of a subject, as described above. The test method of the present invention is characterized in that the expression of RIFIN is measured as a malaria severity marker, and other steps and conditions are not particularly limited. According to the test method of the present invention, it is possible to test the subject's risk of severe malaria. Regarding the test method of the present invention, for example, reference can be made to the description as to the antimalarial drug, treatment method, screening method, and marker of the present invention.
According to the test method of the present invention, for example, it is possible to evaluate the possibility of the progression of the malaria symptom, the possibility of malaria becoming severe, the evaluation of the prognosis, and the like. The malaria is, for example, malaria caused by infection of the Plasmodium falciparum.
Exampes of the subject include humans and non-human animals excluding humans. Examples of the non-human animals include, as described above, mammals such as mice, rats, dogs, monkeys, rabbits, sheep, horses, and the like.
The type of the biological sample is not particularly limited, and examples thereof include body fluids, body fluid-derived cells, organs, tissues, and cells separated from a living organism. The body fluid may be, for example, blood, and specifically, for example, whole blood. The body fluid-derived cells may be, for example, blood-derived cells, and specifically, blood cells such as hemocyte, white blood cells, erythrocytes, lymphocytes, and the like. Since RIFIN is expressed in, for example, erythrocytes in a patient infected with Plasmodium falciparum, the biological sample is preferably a biological sample containing blood or a biological sample containing erythrocytes.
The expression of RIFIN to be measured may be, for example, the expression of mRNA of a RIFIN gene or the expression of a RIFIN protein. With respect to the biological sample, only one of the expression of the mRNA and the expression of the protein or both of them may be measured. These measurement methods are not particularly limited, and known methods can be adopted. As a specific example, the method for measuring the expression of the mRNA may be, for example, a gene amplification method using a reverse transcription reaction such as a reverse transcription (RT)-PCR method. Specifically, for example, the cDNA is synthesized from mRNA by reverse transcription, and cDNA is used as a template to amplify genes. Examples of the method for measuring the expression of the protein include an immuno-antibody method, an ELISA method, a flow cytometry, and a Western blotting method. The expression of a RIFIN protein may also be measured, for example, using a LILRB1 protein. In this case, as a LILRB1 protein, for example, the LILRB1-Fc to be described below can be used. The RIFIN is preferably a LILRB1-binding RIFIN because it is more correlated with the severe malaria, for example.
In the measuring, for example, the presence or absence of the expression of RIFIN may be measured, or the expression level of RIFIN may be measured.
The test method of the present invention further includes testing the subject's risk of severe malaria based on the expression of RIFIN in the biological sample of the subject (hereinafter also referred to as “subject biological sample”), for example. When the measurement result in the measuring is the expression level of RIFIN, the testing includes testing the subject's risk of severe malaria by comparing the expression level of RIFIN in the biological sample of the subject with a reference value, for example. The reference value is not particularly limited, and may be the expression levels of RIFIN in healthy subjects, severe malaria patients, and malaria patients at different severities, for example. For prognostic evaluation, the reference value may be, for example, the expression level of RIFIN after treatment.
The reference value can be obtained, for example, by using a biological sample isolated from a healthy subject and/or a severe malaria patient (hereinafter also referred to as a “reference biological sample”), as described above. In the case of prognostic evaluation, for example, a reference biological sample isolated from the same subject after treatment may be used. The reference value may be measured at the same time as the subject biological sample of the subject, or may be measured in advance, for example. The latter case is preferable because, for example, it is unnecessary to obtain a reference value every time the subject biological sample of the subject is measured. Preferably, the subject biological sample of the subject and the reference biological sample are collected under the same conditions, and RIFIN is measured under the same conditions, for example.
In the testing, the method of evaluating the subject's risk of severe malaria is not particularly limited, and can be appropriately determined, for example, based on the measurement result obtained in the measuring. When the measurement result indicates the presence or absence of the expression of RIFIN, for example, the measurement can be performed as follows. As a specific example, when RIFIN is expressed in the subject biological sample of the subject, the subject can be evaluated as being at a risk of or at a high risk of severe malaria. When RIFIN is not expressed in the subject biological sample of the subject, the subject can be evaluated as having no risk or at a low risk of severe malaria. When the measurement result is the expression level of RIFIN, the evaluation method can be determined as appropriate depending on the type of the reference value, for example. As specific examples, when the expression level of RIFIN in the subject biological sample of the subject is significantly higher than the expression level of RIFIN in the reference biological sample of the healthy subject, when the expression level of RIFIN in the subject biological sample of the subject is the same as the expression level of RIFIN in the reference biological sample of the severe malaria patient (when there is no significant difference therebetween), and/or when the expression level of RIFIN in the subject biological sample of the subject is significantly higher than the expression level of RIFIN in the reference biological sample of the malaria patient, the subject can be evaluate as at a risk or at a high risk of severe malaria. On the other hand, when the expression level of RIFIN in the subject biological sample of the subject is the same as the expression level of RIFIN in the reference biological sample of the healthy subject (when there is no significant difference therebetween), when the expression level of RIFIN in the subject biological sample of the subject is significantly lower than the expression level of RIFIN in the reference biological sample of the healthy subject, and/or when the expression level of RIFIN in the subject biological sample of the subject is significantly lower than the expression level of RIFIN in the reference biological sample of the severe malaria patient, the subject can be evaluated as having no risk or at a low risk of malaria severe malaria. In the testing, the degree of progression of the severity of malaria can be evaluated by comparing the expression level of RIFIN in the subject biological sample of the subject with the expression levels of RIFIN in the reference biological samples of the malaria patients at different severities. Specifically, when the subject biological sample of the subject has an expression level equivalent to that of the reference biological sample of a certain severity (when there is no significant difference therebetween), for example, the subject can be evaluated as having a possibility of being at the certain severity.
In the testing, when the prognostic state is evaluated, for example, the evaluation may be made in the same manner as described above or the evaluation may be made using the expression level of RIFIN in a reference biological sample of the same subject after treatment as a reference value. As a specific example, when the expression level of RIFIN in the subject biological sample of the subject is significantly higher than the reference value, the subject can be evaluated as at a risk of relapse or aggravation (becoming severe) after the treatment. On the other hand, when the expression level of RIFIN in the subject biological sample of the subject is the same as the reference value (when there is no significant difference therebetween) and/or is significantly lower than the reference value, the subject can be evaluated as having no risk or a low risk of relapse after the treatment.
In the present invention, for example, the biological samples of the same subject may be collected over time, and the expression levels of RIFIN in the biological samples may be compared. Thereby, it is possible to judge that the possibility of becoming severe increases if the expression level increases over time, and it is possible to judge that the possibility of becoming severe decreases or that malaria has healed if the expression level decreases over time, for example.
<Test Reagent>
The test reagent of the present invention is characterized in that it includes a reagent for measuring an expression of RIFIN and is for use in the test method of the present invention, as described above. The test method of the present invention is characterized in that the reagent for measuring an expression of RIFIN is used in the test method of the present invention, and other configurations and conditions are not particularly limited. According to the test reagent of the present invention, the test method of the present invention can be easily carried out.
The expression measuring reagent may be any reagent as long as the expression of RIFIN can be measured, and the type of the expression measuring reagent is not particularly limited. The expression measurement reagent of RIFIN may be, for example, a reagent for measuring an expression of a RIFIN protein or a reagent for measuring an expression of mRNA of a RIFIN gene.
The former may be, for example, a binding substance that bind to a RIFIN protein, and specific examples thereof include an antibody and an antigen-binding fragment thereof. In this case, preferably, the test reagent of the present invention further contains, for example, a detection substance for detecting the binding between a RIFIN protein and the antibody or the antigen-binding fragment thereof. The detection substance can be, for example, a combination of a detectable labeled antibody to the antibody or the antigen-binding fragment thereof and a substrate to the label.
The latter may be, for example, a reagent that amplifies mRNA of a RIFIN gene by reverse transcription, and specific examples thereof include poly-dT, random primers, reverse transcriptase, dNTP, DNA polymerases, and primers. The primers can be designed as appropriate based on, for example, the base sequence of a RIFIN gene.
<Diagnostic Method and Diagnostic Reagent for Malaria Severity>
The method for diagnosing the severity of malaria is characterized in that it includes measuring the expression of RIFIN in a biological sample of a subject. The reagent for diagnosing the severity of malaria is characterized in that it includes a reagent for measuring the expression of RIFIN. Regarding the diagnostic method and diagnostic reagent of the present invention, for example, reference can be made to the description as to the test method and test reagent of the present invention.
<Use of Antimalarial Drug>
The present invention relates to a binding inhibitor that inhibits binding between a RIFIN protein and a leukocyte immunoglobulin-like receptor subfamily B member 1 (LILRB1 protein), an inducer of the binding inhibitor, or an expression inhibitor of RIFIN or LILRB1 for use in malaria treatment. The present invention relates to use of a binding inhibitor that inhibits binding between a RIFIN protein and a leukocyte immunoglobulin-like receptor subfamily B member 1 (LILRB1 protein), an inducer of the binding inhibitor, or an expression inhibitor of RIFIN or LILRB1 for producing an antimalarial drug. Regarding the use of the antimalarial drug of the present invention, for example, reference can be made to the description as to the antimalarial drug, treatment method, screening method, marker, test method, and test reagent of the present invention.
The present invention is described below in detail with reference to examples and the like. The present invention, however, is not limited thereto.
The present example examined whether LILRB1 binds to infected erythrocytes infected with Plasmodium falciparum.
(1) Preparation of LILRB1-Fc
A plasmid encoding a LILRB1-Fc fusion protein (LILRB1-Fc expression vector) was prepared in the same manner as described in Reference 1 below. In order to produce a biotinylated LILRB1-Fc protein, a LILRB1-Fc expression vector having an AviTag added to the C-terminal (Avi-LILRB1-Fc expression vector) was produced by inserting a polynucleotide encoding LILRB1-Fc into a pCAGGS expression vector. 293T cells (purchased from RIKEN Cell Bank) were transfected with the LILRB1-Fc and Avi-LILRB1-Fc expression vectors using a transfection reagent (PEI Max, Polysciences Inc.) according to the attached protocols. Then a culture supernatant containing LILRB1-Fc and Avi-LILRB1-Fc was obtained by culturing the 293T cells. Protein A was used to purify LILRB1-Fc and Avi-LILRB1-Fc from the resulting culture supernatant.
(2) Preparation of Clinical Strains of Plasmodium falciparum
Clinical strains of Plasmodium falciparum (hereinafter also referred to as “protozoa”) were prepared by separate on from malaria patients (Patients 1 to 7) resident in Mae Sariang district, Mae Hong Son Province, Thailand, and subjecting to limiting dilution. The clinical strains were cultured in a RPMI-1640 medium (containing 20% AlbuMAXI™, 25 mmol/l HEPES, 0.225% sodium bicarbonate, 0.38 mmol/1 hypoxatine, and 10 g/ml gentamycin) supplemented with human erythrocytes (purchased from the Japanese Red Cross Society, type O, hematocrit 2%). The culture conditions were set under 90% N2, 5% CO2 and 5% O2 atmosphere and at the culture temperature of 37° C.
(3) Culture of Plasmodium falciparum
The protozoa strains 3D7, CDC1, K1, FCR3 and Dd2, and the above-mentioned clinical strains were cultured in a RPMI-1640 medium with 10% human serum that contains human erythrocytes. The recombinant protozoa to be described below were maintained in a RPMI-1640 medium with 10% human serum and 25 ng/ml pyrimethamine (SIGMA). Using 5% D-sorbitol, the protozoa were synchronized to the ring stage. The protozoa in the trophozoite stage and the protozoa in the schizont stage were prepared by the Percoll density gradient centrifugation (GE Healthcare). Each Plasmodium falciparum was examined periodically for mycoplasma contamination by PCR.
(4) Binding Between LILRB1-Fc and Infected Erythrocyte
LILRB1-Fc was mixed with an APC-labeled anti IgG Fc antibody to form a complex. The complex was then incubated with infected erythrocytes infected with a 3D7 strain or with the clinical strain (derived from Patient 1) to stain the infected erythrocytes with the complex. After the staining, the resulting samples were analyzed by flow cytometry. As Control 1, the analysis was performed in the same manner except that the erythrocytes were not infected with protozoa, and as Control 2, the analysis was performed in the same manner except that only an APC-labeled anti IgG Fc antibody was used.
The present example examined whether a LILRB1 protein binds to infected erythrocytes infected with a clinical strain of Plasmodium falciparum.
Infected erythrocytes were stained with a complex of LILRB1-Fc and an APC-labeled anti IgG Fc antibody in the same manner as in Example 1, using the clinical strains of protozoa separated from Patients 1 to 7. The infected erythrocytes were subjected to nuclear staining using a nuclear staining agent (Vybrant® DyeCycle™ Green). After the staining, the resulting samples were analyzed by flow cytometry. As Control 1, the analysis was performed in the same manner except that the erythrocytes were not infected with the clinical strain, and as Control 2, the analysis was performed in the same manner except that a LILRA2-Fc fusion protein was added in place of LILRB1-Fc. LILRA2-Fc was prepared in the same manner as described in Reference 1.
The present example examined whether a LILRB1 protein binds to infected erythrocytes infected with different types of Plasmodium falciparum or at different stages.
Erythrocytes infected with protozoa synchronized to the ring stage, infected erythrocytes infected with protozoa in the trophozoite stage, and infected erythrocytes infected with protozoa in the schizont stage were prepared by the method described in Example 1(3). As the protozoa, protozoa derived from Patient 6 were used. The analysis was performed in the same manner as in Example 2 except that the infected erythrocytes infected with protozoa in the ring stage, trophozoite stage, or schizont stage were used in place of the clinical strain. Furthermore, the analysis was performed in the same manner except that the CDC1 strain, K1 strain, FCR3 strain, and Dd2 strain were used in place of protozoa at different stages. In addition, as a control, the analysis was performed in the same manner except that LILRA2-Fc was added in place of LILRB1-Fc.
The present example examined whether a ligand of a LILRB1 protein is a RIFIN protein. The present example also examined that a LILRB1 protein binds to various RIFIN proteins.
(1) Cloning Using Plasmodium falciparum 3D7 Strains
An APC-labeled anti IgG-Fc antibody was mixed with the LILRB1-Fc fusion protein to be described below to form a complex. The complex was then bonded to infected erythrocytes infected with a 3D7 strain. Then, the infected erythrocytes binding to LILRB1-Fc were enriched. The infected erythrocytes after enrichment were subjected to limiting dilution to obtain monoclonal protozoa. Specifically, the protozoa in the enriched infected erythrocytes were synchronized to the ring stage by culturing in the presence of 5% D-sorbitol. After replacing with a fresh medium and culturing for an additional 48 hours, the protozoa in the late schizont stage were purified by 63 (v/v)% Percoll (manufactured by Amersham Pharmacia Biotech) density gradient centrifugation method. The purified protozoa were mixed with a culture solution (RPMI-1640 medium with 10% O-type human serum, parasitemia: 1%, hematocrit: 2%) and cultured in a T-75 flask. The culture was carried out using a BNP-110 incubator (manufactured by TABAI ESPEC) for 1 hour at 37° C. under 90% N2, 5% CO2, and 5% O2 atmosphere. After the culture, the cap of the flask was tightly closed, and the flask was shaken at 100 rpm using an orbital shaker so as to prevent one cell of erythrocytes from being infected with multiple protozoa. The stage of the protozoa in the flask was checked by Giemsa staining every 2 hours. After confirming that the majority of the protozoa had migrated to the ring stage, the culture solution containing the protozoa was diluted with a fresh culture solution (3% hematocrit). The diluted protozoa (0.5 protozoa/0.2 ml) were seeded in 96-well flat bottom plates. Then, half of the culture solution was changed every 48 hours and cultured for 2 weeks. After the culture, the protozoa were detected in about 20 wells per plate. Whether or not each well contained erythrocytes to which a LILRB1 protein binds was screened by flow cytometric analysis. Thereby, F2 clones in which a LILRB1 protein binds and D11 clones in which a LILRB1 protein does not bind were obtained.
(2) Identification of LILRB1 Ligand
The Avi-LILRB1-Fc obtained in Example 1 (1) was biotinylated with BirA biotin ligase. Next, ghost cells of infected erythrocytes were prepared. Specifically, ghost cells of infected erythrocytes were obtained by mixing infected erythrocytes with a low-permeate (40 times the volume of infected erythrocytes) obtained by diluting a RPMI-1640 medium 5-fold with DDW (twice distilled water), incubating the resultant at 4° C. for 15 minutes followed by centrifugation at 15,000 rpm for 15 minutes and washing three times with the low-permeate. It is to be noted that the ghost cells of the infected erythrocytes were prepared using infected erythrocytes infected with protozoa of the F2 and D11 clones in the schizont stage. Then, ghost cells obtained from the infected erythrocytes infected with protozoa of the F2 and D11 clones in the schizont stage were inclubated with biotinized LILRB1-Fc and then cross-linked with 0.25 mmol/l 3,3-dithiobis (sulfosuccinimidyl propionate) (DTSSP, manufactured by Thermo Scientific). The ghost cells were then washed with a phosphate buffer (PBS) and boiled with a sample buffer without 2-mercaptoethanol. After the boil, a coprecipitate containing LILRB1-Fc was obtained from the post-boil sample using streptavidin sephalose. The coprecipitate was eluted at 50 mmol/l DTT (dithiothreitol), trypsinized and subjected to mass spectrometry (LC-MS/MS). MS/MS spectra were analyzed using software (Mascot). As a control, the analysis was performed in the same manner except that ghost cells not treated with biotinylated LILRB1-Fc were used. A similar analysis was performed one more time independently.
As a result, in any of the two independent analyses, an amino acid sequence (FHEYDER (SEQ ID NO: 36)) that matches a partial sequence of a RIFIN protein was obtained only from the infected erythrocytes infected with the F2 clone. These results showed that a RIFIN protein expressed on erythrocytes due to infection with protozoa serves as a ligand of a LILRB1 protein.
(3) Production of Recombinant Protozoa
It is known that multiple types of RIFIN genes exist in Plasmodium falciparum. Therefore, recombinant protozoa expressing various RIFIN genes were produced, and the binding between a LILRB1 protein and a RIFIN protein was examined. Specifically, in order to produce the recombinant protozoa expressing RIFIN genes, the base sequence of corresponding genomic region was specified on the basis of the amino acid sequence of the RIFIN protein. Based on the base sequence of the genomic region, a primer was designed, and the primer was used to PCR-amplify the entire length of each RIFIN gene including introns from the genome of the 3D7 strain. The obtained amplified fragments were inserted into a PfCEN5 expression vector to obtain a PfCEN5 expression vector into which 19 types of RIFIN genes were inserted. Next, the base sequences of the constant region and the variable region of the inserted RIFIN genes were decoded, and it was examined that the base sequences mach 3D7 genome version 3 (release 32, PlasmoDB, http://plasmodb.org). The base sequence of cDNA of the obtained PfCEN5 expression vector was decoded using primers (5′-TTATCCTTATTTTTTAATAACTGCC-3′ (SEQ ID NO: 37) and 5′-GTTCGTGGCATTCCAC-3′ (SEQ ID NO: 38)) specific for the PfCEN5 expression vector. As a result, the introns were accurately spliced in both of the PfCEN5 expression vectors. The RIFIN genes that have been introduced into the PfCEN5 expression vector are as follows.
(RIFIN genes that have been introduced into PfCEN5 expression vector) PF3D7_1254800, PF3D7_0223100, PF3D7_1100400, PF3D7_0632700, PF3D7_0700200, PF3D7_0100200, PF3D7_0900200, PF3D7_0600300, PF3D7_1480000, PF3D7_0100400, PF3D7_0632200, PF3D7_1254400, PF3D7_1479700, PF3D7_0632400, PF3D7_0101000, PF3D7_1000200, PF3D7_0732200, PF3D7_0937500, PF3D7_1300400
In order to produce recombinant protozoa, fresh erythrocytes were transfected with RIFIN-PfCEN5 expression vectors by electroporation, and then the erythrocytes were caused to be infected with a 3D7 strain. Four days after the infection, the infected erythrocytes were cultured in a pyrimesaminecontain-containing RPMI-1640 medium. The recombinant protozoa were then cultured and maintained in a RPMI-1640 medium with 10% human serum and 25 ng/ml pyrimethamine supplemented with human erythrocytes. Control recombinant protozoa were produced in the same manner except that a PfCEN5 expression vector into which a GFP gene had been inserted was used.
(4) Confirmation of Binding Between a LILRB1 Protein and a RIFIN Protein
The analysis was performed in the same manner as in Example 2 except that the above-described recombinant protozoa were used in place of the clinical strain. As a control, the analysis was performed in the same manner except that the control recombinant protozoa were used in place of the above-described recombinant protozoa.
The present example examined whether a LILRB1 protein binds to the variable region of a RIFIN protein.
A polynucleotide encoding the conserved region on the N-terminal side (an amino acid region extending from 39th to 139th amino acid residues in the amino acid sequence of SEQ ID NO: 4) or the variable region on the C-terminal side (an amino acid region extending from 166th to 275th amino acid residues in the amino acid sequence of SEQ ID NO: 4) of a RIFIN gene (PF3D7_1254800) and a polynucleotide encoding the transmembrane and intracellular region (an amino acid region extending from 196th to 256th amino acid residues in the amino acid sequence of SEQ ID NO: 39) of PILRα were coupled and the resultant was inserted into a pME18s expression vector. Thereby, an expression vector expressing the fusion protein containing the conserved region of a RIFIN gene and an expression vector expressing the fusion protein containing the variable region of a RIFIN gene were produced. The amino acid sequences of a fusion protein (SEQ ID NO: 40) containing a conserved region and a fusion protein (SEQ ID NO: 41) containing a variable region expressed by the expression vector of the fusion protein are as follows.
Next, 293T cells were transfected with the expression vector expressing the fusion protein containing the conserved region or the expression vector expressing the fusion protein containing the variable region using the transfection reagent according to the attached protocols. At the same time, the 293T cells were transfected with the expression vector containing the GFP gene. The analysis was performed in the same manner as in Example 2 except that 293T cells after being transceted with the vector were used in place of the infected erythrocytes. As Control 1, the analysis was performed in the same manner except that an APC-labeled anti FLAG antibody was added in place of LILRB1-Fc. As Control 2, the analysis was performed in the same manner except that the vector-free 293T cells were used.
The present example examined whether a RIFIN protein binds to a LILRB1 protein.
(1) Preparation of Recombinant RIFIN Protein A recombinant RIFIN protein having a His tagged added to the C-terminal was prepared as follows. First, codon-optimization was performed on a polynucleotide encoding the amino acid sequence of the variable region on the C-terminal side (an amino acid region extending from 166th to 275th amino acid residues in the amino acid sequence of SEQ ID NO: 4) of RIFIN (PF3D7_1254800). The codon-optimized polynucleotide was then inserted into a pET-15b expression vector capable of being added with a His-tag on the N-terminal. Using the obtained pET-15b expression vector, transformation of E. Coli BL21 (DE3) was performed by a conventional method. The obtained transformant was cultured in the presence of IPTG (isopropyl-β-thiogalactopyranoside) to express a recombinant RIFIN protein. After the expression, the transformant was collected and crushed, and a recombinant RIFIN protein was purified from the crush using TALON metal affinity chromatography. The purified recombinant RIFIN protein was then refolded.
(2) Preparation of LILRB1 Protein-Expressing 293T Cell
A polynucleotide encoding LILRB1 was inserted into a pMXs expression vector. The 293T cell was then transfected with an expression vector expressing a LILRB1 protein using the transfection reagent accoridng to the attached protocols. Thereby, a LILRB1 protein-expressing 293T cell was obtained.
(3) Binding Between Recombinant RIFIN Protein and LILRB1 Protein
The LILRB1 protein-expressing 293T cells and the purified recombinant RIFIN protein were mixed and incubated. After the incubation, the resultant was stained with an APC-labeled anti-His antibody (clone 28-75, manufactured by WAKO). The resulting sample was analyzed by flow cytometry.
The present example examined whether a RIFIN protein induces a signal through a LILRB1 protein.
(1) Production of Reporter Cell
A LILRB1 reporter cell was prepared in the same manner as in Reference 2 below. Specifically, the LILRB1 reporter cell was obtained by retroviral gene transfer of a fusion protein obtained by fusing PILRβ with the extracellular domains of NFAT-GFP, FLAG-tagged DAP12, and LILRB1 to a murine T-cell hybridoma.
(2) Reporter Assay
The recombinant RIFIN protein (10 μg/ml) produced in Example 6(1) was immobilized on 96-well plates. Then, the LILRB1 reporter cells were seeded so as to be 1×105 cells/wells and cultured for 16 hours. The culture was carried out at 37° C. under 5% CO2 atmosphere. After the culture, the expression of GFP caused by a LILRB1 signal was analyzed by flow cytometry. As Control 1, the analysis was performed in the same manner except that a recombinant RIFIN protein was not immobilized. Furthermore, the analysis was performed in the same manner except that recombinant protozoa transfected with the vector encoding PF3D7_1254800 of Example 4(3) were used in place of the recombinant RIFIN protein. As Control 2, the analysis was performed in the same manner except that no recombinant protozoa were added and only reporter cells were used.
The present example examined whether a RIFIN protein inhibits the activation of B cells through a LILRB1 protein.
(1) Production of RIFIN Protein-Expressing CHO Cell
Using the transfection reagent, CHO cells (puuchased from RIKEN Cell Bank) were transfected with the expression vector expressing the fusion protein containing the variable region of Example 5 and the expression vector expressing CD8 according to the attached protocols. Then, CD8-positive RIFIN-positive cells were isolated using autoMACS® Pro Separator (manufactured by Miltenyi Biotec).
(2) Measurement of B Cell Activity
Peripheral blood mononuclear cells (PBMC) were separated from healthy human blood by Ficoll density gradient centrifugation. Next, PBMC and CD8-positive RIFIN-positive cells were mixed at a ratio of 1:2 (cell number ratio) and cultured for 24 hours. The culture was carried out at 37° C. under 5% CO2 atmosphere. After the culture, PBMC was stimulated with K3CpG (2 μg/ml) for 3 days. After the stimulation, the culture supernatant was collected, and the IgM concentration in the culture supernatant was measured by ELISA. As Control 1, the measurement was performed in the same manner except that the expression vector containing a fusion protein obtained by fusing human MDA5 (human melanoma differentiation-associated protein 5) in place of the transmembrane region of PILRα. In addition, the measurement was performed in the same manner except that PBMC and the infected erythrocytes infected with the recombinant malaria were mixed at a ratio of 1:100 (cell number ratio), and cultured for 16 hours. As Control 2, the measurement was performed in the same manner except that PBMC alone was used and not stimulated with K3CpG. As Control 3, the measurement was performed in the same manner except that GFP-introduced recombinant malaria was used in place of the aforementioned recombinant malaria.
The present example examined whether a RIFIN protein inhibits the activation of NK cells through a LILRB1 protein.
(1) Production of RIFIN Protein-Expressing K562 Cell
K562 cells expressing a RIFIN protein was established by a retroviral gene expression system with reference to Reference 3 below. Specifically, a polynucleotide encoding the fusion protein was excised from an expression vector expressing the fusion protein containing the variable region of Example 5 and inserted into a pMX-expressing vector. The obtained pMX-expressing vector and PLAT-E retroviral packaging cells were used to prepare a retrovirus containing the expression vector. The K562 cells (purchased from the Institute of Aging Medicine, Tohoku University) were caused to be infected with the retrovirus to produce the K562 cells expressing a RIFIN protein (RIFIN-K562).
(2) Measurement of NK Cell Activity
RIFIN-K562 was labeled by culturing at 37° C. for 30 minutes in the presence of a complete medium (composition: phenol-red-free RPMI-1640 medium with 10% heat-inactivated FCS) containing 15 μmol/l Calcein AM (manufactured by Thermo Fisher Scientific). After the labeling, the resultant was washed twice with the complete medium and suspended with the complete medium so that RIFIN-K562 was 5×103/100 μl. The NK cell line NKL cells (obtained from Dr. Lanier, University of California) were washed twice with a complete medium. After the washing, NKL cells and RIFIN-K562 were seeded in 96-well plate at E:T ratios (NK cell line cell number: RIFIN-K562 cell number) of 50:1, 25:1, or 12.5:1. After the seeding, the plate was centrifuged at 100×g for 5 minutes and further cultured at 37° C. under 5% CO2 atmosphere for 4 hours. The plate was then centrifuged at 1500 rpm for 2 minutes and the fluorescence of the culture supernatant was measured by TriStar LB941 (manufactured by Berthold Technologies) (experimental release). Furthermore, the maximum release was measured by treating RIFIN-K562 with 2% Triton X-100-containing complete medium. As a control, the measurement was performed in the same manner except that NKL cells were not seeded (spontaneous release). The NK cell activity (dissolution rate) was calculated by the following formula (1). As a control, the calculation was performed in the same manner except that K562 cells were used in place of RIFIN-K562.
Dissolution rate=(experimental release−spontaneous release)/(maximum release−spontaneous release) (1)
The present example examined whether the binding of LILRB1 protein is higher in a severe malaria patient than in a mild malaria patient (non-severe malaria patient).
Blood was collected from patients with severe malaria (n=9) or with mild malaria (n=30) in Tanzania. After the blood collection, the blood was seeded on a petri dish in which LILRB1-Fc or LILRA2-Fc (control) was immobilized, and cultured. The petri dish was washed within 24 hours after the start of the culture to remove infected erythrocytes infected with protozoa not binding to the petri dish. Infected erythrocytes infected with protozoa binding to petri dish were immobilized by glutaraldehyde, followed by Giemsa staining and counting. For each patient, the number of infected erythrocytes infected with protozoa binding to the petri dish in which LILRA2-Fc was immobilized was subtracted from the number of infected erythrocytes infected with protozoa binding to the petri dish in which LILRB1-Fc was immobilized to calculate the number of infected erythrocytes binding to LILRB1-Fc. The severe malaria patients included those with cerebral malaria and those with severe anemia. The patients with cerebral malaria were defined as Blantyre coma score<3, and the patients with severe anaemia were defined as blood haemoglobin<5 g/dl. Blantyre coma score and blood haemoglobin were calculated by the methods described above. In addition, mild malaria patients were defined as non-severe malaria patients.
The present example examined whether inhibition of the interaction between a LILRB1 protein and a RIFIN protein is critical in preventing malaria from becoming severe.
The recombinant RIFIN proteins were immobilized on beads. Next, the immobilized beads were reacted with plasma from 222 Tanzanians, respectively. After the reaction, the binding of IgG to the immobilized beads was analyzed by Luminex (manufactured by Thermo Fisher Scientific Co., Ltd.), and the percentage of Tanzanians with RIFIN protein-binding IgG was calculated (RIFIN-1). The cut-off value was 2SD of the mean value of 43 European donors who have never had malaria. The calculation was performed in the same manner as described above except that the recombinant RIFIN protein of the variable region of PF3D7_1254200 was used in place of the aforementioned recombinant RIFIN protein (RIFIN-2). The percentage was calculated for each age.
These results suggest that RIFIN proteins are important in the early phase of infection and are important targets in host protective immunity. This led us to presume that the malaria can be prevented from becoming severe by inhibiting the interaction between a LILRB1 protein and a RIFIN protein in living organisms. This presumption does not limit the present invention in any way.
The present example examined whether an anti RIFIN antibody can inhibit the interaction between a LILRB1 protein and a RIFIN protein. The present example also examined whether an anti RIFIN antibody can inhibit a signal through a LILRB1 protein by a RIFIN protein.
(1) Preparation of Anti RIFIN Antibody-Containing Serum
In order to produce an anti RIFIN antibody, the recombinant RIFIN protein was mixed with an adjuvant (manufactured by TiterMax Gold, TiterMax USA, Inc.) and immunized mice threwith (Balb/c (n=2) and ICRs (n=2)). Specifically, each of 6-week-old female Balb/c and ICR mice were immunized with 50 μg of the recombinant RIFIN protein in an animal resource center for infectious diseases. Blood was collected after 2 weeks to obtain a serum.
In order to check the presence or absence of an anti RIFIN antibody in a murine serum, the binding to the beads in which the recombinant RIFIN protein was immobilized (RIFIN beads) was examined. Specifically, RIFIN beads were made by coupling the recombinant RIFIN proteins to Aldehyde/Sulfate Latex beads (3.8 manufactured by invirtogen, Inc., A37304). Then, the RIFIN beads and each serum diluted 100-fold with PBS were incubated for 15 minutes. After washing with PBS, the resultant was stained with an anti mouse IgG antibody (manufactured by Jackson, 5 μg/ml) for 15 minutes. After the staining, RIFIN beads were analyzed by flow cytometry. As a result, it was examined that all sera contained an antibody that binds to a RIFIN protein, i.e., anti RIFIN antibody.
(2) Inhibition of Binding Between RIFIN Protine and LILRB1 Protain by Anti RIFIN Antibody
In order to check whether an anti RIFIN antibodiy inhibits the interaction between a RIFIN and a LILRB1 protein, whether an anti RIFIN antibody-containing serums can inhibit the binding of LILRB1-Fc to RIFIN beads was analyzed. Specifically, the RIFIN beads and each serum diluted 100-fold with PBS were incubated for 15 minutes. After washing with PBS, the resultant was stained with LILRB1-Fc complexed with an APC-labeled anti IgG Fc antibodiy (10 μg/ml) for 15 minutes as described above. After the staining, RIFIN beads were analyzed by flow cytometry. As Control 1, the analysis was performed in the same manner as described above except that the serum was not added. As Control 2, the analysis was performed in the same manner as described above except that a serum from mice not immunized with recombinant RIFIN proteins was used. As Control 3, the analysis was performed in the same manner except that only an APC-labeled anti IgG Fc antibody not forming a complex was used.
(3) Inhibition of Signal Through LILRB1 Protein by RIFIN Protein Due to Anti RIFIN Antibody
The recombinant RIFIN protein (10 μg/ml) was immobilized on 96-well plates. Then, the LILRB1 reporter cells were seeded so as to be 1×105 cells/wells. In addition, the serum was added to each well at 100-fold dilution and cultured for 18 hours. The culture was performed at 37° C. under 5% CO2 atmosphere. After the culture, the expression of GFP caused by a LILRB1 signal was analyzed by flow cytometry. As a control, the analysis was performed in the same manner as described above except that a serum from mice not immunized with recombinant RIFIN proteins was used.
As described above, it is presumed that a RIFIN protein induces the malaria becoming severe by binding to a LILRB1 protein in the living organism. Moreover, it is considered that the severe malaria is due to the fact that a RIFIN protein inhibits the activation of immune cells such as B cells and NK cells by a signal through a LILRB1 protein. As described above, a binding inhibitor that inhibits the binding between a LILRB1 protein and a RIFIN protein such as an anti RIFIN antibody can inhibit a signal through a LILRB1 protein by a RIFIN protein. Therefore, by directly or indirectly inhibiting the binding between a RIFIN protein and a LILRB1 protein by the binding inhibitor, the inducer, and the expression inhibitor according to the present invention, it is possible to inhibit malaria becoming severe in a living organism.
While the present invention has been described above with reference to illustrative embodiments and examples, the present invention is by no means limited thereto. Various changes and variations that may become apparent to those skilled in the art may be made in the configuration and specifics of the present invention without departing from the scope of the present invention.
This application claims priority from Japanese Patent Application No. 2017-228226 filed on Nov. 28, 2017. The entire subject matter of the Japanese Patent Applications is incorporated herein by reference.
(Supplementary Notes)
Some or all of the above embodiments and examples may be described as in the following Supplementary Notes, but are not limited thereto.
As described above, according to the antimalarial drug of the present invention, malaria can be treated. Thus, the present invention is extremely useful, for example, in the clinical field.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2017-228226 | Nov 2017 | JP | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/JP2018/043698 | 11/28/2018 | WO | 00 |
